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The hierarchy 1. Atoms (C, H, O, N, P, S make up about 99% of living matter). 2. Molecules and macromolecules (proteins, nucleic acids, lipids, carbohydrates). 3. Supramolecular complexes (ribosomes, membranes, chromatin). 4. Organelles (nucleus, mitochondria). 5. Cell: the basic unit of life. 6. Tissue: similar cells plus extracellular matrix performing a common function. 7. Organ: several tissues forming a structural and functional unit (stomach, heart). 8. Organ system (apparatus): organs working together (digestive system). 9. Organism. Disciplines - Cytology: the cell. - Histology: tissues. - Microscopic anatomy: how tissues are assembled in organs. - Embryology: development of the organism from the zygote. Properties of living cells - Metabolism (exchange of matter and energy), growth, reproduction, response to stimuli, homeostasis, heredity (genetic information in DNA), evolution. Why size matters for histology - Most cells are 10-30 µm, below the resolution of the naked eye (about 100-200 µm), so we need the light microscope (resolution about 0.2 µm) for cells and tissues, and the electron microscope (about 0.1-1 nm in practice) for organelles. - Units: 1 mm = 1,000 µm; 1 µm = 1,000 nm. A red blood cell is about 7.5 µm, a useful internal ruler on any slide. Exam focus: define tissue, and name the four basic tissues with one defining feature each.

Water - About 70% of cell mass. A polar solvent; hydrogen bonds; high heat capacity. Hydrophobic interactions (the exclusion of non-polar molecules from water) drive membrane and protein folding. Carbohydrates - Monosaccharides: glucose, fructose, galactose (hexoses); ribose and deoxyribose (pentoses). - Oligosaccharides: a few monosaccharides; disaccharides (sucrose, lactose, maltose); the sugar chains on glycoproteins and glycolipids (the glycocalyx). - Polysaccharides: glycogen (animal energy store, seen as granules in liver and muscle), starch, cellulose; glycosaminoglycans of the extracellular matrix (hyaluronic acid, chondroitin sulfate). - Functions: energy, structure, cell recognition. Lipids - Fatty acids: saturated (straight chains, pack tightly) or unsaturated (double bonds cause kinks, increase membrane fluidity). - Triglycerides: glycerol plus three fatty acids; energy store in adipocytes. - Phospholipids: glycerol, two fatty acids, phosphate and a head group. Amphipathic: form bilayers. - Steroids: cholesterol (membranes, precursor of steroid hormones and bile acids). - Histology note: routine processing uses alcohols and xylene, which dissolve lipids. Fat cells therefore appear empty on H&E sections. Proteins - Polymers of 20 amino acids linked by peptide bonds. - Structure levels: primary (sequence), secondary (α-helix, β-sheet), tertiary (3D fold), quaternary (several subunits). - Functions: enzymes, structure (collagen, keratin), transport, signalling, contraction, defence. Nucleic acids - DNA and RNA are polymers of nucleotides (sugar, phosphate, base). - DNA: deoxyribose; A, T, G, C; double helix; the genetic material. - RNA: ribose; U instead of T; usually single-stranded; mRNA, rRNA, tRNA and regulatory RNAs. - Histology note: nucleic acids are acidic, so they bind basic dyes (haematoxylin). Nuclei and ribosome-rich cytoplasm are basophilic (blue-purple); most proteins in the cytoplasm are eosinophilic (pink). Exam focus: explain why a nucleus stains blue and a fat cell looks empty on H&E.

Viruses (10-300 nm) - Genome (DNA or RNA) in a protein capsid, sometimes with an envelope. - No metabolism or ribosomes; they replicate only inside host cells. At the border of life. Detail in Applied Biology. Prokaryotes (bacteria and archaea) - Size about 1-5 µm. - No nucleus: DNA in a nucleoid region; usually one circular chromosome, plus plasmids. - No membrane-bound organelles. 70S ribosomes. - Cell wall (peptidoglycan in bacteria), sometimes capsule, flagella, pili. Eukaryotes (protists, fungi, plants, animals) - Size about 10-100 µm; volume about 1,000 times a bacterium. - Nucleus with a double membrane; linear chromosomes with histones. - Internal membrane system (ER, Golgi, lysosomes, endosomes) and organelles (mitochondria, peroxisomes; chloroplasts in plants). - Cytoskeleton; 80S ribosomes in the cytosol. - Compartmentalisation lets incompatible reactions run in parallel, and supports a larger cell. Origin of eukaryotic compartments - Invagination hypothesis: the plasma membrane of an ancestral cell folded inward. The folds pinched off to form the endomembrane system and the nuclear envelope. This explains why the lumen of the ER and nuclear envelope is topologically equivalent to the outside of the cell. - Endosymbiotic theory: mitochondria (and chloroplasts) descend from engulfed bacteria. Evidence: double membrane, own circular DNA, 70S-like ribosomes, division by fission. Are all cells the same? - No. There are about 200 cell types in the human body, with different shapes and sizes that match their function: - red blood cell about 7.5 µm, biconcave, no nucleus - oocyte about 120 µm, the largest human cell - neurons with axons up to 1 m long - skeletal muscle fibres: multinucleated syncytia several cm long - epithelial cells with microvilli (absorption) - The "typical cell" of textbook diagrams is a composite showing all organelles, not a real cell. - All cells share: a plasma membrane, cytoplasm, genetic material (DNA), ribosomes, and metabolism. Exam focus: list five differences between prokaryotic and eukaryotic cells, and explain the invagination hypothesis.

1. Define a tissue and name the four basic tissues. 2. What does cytology study, and what does histology study? 3. Why are fat cells empty on H&E sections? 4. Why are nuclei basophilic? 5. Name the monomers of the four classes of macromolecules. 6. Why is a virus not considered a cell? 7. Give five differences between a prokaryotic and a eukaryotic cell. 8. What is the invagination hypothesis? 9. What evidence supports the endosymbiotic origin of mitochondria? 10. Why is the "typical cell" a misleading idea?

1. A group of similar cells plus extracellular matrix performing a common function. Epithelial, connective, muscle, nervous. 2. Cytology: the cell. Histology: tissues. 3. Lipids are dissolved by the alcohols and xylene used in processing. 4. Nucleic acids are acidic and bind basic dyes such as haematoxylin. 5. Carbohydrates: monosaccharides. Lipids: fatty acids and glycerol. Proteins: amino acids. Nucleic acids: nucleotides. 6. It has no metabolism or ribosomes and can reproduce only inside a host cell. 7. Any five: nucleus; membrane-bound organelles; size; linear chromosomes with histones; 80S versus 70S ribosomes; cytoskeleton; endomembrane system. 8. Internal membranes and the nuclear envelope arose by inward folding of the plasma membrane of an ancestral cell. 9. Double membrane, own circular DNA, bacterial-type ribosomes, division by fission. 10. Real cells differ widely in shape, size and organelle content according to function (about 200 types).

Structure - Phospholipid bilayer: amphipathic phospholipids with hydrophilic heads outwards and hydrophobic tails inwards. - Cholesterol: in both leaflets; about 1 cholesterol per phospholipid in the plasma membrane. The red cell membrane is the classic model. - Proteins: integral (transmembrane) and peripheral. About 50% of membrane mass. - Carbohydrates: attached to proteins and lipids on the outer face only. - Appearance in TEM: two dark lines (heads bind osmium) separated by a light line (tails). Fluidity - Lipids diffuse laterally quickly within a leaflet; rotate; flip-flop between leaflets is very rare without enzymes (flippases). - Parameters affecting fluidity: - temperature - fatty acid length: longer chains lower fluidity - saturation: double bonds (kinks) raise fluidity - cholesterol: a buffer. It stiffens the membrane at high temperature and prevents crystallisation at low temperature. - Transition temperature (Tm): the temperature at which a membrane changes from a fluid (liquid crystalline) to a rigid gel state. Tm rises with longer, saturated fatty acids and falls with shorter, unsaturated ones. Cells adjust their lipid composition to stay fluid at body temperature. - Discontinuity: lipid rafts, microdomains rich in cholesterol and sphingolipids that gather signalling proteins. Asymmetry - The two leaflets differ: phosphatidylcholine and sphingomyelin mainly outside; phosphatidylserine and phosphatidylethanolamine inside. - Exposure of phosphatidylserine on the outside is an "eat me" signal in apoptosis. Glycocalyx (surface coat) - A carbohydrate-rich coat of glycoproteins, glycolipids and proteoglycans on the outer surface. Stained by PAS (periodic acid-Schiff). - Functions: protection, cell recognition and adhesion, blood group antigens, the enzyme-rich coat of intestinal microvilli. Transport (details in Applied Biology) - Passive: simple diffusion (gases, lipophilic molecules), channels, carriers (facilitated diffusion), down the gradient. - Active: primary (ATP-driven pumps such as Na/K-ATPase, which sends 3 Na⁺ out and 2 K⁺ in, and uses up to about 40% of the cell's energy in some cells); secondary (uses the Na⁺ gradient). - Vesicular transport: endocytosis and exocytosis (see the endomembrane note). Resting membrane potential - About −70 mV (inside negative), mainly because K⁺ leaks out through open K⁺ channels, down the gradient maintained by the Na/K pump. Membrane protein functions: transport, receptors, enzymes, adhesion (cell-cell and cell-matrix), anchoring the cytoskeleton, recognition. Exam focus: explain Tm and how fatty acid length, saturation and cholesterol affect fluidity.

Nuclear features on a slide - Shape: usually round or oval; elongated in smooth muscle and fibroblasts; lobed in neutrophils; flattened in squamous cells. - Position: central in most cells; basal in columnar epithelia; peripheral in skeletal muscle fibres and adipocytes. - Number: usually one; none in mature red cells and platelets; two in some hepatocytes; many in skeletal muscle fibres and osteoclasts. The nuclear envelope - Two membranes separated by a perinuclear space (continuous with the ER lumen). The outer membrane is continuous with the RER and carries ribosomes. - Nuclear lamina: a meshwork of intermediate filaments (lamins A, B, C) lining the inner membrane. Supports the envelope, anchors chromatin, disassembles in mitosis (lamin phosphorylation). - Laminopathies: Hutchinson-Gilford progeria (a mutant lamin A, progerin), causing premature ageing. Nuclear pores - Nuclear pore complexes: about 120 MDa, octagonal symmetry, with cytoplasmic filaments and a nuclear basket. Number varies with activity (about 3,000-4,000 per nucleus). - Molecules smaller than about 9 nm (about 40-60 kDa) diffuse freely; larger ones need signals: NLS for import, NES for export, carried by importins and exportins with the Ran-GTP system. DNA classes by repetitiveness (a slide classification) - Single-copy (unique) DNA: most genes. - Moderately repetitive DNA (repeated about 100-1,000 times): rRNA and histone genes, transposable elements. - Highly repetitive DNA (about 100,000-1,000,000 copies): short satellite sequences at centromeres and telomeres. Chromatin - DNA plus proteins. The total DNA of one human cell is about 2 m long, packed into a nucleus about 5-10 µm across. - Euchromatin: loosely packed, pale, transcriptionally active, mostly single-copy DNA. - Heterochromatin: densely packed, dark, inactive, rich in repetitive DNA. - Constitutive: always condensed (centromeres, telomeres). - Facultative: condensed in some cells only (the inactive X, the Barr body). - Packing levels: 1. Double helix: 2 nm. 2. Nucleosome ("beads on a string", 11 nm): about 147 bp wound 1.7 times around a histone octamer (two each of H2A, H2B, H3, H4), plus linker DNA. 3. 30-nm fibre: nucleosomes coiled together with histone H1 (the linker histone, which binds both linker DNA and the nucleosome). 4. Radial loops (about 300 nm): loops of 30-nm fibre attached to a protein scaffold (non-histone proteins). Loops organise genes into functional domains. 5. Further condensation to the mitotic chromosome (about 700 nm per chromatid; 1,400 nm for the whole chromosome). - During mitosis both eu- and heterochromatin become maximally condensed. Chromosomes and karyotype - A metaphase chromosome: two sister chromatids joined at the centromere; telomeres at the ends. - Types by centromere position: metacentric, submetacentric, acrocentric (detail in Genetics). - Karyotype: the ordered set of chromosomes, 46 in humans. The nucleolus - Not membrane-bound. Forms around the nucleolar organiser regions (rRNA genes on the short arms of acrocentric chromosomes 13, 14, 15, 21, 22). - Three regions in TEM: fibrillar centres (rDNA), dense fibrillar component (rRNA transcription and processing), granular component (assembly of ribosomal subunits). - Disappears at the start of mitosis and reforms in telophase. - Large prominent nucleoli mean high protein synthesis (neurons, active cells, many cancer cells). Ribosomes and polysomes - Eukaryotic 80S ribosome = 60S large subunit (28S, 5.8S, 5S rRNA) plus 40S small subunit (18S rRNA). - Free ribosomes: make cytosolic, nuclear and mitochondrial proteins. - Bound to RER: secreted, membrane and lysosomal proteins. - Polyribosomes (polysomes): several ribosomes translating the same mRNA at once, seen as rosettes or spirals in TEM. - Ribosomes make cytoplasm basophilic. Exam focus: describe the packing levels of chromatin with their sizes, and the three regions of the nucleolus.

Rough endoplasmic reticulum (RER) - Flattened cisternae studded with ribosomes, continuous with the outer nuclear membrane. - Makes secretory, membrane and lysosomal proteins (co-translational import). - Modifications in the lumen: N-linked glycosylation (a precursor oligosaccharide of 14 sugars is transferred from dolichol to asparagine), folding with chaperones, disulfide bonds. Glycosylation also acts as quality control (calnexin cycle); misfolded proteins are exported and degraded. - Abundant in cells that secrete protein: plasma cells, pancreatic acinar cells, fibroblasts. The basophilic cytoplasm is called ergastoplasm; in neurons it is Nissl substance. Smooth endoplasmic reticulum (SER) - Tubular, no ribosomes. Continuous with RER (transitional ER, where vesicles leave for the Golgi). - Functions: - lipid and steroid synthesis (Leydig cells, adrenal cortex, corpus luteum) - detoxification of drugs by cytochrome P450 enzymes (hepatocytes; expands after barbiturates or alcohol) - glycogen metabolism (glucose-6-phosphatase in the liver) - Ca²⁺ storage (in muscle it is the sarcoplasmic reticulum) Golgi apparatus - A stack of flattened curved cisternae (dictyosome) with cis (entry, facing the ER), medial and trans (exit) faces, plus the trans-Golgi network (TGN). - In light microscopy: a pale area next to the nucleus (negative Golgi image, as in plasma cells), or seen with silver impregnation (Golgi's original method, 1898). - Functions: - processing: modifying N-linked sugars; O-linked glycosylation (sugars on serine or threonine, added one at a time in the Golgi); sulfation (proteoglycans); proteolytic cleavage - sorting and packaging: in the TGN proteins go to lysosomes (by the mannose-6-phosphate tag), to constitutive secretion or to regulated secretion (secretory granules) - N- versus O-linked: N in the ER on asparagine, en bloc; O in the Golgi on serine or threonine, one sugar at a time. Vesicular transport - Coats: COPII (ER to Golgi), COPI (Golgi to ER and between cisternae, retrograde), clathrin (from the TGN and plasma membrane). - Targeting and fusion: Rab GTPases and SNAREs (v-SNARE on the vesicle pairs with t-SNARE on the target). - Exocytosis: - constitutive: continuous, unregulated (collagen by fibroblasts, antibodies by plasma cells, membrane components) - regulated: stored in secretory granules, released on a signal (hormones, neurotransmitters, digestive enzymes) - Endocytosis: - phagocytosis ("cell eating") of large particles (bacteria, debris) by pseudopods, in macrophages and neutrophils - pinocytosis ("cell drinking") of fluid - receptor-mediated endocytosis via clathrin-coated pits (LDL, transferrin) - caveolae: flask-shaped invaginations coated by caveolin, abundant in endothelium, adipocytes and smooth muscle Lysosomes - Membrane-bound vesicles with about 50 acid hydrolases (active at pH about 5, kept by a proton pump). The cell's garbage disposal system. - Digest material from three routes: endocytosis, phagocytosis, autophagy (the cell's own organelles). - Seen as dense bodies in TEM; residual bodies and lipofuscin ("age pigment") accumulate in long-lived cells (neurons, cardiac muscle). - Abundant in macrophages, neutrophils and osteoclasts. Peroxisomes - Small vesicles with oxidases that produce H₂O₂ and catalase that breaks it down. In some species there is a crystalline core (urate oxidase). - Functions: β-oxidation of very long-chain fatty acids, plasmalogen (myelin) synthesis, detoxification (about a quarter of ethanol in the liver), bile acid synthesis. - Abundant in liver and kidney. Exam focus: explain how a lysosomal enzyme reaches the lysosome (M6P tag in the Golgi), and compare constitutive and regulated exocytosis.

Structure - Size about 0.5-1 µm wide and 1-10 µm long; visible in light microscopy only with special stains; they make cytoplasm eosinophilic. - Outer membrane: smooth, permeable to small molecules through porins (VDAC). - Intermembrane space: where protons are pumped; contains cytochrome c. - Inner membrane: folded into cristae; very rich in protein (respiratory chain, ATP synthase); impermeable (cardiolipin); requires specific transporters. - Cristae shape varies: shelf-like (lamellar) in most cells; tubular in steroid-secreting cells (adrenal cortex, Leydig cells, corpus luteum). Tubular cristae are a histology clue to steroid synthesis. - Matrix: enzymes of the Krebs cycle and β-oxidation, mtDNA, mitochondrial ribosomes, dense granules (Ca²⁺). - Endosymbiotic origin and mtDNA with maternal inheritance: see Applied Biology. Where they are abundant - Cells with high ATP needs: - cardiac muscle (about 35-40% of cell volume) - skeletal muscle (red, oxidative fibres) - kidney proximal tubule and striated ducts (lined up in basal folds of the membrane, next to the Na/K pumps) - parietal cells of the stomach - sperm midpiece (arranged in a helix around the axoneme) - brown fat (with uncoupling protein 1 for heat) - Position within the cell: near where ATP is used. Mitochondrial dynamics - Mitochondria are not isolated beans. They form a changing network by fusion and fission. - Fusion: mitofusins MFN1 and MFN2 (outer membrane), OPA1 (inner membrane). Mixes contents and dilutes damage. - Fission: DRP1, recruited to sites marked by the ER. Allows distribution to daughter cells and isolation of damaged parts. - Quality control: damaged mitochondria are removed by mitophagy (a form of autophagy, via PINK1 and Parkin; defects are linked to Parkinson disease). - Defects: OPA1 mutations cause dominant optic atrophy; MFN2 mutations cause Charcot-Marie-Tooth type 2A. ER-mitochondria contact sites (MAMs) - Regions where ER membranes lie about 10-30 nm from the outer mitochondrial membrane. - Functions: transfer of Ca²⁺ from ER to mitochondria (regulating metabolism and apoptosis), lipid transfer (phospholipid synthesis), marking fission sites, starting autophagosome formation. Exam focus: name three tissues rich in mitochondria and explain why steroid-secreting cells have tubular cristae.

Functions (from the slides) - Strengthening and support. - Intracellular movement (organelles, vesicles, chromosomes). - Cellular movement (crawling, cilia and flagella, muscle contraction). Microfilaments (actin) - Diameter about 7 nm. Two-stranded helix of globular G-actin subunits forming filamentous F-actin. - Polar: a fast-growing plus (barbed) end and a slow minus (pointed) end. - G-actin binds ATP; after incorporation, ATP is hydrolysed to ADP. - Treadmilling: at steady state, subunits add at the plus end and leave at the minus end, so the filament length stays constant while subunits flow through it. - Locations: - cortex beneath the plasma membrane - core of microvilli (bundles of 20-30 actin filaments cross-linked by fimbrin and villin, anchored in the terminal web) - stress fibres - contractile ring in cytokinesis - sarcomeres of muscle, with myosin - lamellipodia and filopodia of moving cells Microtubules - Diameter 25 nm. Hollow tubes of 13 protofilaments of α/β-tubulin dimers. - Polar: plus end grows; minus end anchored at the MTOC. - Dynamic instability: β-tubulin binds GTP. A GTP cap stabilises the growing plus end; if hydrolysis catches up, the microtubule suddenly shrinks (catastrophe). Rescue restarts growth. - MTOC: the centrosome, a pair of centrioles at right angles (each nine triplets of microtubules) surrounded by pericentriolar material containing γ-tubulin ring complexes, which nucleate microtubules. - Motor proteins: kinesins move toward the plus end (usually outward); dyneins toward the minus end (inward). Axonal transport depends on them. - Cilia and flagella: axoneme with 9 doublets plus 2 central singlets (9+2), with dynein arms that make them bend. Anchored in a basal body (9 triplets, like a centriole). Defects: primary ciliary dyskinesia. - Drugs: colchicine (blocks polymerisation; used for gout and to arrest cells for karyotypes), vinca alkaloids (block assembly), taxanes (stabilise). All block mitosis. Intermediate filaments - Diameter about 10 nm. Rope-like, very strong, not polar, no motors, no nucleotide binding. - Built from rod-shaped monomers, coiled into dimers, antiparallel tetramers, then eight tetramers per filament. - Main classes and where they occur: - keratins: epithelia - vimentin: mesenchymal cells (fibroblasts, endothelium) - desmin: muscle - glial fibrillary acidic protein (GFAP): astrocytes - neurofilaments: neurons - lamins: nuclear lamina (all cells) - Functions: mechanical strength; anchor desmosomes and hemidesmosomes. - Clinical use: the type of intermediate filament identifies the tissue origin of a tumour by immunohistochemistry (keratin for carcinoma, vimentin for sarcoma, desmin for muscle tumour, GFAP for glioma). - Diseases: epidermolysis bullosa simplex (keratin 5/14 mutations: skin blisters with minor trauma). Cell motility - A crawling cell extends a lamellipodium by actin polymerisation at the front, attaches to the substrate by focal adhesions, and pulls the rear forward by actin-myosin contraction. Exam focus: compare the three filaments by diameter, subunit, polarity, motors and function.

1. What does a basophilic cytoplasm tell you about the cell? And a pale, foamy one? 2. What is the transition temperature of a membrane, and what raises it? 3. How does cholesterol affect membrane fluidity? 4. What is the glycocalyx, and how is it stained? 5. What is the nuclear lamina, and which disease results from its defect? 6. Name the three classes of DNA by repetitiveness. 7. List the levels of chromatin packing with their approximate sizes. 8. Distinguish constitutive from facultative heterochromatin. 9. What are the three regions of the nucleolus? 10. Which organelle is abundant in Leydig cells, and why? 11. Distinguish N-linked from O-linked glycosylation. 12. What is the difference between constitutive and regulated exocytosis? Give an example of each. 13. What are caveolae? 14. What is special about the cristae of steroid-secreting cells? 15. Which proteins mediate mitochondrial fusion and fission? 16. What is treadmilling? 17. What is dynamic instability of microtubules? 18. How are intermediate filaments used in tumour diagnosis?

1. Basophilic: much RER and ribosomes, so active protein synthesis. Pale, foamy: lipid droplets and SER, so steroid synthesis. 2. The temperature at which a membrane changes from fluid to gel. Raised by longer and saturated fatty acids. 3. It buffers fluidity: stiffens at high temperature, prevents freezing at low temperature. 4. The carbohydrate coat on the outer surface of the plasma membrane. Stained by PAS. 5. A meshwork of lamins lining the inner nuclear membrane. Progeria (lamin A mutation). 6. Single-copy, moderately repetitive, highly repetitive. 7. Double helix 2 nm; nucleosome fibre 11 nm; 30-nm fibre; radial loops about 300 nm; mitotic chromatid about 700 nm (chromosome 1,400 nm). 8. Constitutive: always condensed (centromeres, telomeres). Facultative: condensed only in some cells or times (the inactive X). 9. Fibrillar centres, dense fibrillar component, granular component. 10. SER, because they synthesise steroids (testosterone). 11. N-linked: in the ER, on asparagine, a preformed oligosaccharide added en bloc. O-linked: in the Golgi, on serine or threonine, sugars added one at a time. 12. Constitutive: continuous, unregulated (collagen from fibroblasts). Regulated: stored in granules and released on a signal (insulin, digestive enzymes). 13. Flask-shaped invaginations coated with caveolin, abundant in endothelium, adipocytes and smooth muscle. 14. They are tubular rather than shelf-like. 15. Fusion: mitofusins (MFN1, MFN2) and OPA1. Fission: DRP1. 16. Actin subunits add at the plus end and leave at the minus end, so the filament keeps its length while subunits flow through it. 17. Microtubules alternate between growth and sudden shrinkage, depending on whether a GTP cap is present at the plus end. 18. Each tissue expresses a characteristic type (keratin in epithelium, vimentin in mesenchyme, desmin in muscle, GFAP in glia), which identifies the tumour's origin.

Apoptosis versus necrosis - Trigger: apoptosis is physiological or pathological, regulated; necrosis is always pathological (ischaemia, toxins, trauma). - Extent: apoptosis affects single cells; necrosis affects groups of cells. - Cell size: apoptosis shrinks the cell; necrosis swells it (oncosis). - Nucleus: apoptosis gives chromatin condensation (pyknosis) at the nuclear edge, then fragmentation (karyorrhexis); necrosis ends with nuclear dissolution (karyolysis). - Membrane: intact in apoptosis (blebs, then apoptotic bodies); ruptured in necrosis. - Contents: in apoptosis enclosed in apoptotic bodies and eaten by phagocytes; in necrosis leak out. - Inflammation: none in apoptosis; present in necrosis. - Energy: apoptosis needs ATP; necrosis does not. - DNA: apoptosis cleaves DNA between nucleosomes into multiples of about 180-200 bp (a "ladder" on gel electrophoresis); necrosis degrades it randomly (a smear). Examples of apoptosis - Physiological: - embryonic development: removal of webbing between fingers; shaping of organs; elimination of excess neurons - hormone-dependent involution: endometrium at menstruation; ovarian follicle atresia; breast after weaning - cell turnover in renewing tissues (intestinal epithelium) - selection of lymphocytes (deletion of self-reactive T cells in the thymus) - killing of infected or tumour cells by cytotoxic T cells - Pathological: DNA damage (radiation, chemotherapy), viral infections, some degenerative diseases, atrophy after duct obstruction. Biochemical features - Caspases: cysteine proteases that cleave after aspartate. Made as inactive procaspases. - Initiator caspases (8, 9) activated by pathway-specific adaptors. - Executioner caspases (3, 6, 7) activated by initiators; they cleave hundreds of proteins: lamins (nuclear collapse), cytoskeletal proteins, the inhibitor of the DNase (so DNA fragmentation). - The caspase cascade amplifies the signal. - Phagocytic recognition: phosphatidylserine flips to the outer leaflet, an "eat me" signal; phagocytes clear apoptotic bodies quickly and silently. Morphological sequence - The cell detaches from neighbours and shrinks; chromatin condenses; the membrane blebs (zeiosis); the cell breaks into membrane-bound apoptotic bodies; phagocytes engulf them. The whole process takes minutes to hours. The two pathways - Extrinsic (death receptor) pathway: - death ligands (FasL, TNF, TRAIL) bind death receptors (Fas/CD95, TNF receptor 1) - the receptors recruit the adaptor FADD - FADD activates procaspase-8 (the death-inducing signalling complex, DISC) - caspase-8 activates caspase-3 - used by cytotoxic T cells and in lymphocyte homeostasis - Intrinsic (mitochondrial) pathway: - triggered by internal stress: DNA damage (through p53), growth factor withdrawal, ER stress, oxidative stress - controlled by the BCL-2 family: anti-apoptotic (BCL-2, BCL-XL) and pro-apoptotic (BAX, BAK, and BH3-only sensors such as BID, BIM, PUMA, NOXA) - BAX/BAK form pores in the outer mitochondrial membrane, and cytochrome c is released - cytochrome c binds APAF-1, forming the apoptosome, which activates caspase-9, then caspase-3 - Link between pathways: caspase-8 cleaves BID to tBID, which activates the mitochondrial pathway. - Inhibitors: IAPs (inhibitors of apoptosis proteins) block caspases. Apoptosis and disease - Too little: cancer (malignant cells often reduce apoptosis, for example by overexpressing BCL-2 in follicular lymphoma, or losing p53); autoimmunity (Fas defects in autoimmune lymphoproliferative syndrome). - Too much: neurodegenerative diseases, ischaemic injury (partly), loss of CD4 cells in HIV. - Drugs: venetoclax (a BCL-2 inhibitor) in chronic lymphocytic leukaemia. Exam focus: tabulate apoptosis versus necrosis, and draw both pathways to caspase-3.

Phases - Interphase: about 90-95% of the cycle; the cell grows and prepares. - G1 (gap 1): growth, protein synthesis, organelle duplication. Very variable length. Decision point: the restriction point (in yeast, START). After it, the cell is committed to divide. - S (synthesis): DNA replication (DNA content goes from 2n to 4n in terms of chromatids) and centrosome duplication. About 8 hours. - G2 (gap 2): preparation for mitosis; checking that replication is complete. - M phase: mitosis (nuclear division) plus cytokinesis. About 1 hour. - G0: a quiescent state outside the cycle. Cells can stay there for years or permanently, or re-enter G1 when stimulated. - A typical proliferating human cell in culture takes about 24 hours per cycle. DNA content through the cycle - G1: 2C (46 chromosomes, each one chromatid). - After S and in G2: 4C (46 chromosomes, each two sister chromatids). - After mitosis: 2C in each daughter. Bizzozero's classification (Giulio Bizzozero, Italian histologist, 1894) - Labile tissues (continuously renewing): cells divide throughout life to replace constant loss. Epidermis, intestinal epithelium, bone marrow haematopoietic cells. - Stable tissues (expanding): cells normally in G0 but can re-enter the cycle when needed. Liver (regeneration after partial hepatectomy), kidney tubules, endocrine glands, fibroblasts, endothelium. - Permanent (perennial) tissues: cells cannot divide after birth. Neurons, cardiac muscle cells, (mostly) skeletal muscle fibres. Damage is repaired by scar. - Clinical relevance: labile tissues are the most sensitive to chemotherapy and radiation (hair loss, mucositis, bone marrow suppression). Permanent tissues cannot regenerate (myocardial infarction and stroke leave scars). Regulation - Cyclins and cyclin-dependent kinases (CDKs): cyclin levels rise and fall through the cycle, activating CDKs at specific phases (cyclin D-CDK4/6 in G1, cyclin E-CDK2 at G1/S, cyclin A in S, cyclin B-CDK1 in mitosis, the M-phase promoting factor). - The retinoblastoma protein (RB) blocks E2F until phosphorylated by cyclin D-CDK4/6 at the restriction point. - CDK inhibitors: p21, p27, p16. Checkpoints 1. G1/S checkpoint: is the DNA intact? Are conditions favourable? p53 stops the cycle (through p21) if DNA is damaged. 2. G2/M checkpoint: is DNA replication complete and undamaged? 3. Mitotic (spindle assembly) checkpoint, in metaphase: are all chromosomes attached to the spindle from both poles? Anaphase is delayed until they are. Failure leads to aneuploidy. Exam focus: give an example of a labile, a stable and a permanent tissue, and name the three checkpoints.

Prophase - Chromatin condenses into visible chromosomes, each made of two sister chromatids joined at the centromere. - The nucleolus disappears. - The two centrosomes move apart to opposite poles, organising the mitotic spindle between them. Prometaphase - The nuclear envelope breaks down (lamins phosphorylated by CDK1). - Kinetochores (protein complexes on each centromere) attach to spindle microtubules. Metaphase - Chromosomes line up at the metaphase (equatorial) plate, each attached to both poles through its two sister kinetochores (bi-orientation). - Chromosomes are most condensed. This is the stage used for karyotyping (colchicine arrests cells here). - The spindle assembly checkpoint waits until all kinetochores are attached. The mitotic spindle: three types of microtubules 1. Kinetochore microtubules: attach to chromosomes. 2. Polar (interpolar) microtubules: overlap at the equator, push the poles apart. 3. Astral microtubules: radiate towards the cell cortex, position the spindle. Anaphase - Cohesin holding sister chromatids together is cleaved by separase (after the anaphase-promoting complex destroys securin), so sister chromatids separate and become independent chromosomes. - Anaphase A: chromosomes move toward the poles as kinetochore microtubules shorten. - Anaphase B: the poles move apart as polar microtubules slide past each other (kinesins) and astral microtubules pull the poles outwards (dynein). Telophase - The chromosomes reach the poles and decondense. - The nuclear envelope reforms around each set; nucleoli reappear. - The spindle disassembles. Cytokinesis - In animal cells: a contractile ring of actin and myosin II forms beneath the membrane at the equator, making a cleavage furrow that deepens until the cell pinches in two. The midbody is the last bridge. - Usually starts in late anaphase or telophase. - Mitosis without cytokinesis produces binucleate or multinucleate cells (some hepatocytes, megakaryocytes are polyploid by endomitosis). Mitosis on a slide - Mitotic figures are counted in pathology (the mitotic index). Many or abnormal (tripolar) mitoses suggest a malignant tumour. Exam focus: describe the events of each phase, and distinguish anaphase A from anaphase B.

Why meiosis is needed - Humans are diploid (2n = 46). Gametes must be haploid (n = 23) so the zygote is diploid again. - One replication then two divisions: meiosis I separates homologous chromosomes (the reductional division); meiosis II separates sister chromatids (the equational division, like mitosis). Meiosis I - Prophase I (long; in human oocytes it lasts from fetal life to ovulation, up to 50 years). Substages: 1. Leptotene: chromosomes begin to condense as thin threads. 2. Zygotene: homologous chromosomes pair (synapsis), joined by the synaptonemal complex. 3. Pachytene: pairing is complete; each pair is a bivalent (tetrad: four chromatids). Crossing over happens: exchange of segments between non-sister chromatids. 4. Diplotene: the synaptonemal complex dissolves; homologues separate except at chiasmata, the visible points of crossover. Oocytes arrest here (dictyotene) before birth. 5. Diakinesis: maximal condensation; the nuclear envelope breaks down. - Metaphase I: bivalents line up at the equator. Each homologue faces one pole, and orientation is random (independent assortment). - Anaphase I: homologues separate to opposite poles; sister chromatids stay together (cohesin at the centromere is protected, by shugoshin). - Telophase I and cytokinesis: two haploid cells, each chromosome still with two chromatids. - Interkinesis: short; no DNA replication. Meiosis II - Prophase II, metaphase II (chromosomes line up singly), anaphase II (sister chromatids separate), telophase II. - Result: four haploid cells with one chromatid per chromosome. In human oocytes meiosis II arrests at metaphase II and is completed only after fertilisation. Mitosis versus meiosis - Divisions: mitosis one; meiosis two. - Pairing of homologues and crossing over: mitosis no; meiosis yes (prophase I). - Products: mitosis two diploid, genetically identical cells; meiosis four haploid, genetically different cells. - Where: mitosis in somatic cells; meiosis in germ cells (testis and ovary). Sources of genetic variation - Independent assortment of homologues at metaphase I: 2²³ (about 8.4 million) combinations per gamete. - Crossing over: new combinations of alleles within each chromosome. - Random fertilisation: any sperm with any egg. Errors - Nondisjunction (failure of homologues or sister chromatids to separate) gives aneuploid gametes (details in Genetics). Exam focus: list the substages of prophase I in order with one event each, and explain why meiosis I is called reductional.

1. Give four morphological differences between apoptosis and necrosis. 2. Why does apoptosis not cause inflammation? 3. What is the role of executioner caspases? Name one. 4. Describe the extrinsic pathway from ligand to caspase-3. 5. What role does cytochrome c play in the intrinsic pathway? 6. Give two physiological examples of apoptosis. 7. What are labile, stable and permanent tissues? Give one example of each. 8. What happens in each phase of interphase? 9. What does the spindle assembly checkpoint check? 10. Name the three types of spindle microtubules. 11. What triggers the separation of sister chromatids in anaphase? 12. Name the substages of prophase I in order. 13. In which substage does crossing over occur? What are chiasmata? 14. What are three sources of genetic variation in sexual reproduction?

1. Any four: shrinkage vs swelling; chromatin condensation and fragmentation vs karyolysis; intact membrane with blebs vs rupture; apoptotic bodies vs spillage; single cells vs groups. 2. The contents stay enclosed in apoptotic bodies, which are quickly eaten by phagocytes. 3. They cleave cellular proteins (lamins, cytoskeleton, DNase inhibitor) and dismantle the cell. Caspase-3 (also 6, 7). 4. Death ligand (FasL, TNF) binds death receptor, which recruits FADD, which activates procaspase-8; caspase-8 activates caspase-3. 5. Released from mitochondria, it binds APAF-1 to form the apoptosome, which activates caspase-9. 6. Any two: removal of interdigital webs; endometrial shedding; thymic deletion of self-reactive T cells; follicle atresia; intestinal cell turnover. 7. Labile: continuously dividing (epidermis). Stable: in G0 but can divide (liver). Permanent: cannot divide (neurons, cardiac muscle). 8. G1: growth, commitment at the restriction point. S: DNA replication and centrosome duplication. G2: preparation for mitosis and checking replication. 9. That every chromosome is attached to both spindle poles before anaphase. 10. Kinetochore, polar (interpolar) and astral. 11. Separase cleaves cohesin, after the anaphase-promoting complex destroys securin. 12. Leptotene, zygotene, pachytene, diplotene, diakinesis. 13. Pachytene. Chiasmata are the visible points where non-sister chromatids have crossed over. 14. Independent assortment, crossing over, random fertilisation.

The seminiferous tubule - Wall: a stratified germinal epithelium (germ cells at all stages plus Sertoli cells) on a basement membrane, surrounded by myoid (peritubular contractile) cells. - Germ cells mature from the basal side toward the lumen. - Between tubules: interstitial tissue with blood vessels and Leydig cells. The stages 1. Spermatogonia: diploid stem cells on the basement membrane. - Type A dark: reserve stem cells, rarely divide. - Type A pale: renewing stem cells, divide by mitosis to maintain the stock and produce type B. - Type B: committed; divide to form primary spermatocytes. - Daughter cells stay connected by cytoplasmic bridges (incomplete cytokinesis), so a clone develops in synchrony. 2. Primary spermatocytes (46 chromosomes, 4C): the largest germ cells; long prophase I (about 22 days), so they are the most often seen stage in sections. 3. Secondary spermatocytes (23 chromosomes, 2C): after meiosis I. Short-lived (hours), rarely seen. 4. Spermatids (23, 1C): after meiosis II. Small round cells near the lumen. 5. Spermatozoa: after spermiogenesis; released into the lumen (spermiation). - From one primary spermatocyte: four spermatozoa. - Duration: about 64-74 days from spermatogonium to spermatozoon; then about 2 weeks of maturation in the epididymis. Spermiogenesis (spermatid to spermatozoon; no division) 1. Golgi phase: Golgi vesicles fuse into the acrosomal vesicle at one pole of the nucleus; the centrioles move to the opposite pole and start the flagellum. 2. Cap phase: the acrosome spreads over the anterior half of the nucleus like a cap. 3. Acrosome phase: the nucleus condenses and elongates (histones replaced by protamines); the cell elongates; mitochondria gather around the proximal flagellum (the midpiece); microtubules form a manchette. 4. Maturation phase: excess cytoplasm is shed as residual bodies, which Sertoli cells phagocytose. - The acrosome is a specialised lysosome with enzymes (hyaluronidase, acrosin) needed to penetrate the egg coats. The spermatozoon - Head: condensed nucleus with the acrosome. - Neck: centriole. - Midpiece: helix of mitochondria around the axoneme (energy). - Principal and end pieces: the flagellum (9+2 axoneme with outer dense fibres). Sertoli cells: functions - Support and nourish germ cells (tall columnar cells from the basement membrane to the lumen, with germ cells in their recesses). - Form the blood-testis barrier. - Phagocytose residual bodies and degenerating germ cells. - Secrete tubular fluid, androgen-binding protein (keeps testosterone high locally), inhibin (negative feedback on FSH), and, in the fetus, anti-Müllerian hormone. - Respond to FSH and testosterone. The blood-testis barrier - Tight junctions between neighbouring Sertoli cells divide the epithelium into a basal compartment (spermatogonia, early primary spermatocytes, exposed to blood) and an adluminal compartment (later spermatocytes and spermatids, isolated). - Why: meiotic and post-meiotic germ cells express new antigens that appear after immune tolerance was set up; the barrier prevents autoimmune attack, and provides a controlled environment. Spermatocytes cross the barrier as it reassembles behind them. - After vasectomy or trauma, antisperm antibodies may form. Leydig cells (interstitial cells) - Polygonal eosinophilic cells in clusters between tubules near capillaries. - Abundant SER, mitochondria with tubular cristae, lipid droplets: steroid-secreting features. Reinke crystals in humans. - Make testosterone under LH stimulation. - In prenatal life: fetal Leydig cells produce testosterone (driven by placental hCG), needed for male development of the genital ducts and external genitalia (via DHT). They regress after birth and reappear at puberty. Hormonal control - GnRH from the hypothalamus drives LH and FSH from the pituitary. - LH acts on Leydig cells (testosterone). - FSH acts on Sertoli cells (with testosterone) to support spermatogenesis. - Feedback: testosterone inhibits GnRH and LH; inhibin inhibits FSH. Exam focus: draw a seminiferous tubule showing all cell types and the blood-testis barrier.

Timeline 1. Fetal life: - primordial germ cells migrate from the yolk sac wall to the gonad (weeks 4-6) - oogonia multiply by mitosis to a peak of about 7 million at 20 weeks - they enter meiosis and arrest in diplotene (dictyotene) of prophase I as primary oocytes - most degenerate (atresia) 2. Birth: about 1-2 million primary oocytes; no oogonia remain (no renewal). 3. Puberty: about 400,000 remain. 4. Reproductive life: each cycle, a cohort of follicles grows; usually one ovulates. About 400-500 are ovulated in a lifetime; the rest undergo atresia. 5. Menopause: follicles are depleted. - Meiosis I resumes only just before ovulation (LH surge): the primary oocyte divides into a secondary oocyte and the first polar body (unequal division: the oocyte keeps almost all the cytoplasm). - The secondary oocyte arrests at metaphase II. Meiosis II is completed only at fertilisation, giving the ovum and the second polar body. - One primary oocyte gives one egg plus two or three polar bodies. Follicle stages 1. Primordial follicle: a primary oocyte (about 30 µm) surrounded by a single layer of flat follicular (pregranulosa) cells. The resting reserve. 2. Primary follicle: the follicular cells become cuboidal (unilaminar), then proliferate into several layers (multilaminar), now called granulosa cells. The zona pellucida (glycoproteins ZP1-ZP3) forms between oocyte and granulosa cells. Stromal cells begin forming the theca. 3. Secondary (antral) follicle: fluid-filled spaces appear among granulosa cells and merge into an antrum with follicular fluid. The theca divides into theca interna (vascular, steroid-secreting: androgens) and theca externa (fibrous). 4. Mature (Graafian, preovulatory) follicle: up to about 20-25 mm, bulging from the ovarian surface. The oocyte sits on the cumulus oophorus, surrounded by the corona radiata (the granulosa cells attached to the zona pellucida). The two-cell, two-gonadotropin model - Theca interna cells (under LH) make androgens; granulosa cells (under FSH) convert them to oestradiol by aromatase. Ovulation - The LH surge triggers: completion of meiosis I, cumulus expansion, local breakdown of the follicle wall (enzymes, prostaglandins), and rupture of the follicle about 36 hours after the surge onset, releasing the secondary oocyte with its corona radiata into the peritoneal cavity. The fimbriae of the uterine tube collect it. After ovulation - The remaining granulosa and theca cells become the corpus luteum (luteinisation): granulosa lutein cells (progesterone, oestrogen) and theca lutein cells (androgens, progesterone). - Without pregnancy: it lives about 14 days, then regresses into a corpus albicans (a white scar). - With pregnancy: hCG from the embryo maintains it as the corpus luteum of pregnancy until the placenta takes over (about week 8-12). Atresia - Most follicles degenerate at some stage: granulosa cells die by apoptosis and the oocyte degenerates. Exam focus: describe the follicle stages in order with one feature of each, and state when meiosis I and II are completed.

The hormonal axis - Hypothalamus: GnRH, released in pulses. - Anterior pituitary: FSH and LH. - Ovary: oestrogens (mainly oestradiol), progesterone, inhibin. The ovarian cycle (typical 28 days) 1. Follicular phase (days 1-14, variable length): - FSH recruits a cohort of antral follicles - granulosa cells make increasing oestradiol; the follicle with most FSH receptors becomes dominant; the others undergo atresia as FSH falls (negative feedback by oestradiol and inhibin) 2. Ovulation (about day 14): - once oestradiol is high for about 36-48 hours, the feedback turns positive, causing the LH surge (and a smaller FSH surge) - ovulation follows about 36 hours after the surge begins 3. Luteal phase (days 15-28, constant about 14 days): - the corpus luteum secretes progesterone and oestrogen - high progesterone plus oestrogen suppresses GnRH, LH and FSH - without hCG the corpus luteum regresses, hormone levels fall, and FSH rises again for the next cycle The uterine (menstrual) cycle - The endometrium has two layers: - stratum functionale: shed at menstruation - stratum basale: stays, regenerates the functionalis - Blood supply: spiral arteries in the functionalis (hormone-sensitive); straight arteries in the basalis. Phases 1. Menstrual phase (days 1-4/5): starts on the first day of bleeding. The functional layer is shed with blood. 2. Proliferative phase (days 5-14): driven by oestrogen from growing follicles. The epithelium and glands regrow from the basalis; glands are straight and narrow; spiral arteries lengthen. The endometrium thickens to about 2-3 mm. 3. Secretory phase (days 15-27): driven by progesterone (and oestrogen) from the corpus luteum. Glands become coiled, then saw-tooth, and secrete glycogen-rich fluid (first sub-nuclear then supranuclear glycogen vacuoles); the stroma becomes oedematous; spiral arteries coil further; stromal cells begin decidualisation. The endometrium reaches about 5-6 mm, ready for implantation (the "implantation window", about days 20-24). 4. Ischaemic phase (about day 28): if there is no pregnancy, progesterone falls; spiral arteries constrict intermittently (prostaglandins), the functional layer becomes ischaemic and necrotic, then is shed, starting menstruation. Cycle with conception - The embryo's trophoblast secretes hCG from about day 8 after fertilisation, rescuing the corpus luteum. Progesterone stays high, the endometrium is kept and becomes the decidua. hCG is the basis of pregnancy tests. Exam focus: match each uterine phase to its ovarian phase and dominant hormone, and explain what triggers the LH surge.

The encounter - Of about 200-300 million sperm ejaculated, only a few hundred reach the ampulla. - The oocyte survives about 12-24 hours; sperm about 3-5 days in the female tract. - Transport: sperm motility, uterine and tubal contractions, and chemotaxis toward signals from the oocyte and cumulus. Capacitation - A series of physiological changes in the female tract (about 5-7 hours) that make sperm able to fertilise. - Removal of the glycoprotein coat and seminal plasma proteins from the sperm surface; cholesterol efflux from the membrane; increased Ca²⁺ entry; hyperactivated motility. - Freshly ejaculated sperm cannot fertilise. IVF procedures capacitate sperm in culture. Phases of fertilisation 1. Penetration of the corona radiata: helped by hyaluronidase on the sperm surface and hyperactivated motility. 2. Binding to the zona pellucida and the acrosome reaction: - the sperm binds ZP3 (and ZP2 in humans), which triggers the acrosome reaction: the acrosomal membrane fuses with the plasma membrane and releases enzymes (acrosin, a serine protease) - the enzymes digest a path through the zona 3. Fusion of sperm and oocyte membranes: - the sperm protein Izumo1 binds the oocyte receptor Juno - the sperm nucleus, centriole and (degraded) mitochondria enter the oocyte Blocks to polyspermy - Fast block (in many species; minor in mammals): depolarisation of the oocyte membrane by an influx of Na⁺. - Cortical reaction (slow block, the main one in humans): - a wave of Ca²⁺ release spreads across the egg - cortical granules beneath the oocyte membrane release their enzymes into the perivitelline space - Zona reaction: the enzymes modify ZP2 and ZP3 so the zona hardens and no longer binds sperm. - Also, Juno is shed from the oocyte membrane. Egg activation (events) 1. Ca²⁺ oscillations triggered by sperm phospholipase C zeta. 2. Cortical reaction. 3. Completion of meiosis II by the oocyte, releasing the second polar body. 4. Metabolic activation: protein synthesis from maternal mRNAs. 5. Formation of pronuclei: the female pronucleus from the oocyte chromosomes; the male pronucleus from the decondensing sperm head (protamines replaced by histones). Within about 12 hours: two pronuclei visible. 6. DNA replication in both pronuclei; they meet but do not fuse. Their envelopes break down and the chromosomes join on the first mitotic spindle (organised by the sperm-derived centrosome). Results of fertilisation - Restoration of the diploid number. - Determination of chromosomal sex (X or Y sperm). - Genetic variation. - Start of cleavage: the zygote divides, forming 2 cells about 30 hours after fertilisation, then continues as a morula (chapter 5). Clinical tip: IVF places oocytes and capacitated sperm together in a dish; ICSI (intracytoplasmic sperm injection) injects a single sperm directly into the oocyte, bypassing capacitation, the zona and membrane fusion. It is used for severe male infertility. Two pronuclei on day 1 confirm normal fertilisation; three suggest polyspermy (triploidy). Exam focus: list the phases of fertilisation in order and explain the cortical and zona reactions.

1. List the cell stages of spermatogenesis in order, with their chromosome number. 2. Why are primary spermatocytes the stage most often seen in tubule sections? 3. What happens in the four phases of spermiogenesis? 4. Give four functions of Sertoli cells. 5. What forms the blood-testis barrier, and why is it needed? 6. What features identify Leydig cells as steroid-secreting cells? 7. At what stage is the human oocyte arrested at birth, and when does it resume meiosis? 8. Describe the follicle from primordial to Graafian. 9. What is the two-cell, two-gonadotropin model? 10. What triggers the LH surge? 11. What happens to the corpus luteum with and without pregnancy? 12. Describe the endometrium in the proliferative and secretory phases. 13. What causes menstruation? 14. What is capacitation? 15. How does the egg prevent polyspermy? 16. What does the presence of two pronuclei tell you in IVF?

1. Spermatogonium (46), primary spermatocyte (46, 4C), secondary spermatocyte (23, 2C), spermatid (23, 1C), spermatozoon (23). 2. Their prophase I is long (about 22 days). 3. Golgi phase: acrosomal vesicle forms; flagellum starts. Cap phase: acrosome covers the nucleus. Acrosome phase: nucleus condenses and elongates, mitochondria form the midpiece. Maturation: excess cytoplasm shed as residual bodies. 4. Any four: support and nourish germ cells; form the blood-testis barrier; phagocytose residual bodies; secrete androgen-binding protein, inhibin and fluid; secrete anti-Müllerian hormone in the fetus. 5. Tight junctions between Sertoli cells. They isolate meiotic and later germ cells, which carry new antigens, from the immune system. 6. Abundant SER, mitochondria with tubular cristae, lipid droplets, eosinophilic cytoplasm. 7. Diplotene (dictyotene) of prophase I. It resumes at the LH surge before ovulation. 8. Primordial: oocyte with flat follicular cells. Primary: cuboidal cells, then multilayered granulosa; zona pellucida forms. Secondary: antrum forms; theca interna and externa. Graafian: large antrum, cumulus oophorus, corona radiata. 9. Theca cells (LH) make androgens; granulosa cells (FSH) aromatise them to oestradiol. 10. Sustained high oestradiol, which switches its feedback from negative to positive. 11. Without pregnancy: regresses after about 14 days to a corpus albicans. With pregnancy: maintained by hCG until the placenta takes over. 12. Proliferative: glands straight and narrow, endometrium regrowing (oestrogen). Secretory: glands coiled and secreting glycogen, oedematous stroma, spiral arteries coiled (progesterone). 13. The fall in progesterone when the corpus luteum regresses, causing spiral artery constriction, ischaemia and shedding of the functional layer. 14. Changes in the female tract that make sperm able to fertilise: removal of coat proteins, cholesterol loss, Ca²⁺ entry, hyperactivation. 15. The cortical reaction releases cortical granule enzymes, which modify the zona (zona reaction) so it no longer binds sperm; also Juno is shed. 16. Normal fertilisation (one male and one female pronucleus). Three suggest polyspermy.

Cleavage - Rapid mitotic divisions without growth: the total size stays the same (still inside the zona pellucida), so blastomeres become smaller. - 2-cell stage: about 30 hours after fertilisation. - 4-cell: about day 2 (second cleavage). - 8-cell: about day 3. - Divisions continue from 2 up to 16-32 cells. - The early embryo relies on maternal mRNAs; the embryonic genome is activated at about the 4-8 cell stage in humans. Compaction (after the 8-cell stage) - Blastomeres flatten against each other and form tight junctions and E-cadherin adhesions, becoming a compact ball. - The first sign of cell polarity: outer cells become polarised; inner cells stay apolar. - The morula (12-32 cells, "mulberry") reaches the uterus about day 3-4. Formation of the blastocyst (day 4-5) - Fluid is pumped into the morula by the outer cells (Na/K pumps), forming a cavity: the blastocoel (blastocyst cavity). - Two cell populations: - trophoblast (trophectoderm): the outer layer; will form the fetal part of the placenta and the chorion - inner cell mass (ICM, embryoblast): a cluster at one pole; will form the embryo proper (plus amnion, yolk sac, allantois) - ICM cells are pluripotent: the source of embryonic stem cells. - The pole with the ICM is the embryonic pole. Hatching (day 5-6) - The blastocyst escapes from the zona pellucida (enzymatic lysis plus expansion). - The zona has prevented premature implantation in the tube; now the trophoblast can contact the endometrium. Clinical tips - Ectopic pregnancy: implantation outside the uterus, most often in the uterine tube (about 95% of cases) because transport is slowed (salpingitis, previous surgery). Risk of rupture and haemorrhage. - Monozygotic twins can arise by splitting of the early embryo: at the 2-cell stage (separate placentas), of the ICM (shared placenta, separate amnions) or later (shared amnion). - IVF embryos are usually transferred at the blastocyst stage (day 5). Exam focus: describe compaction and the two cell populations of the blastocyst and their fates.

Implantation (starting about day 6-8) - The blastocyst attaches by its embryonic pole to the endometrial epithelium, usually on the posterior wall of the uterine body. - The trophoblast differentiates into two layers: - cytotrophoblast: inner, mononucleated, mitotically active cells - syncytiotrophoblast: outer multinucleated mass without cell borders (formed by fusion of cytotrophoblast cells). It invades the endometrium by enzymes and erodes maternal capillaries and glands. - By day 9-10 the blastocyst is completely embedded; a fibrin plug (closing plug) seals the entry point. Interstitial implantation is typical of humans. - The syncytiotrophoblast secretes hCG, which maintains the corpus luteum (the basis of pregnancy tests, positive from about day 10-11 after fertilisation in blood). The bilaminar germ disc - The ICM reorganises into two layers: - epiblast: tall columnar cells facing the future amniotic cavity; will form all three germ layers of the embryo - hypoblast: small cuboidal cells facing the blastocoel; contributes to the yolk sac (extraembryonic) The three cavities 1. Amniotic cavity: forms within the epiblast (day 8); lined by amnioblasts. The floor is the epiblast. 2. Yolk sac: the primary yolk sac forms when hypoblast cells spread to line the blastocoel (Heuser's membrane, about day 9); it is then replaced by a smaller secondary (definitive) yolk sac (day 12-13). 3. Chorionic cavity (extraembryonic coelom): forms by fusion of spaces within the extraembryonic mesoderm (day 12-13). It surrounds the amnion and yolk sac, except at the connecting stalk. Extraembryonic mesoderm - A loose tissue between the trophoblast and the amnion and yolk sac. Splits into: - extraembryonic somatic mesoderm: lines the trophoblast and covers the amnion - extraembryonic splanchnic mesoderm: covers the yolk sac - Connecting stalk: the only link between the embryo and the trophoblast; will become the umbilical cord. - The chorion = the extraembryonic somatic mesoderm plus the two trophoblast layers. Uteroplacental circulation begins - Lacunae form in the syncytiotrophoblast (day 9), fill with maternal blood from eroded capillaries (day 11-12). This is the first exchange with the mother. - Primary villi appear at the end of week 2 (day 13-14). Decidualisation - Under progesterone, stromal cells of the endometrium swell with glycogen and lipid and become decidual cells. The whole endometrium is now the decidua. - Functions: nutrition of the early embryo, limiting trophoblast invasion, immune tolerance of the embryo. Day 14-15 - The prechordal plate appears: a thickening of the hypoblast marking the future cranial end and the site of the mouth. Exam focus: list the "twos" of week 2, and describe the three cavities and when they form. Clinical tip: in hydatidiform mole the trophoblast proliferates abnormally without a normal embryo (complete mole: paternal genome only). hCG is very high; there is a risk of choriocarcinoma, so hCG is monitored after treatment.

Gastrulation (starting day 15) - The primitive streak appears as a groove on the surface of the epiblast at the caudal end of the disc. At its cranial end: the primitive node (Hensen's node) with the primitive pit. - The primitive streak defines the body axes: cranial-caudal, dorsal-ventral, left-right. - Cell movements: epiblast cells move toward the streak, detach (epithelial-to-mesenchymal transition, loss of E-cadherin), and pass inward (invagination, ingression): 1. the first cells displace the hypoblast and form the definitive endoderm 2. the next cells spread between epiblast and endoderm to form the intraembryonic mesoderm 3. the remaining epiblast becomes the ectoderm - So all three germ layers come from the epiblast. - Mesoderm spreads everywhere except at two places where ectoderm and endoderm stay fused: the oropharyngeal membrane (cranial; future mouth) and the cloacal membrane (caudal; future anus). - Mesoderm is patterned in a cranial-to-caudal gradient: cranial structures form first. The streak regresses as the embryo grows. - Clinical: remnants of the primitive streak can give sacrococcygeal teratoma. Disturbed gastrulation can cause caudal dysgenesis (sirenomelia). The notochord - Formed by cells passing through the primitive pit and moving cranially in the midline up to the prechordal plate. A rod of mesodermal cells. - Functions: 1. defines the midline axis 2. provides rigidity to the embryo 3. induces the overlying ectoderm to form the neural plate (by signalling molecules such as sonic hedgehog and inhibitors of BMP) 4. induces the ventral neural tube (floor plate) and the sclerotomes 5. forms the nucleus pulposus of the intervertebral discs (its only adult remnant) - Clinical: chordoma, a tumour of notochord remnants. Neurulation (days 18-28) 1. Neural plate: the ectoderm above the notochord thickens (neuroectoderm). 2. Neural groove: the plate folds; neural folds rise on each side. 3. Neural tube: the folds meet in the midline and fuse, starting in the future neck region and zipping cranially and caudally. Open ends are the neuropores: - cranial neuropore closes about day 25 - caudal neuropore closes about day 27-28 4. The tube separates from the surface ectoderm, which covers it (future epidermis). - The neural tube forms the brain (cranial part) and spinal cord. Neural crest - Cells at the neural plate border (the crest of the neural folds). During closure they undergo epithelial-to-mesenchymal transition and migrate throughout the body. - Derivatives (a very wide range, sometimes called the "fourth germ layer"): - sensory (dorsal root) ganglia and autonomic ganglia - Schwann cells - adrenal medulla chromaffin cells - melanocytes - craniofacial bones, cartilage and connective tissue - parts of the heart outflow tract (aorticopulmonary septum) - odontoblasts, C cells of the thyroid - pia and arachnoid (part) - Clinical: neurocristopathies (Hirschsprung disease, DiGeorge sequence components, neuroblastoma). Clinical tip: neural tube defects (anencephaly when the cranial neuropore fails to close; spina bifida when the caudal one fails) are reduced by folic acid supplementation before conception and in early pregnancy. Maternal serum and amniotic alpha-fetoprotein are raised in open defects. Exam focus: describe gastrulation, list five functions of the notochord, and list six neural crest derivatives.

Differentiation of the mesoderm (from medial to lateral) 1. Paraxial mesoderm: thick columns next to the notochord that segment into somites (the first pair on day 20; about 3 pairs per day, to 42-44 pairs, of which about 37 remain). Somite number is used to stage embryos. - Each somite divides into: - sclerotome (ventromedial): cells lose their adhesion molecules, become mesenchyme, migrate around the notochord and neural tube, and form the vertebrae (vertebral bodies and arches) and ribs - dermatome: dermis of the back - myotome: skeletal muscles of the back, body wall and limbs - Cranially the paraxial mesoderm forms somitomeres: muscles of the head (striated skeletal muscle). 2. Intermediate mesoderm: forms the urogenital system (kidneys, gonads, their ducts). 3. Lateral plate mesoderm: splits into two layers, separated by the intraembryonic coelom (future body cavities): - somatic (parietal) layer, with ectoderm: body wall, parietal serous membranes, limb bones and connective tissue - splanchnic (visceral) layer, with endoderm: wall of the gut (smooth muscle, connective tissue), visceral serous membranes, heart, blood vessels and blood cells Folding of the embryo (week 4) - Driven by the rapid growth of the neural tube and somites, which outgrow the yolk sac. - Folding in the median plane (cranial-caudal): - head fold: the brain grows over the oropharyngeal membrane; the heart and septum transversum move ventrally and caudally to the chest; part of the yolk sac becomes the foregut. - tail fold: the caudal end curves ventrally; part of the yolk sac becomes the hindgut; the connecting stalk and allantois move to the ventral side. - Folding in the horizontal plane (lateral folds): the two sides of the disc roll ventrally and meet in the midline, forming the ventral body wall. Part of the endoderm is incorporated into the embryo as the midgut, which stays connected to the yolk sac by the vitelline (yolk) duct. - Results of folding: 1. the embryo becomes a C-shaped cylinder 2. the gut tube forms (foregut, midgut, hindgut) from endoderm 3. the intraembryonic coelom becomes the closed body cavities 4. the amnion surrounds the embryo; the connecting stalk and yolk duct come together to form the umbilical cord 5. the heart and oropharyngeal membrane move to their ventral positions - Clinical: failure of lateral folding gives ventral body wall defects (gastroschisis; ectopia cordis). Germ-layer derivatives - Ectoderm: - surface ectoderm: epidermis, hair, nails, sweat, sebaceous and mammary glands, enamel of teeth, lens of the eye, inner ear, anterior pituitary (Rathke's pouch) - neuroectoderm: brain, spinal cord, retina, posterior pituitary, pineal gland - neural crest: see week 3 - Mesoderm: - connective tissue, cartilage, bone - skeletal, smooth and cardiac muscle - heart, blood vessels, blood and lymphatic cells, spleen - kidneys, gonads and their ducts - adrenal cortex - serous membranes (pleura, pericardium, peritoneum), dermis - Endoderm: - epithelial lining of the gut tube and respiratory tract - parenchyma of liver, pancreas, thyroid, parathyroids, thymus (epithelial part) - lining of the urinary bladder and urethra (mostly) - lining of the tympanic cavity and auditory tube Exam focus: name the three parts of the somite and their fates, and list three derivatives of each germ layer.

Amnion - A thin membrane (amniotic ectoderm plus extraembryonic somatic mesoderm) enclosing the amniotic cavity. - As the embryo folds, the amnion expands to surround it completely, covering the umbilical cord. By about week 8-12 it fills the chorionic cavity and fuses with the chorion (amniochorionic membrane), which ruptures at birth ("breaking of the waters"). - Amniotic fluid: about 30 mL at week 10, about 1 L by week 34-38. Derived initially from maternal plasma, later mainly from fetal urine. Swallowed by the fetus. - Functions: protection against mechanical shock; free movement (muscle and limb development); lung development (fetal breathing of fluid); temperature control; prevents adhesions of the embryo to the amnion. - Clinical: oligohydramnios (too little fluid; renal agenesis, pulmonary hypoplasia); polyhydramnios (too much; oesophageal atresia, anencephaly, diabetes). Amniocentesis samples fetal cells. Yolk sac - Humans have no yolk; the yolk sac is small but important. - Functions: 1. transfer of nutrients in weeks 2-3, before the placenta circulation starts 2. first site of haematopoiesis (blood islands in its wall, week 3) 3. origin of primordial germ cells, which migrate to the gonads 4. part is incorporated into the gut tube during folding - It then shrinks; the vitelline duct normally closes. Clinical: Meckel diverticulum is a persistent remnant of the yolk duct on the ileum (the "rule of 2s"). Allantois - A small outpouching of the yolk sac (hindgut) into the connecting stalk, about day 16. - Blood vessels form in its wall and become the umbilical arteries and vein. - Proximal part becomes the urachus (connects the bladder to the umbilicus), which becomes the median umbilical ligament. Clinical: a patent urachus leaks urine from the umbilicus. Umbilical cord - Develops from the connecting stalk and contains remnants of the yolk sac (vitelline duct) and allantois. - At term: about 50-60 cm long, 1-2 cm thick. - Contents: two umbilical arteries (deoxygenated blood to the placenta) and one umbilical vein (oxygenated blood to the fetus), in Wharton's jelly (mucous connective tissue rich in hyaluronic acid), covered by amnion. - Clinical: a single umbilical artery (about 1%) is associated with fetal anomalies (cardiac, renal). Chorion - Trophoblast plus extraembryonic somatic mesoderm. Its villi form the fetal part of the placenta (chorion frondosum); elsewhere it becomes smooth (chorion laeve). Exam focus: list the functions of the amnion and yolk sac, and the contents of the umbilical cord.

The decidua (three regions, named by position relative to the embryo) - Decidua basalis: under the embryo, between it and the myometrium. Forms the maternal part of the placenta. - Decidua capsularis: covers the embryo on the luminal side. Thins as the embryo grows. - Decidua parietalis: lines the rest of the uterus. - By about 4-5 months the capsularis fuses with the parietalis and the uterine cavity is obliterated. Chorion - Chorion frondosum (villous chorion): the part facing the decidua basalis, where villi grow and branch: the fetal part of the placenta. - Chorion laeve (smooth chorion): the part facing the decidua capsularis, where villi degenerate. Development of the chorionic villi 1. Primary villi (end of week 2, about day 13): a core of cytotrophoblast covered by syncytiotrophoblast. 2. Secondary villi (about day 16): extraembryonic mesoderm grows into the core. 3. Tertiary villi (end of week 3): blood vessels develop in the mesoderm core and connect with the embryo's circulation through the connecting stalk (heart beating from about day 22). - Anchoring (stem) villi reach the decidua; the cytotrophoblast shell spreads over it and attaches the chorion. Free (terminal) villi branch from them into the intervillous space. - Extravillous cytotrophoblast invades the maternal spiral arteries and remodels them into wide, low-resistance vessels. The placenta at term: gross anatomy - Disc-shaped, about 15-20 cm in diameter, 2-3 cm thick, about 500-600 g (about 1/6 of fetal weight). - Fetal surface: smooth, covered by amnion, with the umbilical cord and chorionic vessels. - Maternal surface: divided by placental septa (from the decidua) into about 15-20 cotyledons (lobes). - The placenta is limited by the chorionic plate (fetal side) and the basal plate (decidua basalis, maternal side). The intervillous space - Filled with maternal blood (about 150 mL, renewed 3-4 times per minute), entering from about 80-100 spiral arteries, bathing the villi, and draining through endometrial veins. - Fetal blood flows inside the villous capillaries: deoxygenated blood in through the umbilical arteries, oxygenated blood out through the umbilical vein. - The human placenta is haemochorial: maternal blood is in direct contact with the chorion (syncytiotrophoblast). The placental barrier (membrane) - Separates fetal from maternal blood. - Early pregnancy (four layers): syncytiotrophoblast, cytotrophoblast, villous connective tissue, fetal capillary endothelium. - After about 20 weeks it thins: the cytotrophoblast becomes discontinuous and capillaries move to the villus surface, forming vasculosyncytial membranes (in places only syncytiotrophoblast and endothelium, about 2-4 µm). - It is not a complete barrier: many drugs, alcohol, some viruses and bacteria cross. Transfer across the placental membrane - Simple diffusion: O₂, CO₂, water, electrolytes, lipid-soluble drugs and gases. - Facilitated diffusion: glucose (GLUT1). - Active transport: amino acids, iron, calcium, vitamins. - Pinocytosis (receptor-mediated transcytosis): maternal IgG via the FcRn receptor, giving the newborn passive immunity for months. IgM does not cross. - Waste: urea, uric acid, bilirubin go to the mother. - Crossing that matters clinically: teratogens (alcohol, isotretinoin, warfarin, some antiepileptics), infections (TORCH: toxoplasma, rubella, cytomegalovirus, herpes, syphilis, HIV, Zika), anti-D IgG (haemolytic disease of the newborn). Placental functions 1. Exchange: respiratory, nutritional, excretory. 2. Endocrine (the syncytiotrophoblast): hCG (maintains the corpus luteum; peaks at about 8-10 weeks), progesterone (placenta takes over from the corpus luteum by week 8-12), oestrogens (mainly oestriol, from fetal adrenal precursors), human placental lactogen (hPL, raises maternal glucose, contributes to insulin resistance), CRH. 3. Barrier function: partial protection against pathogens and toxins. 4. Immunological function: the fetus is a semi-allograft (half paternal antigens), yet it is not rejected. Mechanisms: the syncytiotrophoblast does not express classical HLA class I and II; extravillous trophoblast expresses the non-classical HLA-G, which inhibits NK cells; local immunosuppressive factors; regulatory T cells in the decidua. Clinical tips - Placenta praevia: implantation near or over the cervix, causing painless bleeding late in pregnancy. - Placenta accreta, increta, percreta: villi anchor into, invade or penetrate the myometrium because the decidua basalis is deficient (often at a previous caesarean scar). - Pre-eclampsia: linked to poor remodelling of spiral arteries by the trophoblast. Exam focus: describe the development of primary, secondary and tertiary villi, and the layers of the placental barrier early and late in pregnancy.

1. What happens during compaction, and when? 2. What are the two cell populations of the blastocyst, and what does each form? 3. What is hatching, and why is the zona pellucida important before it? 4. Name the two layers of the trophoblast and describe each. 5. Name the three cavities formed in week 2. 6. What is decidualisation? 7. What is the primitive streak, and what axes does it establish? 8. From which layer do all three germ layers derive? 9. Give four functions of the notochord. 10. Describe neurulation in four steps. 11. Name six neural crest derivatives. 12. Into which three parts does a somite divide, and what does each form? 13. What are the results of folding of the embryo? 14. Give three derivatives of each germ layer. 15. List four functions of the yolk sac. 16. What does the umbilical cord contain? 17. Name the three regions of the decidua. 18. What is the difference between primary, secondary and tertiary villi? 19. List the layers of the placental barrier early in pregnancy. 20. How does the fetus avoid rejection by the mother's immune system?

1. After the 8-cell stage, blastomeres flatten against each other and form tight junctions and E-cadherin adhesions; outer cells become polarised. 2. Trophoblast: the placenta and chorion. Inner cell mass: the embryo (plus amnion, yolk sac, allantois). 3. The blastocyst escapes from the zona (day 5-6). The zona prevents implantation in the tube. 4. Cytotrophoblast: inner, mononucleated, dividing. Syncytiotrophoblast: outer, multinucleated, invasive, secretes hCG. 5. Amniotic cavity, yolk sac, chorionic cavity (extraembryonic coelom). 6. The transformation of endometrial stromal cells into large glycogen- and lipid-rich decidual cells under progesterone. 7. A groove in the caudal epiblast through which cells move inward during gastrulation. It establishes cranial-caudal, dorsal-ventral and left-right axes. 8. The epiblast. 9. Any four: defines the midline; gives rigidity; induces the neural plate; induces the floor plate and sclerotomes; forms the nucleus pulposus. 10. Neural plate, neural groove with folds, fusion into the neural tube, separation from the surface ectoderm (neuropores close about day 25 and 27-28). 11. Any six: sensory and autonomic ganglia, Schwann cells, adrenal medulla, melanocytes, craniofacial skeleton, heart outflow septum, odontoblasts, thyroid C cells. 12. Sclerotome: vertebrae and ribs. Dermatome: dermis of the back. Myotome: skeletal muscles. 13. A C-shaped cylindrical embryo; the gut tube; closed body cavities; the amnion surrounds the embryo; the umbilical cord forms; the heart and mouth move ventrally. 14. Ectoderm: epidermis, nervous system, lens. Mesoderm: bone, muscle, heart, kidney. Endoderm: gut lining, liver, pancreas, thyroid, lung epithelium. 15. Early nutrient transfer, first blood formation, origin of primordial germ cells, incorporation into the gut. 16. Two umbilical arteries and one vein in Wharton's jelly, covered by amnion (plus remnants of the yolk duct and allantois). 17. Decidua basalis, capsularis, parietalis. 18. Primary: cytotrophoblast core. Secondary: plus mesoderm core. Tertiary: plus fetal blood vessels. 19. Syncytiotrophoblast, cytotrophoblast, villous connective tissue, fetal capillary endothelium. 20. The trophoblast lacks classical HLA; HLA-G inhibits NK cells; local immunosuppressive factors; decidual regulatory T cells.

Functions - Protection (epidermis, oesophagus). - Absorption (intestine, kidney tubule). - Secretion (glands, stomach lining). - Excretion (kidney). - Filtration (glomerulus). - Diffusion and exchange (lung alveoli, capillaries). - Sensory reception (taste buds, olfactory epithelium, retina). - Transport along the surface (ciliated airway). - Barrier: controls what enters and leaves the body. Characteristics 1. Cellularity: cells tightly packed, little intercellular space. 2. Polarity: apical (free) surface facing the lumen or outside; lateral surfaces with junctions; basal surface on the basement membrane. Organelles are distributed asymmetrically (nucleus often basal; Golgi above it; secretory granules apical). 3. Attachment: the basal surface is attached to the basement membrane. 4. Avascularity: no blood vessels; nutrients diffuse from capillaries in the underlying connective tissue (the lamina propria in mucosae). This limits epithelial thickness. 5. Innervation: free nerve endings between cells. 6. Regeneration: high turnover in many epithelia (labile tissue); stem cells in the basal layer or crypts. Origin - From all three germ layers: epidermis (ectoderm), gut lining (endoderm), endothelium and mesothelium (mesoderm). Endothelium lines vessels; mesothelium lines serous cavities. The basement membrane - A thin sheet of specialised extracellular matrix between epithelium and connective tissue (also around muscle cells, fat cells and Schwann cells, where it is called the external lamina). - Light microscopy: visible with PAS (because of glycoproteins) or silver stains; often barely visible on H&E. - Electron microscopy: two components: 1. basal lamina (made by the epithelial cells), about 40-60 nm: - lamina lucida (rara): the lighter layer next to the cell membrane, containing laminin and integrin anchors - lamina densa: the dense layer, a network of type IV collagen 2. lamina reticularis (made by connective tissue fibroblasts): type III collagen (reticular fibres), anchored to the lamina densa by type VII collagen anchoring fibrils. - Components: type IV collagen, laminin, nidogen (entactin), perlecan (a heparan sulfate proteoglycan). - Functions: - structural support and attachment of epithelium - selective filtration barrier (thick fused basement membrane in the kidney glomerulus) - boundary: carcinoma becomes invasive when it breaches the basement membrane (carcinoma in situ does not) - scaffold for regeneration and cell migration - signalling for cell polarity, differentiation and survival Clinical tips - Goodpasture syndrome: autoantibodies against type IV collagen in the glomerular and alveolar basement membranes, causing glomerulonephritis and lung haemorrhage. - Alport syndrome: type IV collagen mutations (kidney disease, deafness, eye defects). - Diabetic nephropathy: thickened basement membranes. Exam focus: describe the layers of the basement membrane and their components, and explain why breach of the basement membrane defines invasive carcinoma.

Simple squamous epithelium - One layer of flat cells with flattened nuclei (seen from above: like a fried egg; in section: thin line with bulging nuclei). - Function: diffusion, filtration, secretion of lubricating fluid. - Locations: - endothelium: lines blood and lymphatic vessels and the heart - mesothelium: lines serous cavities (pleura, pericardium, peritoneum) - lung alveoli (type I pneumocytes) - Bowman's capsule (parietal layer), thin loop of Henle Simple cuboidal epithelium - One layer of cube-shaped cells with round central nuclei. - Function: secretion and absorption. - Locations: kidney tubules, thyroid follicles, small ducts of glands, surface of the ovary (germinal "epithelium"), pigment epithelium of the retina. Simple columnar epithelium - One layer of tall cells with oval basal nuclei. - Function: absorption and secretion. - Variants: - non-ciliated with microvilli (brush or striated border) and goblet cells: small and large intestine - secretory: stomach surface (mucous cells, no goblet cells) - ciliated: uterine tubes, uterus, small bronchi, central canal of the spinal cord Pseudostratified columnar epithelium - One layer of cells, all touching the basement membrane, but not all reaching the surface. Nuclei at different heights, so it looks stratified. - Usually ciliated with goblet cells: respiratory epithelium (nasal cavity lining, trachea, bronchi). - Non-ciliated with stereocilia: epididymis, ductus deferens. - Function: secretion of mucus and moving it by cilia (mucociliary clearance). Stratified squamous epithelium - Several layers: cuboidal or columnar basal cells, polygonal cells in the middle, flat surface cells. Named after the surface cell shape. - Function: protection against abrasion. - Non-keratinised (surface cells keep their nuclei, surface kept moist): mouth, oesophagus, vagina, cornea, anal canal (part), ectocervix. - Keratinised (surface layers of dead cells filled with keratin): the epidermis of the skin. Layers of the epidermis (keratinised stratified squamous; deep to superficial) 1. Stratum basale (germinativum): one layer of cuboidal to columnar stem cells on the basement membrane, dividing by mitosis. Also melanocytes and Merkel cells. 2. Stratum spinosum (prickle cell layer): several layers of polygonal cells joined by many desmosomes. After shrinkage in processing they look spiny. Langerhans cells (antigen-presenting) are found here. 3. Stratum granulosum (granular layer): 3-5 layers of flattened cells with keratohyalin granules (basophilic; filaggrin) and lamellar bodies (lipid, secreted to form the water barrier). Cells start to die. 4. Stratum lucidum (clear layer): a thin, clear, eosinophilic layer, only in thick skin (palms, soles). 5. Stratum corneum: about 15-20 (up to 50 in thick skin) layers of dead, non-nucleated flat cells filled with keratin. Shed continuously (desquamation). - Renewal takes about 4 weeks. - Thick skin (palms, soles) has all five layers and no hair; thin skin lacks the stratum lucidum. - Clinical: psoriasis has accelerated turnover, with nuclei retained in the corneum (parakeratosis). Stratified cuboidal and stratified columnar epithelium - Rare. Usually two layers. - Stratified cuboidal: ducts of sweat glands. - Stratified columnar: large ducts of salivary glands, conjunctiva, parts of the male urethra. Transitional epithelium (urothelium) - Found only in the urinary tract: renal calyces and pelvis, ureters, bladder, proximal urethra. - Stratified: basal cuboidal cells, intermediate pear-shaped cells, and large dome-shaped surface cells ("umbrella" cells, sometimes binucleated). - Changes shape with stretching: in an empty bladder about 5-7 layers with domed surface cells; in a full bladder 2-3 layers with flattened surface cells. - The surface cells have a thickened apical membrane with rigid plaques of uroplakins, which fold into the cell when the bladder is empty and form a barrier against hypertonic urine. Metaplasia - The replacement of one mature epithelial type by another in response to chronic stress. It is reversible but may progress to dysplasia and cancer. - Examples: in smokers, respiratory pseudostratified epithelium becomes stratified squamous (squamous metaplasia). In gastro-oesophageal reflux, oesophageal squamous epithelium becomes intestinal-type columnar with goblet cells (Barrett's oesophagus; risk of adenocarcinoma). Exam focus: name each epithelial type with a location and function, and list the layers of the epidermis in order.

The junctional complex - In columnar epithelia (e.g. intestine), the apical-lateral region has a sequence of three junctions visible in TEM, called the junctional complex. In light microscopy it appears as the terminal bar. - From apical to basal: 1. zonula occludens (tight junction) 2. zonula adherens (adherens junction) 3. macula adherens (desmosome) - "Zonula" means a continuous belt around the cell; "macula" means a spot. 1. Zonula occludens (tight junction) - The outer leaflets of adjacent membranes fuse at points, forming a network of sealing strands. - Proteins: claudins and occludin (transmembrane), linked to ZO-1 and the actin cytoskeleton. - Functions: - barrier: seal the intercellular space, preventing paracellular passage of water and solutes (the tightness varies: very tight in the bladder, "leaky" in the proximal tubule) - fence: keep apical and basolateral membrane proteins separate, maintaining polarity - Examples: blood-brain barrier (endothelium), blood-testis barrier (Sertoli cells). 2. Zonula adherens (adherens junction) - A belt just below the tight junction; intercellular space about 15-20 nm. - Proteins: cadherins (E-cadherin; Ca²⁺-dependent), linked by catenins to actin filaments. The belt of actin with myosin (terminal web region) allows shape changes. - Function: cell-cell adhesion; tissue organisation. - Clinical: loss of E-cadherin helps tumour cells invade (and is characteristic of lobular breast carcinoma and diffuse gastric cancer). 3. Macula adherens (desmosome) - A spot weld: two dense plaques on the cytoplasmic side of each membrane, with a dense line in the intercellular space (about 30 nm). - Proteins: desmosomal cadherins (desmogleins and desmocollins) in the space; plaques made of desmoplakin and plakoglobin, which link to intermediate filaments (keratin in epithelia; desmin in cardiac muscle). - Function: strong mechanical attachment; spreads stress across the tissue. Very abundant in the epidermis (stratum spinosum) and heart muscle. - Loss of desmosome function causes blistering diseases: - pemphigus vulgaris: autoantibodies against desmoglein 3 (and 1), causing blisters within the epidermis (acantholysis) and in the mouth - arrhythmogenic cardiomyopathy: mutations in desmosomal proteins 4. Hemidesmosome - Half a desmosome on the basal surface; attaches the cell to the basement membrane. - Proteins: integrins (α6β4) and collagen XVII (BP180) in the membrane; plaques with plectin and BP230 linking to keratin intermediate filaments. - Clinical: bullous pemphigoid: autoantibodies against hemidesmosomal proteins, causing tense blisters below the epidermis. - Focal adhesions are the other cell-matrix junction, linking integrins to actin (important in cell migration). 5. Gap junction (nexus) - Communicating junction: adjacent membranes about 2-3 nm apart, bridged by channels. - Each channel: two connexons (one from each cell), each made of six connexins, with a pore of about 1.5-2 nm. - Allow the passage of ions and small molecules (below about 1 kDa: ions, cAMP, glucose, amino acids) between cells. - Functions: electrical coupling (heart muscle intercalated discs, smooth muscle, electrical synapses); metabolic coupling and coordination (e.g. between osteocytes, granulosa cells and oocyte). - They close when Ca²⁺ rises or pH falls, isolating damaged cells. - Clinical: connexin 26 mutations are the most common cause of hereditary non-syndromic deafness. Summary - Occluding: tight junction. Seals. - Anchoring cell-cell: adherens junction (actin), desmosome (intermediate filaments). - Anchoring cell-matrix: hemidesmosome (intermediate filaments), focal adhesion (actin). - Communicating: gap junction. Exam focus: for each junction give its location, transmembrane proteins, cytoskeletal link, function and one related disease.

Microvilli - Finger-like projections of the plasma membrane, about 1 µm long and 0.08 µm wide. - Core: a bundle of 20-30 actin filaments cross-linked by villin and fimbrin, anchored in the terminal web (a network of actin and spectrin under the apical membrane). - Not motile (may shorten a little). - Function: increase the surface area for absorption (up to 20 times). - Light microscopy: a fuzzy, eosinophilic striated border (intestine) or brush border (proximal kidney tubule), which is PAS-positive due to the glycocalyx. - The glycocalyx on intestinal microvilli contains digestive enzymes (disaccharidases, peptidases). - Clinical: coeliac disease destroys villi and microvilli, causing malabsorption; microvillus inclusion disease is a rare congenital cause of diarrhoea. Cilia - Motile projections, about 5-10 µm long and 0.2 µm wide. About 200-300 per cell in the airway. - Core: axoneme with microtubules in a 9+2 arrangement: 9 peripheral doublets and 2 central singlets inside a central sheath. Radial spokes link doublets to the central pair; nexin links adjacent doublets. - Dynein arms (inner and outer) on each doublet use ATP to make doublets slide past each other. Because the doublets are linked, sliding becomes bending. - Anchored in the cell by a basal body (a centriole-like structure with 9 triplets and no central pair). - Beating: a fast effective stroke and a slow recovery stroke, coordinated in waves (metachronal rhythm). - Function: move materials over epithelia: - airways: move mucus with trapped debris and microbes toward the pharynx (mucociliary escalator) - uterine tube: move the oocyte toward the uterus - brain ventricles (ependyma): move cerebrospinal fluid - Primary cilia: most cells have a single non-motile cilium (9+0, no central pair, no dynein), which acts as a sensor (e.g. flow in kidney tubules). Defects give ciliopathies such as polycystic kidney disease. - Clinical: primary ciliary dyskinesia (Kartagener syndrome when with situs inversus) is caused by dynein arm defects; patients have chronic sinusitis, bronchiectasis and infertility. Smoking paralyses and destroys cilia. - Sperm flagella have the same 9+2 structure but are much longer. Stereocilia - Long, non-motile microvilli (up to 120 µm), often branched, with an actin core but no axoneme. - Locations and functions: - epididymis and ductus deferens: absorption of fluid from the lumen - inner ear hair cells: mechanoreception (they are arranged in rows of increasing height; bending opens mechanosensitive ion channels) - Despite the name, they are not cilia. Comparison - Microvilli: actin core; about 1 µm; not motile; absorption. - Cilia: 9+2 microtubules; 5-10 µm; motile; move material. - Stereocilia: actin core; very long; not motile; absorption or sensing. Exam focus: compare microvilli, cilia and stereocilia by core structure, size, motility and location; describe how dynein causes ciliary bending.

1. List six functions of epithelium. 2. How are epithelial cells nourished? 3. What are the two components of the basement membrane, and who makes each? 4. Name the main molecules of the basal lamina. 5. Why is breach of the basement membrane important in cancer? 6. Give two locations of simple squamous epithelium. 7. Where is pseudostratified ciliated columnar epithelium found, and why does it look stratified? 8. List the layers of the epidermis from deep to superficial. 9. What is special about transitional epithelium? 10. What is metaplasia? Give an example. 11. Name the three components of the junctional complex from apical to basal. 12. What are the two functions of tight junctions? 13. Which junction is attacked in pemphigus vulgaris? In bullous pemphigoid? 14. What passes through gap junctions, and what are they made of? 15. What is the core structure of a microvillus? 16. Describe the axoneme of a cilium. 17. What is primary ciliary dyskinesia? 18. Where are stereocilia found, and what do they do?

1. Any six: protection, absorption, secretion, excretion, filtration, diffusion, sensory reception, transport along the surface. 2. By diffusion from capillaries in the underlying connective tissue; epithelium is avascular. 3. Basal lamina (epithelial cells) and lamina reticularis (fibroblasts). 4. Type IV collagen, laminin, nidogen (entactin), perlecan. 5. Invasive carcinoma is defined by crossing the basement membrane; before that it is carcinoma in situ. 6. Any two: endothelium, mesothelium, lung alveoli, Bowman's capsule. 7. Airways (nasal cavity, trachea, bronchi). All cells touch the basement membrane but not all reach the surface, so nuclei lie at different heights. 8. Basale, spinosum, granulosum, lucidum (thick skin only), corneum. 9. It stretches: surface umbrella cells flatten and the layers decrease when the bladder fills; uroplakin plaques form a barrier to urine. 10. Reversible replacement of one mature epithelial type by another. Barrett's oesophagus, or squamous metaplasia of bronchi in smokers. 11. Zonula occludens, zonula adherens, macula adherens (desmosome). 12. Barrier (block paracellular passage) and fence (keep apical and basolateral membrane proteins separate). 13. Pemphigus: desmosome (desmoglein). Pemphigoid: hemidesmosome. 14. Ions and small molecules below about 1 kDa. Connexons, each of six connexins. 15. A bundle of actin filaments cross-linked by villin and fimbrin, anchored in the terminal web. 16. Nine peripheral microtubule doublets and two central singlets (9+2), with dynein arms, radial spokes and nexin links, anchored in a basal body. 17. An inherited defect (usually of dynein arms) that immobilises cilia, causing chronic airway infections and infertility; with situs inversus it is Kartagener syndrome. 18. Epididymis (absorption) and inner ear hair cells (mechanoreception).

Exocrine versus endocrine - Ducts: exocrine have them; endocrine are ductless. - Destination: exocrine to an epithelial surface or lumen (skin, gut); endocrine into the blood (via fenestrated capillaries) to reach distant targets. - Products: exocrine: sweat, saliva, digestive enzymes, mucus, sebum, milk. Endocrine: hormones. - Organisation: exocrine: secretory units plus ducts. Endocrine: cords, clusters or follicles of cells in a rich capillary network. - Mixed (amphicrine) organs: - pancreas: exocrine acini (enzymes) plus endocrine islets (insulin, glucagon) - liver: bile (exocrine) and plasma proteins released into blood - testis and ovary: gametes (like an exocrine product) plus hormones Unicellular versus multicellular - Unicellular: single secretory cells scattered in a covering epithelium. The main example is the goblet cell. Also diffuse (enteroendocrine) endocrine cells. - Multicellular: organised groups of cells, with or without ducts. Includes secretory sheets (the mucous surface of the stomach: every surface cell secretes) and intraepithelial glands (small groups in the urethra). The goblet cell - A mucus-secreting cell in the simple columnar epithelium of the intestine and the pseudostratified epithelium of the airways (also the conjunctiva). - Shape: the apical part (theca) is swollen with mucinogen granules; the narrow base (stem) holds the nucleus, RER and Golgi. The shape resembles a goblet (wine glass). - H&E: the theca appears pale or empty (mucin is washed out and stains poorly). PAS or alcian blue stain it strongly (magenta or blue). - Apical surface: a few short microvilli. - Secretion: merocrine (exocytosis), both continuous and in bursts in response to irritants. - Mucins are heavily glycosylated proteins (O-linked sugars added in the Golgi) that absorb water to form mucus: lubrication and protection. - Number increases along the intestine (more in the colon) and in the airways of smokers and asthmatics (goblet cell hyperplasia). Exam focus: explain why goblet cells look empty on H&E and which stains reveal their content.

Duct - Simple: one unbranched duct. - Compound: the duct branches repeatedly (like a tree), each branch ending in secretory units. Secretory units - Tubular: the secretory part is a tube. - Acinar (from Latin "acinus", grape): round or flask-shaped with a small lumen. - Alveolar: sac-shaped with a wide lumen. Many authors use acinar and alveolar interchangeably; the slides distinguish them. - Tubuloacinar (tubuloalveolar): both shapes in the same gland. - Branched: several secretory units converge onto one duct (for example 4 secretory units converging on 1 duct). Simple tubular glands - Straight tubes opening directly on the surface. - Examples: intestinal crypts of Lieberkühn (large intestine), glands of the gastric body (fundic glands, which may branch). Simple coiled tubular glands - A long tube whose deep secretory portion is coiled into a ball. - Example: eccrine sweat glands. - Secretory portion in the deep dermis: pale-staining clear cells (water and electrolytes) and dark cells (glycoproteins), with myoepithelial cells around them. - Excretory duct: stratified cuboidal epithelium (two layers of darker cells), reabsorbs Na⁺ and Cl⁻, opens by a spiral path onto the skin surface. - Merocrine secretion; thermoregulation; found all over the skin. Controlled by sympathetic cholinergic fibres. - Clinical: in cystic fibrosis the duct cannot reabsorb Cl⁻, so sweat is salty (the sweat chloride test). - Compare apocrine sweat glands: larger lumen, open into hair follicles (axilla, groin), start working at puberty, secrete a protein-rich fluid that bacteria break down to cause body odour. Despite the name, they probably secrete by merocrine exocytosis. Simple branched tubular glands - Several tubes open into one short duct. - Examples: glands of the stomach (pylorus), uterine glands, Brunner's glands of the duodenum (branched to compound). Compound tubular glands - Branched duct system with tubular secretory units. - Examples: Brunner's (duodenal) glands, bulbourethral glands, mammary glands in some descriptions. Simple (branched) acinar glands - Simple acinar: rare in humans (small glands in the urethra). - Branched acinar: sebaceous glands (several acini open into one short duct that drains into the hair follicle); meibomian (tarsal) glands of the eyelid. Compound acinar glands - Branched duct system with acinar units. - Examples: exocrine pancreas; parotid gland. - Pancreas: purely serous acini with pyramidal cells (basal basophilia, apical eosinophilic zymogen granules). Centroacinar cells (the start of the intercalated duct inside the acinus) are a unique feature. - Parotid: purely serous acini, intercalated and striated ducts, adipocytes in older people. Compound tubuloacinar (tubuloalveolar) glands - Branched ducts ending in both tubular and acinar units. - Examples: submandibular gland (mixed, mostly serous), sublingual gland (mixed, mostly mucous). - Duct system of salivary glands: 1. intercalated ducts: small, low cuboidal cells, drain the acini 2. striated ducts: columnar cells with basal striations (infoldings of the basal membrane with mitochondria) that reabsorb Na⁺ and make saliva hypotonic 3. interlobular and excretory ducts: stratified cuboidal to columnar Compound alveolar glands - Wide saccular secretory units. - Examples: lactating mammary gland; prostate (tubuloalveolar, with irregular, folded alveoli, sometimes containing corpora amylacea; inactive glands have a smaller lumen). Myoepithelial cells - Contractile epithelial cells (with actin, myosin and keratin) between the secretory cells and the basement membrane of sweat, salivary, mammary and lacrimal glands. - Their contraction squeezes the secretion into the ducts (oxytocin triggers milk ejection). Exam focus: give one example each of a simple tubular, simple coiled tubular, branched acinar, compound acinar and compound tubuloacinar gland.

Mode of secretion 1. Merocrine (eccrine) - The product is released by exocytosis of secretory vesicles. No part of the cell is lost. - The most common mode. - Examples: salivary glands, exocrine pancreas, eccrine sweat glands, goblet cells. 2. Apocrine - The product is released together with a small part of the apical cytoplasm, surrounded by membrane. - Example: lipid droplets of milk from the mammary gland (the protein part is merocrine); ceruminous glands of the ear. (Apocrine sweat glands are named for this mode, but most of their secretion is by exocytosis.) 3. Holocrine - The whole cell fills with product, dies and breaks down, and becomes the secretion. The cells must be constantly replaced by division of basal stem cells. - Example: sebaceous glands (sebum). - Clinical: acne involves blocked sebaceous gland ducts, bacterial overgrowth (Cutibacterium acnes) and inflammation. Sebaceous glands are stimulated by androgens at puberty. Nature of the secretory product 1. Serous secretion - A watery fluid rich in proteins, often enzymes (amylase, lipase, proteases). - Serous acini: small round acini with a narrow lumen; pyramid-shaped cells with a round nucleus in the basal third, basophilic basal cytoplasm (RER) and eosinophilic apical zymogen granules. Stain dark. - Examples: parotid gland, exocrine pancreas, lacrimal gland. 2. Mucous secretion - A viscous fluid rich in mucins (glycoproteins) for lubrication and protection. - Mucous acini (usually tubular): larger lumen; cells with pale, foamy or empty-looking cytoplasm (mucin dissolves in processing) and flattened nuclei pushed against the base of the cell. Stain pale on H&E and strongly with PAS and alcian blue. - Examples: sublingual gland (mostly), glands of the palate, Brunner's glands, goblet cells. 3. Mixed (seromucous) secretion - The gland has both serous and mucous units. - Seromucous (mixed) acinus: a mucous tubule capped by a crescent of serous cells called a serous demilune (a half-moon). The demilune is partly an artefact of conventional fixation; with rapid freezing the serous cells line up in the tubule, but the demilune is still the textbook image. - Examples: submandibular gland (mostly serous), sublingual gland (mostly mucous). Differences between serous and mucous acini - Staining on H&E: serous dark (basophilic base, eosinophilic granules); mucous pale. - Nucleus: serous round and basal-central; mucous flattened against the base. - Lumen: serous small; mucous larger. - Cytoplasm: serous granular with zymogen granules; mucous foamy with mucinogen droplets. - PAS: serous weak; mucous strong. - Product: serous watery with enzymes; mucous viscous. The three main salivary glands - Parotid: purely serous; long striated ducts; adipocytes common. - Submandibular: mixed, mostly serous, with some demilunes. - Sublingual: mixed, mostly mucous; short or few striated ducts. Exam focus: describe the three modes of secretion with examples, and draw serous acini, mucous tubules and a serous demilune.

1. What is the main difference between exocrine and endocrine glands? 2. How do exocrine and endocrine glands develop from the covering epithelium? 3. Name an organ that is both exocrine and endocrine. 4. Describe a goblet cell and explain its appearance on H&E. 5. What is the difference between a simple and a compound gland? 6. Give an example of a simple coiled tubular gland. 7. Give an example of a branched acinar gland. 8. Name the three segments of the salivary duct system and one feature of each. 9. What do myoepithelial cells do? 10. Define merocrine, apocrine and holocrine secretion, with an example of each. 11. How do serous acini differ from mucous acini on H&E? 12. What is a serous demilune? 13. Which salivary gland is purely serous? 14. Why is sweat salty in cystic fibrosis?

1. Exocrine glands secrete through ducts onto a surface; endocrine glands are ductless and secrete into the blood. 2. Epithelium grows into connective tissue; if the connection stays it becomes a duct (exocrine); if lost, the cells form cords or follicles near capillaries (endocrine). 3. Pancreas (also liver, testis, ovary). 4. A mucus-secreting cell with a swollen apical theca full of mucinogen granules and a narrow basal stem holding the nucleus. It looks empty on H&E because mucin washes out; PAS or alcian blue stain it. 5. Simple: unbranched duct. Compound: branched duct. 6. Eccrine sweat gland. 7. Sebaceous gland (also meibomian glands). 8. Intercalated (small, low cuboidal), striated (basal striations with mitochondria, reabsorb Na⁺), excretory (stratified). 9. They contract to squeeze secretions out of acini into ducts. 10. Merocrine: exocytosis, no cell loss (salivary glands). Apocrine: loss of apical cytoplasm (milk lipid). Holocrine: the whole cell becomes the secretion (sebaceous glands). 11. Serous: dark, pyramidal cells, round basal nucleus, small lumen, granules. Mucous: pale, foamy, flattened basal nucleus, larger lumen. 12. A crescent of serous cells capping a mucous tubule in mixed glands. 13. The parotid. 14. The sweat duct cannot reabsorb chloride (defective CFTR channel).

Five major functions of hormones 1. Regulation of metabolism and energy balance. 2. Control of growth and development. 3. Reproduction. 4. Homeostasis: water and electrolyte balance, blood glucose, calcium, blood pressure. 5. Response to stress and adaptation. Hormone classification (chemical) 1. Amino acid derivatives: - catecholamines (adrenaline, noradrenaline) from tyrosine: water-soluble - thyroid hormones (T3, T4) from tyrosine: lipid-soluble behaviour (intracellular receptors) - melatonin from tryptophan 2. Peptides and proteins: the largest group (ADH, oxytocin, insulin, glucagon, GH, PTH, calcitonin). Glycoproteins: TSH, LH, FSH, hCG. Made on RER, packaged in the Golgi, stored in granules. 3. Steroids: from cholesterol (cortisol, aldosterone, testosterone, oestrogens, progesterone, vitamin D). Made in SER and mitochondria; not stored, released as made; carried by binding proteins. Hormone action: general - Only cells with receptors respond (target cells). - The response depends on: hormone concentration, number of receptors (up- and down-regulation), affinity, and interactions with other hormones (permissive, synergistic, antagonistic). Action of steroid (lipid-soluble) hormones: summary 1. The hormone detaches from its carrier protein and diffuses through the plasma membrane. 2. It binds an intracellular receptor (in the cytoplasm or nucleus). 3. The hormone-receptor complex binds hormone response elements on DNA. 4. It activates or represses transcription of specific genes. 5. New mRNA and proteins change the cell's activity. - Effects are slower (hours) but long-lasting. Thyroid hormones act the same way (nuclear receptors). Action of protein (water-soluble) hormones: summary 1. The hormone (first messenger) binds a receptor on the plasma membrane. 2. The receptor activates a signal-transduction pathway, e.g. a G protein that activates adenylyl cyclase. 3. A second messenger is produced (cAMP, or IP3 and DAG with Ca²⁺). 4. The second messenger activates protein kinases (e.g. protein kinase A), which phosphorylate enzymes and other proteins. 5. The cell's response changes quickly (seconds to minutes): secretion, enzyme activity, membrane permeability. - The cascade amplifies the signal: one hormone molecule can produce thousands of product molecules. - Some receptors are enzymes themselves (insulin: a tyrosine kinase receptor). Control of hormone secretion - Three types of stimulus: 1. humoral: changing blood levels of ions or nutrients (Ca²⁺ controls PTH; glucose controls insulin) 2. neural: nerve fibres stimulate release (sympathetic fibres on the adrenal medulla) 3. hormonal: hormones from other glands (hypothalamic releasing hormones control the anterior pituitary, which controls the thyroid, adrenal cortex and gonads) - Negative feedback: the hormone's effect (or the hormone itself) inhibits further secretion. Keeps levels in a narrow range. - Example: the hypothalamic-pituitary-thyroid axis: TRH stimulates TSH, which stimulates T3/T4, which inhibit TRH and TSH. - Positive feedback is rare: the LH surge before ovulation; oxytocin during labour. - Rhythms: many hormones have circadian rhythms (cortisol highest in the early morning) or pulsatile release (GnRH). Exam focus: compare the mechanisms of action of steroid and peptide hormones, and give an example of negative feedback.

Development - Adenohypophysis: from Rathke's pouch, an upgrowth of the oral ectoderm (roof of the primitive mouth). - Neurohypophysis: from a downgrowth of the diencephalon (neuroectoderm), the infundibulum. - Clinical: craniopharyngioma arises from remnants of Rathke's pouch. Parts - Adenohypophysis (anterior lobe, about 75%): - pars distalis: the main part - pars intermedia: thin, rudimentary in humans; small colloid-filled cysts - pars tuberalis: wraps around the infundibular stalk - Neurohypophysis (posterior lobe): - pars nervosa: the main part - infundibulum (stalk) and median eminence Link to the hypothalamus - Two routes: 1. Hypophyseal portal system (to the anterior pituitary): neurosecretory cells in the hypothalamus (arcuate and other nuclei) release releasing and inhibiting hormones into a primary capillary plexus in the median eminence; portal veins carry them down the stalk to a secondary capillary plexus (sinusoids) in the pars distalis. The hormones therefore reach the anterior pituitary cells in high concentration. 2. Hypothalamo-hypophyseal tract (to the posterior pituitary): axons of large neurosecretory neurons in the supraoptic and paraventricular nuclei run down the stalk and end on capillaries in the pars nervosa. So the posterior pituitary is under direct nervous control: it stores and releases hormones made in the hypothalamus. The pars distalis: histology - Cords and clusters of epithelial cells between wide fenestrated sinusoids, supported by reticular fibres. - Classic staining (H&E, Azan or trichrome) divides cells into three groups: - acidophils (about 40%): pink or red granules. Somatotrophs (GH) and lactotrophs (prolactin). - basophils (about 10%): blue or purple granules (glycoproteins, PAS-positive). Corticotrophs (ACTH), thyrotrophs (TSH), gonadotrophs (FSH, LH). - chromophobes (about 50%): pale; few granules; degranulated or stem cells. - Mnemonic: basophils secrete B-FLAT (the "B" for basophil, then FSH, LH, ACTH, TSH); acidophils secrete GH and prolactin. - Immunohistochemistry is now used to identify each cell type precisely. Cells and hormones of the anterior pituitary - Somatotrophs (acidophil): GH (growth hormone). Stimulated by GHRH, inhibited by somatostatin. Acts on the liver (IGF-1), bone, muscle. Excess: gigantism in children, acromegaly in adults. - Lactotrophs (acidophil): prolactin. Inhibited by dopamine. Milk production. Excess (prolactinoma) causes galactorrhoea and infertility. - Corticotrophs (basophil): ACTH (and MSH, from the precursor POMC). Stimulated by CRH. Acts on the adrenal cortex (cortisol). Excess: Cushing disease. - Thyrotrophs (basophil): TSH. Stimulated by TRH. Acts on the thyroid. - Gonadotrophs (basophil): FSH and LH. Stimulated by GnRH. Act on the gonads. The neurohypophysis: histology - Unmyelinated axons from hypothalamic neurons; fenestrated capillaries. - Pituicytes: glial cells that support the axons. - Herring bodies: dilations of the axon terminals full of neurosecretory granules (stored hormone with its carrier, neurophysin). - No hormone-producing cells of its own. Hormones of the posterior pituitary - ADH (antidiuretic hormone, vasopressin), made mainly in the supraoptic nucleus: - stimulates water reabsorption in the kidney collecting ducts (by inserting aquaporin-2 channels) - at high levels, constricts arterioles (raises blood pressure) - released when plasma osmolality rises or blood volume falls - deficiency: central diabetes insipidus (large volumes of dilute urine, thirst) - Oxytocin, made mainly in the paraventricular nucleus: - contracts uterine smooth muscle in labour (positive feedback: the Ferguson reflex) - contracts myoepithelial cells of the mammary gland (milk ejection reflex, triggered by suckling) - social bonding and behaviour - used clinically to induce labour and control postpartum bleeding The pineal gland (epiphysis) - A small cone-shaped gland on the roof of the third ventricle (diencephalon). - Composed of pinealocytes (modified neurons that secrete melatonin) and glial cells (astrocyte-like interstitial cells), in lobules with a capsule of pia mater. - Brain sand (corpora arenacea): calcified concretions that increase with age; visible on radiographs, a midline marker. - Melatonin: made from serotonin; secreted at night in darkness. Light on the retina inhibits it via the suprachiasmatic nucleus and a sympathetic pathway (superior cervical ganglion). - Functions: regulates circadian rhythms and sleep; seasonal and reproductive rhythms in animals; antioxidant. Used to treat jet lag and some sleep disorders. Exam focus: explain how the hypothalamus controls the anterior and posterior pituitary differently, and list each anterior pituitary cell type with its hormone and staining.

Thyroid: structure - Two lobes joined by an isthmus, in front of the trachea below the larynx; about 20-25 g. Covered by a capsule sending septa inward. - Developed from the floor of the pharynx (endoderm), descending by the thyroglossal duct. Clinical: thyroglossal duct cysts in the midline of the neck. - The functional unit is the follicle: a sphere of simple epithelium (follicular cells) around a lumen filled with colloid (thyroglobulin), surrounded by a basement membrane and a dense network of fenestrated capillaries. - Follicle size: 0.2-0.9 mm. - The height of the epithelium reflects activity: - inactive: flat (squamous) cells, large follicles full of colloid - active: cuboidal to columnar cells, less colloid with scalloped edges (resorption vacuoles) Thyroid hormone synthesis (follicular cells utilise iodine) 1. Thyroglobulin is synthesised on RER, glycosylated in the Golgi and secreted into the colloid. 2. Iodide is actively taken up from the blood by the Na⁺/I⁻ symporter on the basal membrane. 3. Iodide moves to the apical membrane (via pendrin) and is oxidised by thyroid peroxidase (TPO). 4. Iodine is attached to tyrosine residues of thyroglobulin in the colloid, forming MIT and DIT. 5. Coupling: DIT + DIT = T4; MIT + DIT = T3. The hormones stay attached to thyroglobulin in the colloid (storage for weeks). 6. Under TSH stimulation, follicular cells take up colloid by endocytosis; lysosomes digest thyroglobulin and release T4 and T3, which leave through the basal membrane into capillaries. - The thyroid secretes mostly T4 (about 90%); T3 is the more active form, made mainly by conversion of T4 in peripheral tissues (deiodinases). Functions of thyroid hormones (T4 and T3) - Increase basal metabolic rate and O₂ consumption (more Na/K pumps, more mitochondria). - Heat production (thermogenesis). - Growth and development: essential for brain development and skeletal maturation in the fetus and infant; work with GH. - Cardiovascular: increase heart rate and contractility (more β-adrenergic receptors). - Metabolism: increase glucose absorption, glycogenolysis, lipolysis; lower cholesterol. - Nervous system: alertness, reflexes. Physiological effects summary - Too much (hyperthyroidism, e.g. Graves disease): weight loss, heat intolerance, tachycardia, tremor, anxiety, exophthalmos in Graves. - Too little (hypothyroidism, e.g. Hashimoto thyroiditis): weight gain, cold intolerance, bradycardia, fatigue, dry skin, myxoedema. Thyroid disorders on the slides - Simple (endemic) goitre: enlargement of the thyroid due to iodine deficiency. Low T3 and T4 cause high TSH (loss of negative feedback), which makes the gland grow. Prevented by iodised salt. - Cretinism (congenital hypothyroidism): thyroid hormone deficiency in the fetus or newborn; causes intellectual disability and short stature. Prevented by newborn screening (heel-prick TSH) and early treatment with thyroxine. Parafollicular cells (C cells) - Large pale cells between the follicular cells (inside the basement membrane) or in small groups between follicles. Derived from neural crest (via the ultimobranchial body). - Secrete calcitonin in response to high blood Ca²⁺. - Calcitonin: lowers blood calcium by inhibiting osteoclasts (bone resorption) and increasing renal calcium excretion. A minor role in adult humans. - Clinical: medullary thyroid carcinoma arises from C cells; calcitonin is its tumour marker (MEN2 syndromes). Parathyroid glands - Usually four small glands (about 30-50 mg each) on the posterior surface of the thyroid. From the third and fourth pharyngeal pouches. - Consist of two types of cells in cords with rich capillaries: 1. chief (principal) cells: small, many, pale-staining; secrete PTH 2. oxyphil cells: larger, eosinophilic, full of mitochondria; appear at puberty and increase with age; function unclear - With age the parathyroid glands accumulate adipocytes (up to 50% of the gland). Parathyroid hormone (PTH) - Secreted in response to hypocalcaemia (low blood calcium), sensed by the calcium-sensing receptor on chief cells. - Raises blood calcium by: 1. bone: stimulating osteoclast activity indirectly (PTH acts on osteoblasts, which increase RANKL), releasing Ca²⁺ and phosphate 2. kidney: increasing Ca²⁺ reabsorption, decreasing phosphate reabsorption, and activating vitamin D (1α-hydroxylase) 3. intestine: indirectly, by active vitamin D, increasing calcium absorption - Hypocalcaemia effects: neuromuscular excitability, tetany, muscle cramps, tingling, seizures (e.g. after accidental removal of the parathyroids during thyroidectomy). - Hyperparathyroidism: hypercalcaemia ("stones, bones, groans and psychic moans"). Exam focus: describe thyroid hormone synthesis step by step, and compare the actions of PTH and calcitonin.

General structure - Weight about 4-5 g each. Covered by a thick capsule of dense connective tissue. - Cortex: about 80-90% of the gland; from mesoderm (coelomic epithelium). Yellow in fresh tissue (lipids). - Medulla: about 10-20%; from neural crest. - Blood supply: capsular arteries, then sinusoids flowing from the cortex to the medulla. So medullary cells receive blood rich in cortisol, which induces the enzyme that converts noradrenaline to adrenaline (PNMT). Adrenal cortex: three zones (from outside in) Mnemonic: "GFR" for the zones and "salt, sugar, sex" for their products; "the deeper you go, the sweeter it gets". 1. Zona glomerulosa (about 15%) - Just under the capsule. Cells arranged in rounded clusters (arches). - Small cells with dark nuclei and few lipid droplets. - Secretes mineralocorticoids, mainly aldosterone. - Aldosterone acts on the kidney (distal tubule and collecting duct) to retain Na⁺ (and water) and excrete K⁺ and H⁺. Controls blood volume and pressure. - Controlled mainly by the renin-angiotensin system (angiotensin II) and plasma K⁺; ACTH has only a minor effect. 2. Zona fasciculata (about 75-80%, the thickest) - Cells arranged in long straight cords (fascicles) one or two cells thick, running radially, separated by sinusoids. - Large polyhedral cells full of lipid droplets, which dissolve in processing, giving a pale, foamy, vacuolated look (spongiocytes). Abundant SER; mitochondria with tubular cristae. - Secretes glucocorticoids, mainly cortisol. - Controlled by ACTH. 3. Zona reticularis (about 5-10%) - Innermost zone. Cells arranged in an irregular network (anastomosing cords) with sinusoids. - Smaller cells, more eosinophilic, fewer lipid droplets; lipofuscin pigment. - Secretes gonadocorticoids (weak androgens: DHEA, androstenedione) and some glucocorticoids. - Controlled by ACTH. Androgens contribute to pubic and axillary hair in women (adrenarche). Cortisol (the stress hormone) - Effects: - metabolism: raises blood glucose (gluconeogenesis in the liver), protein breakdown in muscle, lipolysis (with redistribution of fat) - anti-inflammatory and immunosuppressive (the basis of corticosteroid drugs) - helps the body resist long-term stress; permissive for catecholamine effects on vessels - bone: reduces formation (osteoporosis with long-term steroid use) - Circadian rhythm: cortisol peaks early in the morning around waking, and is lowest around midnight. This is why steroid doses are given in the morning and why cortisol is tested at specific times. - Excess: Cushing syndrome (central obesity, moon face, striae, hypertension, hyperglycaemia). Deficiency: Addison disease (weakness, hypotension, hyperpigmentation from high ACTH/MSH). Adrenal medulla - Clusters and cords of large chromaffin cells (they stain brown with chromium salts because catecholamines oxidise), around sinusoids and large veins. - Chromaffin cells are modified postganglionic sympathetic neurons without axons. They are innervated by preganglionic sympathetic fibres (acetylcholine). - Secrete catecholamines: adrenaline (epinephrine, about 80%) and noradrenaline (norepinephrine, about 20%), stored in dense-core granules. - The fight-or-flight response (stimulated in stress, exercise, fear): raised heart rate and contractility, raised blood pressure, bronchodilation, raised blood glucose (glycogenolysis) and fatty acids, dilated pupils, redistribution of blood to skeletal muscle, reduced digestion. - Clinical: phaeochromocytoma, a chromaffin cell tumour causing episodes of hypertension, headache, sweating and palpitations. Neuroblastoma in children arises from neural crest cells of the medulla. Stress response: two phases - Short-term (seconds): the sympathetic system and adrenal medulla (catecholamines). - Long-term (hours to days): the hypothalamus-pituitary-adrenal axis (CRH, ACTH, cortisol). Exam focus: draw the adrenal gland with the three cortical zones and medulla, and give the arrangement, product and control of each zone.

Pancreatic islets (islets of Langerhans) - About 1-2 million islets, together about 1-2% of the pancreas; more in the tail. - Pale-staining clusters of cells (about 100-200 µm) among darker serous acini, with a rich network of fenestrated capillaries. - Cell types in each islet: - β cells (about 60-70%, mostly central in rodents, scattered in humans): insulin (and amylin). - α cells (about 15-20%, more peripheral): glucagon. - δ cells (about 5-10%): somatostatin, which inhibits insulin and glucagon secretion locally (paracrine). - PP (F) cells (few): pancreatic polypeptide (inhibits exocrine secretion). - ε cells (very few): ghrelin. - Identified by immunohistochemistry (on H&E they look similar). - Insulin lowers blood glucose: uptake by muscle and fat (GLUT4), glycogen, fat and protein synthesis. - Glucagon raises blood glucose: glycogenolysis and gluconeogenesis in the liver. - Clinical: - type 1 diabetes: autoimmune destruction of β cells (insulitis: lymphocytes in islets) - type 2 diabetes: insulin resistance with progressive β-cell failure; amyloid deposits (amylin) in islets - insulinoma: β-cell tumour causing hypoglycaemia Interstitial glands - Clusters of steroid-producing cells in the connective tissue between other structures in the gonads. - Leydig cells (testis): between seminiferous tubules, near capillaries. Eosinophilic cytoplasm, lipid droplets, abundant SER, mitochondria with tubular cristae, Reinke crystals. Produce testosterone under LH (see Gametogenesis). - Theca interna cells of the ovary, including those of atretic follicles: produce androgens (converted to oestrogens by granulosa cells). In some species, the theca of atretic follicles persists as an "interstitial gland" of the ovary; in humans it is limited. - Hilus cells of the ovary: similar to Leydig cells. Isolated (diffuse) endocrine cells - Single endocrine cells scattered in epithelia; together they form the diffuse neuroendocrine system (in older texts, the APUD system). - Enteroendocrine cells of the gut (the largest endocrine "organ" by cell number): at the base of glands, with granules at the basal side (they secrete toward the capillaries, the opposite of exocrine cells). Examples: - G cells (gastric antrum): gastrin, stimulated by protein in the lumen and gastrin-releasing peptide from vagal nerves; stimulates acid secretion - I cells (duodenum): cholecystokinin (gallbladder contraction, pancreatic enzymes) - S cells (duodenum): secretin (bicarbonate from the pancreas) - K cells: GIP; L cells: GLP-1 (incretins that boost insulin release; the targets of GLP-1 agonist drugs) - enterochromaffin cells: serotonin - D cells: somatostatin - Neuroendocrine cells in the airways (Kulchitsky cells), which are the origin of small cell lung cancer and carcinoid tumours. - Other hormone-producing tissues: heart (atrial natriuretic peptide), kidney (erythropoietin, renin), liver (IGF-1, angiotensinogen), adipose tissue (leptin), placenta. Exam focus: list the islet cell types with their hormone and proportion, and give three examples of enteroendocrine cells.

1. What histological features do all endocrine glands share? 2. Name the three chemical classes of hormones, with one example of each. 3. Describe how a steroid hormone acts on its target cell. 4. Describe how a peptide hormone acts through cAMP. 5. What is negative feedback? Give an example. 6. What are the embryological origins of the adeno- and neurohypophysis? 7. Explain the hypophyseal portal system. 8. Which anterior pituitary cells are acidophils, and which are basophils? 9. Where are ADH and oxytocin made, and what does each do? 10. What are Herring bodies and pituicytes? 11. What does the pineal gland secrete, and what controls it? 12. Describe the thyroid follicle and how its appearance reflects activity. 13. List the steps of thyroid hormone synthesis. 14. What causes simple goitre? 15. What do C cells secrete, and what is its effect? 16. Name the two cell types of the parathyroid gland. 17. How does PTH raise blood calcium? 18. Name the three zones of the adrenal cortex, their arrangement and products. 19. What is special about cortisol secretion over the day? 20. What are chromaffin cells? 21. Name the islet cell types and their hormones. 22. What is an interstitial gland? Give an example. 23. Give two examples of enteroendocrine cells and their hormones.

1. No ducts; cells in cords, clusters or follicles; rich fenestrated capillaries; secretory granules or lipid droplets and SER. 2. Amino acid derivatives (adrenaline, T4), peptides and proteins (insulin), steroids (cortisol). 3. It crosses the membrane, binds an intracellular receptor; the complex binds DNA response elements and changes gene transcription and protein synthesis. 4. It binds a membrane receptor, activates a G protein and adenylyl cyclase; cAMP activates protein kinase A, which phosphorylates target proteins. 5. The hormone's effect inhibits its own secretion. TRH, TSH, T3/T4 (T3/T4 inhibit TRH and TSH). 6. Adenohypophysis: Rathke's pouch (oral ectoderm). Neurohypophysis: downgrowth of the diencephalon (neuroectoderm). 7. Hypothalamic releasing and inhibiting hormones go into capillaries in the median eminence, then down portal veins to the sinusoids of the anterior pituitary. 8. Acidophils: somatotrophs (GH) and lactotrophs (prolactin). Basophils: corticotrophs (ACTH), thyrotrophs (TSH), gonadotrophs (FSH, LH). 9. In hypothalamic neurons (ADH mainly supraoptic, oxytocin mainly paraventricular). ADH: water reabsorption in collecting ducts; vasoconstriction. Oxytocin: uterine contraction in labour and milk ejection. 10. Herring bodies: axon dilations storing neurosecretory granules. Pituicytes: glial cells of the neurohypophysis. 11. Melatonin, secreted at night; inhibited by light via the retina and suprachiasmatic nucleus. 12. A sphere of simple epithelium around colloid. Inactive: flat cells, much colloid. Active: tall cells, less colloid, resorption vacuoles. 13. Thyroglobulin synthesis and secretion; iodide uptake; oxidation by thyroid peroxidase; iodination of tyrosines (MIT, DIT); coupling to T3 and T4; endocytosis and lysosomal release under TSH. 14. Iodine deficiency: low T3/T4 raise TSH, which stimulates thyroid growth. 15. Calcitonin; lowers blood calcium by inhibiting osteoclasts. 16. Chief (principal) cells and oxyphil cells. 17. Bone resorption (indirectly via osteoblasts and RANKL), renal Ca²⁺ reabsorption and phosphate excretion, and activation of vitamin D (more intestinal absorption). 18. Glomerulosa: rounded clusters; aldosterone. Fasciculata: straight cords of lipid-rich cells; cortisol. Reticularis: network; androgens. 19. It follows a circadian rhythm, peaking in the early morning and lowest at midnight. 20. Catecholamine-secreting cells of the adrenal medulla; modified postganglionic sympathetic neurons. 21. β: insulin. α: glucagon. δ: somatostatin. PP: pancreatic polypeptide. ε: ghrelin. 22. A cluster of steroid-secreting cells in gonadal connective tissue. Leydig cells (testosterone); theca interna (androgens). 23. Any two: G cells (gastrin), I cells (CCK), S cells (secretin), L cells (GLP-1), enterochromaffin cells (serotonin).

Where is it from? - Mesenchyme: loosely arranged, stellate, pluripotent cells in a gel-like matrix with few fibres. It forms all connective tissues, including bone, cartilage, blood, and smooth muscle of vessels. - Mesodermal origin for most of the body; neural crest (ectomesenchyme) forms much of the connective tissue and bones of the face and neck. Functions 1. Structural support and binding: tendons, ligaments, capsules of organs, stroma. 2. Mechanical protection: bone, cartilage, fat pads. 3. Medium of exchange: nutrients and waste pass between blood and cells through the ground substance. 4. Defence: immune cells (macrophages, mast cells, plasma cells) and the physical barrier of the matrix against spread of microbes. 5. Energy storage and insulation: adipose tissue. 6. Transport: blood. 7. Repair: fibroblasts form scar tissue after injury. 8. Heat production: brown fat. Structural organisation - Cells: resident (fixed) and wandering (transient). - Extracellular matrix: - fibres: collagen, reticular, elastic - ground substance: glycosaminoglycans, proteoglycans, structural (adhesive) glycoproteins, plus tissue fluid - The balance of fibres and ground substance defines the tissue: more fibres give strength (tendon); more ground substance allows diffusion and cushioning (loose tissue); mineralised matrix gives rigidity (bone). Connective tissue versus epithelium - Cells: few and separated in connective tissue; many and tightly joined in epithelium. - Matrix: abundant versus scant. - Vessels: usually vascular versus avascular. - Basement membrane: none (except around some cells) versus always present. - Surface: no free surface versus apical free surface. Exam focus: list six functions of connective tissue and describe mesenchyme.

Glycosaminoglycans (GAGs) - Long unbranched polysaccharides of repeating disaccharide units (an amino sugar plus a uronic acid, usually). - Highly negatively charged (carboxyl and sulfate groups), so they attract cations (Na⁺) and water, forming a hydrated gel that resists compression. - Types: - hyaluronic acid (hyaluronan): the largest; not sulfated; not bound to a core protein; synthesised at the cell surface. Found in loose connective tissue, synovial fluid, vitreous body, cartilage. - chondroitin sulfate: cartilage, bone, cornea. - dermatan sulfate: skin, tendons, blood vessels. - heparan sulfate: basement membranes (perlecan), cell surfaces. - keratan sulfate: cartilage, cornea. - heparin: in mast cell granules (anticoagulant). - Stained by alcian blue, and metachromatically with toluidine blue. Proteoglycans - A core protein with GAG chains (except hyaluronic acid) attached covalently; like a bottle brush. - Aggregates of proteoglycans: many aggrecan molecules bind by link proteins to one long hyaluronic acid molecule, forming huge aggregates (the main feature of cartilage). - Examples: - aggrecan: cartilage (compressive resistance) - decorin: binds collagen fibrils and regulates their assembly (tendons, skin) - perlecan: basement membranes - syndecan: a cell-surface proteoglycan (binds growth factors, matrix) - Functions: hydration and resistance to compression; molecular sieve (filtration, diffusion); binding and storing growth factors; regulating collagen fibril assembly; binding of cells. - Clinical: mucopolysaccharidoses (such as Hurler syndrome) are lysosomal storage diseases in which GAGs cannot be degraded. Differences between glycoproteins and proteoglycans - Proteoglycans: mostly carbohydrate (up to 95%), long unbranched GAG chains, highly charged; mainly structural and hydration role. - Glycoproteins: mostly protein, short branched oligosaccharides; mainly adhesion and signalling. Structural (adhesive) glycoproteins - Link cells to the matrix and matrix components to each other. Cells bind them through integrins. - Fibronectin: a dimer with binding sites for integrins (the RGD sequence), collagen, heparin and fibrin. Made by fibroblasts. Guides cell migration in development and wound healing. Plasma fibronectin (from the liver) helps clot formation. - Laminin: the main glycoprotein of basement membranes (cross-shaped); binds type IV collagen, perlecan and integrins. - Others: chondronectin (cartilage), osteonectin and osteopontin (bone), tenascin, nidogen (entactin). Tissue fluid - Plasma filtered from capillaries at the arterial end, reabsorbed at the venous end and by lymphatics. - Clinical: oedema is an excess of tissue fluid (high capillary pressure, low plasma proteins, lymphatic block, increased permeability). Exam focus: list the main GAGs and where they are found, and explain how aggrecan aggregates resist compression.

Collagen fibres - Light microscopy: thick, wavy, eosinophilic (pink) bundles on H&E; blue or green with trichrome (Masson); red with picrosirius red. - Hierarchy of structure: 1. Tropocollagen (the collagen molecule): three polypeptide α-chains wound into a triple helix, about 300 nm long and 1.5 nm wide. 2. Every third amino acid is glycine (the smallest amino acid, which fits into the centre of the helix): the repeat Gly-X-Y, where X is often proline and Y often hydroxyproline. 3. Fibrils: tropocollagen molecules line up in a staggered pattern (quarter-stagger), with gaps; this gives a banding of 67 nm (D-period) in the electron microscope. 4. Fibres: fibrils grouped together. 5. Bundles: fibres grouped together (e.g. in tendons). - Why so strong: the triple helix, the staggered arrangement and covalent cross-links between molecules (by lysyl oxidase, a copper-dependent enzyme). Collagen I is stronger than steel of the same weight. Main collagen types - Type I: about 90% of body collagen. Thick fibres. Skin (dermis), bone, tendons, ligaments, organ capsules, fibrous cartilage, cornea. Resists tension. - Type II: thin fibrils. Hyaline and elastic cartilage, vitreous body, nucleus pulposus. Resists pressure (with proteoglycans). - Type III: reticular fibres. Stroma of lymphoid organs, bone marrow, liver, around smooth muscle, blood vessels, early granulation tissue and scar, fetal skin. - Type IV: non-fibrillar network. Basement membranes (lamina densa). - Type VII: anchoring fibrils of the basement membrane to the underlying tissue. - Mnemonic: "Be (I, bone) So (II, cartilage) Totally (III, reticular) Cool, Read Books (IV, basement membrane)". Or "one: bONE; two: carTWOlage; three: reTHREEticular; four: under the FLOOR (basement membrane)". - Non-fibrillar collagens also include FACIT collagens (e.g. type IX in cartilage) that link fibrils. Collagen synthesis Intracellular steps (fibroblast, osteoblast, chondroblast): 1. Translation of pro-α chains on RER, with signal peptide. 2. Hydroxylation of proline and lysine residues in the RER (prolyl and lysyl hydroxylases, which need vitamin C as a cofactor). 3. Glycosylation of some hydroxylysines. 4. Assembly of three pro-α chains into a triple helix (procollagen), starting from the C-terminal propeptides, with disulfide bonds. 5. Transport through the Golgi and secretion by exocytosis. Extracellular steps: 6. Cleavage of the N- and C-terminal propeptides by procollagen peptidases, producing tropocollagen. 7. Self-assembly of tropocollagen into fibrils (collagen I fibril assembly), guided by decorin and other molecules. 8. Covalent cross-linking by lysyl oxidase. Clinical: defects of collagen - Scurvy: vitamin C deficiency. Hydroxylation fails, so the triple helix is unstable. Bleeding gums, poor wound healing, easy bruising, loose teeth. - Osteogenesis imperfecta: type I collagen mutations (often glycine substitutions). Brittle bones, blue sclerae, hearing loss. - Ehlers-Danlos syndromes: various collagen defects (type V, III, procollagen processing). Hyperelastic skin, hypermobile joints; vascular type III with arterial rupture. - Alport syndrome: type IV collagen. - Keloids: excessive collagen deposition in scars. Reticular fibres - Thin (0.5-2 µm), branched fibres of type III collagen, heavily glycosylated. - Not visible on H&E; seen with silver impregnation (black, "argyrophilic") and PAS. - Form delicate supporting networks (stroma) for cells: lymph nodes, spleen, bone marrow, liver sinusoids, around adipocytes, smooth muscle and nerve fibres, basement membranes (lamina reticularis). - Made by fibroblasts, reticular cells (in lymphoid organs), smooth muscle cells and Schwann cells. Elastic fibres - Thinner than collagen, branching and anastomosing; stretch up to about 150% of their length and recoil. - Structure: an amorphous core of elastin (rich in glycine, proline, and the unique cross-links desmosine and isodesmosine) surrounded by microfibrils of fibrillin (which form the scaffold first). - Elastin is hydrophobic, randomly coiled, cross-linked; it straightens when stretched and recoils like rubber. - Stains: pale on H&E; dark with orcein, resorcin-fuchsin or Verhoeff stain. - Locations: skin, lungs, elastic arteries (fenestrated elastic lamellae in the aorta), elastic cartilage, vocal cords, ligamenta flava. - Made by fibroblasts and smooth muscle cells (in vessels). - Clinical: Marfan syndrome, a fibrillin-1 mutation: tall stature, long limbs, lens dislocation, aortic dilatation and dissection. Loss of elastic fibres in the lung contributes to emphysema. Sun-damaged skin shows solar elastosis. Wrinkles with ageing reflect elastic fibre loss. Exam focus: describe collagen synthesis, the role of vitamin C and name four collagen types with locations.

Resident (fixed) cells Fibroblasts - The most common cell in connective tissue proper. - Active fibroblasts: large, spindle or star-shaped cells with long processes, a large oval pale nucleus with a prominent nucleolus, and basophilic cytoplasm (abundant RER and Golgi). - Synthesise all matrix components: collagen, elastin, reticular fibres, GAGs, proteoglycans, glycoproteins. Also enzymes (metalloproteinases) that degrade the matrix, so they remodel it. - Key role in wound healing: they proliferate and make new collagen (scar). - Myofibroblasts: fibroblasts with actin and myosin bundles (α-smooth muscle actin); they contract to close wounds. Excess causes contractures (Dupuytren). Fibrocytes - Inactive (quiescent) fibroblasts: smaller, flattened, with a dark elongated nucleus and little cytoplasm. Can become active again when needed (injury). Reticular cells - Fibroblast-like cells that make reticular fibres and form the stroma of lymphoid organs and bone marrow. Adipocytes - Fat cells: single or in groups (see Adipose tissue). Mesenchymal (stem) cells - Undifferentiated cells, often near blood vessels (pericytes). Can differentiate into fibroblasts, adipocytes, smooth muscle, chondroblasts, osteoblasts. Wandering (transient) cells Macrophages (histiocytes) - Derived from blood monocytes that enter tissues. - Irregular shape, kidney-shaped or indented nucleus, many lysosomes and phagocytic vacuoles. Hard to see on H&E; revealed by uptake of dyes (trypan blue, carbon) or by immunohistochemistry (CD68). - Functions: phagocytosis of microbes, dead cells and debris; antigen presentation to T cells; secretion of cytokines (IL-1, TNF) that drive inflammation; tissue repair. - Tissue-specific macrophages form the mononuclear phagocyte system: Kupffer cells (liver), alveolar macrophages ("dust cells", lung), microglia (CNS), osteoclasts (bone, by fusion), Langerhans cells (skin, related), splenic and lymph node macrophages. - Fusion of macrophages forms multinucleated giant cells (foreign body giant cells, Langhans cells in tuberculous granulomas). Mast cells - Large oval cells, often near small blood vessels, packed with basophilic granules that hide the round central nucleus. The granules stain metachromatically with toluidine blue (purple-red). - Derived from bone marrow precursors; mature in tissues. Similar to basophils but a separate lineage. - Granule contents: histamine (vasodilation, increased permeability, bronchoconstriction), heparin (anticoagulant), proteases (tryptase), chemotactic factors for eosinophils and neutrophils. Newly made mediators: leukotrienes, prostaglandins. - They have high-affinity receptors for IgE. When an allergen cross-links IgE on the mast cell surface, the cell degranulates. - Role: immediate (type I) hypersensitivity: allergic rhinitis, asthma, urticaria, anaphylaxis. Also defence against parasites. - Clinical: antihistamines block H1 receptors; mast cell stabilisers (cromoglycate) prevent degranulation; adrenaline treats anaphylaxis. Plasma cells - Terminally differentiated B lymphocytes that produce antibodies. - Oval cell with an eccentric round nucleus with a "clock-face" or "cart-wheel" chromatin pattern; strongly basophilic cytoplasm (abundant RER); a pale perinuclear area (negative Golgi image). - Most numerous in the lamina propria of the gut and airways, and in chronic inflammation. - Clinical: multiple myeloma, a malignant plasma cell tumour. Leukocytes - Neutrophils, eosinophils, lymphocytes, monocytes leave the blood (diapedesis) into connective tissue, especially in inflammation. Eosinophils are normally present in the lamina propria of the gut. Exam focus: describe the appearance and function of fibroblasts, macrophages, mast cells and plasma cells, and explain mast cell degranulation in allergy.

Embryonic: mucous connective tissue - Star-shaped fibroblasts in an abundant jelly-like ground substance rich in hyaluronic acid, with few fine collagen fibres. - Example: Wharton's jelly of the umbilical cord; also the pulp of young teeth. A source of mesenchymal stem cells. Loose (areolar) connective tissue - Many cells of all types; abundant ground substance; loosely arranged thin collagen, elastic and reticular fibres in all directions. - Distribution: the lamina propria of mucous membranes (gut, airways), under epithelia (papillary dermis), around blood vessels and nerves, between muscles (endomysium), in the submucosa, the stroma of glands, the hypodermis with fat. - Functions: supports epithelium; site of immune responses and inflammation (oedema forms here); diffusion of nutrients and oxygen to epithelium; allows movement. Dense connective tissue - More fibres (mostly collagen type I), fewer cells (mainly fibrocytes), little ground substance. Strength rather than flexibility. - Well seen with trichrome stains (Masson's trichrome makes collagen blue or green). - Dense irregular connective tissue: - collagen bundles run in all directions, in a woven pattern - resists tension from many directions - locations: reticular dermis of the skin, capsules of organs (liver, kidney, lymph nodes), periosteum, perichondrium, submucosa of the gut (in parts), dura mater, sclera, heart valves - Dense regular connective tissue: - collagen bundles are parallel, closely packed, with rows of flattened fibrocytes (tendon cells, tenocytes) between them - very strong along one axis; poor blood supply, so slow healing - locations: tendons, most ligaments, aponeuroses, cornea (orthogonal layers of parallel fibres) - variant: dense regular elastic tissue (ligamenta flava of the vertebrae, suspensory ligament of the penis, vocal ligaments) Reticular connective tissue - A network of reticular fibres (type III collagen) and reticular cells, supporting free cells. - Visible only with silver stains. - Locations: stroma of lymph nodes, spleen, bone marrow, liver. Elastic connective tissue - Dominated by elastic fibres or fenestrated elastic sheets. - Locations: walls of large elastic arteries (aorta: 40-70 concentric elastic lamellae), ligamenta flava, vocal cords, lung. - Function: stretch and recoil (keeps blood flowing during diastole: the Windkessel effect). Exam focus: distinguish dense regular and dense irregular connective tissue with two locations each, and give the location and stain for reticular tissue.

White adipose tissue (unilocular) - The main type in adults: about 15-20% of body weight in men and 20-25% in women. - Adipocytes: large (up to 100-120 µm) round cells; each cell fills up with a single, large triglyceride droplet that pushes the nucleus and a thin rim of cytoplasm to the edge ("signet ring" appearance). - On H&E the lipid is dissolved during processing, so cells appear empty, like a chicken-wire or honeycomb network. Lipids can be shown on frozen sections with Sudan or Oil Red O stains. - Each adipocyte is surrounded by an external lamina and reticular fibres; lobules are separated by septa with rich capillaries and sympathetic nerves. - Functions: 1. energy storage (the largest energy reserve of the body; about 9 kcal/g of fat) 2. thermal insulation (subcutaneous fat) 3. mechanical cushioning (around kidneys, in the palms, soles, orbit) 4. endocrine: secretion of adipokines - Lipid storage (lipogenesis): after a meal, insulin stimulates glucose uptake and lipoprotein lipase (on capillary endothelium), which frees fatty acids from chylomicrons and VLDL; adipocytes re-esterify them into triglycerides. - Lipid mobilisation (lipolysis): in fasting and stress, noradrenaline (sympathetic nerves), adrenaline and glucagon activate hormone-sensitive lipase; fatty acids and glycerol are released into the blood. Insulin inhibits lipolysis. Distribution: determining factors - Age, sex (sex hormones), genetics, diet. - Men: more in the abdomen (visceral, "apple" pattern). Women: more in the hips, thighs and breasts (subcutaneous, "pear" pattern), because of oestrogen. - Visceral fat is more metabolically active and linked to insulin resistance, type 2 diabetes and cardiovascular disease. Adipose tissue as an endocrine organ - Adipocytes secrete leptin, adiponectin, resistin, oestrogens (by aromatase), angiotensinogen, and inflammatory cytokines (TNF-α, IL-6) that come from macrophages in fat. - Leptin: - secreted in proportion to fat mass - acts on the hypothalamus (arcuate nucleus) to reduce appetite (inhibits NPY/AgRP neurons, stimulates POMC neurons) and increase energy expenditure: the satiety signal - also needed for normal reproductive function (low leptin in very lean athletes causes amenorrhoea) - obese people usually have high leptin but are resistant to its effects (leptin resistance); rare leptin gene mutations cause severe early obesity - Adiponectin: increases insulin sensitivity; lower in obesity. - Ghrelin (the hunger hormone, from the stomach, not fat): rises before meals and stimulates hunger via the hypothalamus; falls after eating. Leptin and ghrelin are opposite partners in the hunger response. - Energy balance: the hypothalamus integrates leptin, ghrelin, insulin and gut hormones; weight is stable when intake equals expenditure. Brown adipose tissue (multilocular) - Cells are smaller, polygonal, with many small lipid droplets and a central round nucleus, and very many mitochondria (with many cristae), which give the brown colour (cytochromes). Richly vascularised and innervated by sympathetic nerves. - Function: non-shivering thermogenesis. Uncoupling protein 1 (UCP1, thermogenin) in the inner mitochondrial membrane lets protons leak back into the matrix without making ATP, so the energy is released as heat. - Abundant in newborns (about 2-5% of body weight: between the shoulder blades, around the neck, kidneys and great vessels) to protect against cold. Present in small amounts in adults (neck, supraclavicular region), more in lean people; activated by cold. - Beige (brite) adipocytes: brown-like cells appearing within white fat after cold exposure or exercise. Obesity - Characterisation: body mass index (BMI) = weight (kg) / height (m)². Normal 18.5-24.9; overweight 25-29.9; obesity 30 or more (class I 30-34.9, class II 35-39.9, class III 40 or more). Waist circumference better reflects visceral fat. - The obesity trend is rising worldwide; projections suggested that nearly half of American adults would be obese by 2030. - Hypertrophy (bigger adipocytes) is the main mechanism in adults; hyperplasia (more adipocytes) occurs mainly in childhood and in severe obesity. - Consequences: type 2 diabetes, hypertension, dyslipidaemia, cardiovascular disease, fatty liver (MASLD), sleep apnoea, osteoarthritis, some cancers. - Liposarcoma and lipoma are tumours of adipocytes (malignant and benign). Exam focus: compare white and brown adipose tissue (cell structure, location, function), and explain the roles of leptin and ghrelin.

1. What is mesenchyme? 2. What are the two components of the extracellular matrix? 3. Why do GAGs attract water? 4. How is hyaluronic acid different from other GAGs? 5. What is the function of fibronectin? 6. Describe the structure of collagen from molecule to bundle. 7. Why is glycine every third amino acid in collagen? 8. What is the role of vitamin C in collagen synthesis? 9. Name the main locations of collagen types I, II, III and IV. 10. How are reticular fibres stained, and where are they found? 11. What protein forms the scaffold of elastic fibres, and which disease involves it? 12. How do you tell an active fibroblast from a fibrocyte? 13. Name four tissue-specific macrophages. 14. What happens when an allergen binds to a mast cell? 15. Describe a plasma cell on H&E. 16. Where is dense regular connective tissue found? 17. Why does white fat look empty on H&E? 18. What does leptin do? 19. What makes brown fat produce heat? 20. How is obesity defined by BMI?

1. Embryonic connective tissue of stellate pluripotent cells in a gel matrix; the source of all connective tissues. 2. Fibres and ground substance. 3. They are highly negatively charged, so they attract Na⁺ and water. 4. It is not sulfated, not bound to a core protein, and is made at the cell surface. 5. It links cells (via integrins) to matrix components (collagen, fibrin) and guides cell migration. 6. Triple-helical tropocollagen molecules, staggered into banded fibrils (67 nm), fibrils into fibres, fibres into bundles. 7. It is the smallest amino acid and fits into the crowded centre of the triple helix. 8. Cofactor for prolyl and lysyl hydroxylases; without it the triple helix is unstable (scurvy). 9. I: skin, bone, tendon. II: cartilage. III: reticular fibres (lymphoid organs, liver). IV: basement membranes. 10. Silver impregnation (black) and PAS. Lymph nodes, spleen, bone marrow, liver. 11. Fibrillin. Marfan syndrome. 12. Active: large, pale oval nucleus, prominent nucleolus, basophilic cytoplasm. Fibrocyte: small, dark flat nucleus, little cytoplasm. 13. Kupffer cells, alveolar macrophages, microglia, osteoclasts (also Langerhans cells). 14. The allergen cross-links IgE bound to receptors, causing degranulation and release of histamine and other mediators (type I hypersensitivity). 15. Oval cell, eccentric clock-face nucleus, basophilic cytoplasm, pale perinuclear Golgi area. 16. Tendons, ligaments, aponeuroses (cornea). 17. The lipid is dissolved by the alcohols and xylene used in processing. 18. It signals fat stores to the hypothalamus, reducing appetite and increasing energy expenditure. 19. UCP1 (thermogenin) in the mitochondria lets protons leak back without making ATP, releasing energy as heat. 20. BMI 30 or more.

Extracellular matrix (ECM) - Collagen: mainly type II (thin fibrils, not visible in light microscopy in hyaline cartilage, which is why the matrix looks glassy and uniform). Minor types IX, X (hypertrophic cartilage), XI. - Proteoglycans: aggrecan (core protein with many chondroitin sulfate and keratan sulfate chains). Up to about 100 aggrecan molecules bind to a single hyaluronic acid molecule, each attached by a link protein, forming huge aggregates (up to several µm). - The aggregates trap water; when compressed, water is pushed out, and when released it flows back. This gives cartilage its resilience and shock absorption and allows diffusion of nutrients. - The collagen fibrils form a network that resists tension and holds the swollen proteoglycans. - Glycoproteins: chondronectin (links chondrocytes to collagen II). - TEM: thin collagen fibrils without clear banding and fine granules (proteoglycans). - Staining: the matrix is basophilic (sulfated GAGs), PAS-positive, and metachromatic with toluidine blue. Distribution of matrix components - Pericellular (capsular) matrix: a thin rim (about 1-3 µm) right around each lacuna, rich in type VI collagen, hyaluronic acid and proteoglycans. Stains most intensely. - Territorial matrix: around each isogenous group; strongly basophilic (many GAGs, fewer collagen fibrils). - Interterritorial matrix: between groups; paler (more collagen, fewer proteoglycans). Chondroblasts and chondrocytes - Chondroblasts: young cells derived from mesenchymal cells or from the chondrogenic layer of the perichondrium. Flattened or oval, basophilic (RER), actively secreting matrix. - Chondrocytes: mature cells, trapped in cavities of the matrix called lacunae. Round to oval; their cytoplasm contains RER, Golgi, glycogen and lipid droplets. Chondrocytes completely fill the lacunae in living tissue; the space around them in sections is shrinkage artefact. - They maintain the matrix. Because there are no vessels, chondrocytes use mainly anaerobic glycolysis (glycogen stores). - Isogenous groups (cell nests): clusters of 2, 4 or more chondrocytes produced by mitotic division of a single chondrocyte, each still in its own lacuna, the result of interstitial growth. - In articular cartilage the arrangement varies with depth: flattened cells in the superficial (tangential) zone; round cells in the middle; columns in the deep zone. Perichondrium - A layer of dense irregular connective tissue covering most hyaline and elastic cartilage (absent at articular surfaces and in fibrocartilage). - Two layers: - outer fibrous layer: type I collagen, fibroblasts, blood vessels - inner chondrogenic (cellular) layer: chondroprogenitor cells that become chondroblasts - Functions: nutrition (vessels), attachment, growth and (limited) repair. Cartilage growth 1. Appositional growth: new chondroblasts from the chondrogenic layer of the perichondrium add matrix to the surface. Increases width. Continues in adults (limited). 2. Interstitial growth: chondrocytes within the cartilage divide and secrete new matrix from within (forming isogenous groups), expanding the cartilage from the inside. Important in young cartilage, the growth plates and articular cartilage. Possible because the matrix is still deformable. - Hormones: growth hormone (via IGF-1), thyroid hormone and testosterone stimulate growth; cortisol inhibits. Clinical - Cartilage heals poorly because of the lack of blood vessels and low cell turnover. Damaged articular cartilage (e.g. osteoarthritis) is not regenerated; defects fill with fibrocartilage. - With age, hyaline cartilage may calcify (costal, laryngeal) and show degenerative changes. Asbestiform (amianthoid) degeneration: thick, parallel collagen fibres appear in the matrix of ageing hyaline cartilage (costal cartilage), making it look like asbestos fibres. Exam focus: describe the proteoglycan aggregate, the three matrix regions, and the two types of growth.

Hyaline cartilage - The most common type. Glassy, bluish-white and translucent in fresh tissue ("hyalos" means glass). - Matrix: homogeneous, basophilic; type II collagen fibrils not visible in light microscopy. - Cells: chondrocytes in lacunae, in isogenous groups of 2 or 4. - Perichondrium: present (except at articular surfaces). - Locations: - fetal skeleton (model for endochondral ossification) - epiphyseal growth plates - articular surfaces of synovial joints - costal cartilages - nose, larynx (thyroid, cricoid, most of the arytenoid), trachea (C-shaped rings) and bronchi - Trachea histology: a C-shaped ring of hyaline cartilage with perichondrium, open posteriorly (trachealis smooth muscle), under the respiratory mucosa; the oesophagus is behind it. - Function: smooth low-friction surfaces, support of airways, flexibility, bone growth. Articular cartilage - A special hyaline cartilage covering joint surfaces, about 2-5 mm thick. - No perichondrium; nourished by synovial fluid (by diffusion, helped by joint loading and unloading) and from subchondral bone vessels in deep layers. - Zones (superficial to deep): 1. superficial (tangential) zone: flattened chondrocytes; collagen fibrils parallel to the surface; resists shear. The articular surface itself is smooth and slippery (lubricin). 2. middle (transitional) zone: round chondrocytes; oblique fibrils. 3. deep (radial) zone: chondrocytes in vertical columns; fibrils perpendicular to the surface. 4. calcified zone: calcified matrix separated from the deep zone by a wavy line (the tidemark), anchoring cartilage to the subchondral bone. - Collagen arcades (Benninghoff): fibrils run up from the deep zone and turn parallel at the surface. - Clinical: osteoarthritis: loss of proteoglycans, fibrillation and fissuring of the surface, loss of cartilage, exposure of bone (eburnation), osteophytes. Elastic cartilage - Like hyaline cartilage plus a dense network of branching elastic fibres in the matrix (stained dark with orcein or Verhoeff). - Chondrocytes are more numerous and larger, often single or in pairs; less matrix. - Perichondrium: present. - Does not calcify with age. - Location and function: - external ear (auricle), external auditory canal, auditory (Eustachian) tube - epiglottis - corniculate and cuneiform cartilages of the larynx - gives flexible support: returns to shape after bending. Fibrocartilage - A combination of dense regular connective tissue and hyaline cartilage. - Thick bundles of type I collagen (visible, eosinophilic) arranged in parallel rows, with small chondrocytes in rows or small groups between them; scant ground substance. - No perichondrium. - Location: - intervertebral discs (annulus fibrosus) - pubic symphysis - menisci of the knee - articular discs of the temporomandibular and sternoclavicular joints - glenoid and acetabular labra - insertion of some tendons and ligaments into bone - Function: resists compression and tension; strong shock absorber; transition between dense connective tissue and hyaline cartilage. - The intervertebral disc: an outer annulus fibrosus (concentric lamellae of fibrocartilage) around a central nucleus pulposus (a gel rich in hyaluronic acid and water, a remnant of the notochord in children). - Clinical: disc herniation (hernia): the nucleus pulposus bulges or herniates through a tear in the annulus, usually posterolaterally, compressing spinal nerve roots (e.g. L4-L5 or L5-S1, causing sciatica). Meniscal tears heal poorly because they are largely avascular. Comparison - Hyaline: collagen II; glassy matrix; perichondrium yes; articular cartilage, trachea, costal cartilage. - Elastic: collagen II plus elastic fibres; perichondrium yes; ear, epiglottis. - Fibrocartilage: collagen I bundles; perichondrium no; discs, symphysis, menisci. Chordoid tissue (pseudo-cartilage) - A cartilage-like tissue with large vacuolated cells, packed closely together with very little matrix, and without typical cartilage features. - Also called pseudo-cartilage or chondroid tissue. Seen in the notochord (vacuolated cells), in some sesamoid tissues and tendon attachments in animals. - In humans its main remnants are in the nucleus pulposus. Clinical: chordoma (tumour of notochord remnants) has physaliferous (bubbly) cells. Exam focus: compare the three types of cartilage (fibres, perichondrium, locations) and describe the zones of articular cartilage.

1. Why does cartilage heal poorly? 2. What type of collagen is found in hyaline cartilage? 3. Describe the proteoglycan aggregate of cartilage. 4. How does cartilage absorb shocks? 5. What is the difference between territorial and interterritorial matrix? 6. What is an isogenous group? 7. What are the two layers of the perichondrium? 8. Compare appositional and interstitial growth. 9. Where is hyaline cartilage found? 10. What are the zones of articular cartilage? What is the tidemark? 11. How does elastic cartilage differ from hyaline cartilage? 12. Which cartilage has no perichondrium? 13. Describe the structure of an intervertebral disc and what happens in a herniation. 14. What is asbestiform degeneration? 15. What is chordoid tissue?

1. It is avascular, its cells divide little, and they are trapped in lacunae. 2. Type II. 3. Many aggrecan molecules (core protein with chondroitin and keratan sulfate) attached by link proteins to one hyaluronic acid molecule. 4. The aggregates trap water; compression pushes water out, and it flows back when released. 5. Territorial matrix: around each group, rich in GAGs, darker. Interterritorial: between groups, more collagen, paler. 6. A cluster of 2-4 chondrocytes derived from one parent cell by division. 7. Outer fibrous layer and inner chondrogenic layer. 8. Appositional: new chondroblasts from the perichondrium add matrix at the surface. Interstitial: chondrocytes divide within the cartilage and expand it from inside. 9. Articular surfaces, costal cartilage, nose, larynx, trachea and bronchi, growth plates, fetal skeleton. 10. Superficial (tangential), middle, deep (radial), calcified. The tidemark is the wavy line between the deep and calcified zones. 11. It has a dense network of elastic fibres, more cells, is more flexible and does not calcify with age. 12. Fibrocartilage (and articular cartilage). 13. Outer annulus fibrosus (fibrocartilage lamellae) and inner nucleus pulposus (gel). In herniation the nucleus pushes through a tear in the annulus and may compress nerve roots. 14. The appearance of thick parallel collagen fibres in ageing hyaline cartilage, looking like asbestos. 15. A cartilage-like tissue of large vacuolated cells with little matrix (notochord; nucleus pulposus remnants).

Functions of the skeletal system 1. Support: framework for the body. 2. Protection of internal organs (skull, rib cage, vertebrae). 3. Movement: levers for muscles. 4. Mineral storage: about 99% of the body's calcium and 85% of its phosphate. Bone releases or takes up Ca²⁺ to keep blood levels constant. 5. Haematopoiesis: red bone marrow. 6. Energy storage: yellow marrow (fat). 7. Endocrine: osteocalcin (influences insulin secretion), FGF23 (from osteocytes; regulates phosphate). 8. Acid-base buffering. Composition of bone tissue - Cells. - Extracellular matrix: - organic portion (about 30-35% of dry weight): osteoid - inorganic portion (about 65-70%): mineral - water (about 10% of total weight) Organic matrix - Collagen fibres: about 90% of the organic matrix, type I (with small amounts of V). Gives tensile strength and flexibility. - Ground substance (about 10%): - proteoglycans: small ones such as decorin and biglycan (proteoglycan I), with chondroitin sulfate and keratan sulfate - multi-adhesive glycoproteins: osteonectin (binds collagen and hydroxyapatite), osteopontin and bone sialoprotein (anchor cells to the matrix via integrins, important for osteoclast attachment) - vitamin K-dependent proteins: osteocalcin (the most abundant non-collagen protein, made only by osteoblasts, a marker of bone formation; binds calcium) - growth factors and cytokines stored in the matrix (TGF-β, IGF, BMPs), released during resorption - Osteoid: the newly deposited, unmineralised organic matrix, seen as a pale pink seam between osteoblasts and mineralised bone. It is mineralised after about 10 days. Inorganic matrix - Calcium hydroxyapatite crystals, Ca₁₀(PO₄)₆(OH)₂: thin plates about 40 nm long, deposited along and within collagen fibrils (in the gaps between tropocollagen molecules). - Also citrate, carbonate, magnesium, sodium, fluoride (fluoride substitutes for OH, making crystals more resistant). - Gives hardness and resistance to compression. Bone mineralisation - Starts in matrix vesicles: small membrane-bound vesicles that bud off from osteoblasts into the osteoid. - Matrix vesicles contain: - alkaline phosphatase (increases local phosphate concentration by cleaving phosphate esters and pyrophosphate, an inhibitor of mineralisation) - calcium-binding molecules (annexins, phosphatidylserine) and pumps - Calcium and phosphate concentrate in the vesicles, crystals form, the vesicle breaks, and crystals spread along the collagen fibrils (crystal growth then continues). - Needs adequate calcium, phosphate and vitamin D. - Clinical: raised serum alkaline phosphatase indicates increased osteoblast activity (growth, healing fractures, Paget disease, bone metastases). Exam focus: list the organic and inorganic components of bone matrix, and describe the role of matrix vesicles in mineralisation.

Osteoprogenitor (osteogenic) cells - Flattened stem cells derived from mesenchymal stem cells; found in the inner cellular layer of the periosteum, the endosteum, and lining Haversian canals. - Differentiate into osteoblasts under the control of the transcription factor RUNX2 (and osterix), stimulated by BMPs and Wnt signalling. Osteoblasts - Cuboidal or columnar cells in a single layer on the surface of forming bone, like an epithelium (but not joined by tight junctions). - Basophilic cytoplasm (much RER), a prominent Golgi (pale perinuclear area), and a nucleus away from the bone surface. They are polarised toward the osteoid. - Functions: - synthesise and secrete osteoid: type I collagen, osteocalcin, osteonectin, proteoglycans - control mineralisation (alkaline phosphatase, matrix vesicles) - regulate osteoclasts: express RANKL and M-CSF (stimulating osteoclasts) and OPG (inhibiting them) - have receptors for PTH, vitamin D and oestrogen - Fate: some become trapped in the matrix as osteocytes (osteoblasts become osteocytes), some become flat inactive bone-lining cells, and many die by apoptosis. Osteocytes - The most numerous bone cells (over 90%); mature, long-lived cells. - Each osteocyte lies in a small cavity in the matrix called a lacuna, with a flattened almond-shaped body. - Many long, thin cytoplasmic processes run through tiny channels in the matrix called canaliculi. The processes of neighbouring osteocytes touch through gap junctions. The canaliculi form a network connecting osteocytes to each other and to the central canal, through which nutrients and signals diffuse. - Functions: - maintain the matrix - mechanosensation: sense mechanical strain (fluid flow in canaliculi) and signal osteoblasts and osteoclasts to adapt bone to the load (Wolff's law) - secrete sclerostin (inhibits bone formation by blocking Wnt; less sclerostin when loaded) - secrete FGF23 (phosphate regulation) and RANKL - can release calcium from the surrounding matrix (osteocytic osteolysis, minor) - When osteocytes die, empty lacunae remain (e.g. in dead bone, necrosis). Osteoclasts - Large (up to 100 µm) multinucleated cells (5-50 nuclei) with eosinophilic, foamy cytoplasm. They come from the fusion of monocyte-macrophage precursors in the bone marrow (not from osteoprogenitor cells). - Lie in shallow pits they have dug on the bone surface: resorption bays called Howship's lacunae. - Polarised when active (unpolarised when resting). Structure of an active osteoclast: 1. ruffled border: deeply folded plasma membrane facing the bone, the site of resorption 2. clear (sealing) zone: a ring of actin filaments with integrins (αvβ3) that attaches tightly to the bone (via osteopontin) and seals the resorption space 3. basolateral region: exocytosis of degraded products 4. vesicular region, many mitochondria and lysosomes - Bone resorption: 1. carbonic anhydrase makes H⁺, which proton pumps (V-ATPase) in the ruffled border secrete into the sealed space; Cl⁻ follows. The pH falls to about 4.5, dissolving hydroxyapatite. 2. lysosomal enzymes, especially cathepsin K, and matrix metalloproteinases digest the exposed collagen. 3. degradation products are taken up by endocytosis and transported across the cell (transcytosis) and released on the other side. - Clinical: osteopetrosis (marble bone disease), defective osteoclasts (carbonic anhydrase II or proton pump mutations): dense but brittle bones, marrow failure. Regulation of osteoclast function (finely regulated by hormonal and local factors) - RANKL (receptor activator of nuclear factor κB ligand): expressed by osteoblasts, osteocytes and stromal cells. Binds RANK on osteoclast precursors, promoting their fusion, differentiation and activity. M-CSF is also needed. - OPG (osteoprotegerin): a decoy receptor secreted by osteoblasts that binds RANKL and blocks it. The RANKL/OPG ratio determines the amount of resorption. - Clinical: denosumab is a monoclonal antibody against RANKL, used to treat osteoporosis and bone metastases. Regulation by hormonal feedback - When blood calcium declines, parathyroid hormone (PTH) is released: - PTH acts on osteoblasts (osteoclasts have no PTH receptors), which increase RANKL and reduce OPG, activating osteoclasts; calcium and phosphate are released from bone - PTH also increases renal Ca²⁺ reabsorption and vitamin D activation - blood calcium rises; negative feedback turns off PTH - paradox: intermittent low doses of PTH (teriparatide) stimulate bone formation and are used for severe osteoporosis - When blood calcium rises, calcitonin (from thyroid C cells) is released: it acts directly on osteoclasts (they have calcitonin receptors), making them retract and stop resorbing. Calcium is deposited in bone and blood calcium falls. - Vitamin D (calcitriol): increases intestinal absorption of Ca²⁺ and phosphate (for mineralisation) and, at high levels, stimulates resorption via RANKL. - Oestrogens: inhibit osteoclasts (reduce RANKL, increase OPG, promote osteoclast apoptosis) and stimulate osteoblasts. Their fall at menopause causes rapid bone loss. - Glucocorticoids: reduce bone formation and increase resorption. Bone deposition versus resorption - In young adults the two are balanced (bone mass constant). In growth, deposition dominates. After about 35-40 years, and especially after menopause, resorption slightly exceeds deposition, so bone mass declines. Exam focus: describe the ruffled border and sealing zone of the osteoclast and how it resorbs bone, and explain the RANKL-OPG system and PTH action.

Woven bone (primary, immature) - Collagen fibres in an irregular, interwoven arrangement. - More cells (osteocytes) with random distribution, less mineral, more ground substance. - Formed rapidly: in the fetus, in the first stages of ossification, in fracture repair (callus), in some diseases (Paget disease, tumours). - Mechanically weaker. Replaced by lamellar bone in remodelling. In adults found only at tooth sockets (alveolar bone), some tendon insertions and sutures. Lamellar bone (secondary, mature) - Collagen fibres arranged in parallel layers (lamellae) about 3-7 µm thick. Within each lamella the fibres are parallel; in adjacent lamellae they run in different directions (like plywood), giving strength. - Osteocytes in lacunae between or within lamellae, regularly arranged. - Found in compact and spongy bone of the adult skeleton. - In polarised light, an osteon shows alternating light and dark lamellae because of the changing collagen orientation. Compact (cortical) bone - Dense, solid-looking; about 80% of skeletal mass; forms the outer shell of all bones and the diaphysis (shaft) of long bones. - The structural unit is the osteon (Haversian system): - a cylinder (about 200-300 µm wide, several mm long) running parallel to the long axis of the bone - central (Haversian) canal: contains blood vessels (a capillary or arteriole and venule), nerves, and loose connective tissue, lined by endosteum - concentric lamellae: 4-20 layers around the canal - osteocytes in lacunae between the lamellae, connected by canaliculi radiating toward the central canal - cement line: a thin, mineral-rich, collagen-poor boundary at the outer edge of each osteon; canaliculi do not cross it - Perforating (Volkmann) canals: run transversely or obliquely, connect Haversian canals to each other and to the periosteum and marrow cavity. Not surrounded by concentric lamellae. Types of lamellae in compact bone 1. Osteonal (concentric, Haversian) lamellae: around the central canal of each osteon. 2. Interstitial lamellae: fragments of older osteons between current osteons, left after remodelling. 3. Outer circumferential lamellae: under the periosteum, running around the whole bone. 4. Inner circumferential lamellae: next to the endosteum and marrow cavity. - New osteons are made by remodelling (see the remodelling note): each new set of lamellae narrows the canal, so younger osteons have wider canals. Ground section versus decalcified section - Ground (undecalcified) section: bone is sawed and ground thin; cells and soft tissue are lost, but lacunae, canaliculi and canals appear black (filled with debris or air), showing osteon architecture beautifully. - Decalcified section: minerals removed by acids or EDTA; stained with H&E; shows cells, periosteum and marrow. Spongy (cancellous, trabecular) bone - A three-dimensional lattice of thin plates and rods called trabeculae (spicules), with spaces filled with red or yellow bone marrow. - Found inside flat bones (the diploë of the skull), vertebrae, the epiphyses and metaphyses of long bones, and ribs. - Trabeculae consist of lamellae with osteocytes; they have no osteons (they are thin enough to be nourished from the marrow by diffusion through canaliculi). - Trabeculae align along lines of mechanical stress, giving maximum strength with minimum weight (e.g. in the femoral neck). - Lighter, with a larger surface area; remodels faster (about 25% per year versus about 3% for compact bone). Therefore it is affected first by osteoporosis (vertebral and hip fractures). Periosteum - Covers the outer surface of bones (except articular surfaces and tendon insertions). - Two layers: - outer fibrous layer: dense irregular connective tissue, vessels and nerves (periosteal pain) - inner cellular (osteogenic, cambium) layer: osteoprogenitor cells and osteoblasts, active in growth and fracture repair - Attached to the bone by Sharpey's (perforating) fibres, bundles of collagen that enter the outer circumferential lamellae; tendons and ligaments also insert this way. Endosteum - A thin layer of osteoprogenitor cells and bone-lining cells covering all inner surfaces: the marrow cavity, trabeculae, and Haversian and Volkmann canals. Structure of a long bone - Diaphysis: shaft of compact bone around the marrow (medullary) cavity. - Epiphyses: ends, spongy bone covered by thin compact bone and articular cartilage. - Metaphysis: the region between, next to the growth plate (epiphyseal plate) in children; the epiphyseal line in adults. - Blood supply: nutrient artery (enters the diaphysis), metaphyseal and epiphyseal arteries, periosteal arteries. Exam focus: draw an osteon with all its parts, name the four types of lamellae, and compare compact and spongy bone.

Intramembranous ossification - Forms: the flat bones of the skull (frontal, parietal, parts of the occipital and temporal), the mandible (mostly), maxilla, clavicle (partly). - Steps: 1. Mesenchymal cells condense in a richly vascularised area and differentiate into osteoprogenitor cells and then osteoblasts, forming centres of ossification. 2. Osteoblasts secrete osteoid, which mineralises within days; osteoblasts trapped in the matrix become osteocytes. 3. Trabeculae (spicules) of woven bone form and fuse into a network (spongy bone); vascular mesenchyme between them becomes red marrow. 4. Mesenchyme on the surface condenses into the periosteum. 5. Compact bone plates form on the surfaces (the inner and outer tables of the skull), with spongy bone (diploë) in between; woven bone is remodelled into lamellar bone. - The fontanelles are unossified membrane areas in the infant skull, which allow moulding during birth and brain growth; the anterior fontanelle closes at about 18-24 months. - Mantellar (appositional) ossification: the subperiosteal formation of bone on the surface of a cartilage model (the bone collar) is intramembranous in type. Endochondral ossification - Forms: most bones of the skeleton, especially long bones (limbs), vertebrae, pelvis, base of the skull. - Steps in a long bone: 1. Cartilage model: mesenchyme forms a hyaline cartilage model of the bone, covered by perichondrium (week 6-8). 2. Bone collar: the perichondrium around the middle of the shaft becomes periosteum, and its osteoblasts form a thin collar of bone by intramembranous-type ossification. 3. Hypertrophy and calcification: chondrocytes in the centre of the shaft enlarge (hypertrophy), make type X collagen and alkaline phosphatase, the matrix calcifies, and the chondrocytes die by apoptosis, leaving empty lacunae. 4. Primary ossification centre (by about week 12 in the fetus; fetus 12 weeks: most long bones have one): a periosteal bud (blood vessels, osteoprogenitor cells and osteoclast precursors) enters the calcified cartilage. Osteoblasts lay down bone on the remaining calcified cartilage spicules (mixed spicules: a cartilage core covered by bone, bluish core with pink bone on H&E). 5. Spread: ossification spreads toward both ends; osteoclasts resorb the central spongy bone to form the marrow cavity. 6. Secondary ossification centres: appear in the epiphyses, mostly after birth (the distal femur at birth is a useful sign of a mature fetus). Here too, cartilage is replaced by bone, except for: - the articular cartilage (permanent) - the epiphyseal (growth) plate between epiphysis and diaphysis (until growth ends) Growth plate: zones (from the epiphysis side toward the diaphysis) 1. Zone of resting (reserve) cartilage: small scattered chondrocytes; anchors the plate to the epiphysis; a stem cell reserve. 2. Zone of proliferation: chondrocytes divide rapidly and form stacks of flat cells in columns parallel to the long axis ("stacks of coins"). The driver of growth in length. 3. Zone of hypertrophy (maturation): chondrocytes enlarge greatly, accumulate glycogen, and the matrix between columns becomes thin. 4. Zone of calcification: hypertrophic chondrocytes calcify the matrix and die by apoptosis. 5. Zone of ossification: blood vessels and osteoprogenitor cells invade the empty lacunae; osteoblasts deposit bone on the calcified cartilage spicules; osteoclasts remodel. - Mnemonic for the zones: "Real People Have Career Options" (Resting, Proliferation, Hypertrophy, Calcification, Ossification). Growth in length - The plate stays about the same thickness: cartilage is added at the epiphysis side (proliferation) and replaced by bone at the diaphysis side at the same rate. The bone lengthens as the epiphysis is pushed away. - At the end of puberty (under high oestrogen in both sexes), proliferation stops, the plate is completely replaced by bone, and only the epiphyseal line remains: the epiphyses are "closed" (about 18 years in females, 21 in males). - Hormones: growth hormone via IGF-1 (stimulates proliferation), thyroid hormones, sex hormones (growth spurt then closure), vitamin D; excess glucocorticoids inhibit. - Clinical: - achondroplasia: a gain-of-function mutation of FGFR3 inhibits proliferation in the growth plate, causing short-limbed dwarfism (autosomal dominant) - gigantism: excess GH before plate closure; acromegaly: after closure (bones thicken, no growth in length) - rickets: lack of vitamin D in children; the growth plate widens with uncalcified cartilage and osteoid - fractures through the growth plate (Salter-Harris) can stop growth - bone age on hand X-rays estimates maturity Growth in thickness (width): appositional growth 1. Osteoblasts beneath the periosteum lay down new bone at the outer surface, forming ridges around periosteal blood vessels. 2. The ridges fuse around the vessel, enclosing it in a tunnel. 3. Osteoblasts in the tunnel lay down concentric lamellae, forming a new osteon. 4. Circumferential lamellae are added under the periosteum. - At the same time osteoclasts on the endosteal surface resorb bone, enlarging the marrow cavity so the bone does not become too heavy. Types of growth: summary - Cartilage: appositional and interstitial. - Bone: only appositional (bone cannot grow interstitially because its matrix is rigid). Exam focus: describe endochondral ossification step by step, and name the zones of the growth plate with what happens in each.

Bone remodelling - Carried out by the basic multicellular unit (BMU): osteoclasts in front, followed by osteoblasts. - In compact bone, a cutting cone (osteoclasts) bores a tunnel through the bone; a closing cone behind it (osteoblasts) fills it with concentric lamellae, producing a new osteon with a central canal. Remnants of old osteons become interstitial lamellae. - In spongy bone, remodelling occurs on the trabecular surfaces. - Phases: activation, resorption (about 2-3 weeks), reversal, formation (about 3 months), quiescence. - Functions: 1. adapt bone to mechanical stress (Wolff's law: bone is laid down where needed and removed where not; the reason for bone loss in bed rest and space flight, and gain with weight-bearing exercise) 2. repair microdamage (microcracks) 3. calcium homeostasis 4. replace woven bone with lamellar bone Factors that affect bone growth and maintenance - Mechanical: weight-bearing exercise and muscle pull stimulate formation (via osteocytes). - Nutritional: - calcium and phosphate - vitamin D: calcium absorption - vitamin C: collagen synthesis (lack: scurvy, poor bone matrix) - vitamin K: osteocalcin carboxylation - vitamin A: osteoblast and osteoclast activity; excess weakens bone - protein - Hormonal: - growth hormone and IGF-1: growth at the growth plates - thyroid hormones: growth and maturation - sex hormones: growth spurt at puberty, then closure of growth plates; maintain bone mass in adults. Oestrogens stimulate the proliferation of osteoblasts and the apoptosis of osteoclasts. - PTH, calcitonin, vitamin D: calcium balance - glucocorticoids in excess: bone loss - insulin: growth - How they work together: in childhood GH, thyroid and nutrition drive growth; at puberty sex hormones cause the growth spurt and then end growth; in adulthood sex hormones and exercise maintain mass. Bone repair (fracture healing) 1. Haematoma formation (hours): torn vessels bleed and form a clot; bone cells at the fracture ends die; inflammation begins (swelling, pain). 2. Soft (fibrocartilaginous) callus (days to about 3 weeks): capillaries grow into the haematoma; fibroblasts produce collagen; osteoprogenitor cells from the periosteum and endosteum become chondroblasts (away from vessels, where oxygen is low) and osteoblasts. The callus bridges the gap with fibrocartilage and hyaline cartilage. 3. Hard (bony) callus (weeks 3-12): osteoblasts form trabeculae of woven bone, which replace the soft callus (by endochondral ossification of the cartilage and direct ossification). The callus is a bony swelling that joins the fragments firmly. 4. Remodelling (months to years): osteoclasts remove excess callus, woven bone is replaced by lamellar compact bone, and the shape is restored along lines of stress. - Primary (direct) healing: with rigid surgical fixation and close contact of fragments, osteons cross the fracture directly without a callus. - Healing needs a good blood supply, immobilisation, and nutrition. Poor healing: non-union (smoking, diabetes, infection, instability). Bone disorders - Osteoporosis: reduced bone mass and deteriorated microarchitecture (thinner, fewer trabeculae), with normal mineralisation of the remaining bone. Increased risk of fractures (vertebrae, hip, wrist). Causes: postmenopausal oestrogen loss, ageing, glucocorticoids, immobility. Diagnosis: bone densitometry (DEXA, T-score -2.5 or lower). Treatment: calcium and vitamin D, weight-bearing exercise, bisphosphonates (inhibit osteoclasts), denosumab, teriparatide. - Osteomalacia: demineralisation of bone due to lack of vitamin D (or phosphate) in adults; osteoid is not mineralised. Soft bones, pain, pseudofractures. In children, the same defect is rickets: bowed legs, widened growth plates, rachitic rosary (swelling of rib costochondral junctions). - Paget disease of bone: excessive, disorganised remodelling (overactive giant osteoclasts, then chaotic formation); thick but weak bones with a mosaic pattern of cement lines; high alkaline phosphatase. - Osteogenesis imperfecta: defective type I collagen, brittle bones. - Osteopetrosis: defective osteoclasts, dense brittle bones. - Osteomyelitis: infection of bone, usually Staphylococcus aureus. - Bone tumours: osteosarcoma (malignant osteoid-forming tumour in adolescents, at the metaphysis around the knee); metastases (breast, prostate, lung, kidney, thyroid) are much more common than primary tumours. Exam focus: describe the four stages of fracture healing, and compare osteoporosis and osteomalacia.

1. List six functions of bone. 2. What makes up the organic and inorganic parts of bone matrix? 3. What is osteoid? 4. What is the role of matrix vesicles and alkaline phosphatase? 5. Name the four bone cell types and their origins. 6. Describe osteocytes and their canaliculi. 7. Describe the structure of an active osteoclast. 8. How does an osteoclast dissolve bone? 9. What are RANKL and OPG? 10. How does PTH activate osteoclasts if osteoclasts have no PTH receptors? 11. What is the effect of calcitonin on bone? 12. Compare woven and lamellar bone. 13. Describe an osteon. 14. What is the difference between Haversian and Volkmann canals? 15. Name the four types of lamellae in compact bone. 16. Why does spongy bone have no osteons? 17. Describe the layers of the periosteum and Sharpey's fibres. 18. Which bones form by intramembranous ossification? 19. List the steps of endochondral ossification. 20. Name the zones of the growth plate. 21. How does a bone grow in width? 22. What are the stages of fracture healing? 23. Compare osteoporosis and osteomalacia.

1. Any six: support, protection, movement, mineral storage, haematopoiesis, energy storage, endocrine, acid-base buffering. 2. Organic: type I collagen and ground substance (proteoglycans, osteocalcin, osteonectin, osteopontin). Inorganic: hydroxyapatite crystals. 3. Unmineralised organic bone matrix newly laid down by osteoblasts. 4. Matrix vesicles from osteoblasts concentrate calcium and phosphate and start crystal formation; alkaline phosphatase raises local phosphate and removes pyrophosphate. 5. Osteoprogenitor cells (mesenchymal stem cells), osteoblasts (from osteoprogenitor cells), osteocytes (trapped osteoblasts), osteoclasts (fused monocyte-macrophage precursors). 6. Mature cells in lacunae whose processes run through canaliculi and connect by gap junctions to neighbours and to the central canal; they sense strain. 7. Multinucleated cell in a Howship's lacuna with a ruffled border, a sealing (clear) zone, a vesicular region and a basolateral secretory domain. 8. It pumps H⁺ into the sealed space to dissolve mineral and secretes cathepsin K and MMPs to digest collagen. 9. RANKL (from osteoblasts) binds RANK on osteoclast precursors to activate osteoclastogenesis; OPG is a decoy receptor that blocks RANKL. 10. PTH acts on osteoblasts, which increase RANKL and reduce OPG, activating osteoclasts. 11. It inhibits osteoclasts directly, reducing resorption. 12. Woven: random collagen, many cells, less mineral, immature, fast-formed. Lamellar: collagen in parallel lamellae, mature, strong. 13. A cylinder of concentric lamellae around a central canal with vessels and nerves, with osteocytes in lacunae connected by canaliculi, bounded by a cement line. 14. Haversian canals run longitudinally in osteons; Volkmann canals run transversely and connect them to each other and to periosteum and marrow. 15. Osteonal (concentric), interstitial, outer circumferential, inner circumferential. 16. Trabeculae are thin enough to be nourished by diffusion from the marrow through canaliculi. 17. Outer fibrous layer and inner osteogenic layer. Sharpey's fibres are collagen bundles anchoring periosteum (and tendons) into bone. 18. Flat bones of the skull, mandible, maxilla, clavicle (partly). 19. Cartilage model; bone collar; chondrocyte hypertrophy and calcification; periosteal bud and primary ossification centre; spread and marrow cavity; secondary centres in the epiphyses; articular cartilage and growth plate remain. 20. Resting, proliferation, hypertrophy, calcification, ossification. 21. Appositional growth: periosteal osteoblasts add bone at the surface (forming new osteons around vessels), while endosteal osteoclasts enlarge the marrow cavity. 22. Haematoma, soft (fibrocartilaginous) callus, hard (bony) callus, remodelling. 23. Osteoporosis: less bone with normal mineralisation. Osteomalacia: normal amount of matrix but poorly mineralised (vitamin D deficiency).

Blood functions 1. Transport: O₂ and CO₂; nutrients; waste (urea to the kidney); hormones; heat. 2. Regulation: pH (buffers), body temperature, fluid balance (osmotic pressure). 3. Protection: immunity (leukocytes, antibodies, complement); haemostasis (platelets, clotting factors). Plasma composition - Water: about 90-92%. - Plasma proteins: about 7% (60-80 g/L), mostly made by the liver: - albumin (about 55-60%): main contributor to colloid osmotic (oncotic) pressure, keeps fluid in vessels; carries fatty acids, bilirubin, hormones, drugs. Low albumin causes oedema (liver disease, nephrotic syndrome). - globulins (about 35-38%): - α and β globulins: transport proteins (transferrin, ceruloplasmin, lipoproteins, hormone-binding proteins) - γ globulins: antibodies (immunoglobulins), made by plasma cells, not the liver - fibrinogen (about 4-7%): clotting factor I, converted to fibrin - others: complement, clotting factors, enzymes, hormones - Electrolytes: Na⁺ (main cation), K⁺, Ca²⁺, Mg²⁺, Cl⁻, HCO₃⁻, phosphate. - Nutrients: glucose, amino acids, lipids (as lipoproteins), vitamins. - Gases: O₂, CO₂, N₂. - Waste: urea, creatinine, uric acid, bilirubin. - Serum is plasma without clotting factors (the fluid left after blood clots): plasma minus fibrinogen. Haematocrit - The percentage of blood volume occupied by red blood cells, measured by centrifuging anticoagulated blood. - Normal: about 40-50% in men, 36-46% in women. - Low: anaemia, haemodilution. High: polycythaemia, dehydration. - After centrifugation: plasma on top (55%), buffy coat (leukocytes and platelets, under 1%), red cells at the bottom (45%). The blood smear - A drop of blood spread thin on a slide, dried and stained with a Romanowsky-type stain (Wright, Giemsa, May-Grünwald-Giemsa). These contain: - methylene blue (basic dye): stains acidic structures blue (nuclei, RNA-rich cytoplasm, basophil granules) - eosin (acid dye): stains basic structures pink-orange (haemoglobin, eosinophil granules) - azure dyes: purple (azurophilic granules) - Used to count and identify leukocytes (differential count), examine red cell shape and size, and detect parasites (malaria). Normal counts (adult) - Red cells: about 4.5-5.5 × 10¹²/L (men), 4.0-5.0 × 10¹²/L (women). - Haemoglobin: about 130-170 g/L (men), 120-150 g/L (women). - White cells: 4-11 × 10⁹/L. - Platelets: 150-400 × 10⁹/L. Exam focus: list the main plasma proteins with their functions, and define haematocrit and serum.

Structure - Biconcave disc, about 7.5 µm in diameter, 2.6 µm thick at the rim and 0.8 µm at the centre (pale central area in smears, about 1/3 of the diameter). - The shape increases the surface area to volume ratio (faster gas exchange) and makes them very deformable. - No nucleus, no mitochondria, no ribosomes: they cannot divide or make proteins, and obtain ATP from anaerobic glycolysis (with the pentose phosphate pathway for protection against oxidants). - About 33% of their mass is haemoglobin (about 280 million molecules per cell). - Used as a size reference (7.5 µm) on tissue slides. - In capillaries they may line up in single file; in slow flow they stack into rouleaux. Cell membrane and cytoskeleton - The membrane is supported by a flexible network (membrane skeleton) on its inner surface: - spectrin (α and β chains forming a mesh) - short actin filaments, protein 4.1, adducin (junctional complexes) - ankyrin: links spectrin to band 3 (the anion exchanger, Cl⁻/HCO₃⁻) - glycophorins: glycoproteins carrying blood group antigens and a negative charge (prevents clumping) - Clinical: hereditary spherocytosis (spectrin or ankyrin defects): round, fragile spherocytes that are destroyed in the spleen (haemolytic anaemia, splenomegaly). Haemoglobin - A tetramer: four globin chains (in adults, HbA is α₂β₂) each with a haem group: a porphyrin ring holding one iron atom in the ferrous (Fe²⁺) state. Each haem binds one O₂, so one Hb carries four O₂. - Other forms: HbA2 (α₂δ₂, about 2-3%), HbF (fetal haemoglobin, α₂γ₂): higher O₂ affinity, taking O₂ from maternal blood. - Oxygen binding is cooperative (sigmoidal O₂ dissociation curve). - Effects shifting the curve to the right (lower affinity, more O₂ released to tissues): lower pH, higher CO₂ (the Bohr effect), higher 2,3-bisphosphoglycerate (2,3-BPG, from red cell glycolysis, rising at altitude and in anaemia), higher temperature. - CO₂ transport: about 70% as bicarbonate (made by carbonic anhydrase in RBCs; exchanged for Cl⁻ by band 3, the chloride shift), about 20-23% bound to Hb (carbaminohaemoglobin), about 7-10% dissolved. - Carbon monoxide binds Hb about 200-250 times more strongly than O₂ (carboxyhaemoglobin): CO poisoning. - Methaemoglobin: iron oxidised to Fe³⁺, cannot carry O₂. - Clinical: sickle cell disease (HbS, a glutamate-to-valine substitution in β-globin; HbS polymerises when deoxygenated and red cells sickle); thalassaemias (reduced α or β chain synthesis). Life cycle - Produced in the red bone marrow (erythropoiesis, stimulated by erythropoietin); released as reticulocytes (about 1% of circulating RBCs, mature in 1-2 days). - Lifespan about 120 days. Old, rigid RBCs are removed by macrophages mainly in the spleen (the "graveyard" of red cells) and liver. - Breakdown: globin into amino acids; iron recycled (bound to transferrin, stored as ferritin); haem to biliverdin then bilirubin, carried by albumin to the liver, conjugated and excreted in bile. Excess breakdown causes jaundice. ABO blood types - Determined by carbohydrate antigens on the RBC surface (on glycoproteins and glycolipids). - Type A: A antigen; anti-B antibodies in plasma. - Type B: B antigen; anti-A. - Type AB: both antigens; no antibodies (the "universal recipient" for red cells). - Type O: no antigens (only H); both anti-A and anti-B (the "universal donor" of red cells). - The antibodies are IgM, formed naturally in infancy (from gut bacteria with similar antigens). - Incompatible transfusion causes agglutination and intravascular haemolysis: a life-threatening transfusion reaction. Cross-matching is always done before transfusion. Rh factor - The D antigen of the Rh system. Rh-positive (about 85% of Europeans) or Rh-negative. - Anti-D antibodies are not natural; they form only after exposure (transfusion or pregnancy). They are IgG and cross the placenta. - Haemolytic disease of the newborn (erythroblastosis fetalis): an Rh-negative mother with an Rh-positive fetus becomes sensitised at the first delivery; in a later Rh-positive pregnancy her anti-D IgG crosses the placenta and destroys fetal red cells. Prevented by giving anti-D immunoglobulin (RhoGAM) to the mother during pregnancy and after delivery. Anaemia - A reduced oxygen-carrying capacity of blood: low haemoglobin, haematocrit or RBC count. - Causes: 1. blood loss (acute haemorrhage; chronic, e.g. gastrointestinal bleeding or heavy menstruation, leading to iron deficiency) 2. reduced production: iron deficiency (microcytic, hypochromic cells; the most common worldwide), vitamin B12 or folate deficiency (megaloblastic, macrocytic; pernicious anaemia), aplastic anaemia (marrow failure), renal failure (lack of erythropoietin), chronic disease 3. increased destruction (haemolytic): sickle cell disease, thalassaemia, spherocytosis, G6PD deficiency, autoimmune haemolysis, malaria, incompatible transfusion - Symptoms: fatigue, pallor, breathlessness on exertion, tachycardia. Defective erythrocytes: shapes on a smear - Microcytes (small; iron deficiency), macrocytes (large; B12 or folate deficiency), spherocytes, sickle cells (drepanocytes), target cells (codocytes; thalassaemia, liver disease), schistocytes (fragments; microangiopathic haemolysis), teardrop cells (marrow fibrosis). - Anisocytosis: variation in size. Poikilocytosis: variation in shape. - Polycythaemia: too many RBCs (high altitude, chronic hypoxia, polycythaemia vera). - On average RBCs have an MCV (mean corpuscular volume) of about 80-100 fL. Exam focus: describe the structure of the RBC and why each feature matters, and explain the cause and prevention of haemolytic disease of the newborn.

General features - Normal count: 4-11 × 10⁹/L. Leukocytosis: above normal (infection, inflammation, leukaemia). Leukopenia: below normal (chemotherapy, some viral infections). - They are true cells with a nucleus and organelles. - They leave the blood by diapedesis (squeezing between endothelial cells) and move by amoeboid movement, guided by chemical signals (chemotaxis). - All have azurophilic (non-specific) granules, which are lysosomes. Granulocytes also have specific granules with distinctive staining. Leukocyte classification - Granulocytes (polymorphonuclear, with lobed nuclei; short-lived): neutrophils, eosinophils, basophils. - Agranulocytes (mononuclear, unlobed nuclei): lymphocytes, monocytes. Leukocyte formula (differential count, approximate percentages) - Neutrophils: 50-70% - Lymphocytes: 20-40% - Monocytes: 2-8% - Eosinophils: 1-4% - Basophils: 0.5-1% - Mnemonic: "Never Let Monkeys Eat Bananas" (from most to least common). Neutrophils - Size 12-15 µm. Nucleus with 3-5 lobes joined by thin strands (older cells have more lobes). In women, a small drumstick appendage (the Barr body) may be seen on some nuclei. - Cytoplasm: pale pink with fine, small, lilac specific granules (lysozyme, lactoferrin, collagenase) and azurophilic granules (myeloperoxidase, defensins, elastase). Tertiary granules contain gelatinase. - Band (stab) forms: immature neutrophils with a horseshoe nucleus; a rise ("left shift") indicates acute bacterial infection. - Life: about 6-12 hours in blood, then 1-4 days in tissues. - Function: the first line of cellular defence. Neutrophils arrive first at sites of acute inflammation and phagocytose bacteria. Killing by: - the respiratory burst: NADPH oxidase produces superoxide, H₂O₂; myeloperoxidase produces hypochlorous acid (HOCl, bleach) - granule enzymes and antimicrobial peptides - neutrophil extracellular traps (NETs) of DNA and proteins - Dead neutrophils and bacteria form pus. - Clinical: chronic granulomatous disease (NADPH oxidase deficiency), recurrent infections. Recruitment of neutrophils into tissues (margination and emigration) 1. Margination: in inflamed vessels, blood flow slows and leukocytes move to the edge of the vessel. 2. Rolling: selectins on the endothelium (E- and P-selectin, induced by cytokines) bind weakly to carbohydrate ligands on the leukocyte, so it rolls along. 3. Firm adhesion: chemokines activate leukocyte integrins (LFA-1), which bind ICAM-1 on the endothelium (the LFA-1-ICAM-1 interaction). 4. Transmigration (diapedesis): the leukocyte squeezes between endothelial cells (PECAM-1) and crosses the basement membrane. 5. Chemotaxis: movement toward bacteria along gradients of chemoattractants (bacterial products, C5a, leukotriene B4, IL-8). - Clinical: leukocyte adhesion deficiency (defective integrins): recurrent infections without pus, high blood neutrophils. Eosinophils - Size 12-17 µm. Nucleus usually bilobed ("spectacles"). - Cytoplasm full of large, bright orange-red (eosinophilic) specific granules. In TEM, the granules have a crystalline core (major basic protein) in a lighter matrix (eosinophil cationic protein, eosinophil peroxidase, neurotoxin). - Functions: - defence against parasites (helminths): they attach to IgE- or IgG-coated worms and release toxic granule proteins - modulation of allergic reactions: they degrade histamine and leukotrienes, but also contribute to tissue damage in asthma - phagocytosis of antigen-antibody complexes - Normally numerous in the lamina propria of the gut and respiratory tract. - Eosinophilia: parasitic infections, allergies (asthma, hay fever, eczema), some drugs and cancers. Basophils - Size 10-14 µm; the rarest leukocyte. Bilobed or S-shaped nucleus usually hidden by large, dark blue-purple (basophilic, metachromatic) granules. - Granules contain histamine, heparin, chondroitin sulfate; they also make leukotrienes. - Have high-affinity IgE receptors like mast cells. - Function: heighten the inflammatory response and allergic reactions (type I hypersensitivity): on the first exposure to an allergen, B cells make IgE, which binds to basophils and mast cells (sensitisation). On re-exposure, the allergen cross-links the IgE and the cells release histamine: vasodilation, oedema, itch, bronchoconstriction. - Different from mast cells (separate lineages; mast cells live in tissues). Monocytes - The largest leukocytes (15-20 µm). Kidney-shaped or horseshoe nucleus with fine, lacy chromatin (paler than lymphocytes); abundant grey-blue cytoplasm ("ground glass") with fine azurophilic granules and sometimes vacuoles. - Circulate for 1-3 days, then enter tissues and differentiate into macrophages (monocyte to macrophage), which live months to years. - Macrophages: phagocytosis, antigen presentation, cytokine secretion (IL-1, TNF-α). They form the mononuclear phagocyte system (Kupffer cells, alveolar macrophages, microglia, osteoclasts). Monocytes also give some dendritic cells. - Monocytosis: chronic infections (tuberculosis), recovery from acute infection. Lymphocytes - Small lymphocytes (6-9 µm, most common in blood): round, dark nucleus filling almost the whole cell, a thin rim of pale blue cytoplasm. Large lymphocytes (9-15 µm): more cytoplasm, activated cells or NK cells. - The only leukocytes that return from tissues to the blood (recirculate through lymph nodes). Some live for years (memory cells). - Three functional types (not distinguishable by morphology; identified by surface markers, CD antigens): - T lymphocytes (about 70-80%): mature in the thymus; cell-mediated immunity. Helper T cells (CD4) coordinate the immune response; cytotoxic T cells (CD8) kill infected and tumour cells; regulatory T cells suppress responses. - B lymphocytes (about 10-15%): mature in bone marrow; humoral immunity; become plasma cells that secrete antibodies. - NK (natural killer) cells (about 5-10%): large granular lymphocytes; innate immunity; kill virus-infected and tumour cells without prior sensitisation (by perforin and granzymes, detecting missing MHC class I). - Lymphocytosis: viral infections (infectious mononucleosis with atypical lymphocytes), chronic lymphocytic leukaemia. Lymphopenia: HIV (CD4 loss), corticosteroids. - Detail in the Immune System chapter. Exam focus: describe each leukocyte (size, nucleus, granules, percentage, function), and list the steps of neutrophil recruitment with the molecules involved.

Platelets - Small (2-4 µm), anucleate, disc-shaped fragments of megakaryocyte cytoplasm. - Count 150-400 × 10⁹/L; lifespan about 7-10 days; removed by the spleen, which also holds about 1/3 of them. - Regulated by thrombopoietin (TPO, from the liver). - Structure in a smear: a pale blue peripheral zone (hyalomere) and a central purple granular zone (granulomere). - Ultrastructure: - peripheral zone: glycocalyx with receptors (glycoprotein Ib for von Willebrand factor; GP IIb/IIIa for fibrinogen) - structural zone: a marginal band of microtubules keeping the disc shape; actin and myosin (for contraction) - open canalicular system (invaginations of the plasma membrane, increasing surface and exits for release) and the dense tubular system (Ca²⁺ store, like SER) - organelle zone with storage organelles Platelet storage organelles - α-granules (the most numerous): fibrinogen, von Willebrand factor, factor V, platelet-derived growth factor (PDGF), platelet factor 4, fibronectin, P-selectin. - Dense (δ) granules: ADP, ATP, serotonin, Ca²⁺. - Lysosomes (λ granules): hydrolytic enzymes. - Also mitochondria and glycogen. Haemostasis: basic steps 1. Vascular spasm - Immediate constriction of the damaged vessel (by smooth muscle reflexes, local injury, and later by thromboxane A2 and serotonin from platelets, endothelin from endothelium). - Reduces blood loss for minutes; enough for very small vessels. 2. Platelet plug formation (primary haemostasis), in 2 steps: a. Adhesion and activation: - injury exposes subendothelial collagen; von Willebrand factor (vWF) binds collagen and platelet GP Ib, so platelets stick - platelets also bind collagen directly (GP VI) - activation: shape change: the discs become spheres with long pseudopods (spiky), which makes them more adhesive b. Platelet release reaction and aggregation: - activated platelets release granule contents: ADP and thromboxane A2 (made from arachidonic acid by cyclooxygenase) activate more platelets; serotonin constricts vessels - GP IIb/IIIa changes shape and binds fibrinogen, which bridges adjacent platelets: aggregation - positive feedback builds a loose platelet plug within minutes - the activated platelet membrane exposes phosphatidylserine, a surface for clotting reactions - Healthy endothelium prevents platelet adhesion by prostacyclin (PGI2), nitric oxide and ADPase. - Drugs: aspirin irreversibly inhibits COX, blocking thromboxane A2 for the platelet's life (7-10 days); clopidogrel blocks the ADP receptor (P2Y12); GP IIb/IIIa antagonists. 3. Blood clotting (coagulation, secondary haemostasis) - A cascade of clotting factors (mostly proteases made by the liver) that converts soluble fibrinogen into an insoluble mesh of fibrin, which stabilises the platelet plug and traps red cells. - Initiation (extrinsic, tissue factor pathway): damaged tissue exposes tissue factor (factor III), which binds factor VIIa; this activates factor X (and IX). A small amount of thrombin is made. - Amplification: thrombin activates platelets and factors V, VIII and XI. - Propagation (on the platelet surface): the tenase complex (IXa + VIIIa) activates much more factor X; the prothrombinase complex (Xa + Va) converts prothrombin (factor II) into thrombin (IIa) in a large burst. - The intrinsic pathway (contact activation: XII, XI, IX, VIII) is triggered by contact with negatively charged surfaces; it is important in laboratory tests and amplification. - Common pathway: factor X to Xa; prothrombin to thrombin; thrombin cleaves fibrinogen to fibrin monomers, which polymerise; factor XIIIa cross-links the fibrin, making a stable clot. - Ca²⁺ (factor IV) is needed at many steps (this is why citrate or EDTA stop blood clotting in test tubes). - Vitamin K is needed by the liver to make functional factors II, VII, IX and X (and proteins C and S); warfarin blocks vitamin K recycling. - Tests: PT/INR (extrinsic and common pathway; warfarin monitoring), aPTT (intrinsic and common; heparin monitoring). - Natural anticoagulants limit clotting to the site: antithrombin (enhanced by heparin), protein C and S (inactivate Va and VIIIa), tissue factor pathway inhibitor. Clot retraction and repair - Platelets contract (actin and myosin), pulling the edges of the wound together and squeezing serum out of the clot (30-60 minutes). - PDGF stimulates fibroblasts and smooth muscle to repair the vessel. Clot dissolution (fibrinolysis) - Plasminogen, incorporated in the clot, is activated to plasmin by tissue plasminogen activator (tPA, from endothelium) and urokinase. - Plasmin digests fibrin into fibrin degradation products, including D-dimers (a test for thrombosis). - Drugs: thrombolytics (alteplase, a recombinant tPA) dissolve clots in stroke, myocardial infarction and pulmonary embolism; tranexamic acid inhibits fibrinolysis in bleeding. Disorders - Thrombocytopenia: low platelets (below about 150 × 10⁹/L; bleeding risk when below about 20-50): petechiae, purpura, mucosal bleeding. Causes: immune thrombocytopenia, marrow failure, hypersplenism, heparin-induced. - von Willebrand disease: the most common inherited bleeding disorder; defective platelet adhesion. - Haemophilia: X-linked recessive clotting factor deficiencies: - haemophilia A: factor VIII deficiency (about 80%) - haemophilia B (Christmas disease): factor IX deficiency - affects mainly males; causes bleeding into joints (haemarthrosis) and muscles, prolonged bleeding after trauma or surgery; aPTT prolonged - treated with recombinant factor concentrates, emicizumab, and gene therapy - The royal disease: haemophilia B spread through Queen Victoria's descendants (a carrier) to the royal families of Europe, including the Russian Tsarevich Alexei. - Thrombosis: inappropriate clotting in an intact vessel (deep vein thrombosis, arterial thrombosis in heart attack and stroke). Risk factors: Virchow's triad (stasis, endothelial injury, hypercoagulability). Exam focus: describe the three steps of haemostasis, the steps of platelet plug formation with the receptors involved, and the genetics of haemophilia.

1. What are the three main groups of plasma proteins and their functions? 2. What is the difference between plasma and serum? 3. Define haematocrit and give normal values. 4. Why is a red cell biconcave and without a nucleus? 5. What links the RBC membrane to its skeleton, and what disease results from defects? 6. What is the structure of adult haemoglobin? 7. Name four factors that shift the O₂ dissociation curve to the right. 8. How is most CO₂ carried in the blood? 9. What happens to haem when red cells are broken down? 10. Which antibodies does a type A person have? 11. Explain haemolytic disease of the newborn and how it is prevented. 12. Give three causes of anaemia. 13. List the leukocytes in order of frequency, with their percentages. 14. How do you recognise a neutrophil on a smear? 15. How do neutrophils kill bacteria? 16. List the steps of leukocyte recruitment and the molecules involved. 17. What are the functions of eosinophils? 18. What happens to basophils on re-exposure to an allergen? 19. What do monocytes become in the tissues? 20. Name the three types of lymphocytes and their functions. 21. What are the two main types of platelet granules and their contents? 22. List the three steps of haemostasis. 23. What does aspirin do to platelets? 24. What is the role of vitamin K in coagulation? 25. What is haemophilia A, and how is it inherited?

1. Albumin (oncotic pressure, transport), globulins (transport and antibodies), fibrinogen (clotting). 2. Serum is plasma without fibrinogen and clotting factors (the fluid after clotting). 3. The percentage of blood volume made of red cells. About 40-50% in men and 36-46% in women. 4. Biconcavity gives more surface for gas exchange and flexibility; no nucleus or organelles leaves more room for haemoglobin, and the cell cannot use the O₂ it carries. 5. Ankyrin links spectrin to band 3. Hereditary spherocytosis. 6. Two α and two β globin chains, each with a haem containing Fe²⁺. 7. Lower pH, higher CO₂, higher 2,3-BPG, higher temperature. 8. As bicarbonate (about 70%), made by carbonic anhydrase in red cells. 9. Iron is recycled; haem becomes biliverdin, then bilirubin, which is conjugated in the liver and excreted in bile. 10. Anti-B. 11. An Rh-negative mother sensitised by an Rh-positive fetus makes anti-D IgG, which crosses the placenta in a later pregnancy and destroys fetal red cells. Prevented by anti-D immunoglobulin. 12. Any three: blood loss, iron deficiency, B12 or folate deficiency, marrow failure, kidney failure, haemolysis. 13. Neutrophils 50-70%, lymphocytes 20-40%, monocytes 2-8%, eosinophils 1-4%, basophils 0.5-1%. 14. Nucleus with 3-5 lobes; pale cytoplasm with fine lilac granules; 12-15 µm. 15. Phagocytosis, respiratory burst (superoxide, H₂O₂, HOCl by myeloperoxidase), granule enzymes and peptides, NETs. 16. Margination, rolling (selectins), firm adhesion (integrins LFA-1 on ICAM-1), transmigration (PECAM-1), chemotaxis (C5a, IL-8, LTB4). 17. Defence against parasites, modulation of allergic reactions, phagocytosis of immune complexes. 18. The allergen cross-links IgE on their surface, causing release of histamine and other mediators. 19. Macrophages (and some dendritic cells). 20. T cells: cell-mediated immunity. B cells: antibodies via plasma cells. NK cells: innate killing of infected and tumour cells. 21. α-granules (fibrinogen, vWF, factor V, PDGF) and dense granules (ADP, ATP, serotonin, Ca²⁺). 22. Vascular spasm, platelet plug formation, coagulation. 23. It irreversibly inhibits cyclooxygenase, blocking thromboxane A2 for the platelet's lifespan. 24. The liver needs it to make functional factors II, VII, IX and X (and proteins C and S). 25. Factor VIII deficiency; X-linked recessive, so it mostly affects males.

Prenatal phases of haematopoiesis 1. Mesoblastic (yolk sac) phase: from about week 3. Blood islands in the wall of the yolk sac (and in the chorionic villi and connecting stalk) form primitive nucleated erythrocytes with embryonic haemoglobins. 2. Hepatic phase: from about week 5-6. The liver becomes the main site until about month 7; it makes mainly red cells (still some nucleated) with fetal haemoglobin (HbF), plus some granulocytes and platelets. Definitive HSCs arise in the aorta-gonad-mesonephros region and seed the liver. 3. Splenic phase: from about month 3-4, a minor contribution alongside the liver. 4. Medullary (myeloid, bone marrow) phase: from about month 5; the marrow becomes the main site in the last trimester and after birth. - Clinical: in adults, extramedullary haematopoiesis can restart in the liver and spleen when the marrow is replaced or fails (myelofibrosis, thalassaemia), causing hepatosplenomegaly. Bone marrow - Red marrow: active haematopoiesis. In children it fills almost all bones. In adults it is found in flat bones (sternum, ribs, vertebrae, pelvis, skull) and the proximal ends of femur and humerus. - Yellow marrow: fat cells; inactive but can revert to red marrow when demand rises (after haemorrhage). - Structure: - stroma: reticular cells and reticular fibres (type III collagen), adipocytes, macrophages, endothelium; stromal cells make growth factors (SCF, CXCL12) that form the stem cell niche - haematopoietic cords: islands of developing cells between the sinusoids. Erythroblasts form erythroblastic islands around a central macrophage (nurse cell, which supplies iron and eats the extruded nuclei); megakaryocytes lie next to sinusoids - sinusoids: wide, thin-walled capillaries lined by discontinuous endothelium; mature cells cross the wall to enter the blood - Examination: bone marrow aspirate (smear) and trephine biopsy from the posterior iliac crest (or sternum for aspirate in adults). The stem cell hierarchy - Haematopoietic stem cell (HSC): rare (about 1 in 10,000 marrow cells), CD34-positive, self-renewing, multipotent. Mostly quiescent. - Multipotent progenitors give two main branches: - common myeloid progenitor (CMP, CFU-GEMM: granulocyte, erythrocyte, monocyte, megakaryocyte), which gives: - megakaryocyte-erythroid progenitor: CFU-E (erythrocytes) and CFU-Meg (platelets) - granulocyte-monocyte progenitor (CFU-GM): CFU-G (neutrophils), CFU-M (monocytes); separate CFU-Eo and CFU-Ba for eosinophils and basophils - common lymphoid progenitor (CLP, CFU-L): B cells, T cells (after migration to the thymus), NK cells - CFU: colony-forming unit, a progenitor defined by the colonies it forms in culture. CFU-LM refers to a lympho-myeloid multipotent progenitor. - Progenitors cannot self-renew indefinitely; they amplify cell numbers. Precursors (blasts) are the first recognisable cells in a smear. - Growth factors (cytokines): erythropoietin (red cells), thrombopoietin (platelets), G-CSF (neutrophils), M-CSF (monocytes), GM-CSF, IL-3 (multi-lineage), IL-5 (eosinophils), IL-7 (lymphocytes), stem cell factor. - Clinical: - G-CSF (filgrastim) raises neutrophils after chemotherapy and mobilises stem cells into blood for collection - HSC transplantation (bone marrow transplant) treats leukaemias, aplastic anaemia, some genetic diseases - leukaemias are clonal expansions of blocked progenitors (myeloid or lymphoid, acute or chronic) Common characteristics of maturation (for all lineages) - Cell size decreases. - Nucleus becomes smaller and denser (chromatin condenses); nucleoli disappear. In red cells the nucleus is extruded; in granulocytes it becomes lobed. - Cytoplasm: basophilia (ribosomes) decreases as the cell makes its specific products (haemoglobin, granules). - Ability to divide is lost in the late stages. Exam focus: list the prenatal sites of haematopoiesis in order, describe the marrow structure, and draw the stem cell tree.

Stages (in order) 1. Proerythroblast (pronormoblast): large (14-19 µm), round nucleus with fine chromatin and nucleoli, intensely basophilic cytoplasm (ribosomes). Begins making haemoglobin. 2. Basophilic erythroblast (normoblast): smaller (12-16 µm), chromatin coarser, nucleoli gone, very basophilic cytoplasm (ribosomes making globin). 3. Polychromatophilic erythroblast (normoblast): 10-12 µm; cytoplasm grey-blue to lilac (a mixture of blue ribosomes and pink haemoglobin); chromatin in a checkerboard pattern. The last stage that can divide. 4. Orthochromatic erythroblast (normoblast): 8-10 µm; cytoplasm almost pink like a mature RBC; small, dense (pyknotic) nucleus that is extruded (the nucleus is eaten by the macrophage of the erythroblastic island). 5. Reticulocyte: no nucleus, slightly larger than an RBC, remaining ribosomes seen as a network (reticulum) with supravital stains (new methylene blue). Spends 1-2 days in the marrow and about 1 day in blood. Normally about 0.5-2% of red cells. 6. Mature erythrocyte. - Mnemonic: "Pro Baseball Players Obviously Rarely Error" (proerythroblast, basophilic, polychromatophilic, orthochromatic, reticulocyte, erythrocyte). - The earlier progenitors are BFU-E (burst-forming unit) and CFU-E (the main target of EPO). Erythropoietin (EPO) - A glycoprotein hormone made mainly (about 90%) by peritubular interstitial fibroblast-like cells of the renal cortex; about 10% by the liver. - Why the kidney? The kidney is an ideal oxygen sensor: its blood flow is set by filtration needs rather than oxygen demand, so the local O₂ level reflects the O₂-carrying capacity of blood (haemoglobin), not the kidney's own metabolism. - Hypoxia stabilises the transcription factor HIF (hypoxia-inducible factor), which increases EPO gene transcription. - EPO acts on CFU-E and early erythroblasts: prevents their apoptosis and stimulates proliferation and differentiation. - Stimuli: anaemia, blood loss, high altitude, lung disease, heart failure. - Negative feedback: more red cells raise O₂ delivery, reducing EPO. - Clinical: - chronic kidney disease: EPO deficiency causes normocytic anaemia; treated with recombinant EPO - doping with EPO raises haematocrit (risk of thrombosis) - EPO-secreting tumours (renal cell carcinoma) cause secondary polycythaemia Requirements for erythropoiesis - Iron (haem): absorbed in the duodenum, transported by transferrin, stored as ferritin. Deficiency: microcytic hypochromic anaemia. - Vitamin B12 and folate (DNA synthesis): deficiency blocks nuclear maturation while haemoglobin synthesis continues: large megaloblasts in marrow and macrocytes in blood. - Also protein, vitamin B6, copper, vitamin C, and hormones (thyroid, androgens stimulate). - Clinical: the reticulocyte count shows the marrow's response. High after bleeding or haemolysis, or after starting iron or B12 treatment; low in marrow failure or deficiencies. Exam focus: list the stages of erythropoiesis with the change in each, and explain why EPO comes from the kidney.

Granulopoiesis (neutrophils, eosinophils, basophils): about 2 weeks 1. Myeloblast: about 20 µm; large round nucleus with fine chromatin and 2-5 nucleoli; basophilic cytoplasm without granules. 2. Promyelocyte: the largest cell of the series (about 18-24 µm); round or oval nucleus; basophilic cytoplasm with many purple azurophilic (primary) granules (myeloperoxidase). The last stage with nucleoli. 3. Myelocyte: 10-15 µm; eccentric round or oval nucleus without nucleoli; specific (secondary) granules appear, so the three granulocyte types first become distinguishable here (neutrophilic, eosinophilic, basophilic myelocytes). Last stage that can divide. 4. Metamyelocyte: 10-12 µm; kidney-shaped (indented) nucleus; many specific granules; no longer divides. 5. Band (stab) cell: horseshoe or C-shaped nucleus of even width. 6. Mature segmented granulocyte. - Compartments: the mitotic compartment (myeloblast to myelocyte), the maturation compartment (metamyelocyte to band), the marrow storage pool (a large reserve of mature neutrophils, released quickly in infection), and in blood the circulating and marginating pools. - Regulation: G-CSF (neutrophils), IL-5 (eosinophils), IL-3 and GM-CSF. - Clinical: a "left shift" (bands and metamyelocytes in blood) is typical of acute bacterial infection. Chronic myeloid leukaemia shows the whole series in the blood. Neutropenia after chemotherapy raises infection risk. Monocyte maturation (monocytopoiesis) 1. Monoblast: large, basophilic, difficult to distinguish from a myeloblast. 2. Promonocyte: large, with an indented nucleus and fine azurophilic granules. 3. Monocyte: released into the blood after about 2-3 days; circulates 1-3 days and enters tissues to become a macrophage or dendritic cell. - Shares the CFU-GM progenitor with neutrophils; regulated by M-CSF and GM-CSF. Thrombopoiesis (platelets) 1. Megakaryoblast: large (25-50 µm), single nucleus, basophilic cytoplasm without granules. 2. Promegakaryocyte. 3. Megakaryocyte: a giant cell (50-100 µm, the largest cell in the marrow) with a single, large, multilobed polyploid nucleus. The cell replicates DNA without dividing (endomitosis), reaching up to 64N. The cytoplasm fills with granules and demarcation membranes (invaginations of the plasma membrane that outline future platelets). 4. Megakaryocytes lie next to sinusoids and extend long cytoplasmic processes (proplatelets) through the endothelium; the flowing blood fragments them into platelets. Each megakaryocyte releases about 1,000-5,000 platelets. The remaining naked nucleus is phagocytosed. - Regulated by thrombopoietin (TPO), made at a constant rate by the liver. Platelets bind and remove TPO, so fewer platelets mean more free TPO (a neat feedback). - Takes about 5 days. Clinical: TPO receptor agonists treat immune thrombocytopenia. Lymphopoiesis - From the common lymphoid progenitor in the marrow. - B cells: develop in the bone marrow (pro-B, pre-B, immature B cell with surface IgM), undergo negative selection there, then migrate to secondary lymphoid organs, where antigen contact makes them plasma cells and memory cells. - T cells: progenitors leave the marrow and complete development in the thymus (see the thymus note). - NK cells: develop mostly in the marrow. - Lymphocytes are the only white cells that can divide again after they leave the marrow (when activated by antigen). - Lymphoblast: the large activated or immature lymphoid cell. Clinical: acute lymphoblastic leukaemia is the most common childhood cancer. Exam focus: list the stages of granulopoiesis and state where the specific granules appear, and describe how megakaryocytes produce platelets.

1. List the prenatal sites of haematopoiesis in order. 2. Where is red marrow found in adults? 3. What is an erythroblastic island? 4. What two properties define a haematopoietic stem cell? 5. What are the two main branches from the multipotent progenitor? 6. List the general trends of blood cell maturation. 7. List the stages of erythropoiesis. 8. Why does the cytoplasm change from blue to pink during erythropoiesis? 9. What is a reticulocyte and what does its count tell you? 10. Where is EPO made, and what stimulates it? 11. Why does B12 deficiency cause large red cells? 12. List the stages of granulopoiesis. 13. At which stage can you first tell neutrophils, eosinophils and basophils apart? 14. What is a left shift? 15. How are platelets made? 16. How does thrombopoietin regulate platelet counts? 17. Where do B and T cells mature?

1. Yolk sac, liver (with spleen), bone marrow. 2. Flat bones (sternum, ribs, vertebrae, pelvis, skull) and the proximal femur and humerus. 3. A cluster of erythroblasts around a central macrophage that supplies iron and eats extruded nuclei. 4. Self-renewal and multipotency. 5. Common myeloid progenitor and common lymphoid progenitor. 6. Cells get smaller; the nucleus condenses (and is lost or lobed); nucleoli disappear; cytoplasmic basophilia decreases; ability to divide is lost. 7. Proerythroblast, basophilic, polychromatophilic, orthochromatic erythroblast, reticulocyte, erythrocyte. 8. Ribosomes (blue) decrease while haemoglobin (pink) accumulates. 9. A young anucleate red cell with residual ribosomes. Its count shows how actively the marrow is making red cells. 10. Mainly peritubular fibroblasts of the renal cortex. Hypoxia (via HIF). 11. DNA synthesis is slowed, so nuclear maturation lags while cytoplasm keeps growing: megaloblasts. 12. Myeloblast, promyelocyte, myelocyte, metamyelocyte, band, segmented granulocyte. 13. The myelocyte, when specific granules appear. 14. Immature neutrophils (bands, metamyelocytes) in the blood, typical of acute bacterial infection. 15. Polyploid megakaryocytes next to sinusoids extend proplatelets into the blood, which fragment into platelets. 16. Platelets bind and remove TPO; with fewer platelets more free TPO stimulates megakaryocytes. 17. B cells in the bone marrow; T cells in the thymus.

First line: surface barriers - Skin: keratinised epithelium, acidic pH (about 3-5), sebum, dermcidin, defensins. - Mucous membranes: mucus traps microbes; cilia sweep it away (mucociliary escalator); lysozyme in tears and saliva; stomach acid; normal microbiota compete with pathogens. Second line: internal defences 1. Pattern recognition: innate cells detect pathogen-associated molecular patterns (PAMPs, such as LPS, flagellin, viral RNA) with pattern recognition receptors (Toll-like receptors, TLRs). 2. Phagocytes: neutrophils (first to arrive) and macrophages engulf and kill microbes. Opsonins (IgG, C3b) coat microbes and make phagocytosis more efficient. 3. Natural killer (NK) cells: large granular lymphocytes that kill virus-infected and cancer cells that have lost MHC class I ("missing self"). They release perforin (forms pores) and granzymes (trigger apoptosis). They also secrete interferon-γ, which activates macrophages. 4. Inflammation (see below). 5. Antimicrobial proteins: interferons and complement. 6. Fever. Interferons (IFNs) - Type I (IFN-α and IFN-β): secreted by virus-infected cells. They diffuse to neighbouring cells and induce an antiviral state (enzymes that block viral protein synthesis and degrade viral RNA). They also activate NK cells and increase MHC class I. - Type II (IFN-γ): from T cells and NK cells; activates macrophages. - Clinical: IFN-α was used for hepatitis B and C; IFN-β for multiple sclerosis. Complement system - About 20-30 plasma proteins (the main components named C1 to C9, plus factors B, D, and regulatory proteins) made by the liver, circulating inactive. Once activated they form an enzyme cascade. - Three pathways converge on cleaving C3: - classical: C1 binds antigen-antibody complexes (IgG, IgM) - lectin: mannose-binding lectin binds microbial sugars - alternative: C3 binds directly to microbial surfaces - Effects: 1. opsonisation: C3b coats microbes, enhancing phagocytosis 2. inflammation: C3a and C5a (anaphylatoxins) cause mast cell degranulation, vasodilation; C5a attracts neutrophils (chemotaxis) 3. lysis: C5b-C9 forms the membrane attack complex (MAC), a pore that lyses bacteria (especially Neisseria) 4. clearance of immune complexes - Regulation protects host cells (CD55/DAF, CD59, C1 inhibitor). Clinical: C1 inhibitor deficiency causes hereditary angioedema; deficiency of C5-C9 increases Neisseria infections. Inflammatory response - Triggered by tissue injury or infection; signs: redness, heat, swelling, pain, and loss of function. - Steps: release of mediators (histamine, kinins, prostaglandins, complement, cytokines); vasodilation and increased permeability (exudate brings proteins and clotting factors); phagocyte mobilisation (margination, diapedesis, chemotaxis); removal of debris and repair. Fever - An abnormally high body temperature, a systemic response to infection. - Pyrogens: exogenous (LPS) and endogenous cytokines (IL-1, IL-6, TNF-α) from macrophages act on the hypothalamus, where prostaglandin E2 raises the thermostat set point. - Benefits: moderate fever makes the liver and spleen sequester iron and zinc (needed by bacteria) and speeds up tissue repair and immune reactions. Very high fever (above about 41 °C) denatures proteins and is dangerous. - Antipyretics (paracetamol, NSAIDs) block prostaglandin synthesis. Exam focus: explain the actions of interferons and the three main effects of complement, and describe how fever is produced.

B cells - Develop in the bone marrow. Each B cell makes one kind of B-cell receptor (BCR): a membrane-bound antibody (IgM and IgD) specific for one antigen, generated by random gene rearrangement (V(D)J recombination). - B cells that bind self-antigen strongly are removed (negative selection, tolerance). - Mature naive B cells circulate and settle in the follicles of secondary lymphoid organs. Activation of B cells (clonal selection) 1. Antigen binds the BCR and is internalised, processed and presented on MHC class II. 2. A helper T cell (CD4) that recognises the same antigen binds and gives signals (CD40L-CD40 and cytokines). 3. The B cell proliferates, forming a clone; in the germinal centres of lymphoid follicles the clone undergoes: - somatic hypermutation and affinity maturation (antibodies bind more tightly) - class switching (from IgM to IgG, IgA or IgE) 4. The clone gives: - plasma cells: antibody factories (secrete about 2,000 molecules per second), live days (or years in the marrow) - memory B cells: long-lived, respond quickly to re-exposure Antibody structure - Immunoglobulins: Y-shaped glycoproteins of two identical heavy chains and two identical light chains (κ or λ) joined by disulfide bonds. - Variable regions (at the tips of the arms): the antigen-binding sites (Fab region). Each antibody has two binding sites. - Constant region of the heavy chain (Fc, the stem): determines the class and the effector functions (binding complement, Fc receptors on phagocytes and mast cells). Classes of antibodies (mnemonic: GAMED) - IgG: monomer; about 75-80% of antibodies in plasma; the main antibody of the secondary response; crosses the placenta (passive immunity for the newborn); activates complement; opsonises. Longest half-life (about 3 weeks). - IgA: monomer in plasma; a dimer (secretory IgA, with a J chain and secretory component) in secretions: saliva, tears, mucus, breast milk (colostrum). Protects mucous membranes by neutralising microbes before they attach. - IgM: pentamer (10 binding sites); the first antibody made in a primary response (a marker of recent infection); very good at agglutination and complement activation. Monomeric IgM is a BCR. Too big to cross the placenta. ABO antibodies are IgM. - IgD: monomer; mainly a BCR on naive B cells; function in plasma unclear. - IgE: monomer; very low levels in plasma; binds Fc receptors on mast cells and basophils; triggers the release of histamine in allergies (to food allergens such as peanuts, pollen); raised in parasitic infections. Primary and secondary responses - Primary response (first exposure): a lag of about 3-6 days before antibodies appear; peak at about 10-14 days; mostly IgM, then some IgG; low levels. - Secondary response (re-exposure): memory cells respond within hours to 2-3 days; much higher levels (100-1000 times), mainly IgG with higher affinity; lasts longer. - This is the basis of vaccination and booster doses. - Clinical: IgM against a pathogen suggests acute (current) infection; IgG alone suggests past infection or vaccination. Mechanisms of antibody action (antibodies do not destroy antigens themselves; they mark them) 1. Neutralisation: blocking the sites of viruses or toxins that bind to cells. 2. Agglutination: cross-linking particulate antigens (cells) into clumps (the basis of blood typing). 3. Precipitation: cross-linking soluble antigens into insoluble complexes cleared by phagocytes. 4. Complement activation (classical pathway), especially by IgM and IgG, leading to lysis and inflammation. 5. Opsonisation: coating antigens for phagocytosis (via Fc receptors). 6. Antibody-dependent cell-mediated cytotoxicity (ADCC): NK cells kill antibody-coated cells. - Mnemonic: "PLAN" (Precipitation, Lysis via complement, Agglutination, Neutralisation), plus opsonisation. Active and passive immunity - Active: the body makes its own antibodies. Natural (infection) or artificial (vaccine). Memory, long-lasting. - Passive: antibodies are received. Natural (IgG across the placenta, IgA in milk) or artificial (antiserum, immunoglobulin, monoclonal antibodies). Immediate but short-lived, no memory. Exam focus: describe the structure and function of each antibody class, and compare primary and secondary responses.

MHC (major histocompatibility complex) - In humans called HLA (human leukocyte antigens), coded on chromosome 6. Extremely polymorphic: each person has a different set (the reason for transplant rejection and the need for matching). - MHC class I: - expressed on all nucleated cells (not on red cells) - presents peptides from inside the cell (endogenous antigens: viral or tumour proteins made in the cytoplasm) - recognised by CD8 cytotoxic T cells - MHC class II: - expressed only on professional antigen-presenting cells (dendritic cells, macrophages, B cells) and thymic epithelium - presents peptides from outside the cell (exogenous antigens taken up by endocytosis or phagocytosis) - recognised by CD4 helper T cells - Rule of 8: CD4 × MHC II = 8; CD8 × MHC I = 8. Antigen-presenting cells (APCs) - Dendritic cells: the most effective APCs. They capture antigens in tissues (Langerhans cells of the epidermis), migrate through lymphatics to lymph nodes and activate naive T cells. - Macrophages: present to already activated T cells and are activated by them in return. - B cells: present the antigen they bound through their BCR to helper T cells. Antigen processing pathways - Cytosolic (endogenous, MHC I) pathway: 1. cytoplasmic proteins (e.g. viral) are tagged with ubiquitin and cut into peptides by the proteasome 2. peptides are carried into the RER by the TAP transporter 3. they load onto MHC class I molecules in the RER 4. the complex goes through the Golgi to the cell surface, presenting to CD8 T cells - Endocytic (exogenous, MHC II) pathway: 1. extracellular antigens are taken up by phagocytosis or endocytosis 2. degraded by lysosomal proteases in endosomes into peptides 3. MHC class II molecules, made in the RER with an invariant chain blocking the groove, travel to the endosome, where the invariant chain is removed and the peptide loads 4. the complex reaches the surface, presenting to CD4 T cells - Cross-presentation: dendritic cells can present exogenous antigens on MHC I, needed to activate CD8 cells against viruses that do not infect dendritic cells. T cell activation - Two signals are needed: 1. antigen recognition: the T-cell receptor (TCR) binds the peptide-MHC complex; CD4 or CD8 binds MHC as a co-receptor 2. co-stimulation: CD28 on the T cell binds B7 (CD80/86) on the APC - Without co-stimulation the T cell becomes unresponsive (anergy), protecting against self-reactivity. - Cytokines (IL-2) drive proliferation (clonal expansion) into effector and memory T cells. - Clinical: checkpoint inhibitors block inhibitory receptors (CTLA-4, PD-1) to unleash T cells against cancer. Types of T cells - Helper T cells (CD4⁺, TH): the "managers" of the immune response. They release cytokines that: - activate B cells (and allow class switching) - activate macrophages (IFN-γ) - help CD8 T cells - subsets: TH1 (intracellular microbes, macrophage activation), TH2 (parasites, IgE, allergy), TH17 (extracellular bacteria and fungi, neutrophils), TFH (follicular help to B cells) - Cytotoxic T cells (CD8⁺, TC): the only T cells that kill directly. They recognise antigen on MHC class I of virus-infected, tumour or foreign (graft) cells, and kill them by: - perforin and granzymes (inducing apoptosis) - Fas ligand binding Fas on the target cell - Regulatory T cells (Treg; CD4⁺CD25⁺, FOXP3⁺): suppress immune responses (IL-10, TGF-β) and prevent autoimmunity. Clinical: FOXP3 mutation causes IPEX syndrome. - Memory T cells: long-lived, respond quickly to re-exposure. CD4 versus CD8: summary - CD4: helper; recognises MHC II; on APCs; coordinates. Target of HIV. - CD8: cytotoxic; recognises MHC I; on all nucleated cells; kills. Exam focus: compare the MHC class I and class II pathways and explain the two-signal model of T cell activation.

Hypersensitivity (Gell and Coombs classification) - Type I (immediate, IgE-mediated): - first exposure sensitises (IgE binds mast cells and basophils); re-exposure cross-links IgE and triggers degranulation within minutes - examples: hay fever (allergic rhinitis), asthma, urticaria, food allergy, anaphylaxis (systemic vasodilation, bronchospasm, shock; treated with intramuscular adrenaline) - Type II (antibody-mediated, cytotoxic): IgG or IgM against antigens on cells or tissues cause lysis or dysfunction. - examples: transfusion reactions, haemolytic disease of the newborn, autoimmune haemolytic anaemia, Goodpasture syndrome, Graves disease and myasthenia gravis (antibodies against receptors) - Type III (immune complex-mediated): antigen-antibody complexes deposit in vessel walls, kidneys and joints, activate complement and cause inflammation. - examples: systemic lupus erythematosus, post-streptococcal glomerulonephritis, serum sickness, Arthus reaction - Type IV (delayed, T-cell-mediated): takes 1-3 days; mediated by TH1 cells and macrophages or by CD8 cells. - examples: tuberculin (Mantoux) skin test, contact dermatitis (nickel, poison ivy), type 1 diabetes, transplant rejection - Mnemonic: "ACID" (Anaphylactic, Cytotoxic, Immune complex, Delayed). Autoimmunity - Loss of self-tolerance: the immune system attacks self-antigens. - Mechanisms: failure of central tolerance (thymus, marrow) or peripheral tolerance (Tregs, anergy); molecular mimicry (microbial antigens resembling self, such as streptococcal M protein and heart valves in rheumatic fever); release of hidden antigens; genetic factors (HLA types, e.g. HLA-B27 with ankylosing spondylitis); more common in women. - Examples: type 1 diabetes, multiple sclerosis, rheumatoid arthritis, SLE, Hashimoto thyroiditis, Graves disease, coeliac disease, myasthenia gravis. Immunodeficiency - Primary (congenital, genetic): - severe combined immunodeficiency (SCID): absent T and B function (e.g. X-linked IL-2 receptor γ-chain or ADA deficiency). Fatal without bone marrow transplant or gene therapy. - X-linked agammaglobulinaemia (Bruton): no B cells, no antibodies - DiGeorge syndrome: thymic hypoplasia (22q11 deletion), few T cells - selective IgA deficiency: the most common - complement and phagocyte defects (chronic granulomatous disease) - Secondary (acquired): - HIV/AIDS: HIV infects CD4 T cells (binding CD4 and CCR5 or CXCR4) and destroys them; when CD4 cells fall below 200/µL, opportunistic infections (Pneumocystis pneumonia, Candida, CMV, toxoplasmosis) and cancers (Kaposi sarcoma, lymphoma) appear. Antiretroviral therapy controls it. - malnutrition, chemotherapy, immunosuppressive drugs (transplants), corticosteroids, splenectomy (risk of encapsulated bacteria), ageing, diabetes, cancers of the immune system (leukaemia, lymphoma) Exam focus: give the mechanism and two examples of each type of hypersensitivity, and explain how HIV causes immunodeficiency.

Functions of the lymphatic system 1. Return of fluid and proteins: about 3 L/day of interstitial fluid that is not reabsorbed by capillaries. 2. Absorption of dietary fats via lacteals in the small intestine villi (chyle). 3. Immune surveillance: lymph passes through lymph nodes, where antigens are detected. Lymphatic vessels - Lymphatic capillaries: blind-ended; larger and more irregular than blood capillaries; a single layer of overlapping endothelial cells with no continuous basement membrane; anchored to surrounding tissue by anchoring filaments. When interstitial pressure rises, the filaments pull the overlapping edges apart (flap valves), letting fluid, proteins, cells and even bacteria in. - Absent from: bone marrow, epidermis, cartilage, cornea, and (for classic vessels) the CNS, which has meningeal lymphatics and a glymphatic system. - Collecting vessels: like thin veins with three thin layers and many valves (a beaded appearance when full). - Trunks and ducts: the thoracic duct drains most of the body (the whole lower half and the left side of the upper half) into the left venous angle (junction of left internal jugular and subclavian veins); the right lymphatic duct drains the right upper quadrant into the right venous angle. The cisterna chyli is a sac at the start of the thoracic duct. - Lymph flow: low pressure; driven by skeletal muscle pump, pressure changes in breathing, rhythmic contraction of smooth muscle in the vessel walls, and valves that prevent backflow. Lymphoedema - Swelling due to impaired lymph drainage, rich in protein, leading to fibrosis. - Primary (congenital lymphatic abnormalities, e.g. Milroy disease) or secondary: surgical removal of lymph nodes (axillary clearance in breast cancer), radiotherapy, tumours, infection (filariasis, Wuchereria bancrofti, causing elephantiasis). - Treatment: compression, manual lymphatic drainage, elevation, exercise. Primary lymphoid organs Bone marrow - Site of B-cell maturation (and origin of all lymphocyte precursors). - B-cell maturation stages: common lymphoid progenitor, pro-B cell (heavy chain gene rearrangement), pre-B cell (heavy chain with surrogate light chain, the pre-BCR), immature B cell (complete IgM BCR; tested for self-reactivity: strongly self-reactive cells are deleted or edit their receptor), mature naive B cell (IgM and IgD) leaving for the periphery. Thymus - Located in the anterior mediastinum, behind the sternum. Large in children, it reaches its maximum weight at puberty and then involutes (replaced by fat), although it still functions at a low level in adults. - Develops from the endoderm of the third pharyngeal pouch (epithelium), populated by T-cell precursors from the marrow. - Structure: - a connective tissue capsule sends septa (trabeculae) into the organ, dividing it into incomplete lobules - each lobule has a dark outer cortex (densely packed T cells, thymocytes) and a pale inner medulla (fewer thymocytes, more epithelial cells). The medulla is continuous between lobules. - the framework is not reticular fibres but a network of thymic epithelial reticular cells (joined by desmosomes), which make thymic hormones (thymosin, thymulin, thymopoietin) - Hassall's (thymic) corpuscles in the medulla: concentric whorls of flattened, keratinising epithelial cells; they produce cytokines (TSLP) that help Treg development. They are the key identifying feature of the thymus. - no lymphoid follicles (unlike other lymphoid organs) - Blood-thymus barrier in the cortex: continuous endothelium with a thick basal lamina, a sheath of pericytes and macrophages, and epithelial reticular cells. It keeps circulating antigens away from developing T cells. - T-cell selection (thymic education): 1. precursors enter at the corticomedullary junction and migrate to the outer cortex; they are double negative (CD4⁻CD8⁻) 2. they rearrange their TCR genes and become double positive (CD4⁺CD8⁺) 3. positive selection (cortex): cortical epithelial cells present self-MHC; thymocytes whose TCR can bind self-MHC survive; those that cannot die by neglect. Survivors become single positive: CD4 if they bound MHC II, CD8 if MHC I (MHC restriction). 4. negative selection (medulla): medullary epithelial cells (which express many tissue antigens under the control of AIRE) and dendritic cells present self-antigens; thymocytes that bind strongly are deleted by apoptosis (central tolerance) or become Tregs 5. about 95-98% of thymocytes die; macrophages clear them (tingible body macrophages) 6. mature naive T cells leave via medullary venules - Clinical: DiGeorge syndrome (thymic aplasia); AIRE mutation causes autoimmune polyendocrine syndrome type 1; thymoma associated with myasthenia gravis. Exam focus: describe the structure of lymphatic capillaries, the thymic cortex versus medulla, and the steps of T-cell selection.

Lymphoid follicles (nodules) - Primary follicle: a dense, round cluster of naive B cells (and follicular dendritic cells), without a germinal centre. - Secondary follicle: after antigen stimulation, a pale germinal centre develops, surrounded by a mantle zone of small dark naive B cells. - Germinal centre: proliferating B cells undergo somatic hypermutation, affinity maturation and class switching. It has a dark zone (proliferating centroblasts) and a light zone (centrocytes selected by follicular dendritic cells and TFH cells). Tingible body macrophages eat apoptotic B cells. Lymph node - Small bean-shaped organs (1-25 mm) along lymphatic vessels, clustered in the neck, axilla, groin, mediastinum and mesentery. - Structure: - capsule of dense connective tissue with trabeculae; stroma of reticular fibres and reticular cells - afferent lymphatic vessels (several) enter on the convex surface; efferent vessels (one or two) leave at the hilum, where the artery enters and the vein leaves - sinuses (lined by endothelium with macrophages): subcapsular sinus, then trabecular (cortical) sinuses, then medullary sinuses to the efferent vessel - outer cortex: B-cell follicles (primary and secondary) - deep cortex (paracortex): T cells and dendritic cells. Contains high endothelial venules (HEVs), with cuboidal endothelium, through which blood lymphocytes enter the node. The thymus-dependent zone (depleted in DiGeorge syndrome). - medulla: medullary cords (plasma cells, macrophages, B cells) and medullary sinuses - Functions: filtering lymph (macrophages remove microbes and particles); antigen presentation; activation and proliferation of lymphocytes; antibody production. - Clinical: - lymphadenopathy: reactive enlargement in infection (tender) versus metastatic cancer or lymphoma (hard, painless, fixed) - sentinel lymph node: the first node draining a tumour; biopsied to stage breast cancer and melanoma - cancers spread along lymphatic drainage (Virchow's node in the left supraclavicular fossa suggests abdominal cancer) Spleen - The largest lymphoid organ, in the left upper quadrant (under ribs 9-11), about 150 g; filters blood, not lymph. No afferent lymphatics. - Capsule of dense connective tissue (with some smooth muscle) and trabeculae carrying trabecular arteries and veins. - Parenchyma (pulp): - white pulp (about 20%): lymphoid tissue around the arteries - periarteriolar lymphoid sheath (PALS): T cells surrounding the central arteriole - lymphoid follicles (splenic nodules, Malpighian corpuscles) of B cells, often with germinal centres, with an eccentric central arteriole - marginal zone: between white and red pulp; macrophages and marginal zone B cells that respond to blood-borne antigens, especially polysaccharides of encapsulated bacteria - red pulp (about 75-80%): filters and destroys old red cells - splenic cords (of Billroth): reticular tissue with macrophages, red cells, platelets, leukocytes, plasma cells - splenic sinusoids: wide vessels lined by elongated stave cells with slits between them and a discontinuous basement membrane (like barrel hoops) - Circulation: splenic artery, trabecular arteries, central arterioles (in white pulp), penicillar arterioles, then either: - open circulation (most in humans): blood enters the cords and must squeeze through slits between stave cells into sinusoids; old, rigid or abnormal red cells cannot and are eaten by macrophages - closed circulation: directly into sinusoids - Functions: 1. immune response to blood-borne antigens 2. removal of old red cells and platelets (culling), removing inclusions from red cells (pitting), iron recycling 3. reservoir of platelets (about 1/3) and monocytes 4. haematopoiesis in the fetus (and in adults with marrow failure) - Clinical: - splenomegaly: portal hypertension, haemolytic anaemias, infections (malaria, mononucleosis), leukaemias and lymphomas - rupture after trauma (a common abdominal emergency) - after splenectomy: Howell-Jolly bodies in red cells and a lifelong risk of overwhelming sepsis by encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis). Patients are vaccinated. MALT (mucosa-associated lymphoid tissue) - Unencapsulated lymphoid tissue in the mucosa (lamina propria) of the digestive, respiratory, urinary and reproductive tracts. It guards the mucosal surfaces where most antigens enter. Plasma cells here secrete IgA. - Forms: - diffuse lymphocytes and plasma cells in the lamina propria - solitary lymphoid follicles - aggregated follicles: Peyer's patches in the ileum (GALT). Covered by a dome epithelium with M cells that transport antigens from the lumen to the lymphoid tissue beneath. - the appendix (lymphoid follicles filling the mucosa and submucosa) - BALT (bronchus-associated) in the airways - Clinical: MALT lymphoma of the stomach, linked to Helicobacter pylori (can regress after eradication). Tonsils - Partially encapsulated aggregates of lymphoid follicles in the mucosa of the pharynx: they form Waldeyer's ring at the entrance to the digestive and respiratory tracts. - Palatine tonsils (paired, between the palatoglossal and palatopharyngeal arches): - covered by stratified squamous non-keratinised epithelium - 10-20 deep invaginations called crypts, lined by the same epithelium (often infiltrated by lymphocytes), trap bacteria and food debris - many follicles with germinal centres along the crypts - a dense connective tissue capsule on the deep side only - Pharyngeal tonsil (adenoid; single, on the posterior wall of the nasopharynx): covered by pseudostratified ciliated columnar epithelium; no crypts but folds; partial capsule. Enlarged adenoids cause mouth breathing and middle ear effusion in children. - Lingual tonsils (at the base of the tongue): stratified squamous epithelium, one crypt each. - Clinical: tonsillitis (streptococcal pharyngitis); peritonsillar abscess (quinsy). Exam focus: draw a lymph node and label the B- and T-cell zones and the sinuses; describe the white and red pulp of the spleen; distinguish the palatine tonsil from the thymus on a slide.

1. Give three differences between innate and adaptive immunity. 2. How do NK cells recognise target cells? 3. What do type I interferons do? 4. Name the three pathways and three effects of complement. 5. How is fever produced, and what are its benefits? 6. Describe the structure of an antibody. 7. Which antibody crosses the placenta? Which is found in secretions? Which is made first? 8. Compare the primary and secondary antibody responses. 9. List four mechanisms of antibody action. 10. Which cells express MHC class I and class II? 11. Describe the MHC class I antigen processing pathway. 12. What two signals does a T cell need to be activated? 13. How do cytotoxic T cells kill? 14. Give an example of each type of hypersensitivity. 15. Why does HIV cause opportunistic infections? 16. How is a lymphatic capillary adapted to take up fluid? 17. Where does the thoracic duct drain? 18. What are Hassall's corpuscles? 19. Describe positive and negative selection in the thymus. 20. Where are the B-cell and T-cell zones of a lymph node? 21. What are high endothelial venules? 22. What is the difference between white and red pulp? 23. Why are splenectomy patients vaccinated against encapsulated bacteria? 24. What are M cells? 25. How do you recognise a palatine tonsil?

1. Innate: from birth, non-specific (patterns), fast, no memory. Adaptive: acquired, specific (antigen receptors), slower first time, memory. 2. By the loss of MHC class I ("missing self") and by stress ligands; also antibody-coated cells (ADCC). 3. Induce an antiviral state in neighbouring cells, activate NK cells, increase MHC I. 4. Classical, lectin, alternative. Opsonisation (C3b), inflammation and chemotaxis (C3a, C5a), lysis by the membrane attack complex (C5b-C9). 5. IL-1, IL-6 and TNF-α act on the hypothalamus via PGE2, raising the set point. Benefits: iron and zinc sequestration, faster immune reactions and repair. 6. Two heavy and two light chains joined by disulfide bonds; variable regions form two antigen-binding sites (Fab), the constant region (Fc) sets class and effector functions. 7. IgG. IgA. IgM. 8. Primary: lag of days, mainly IgM, low levels. Secondary: fast, much higher, mainly high-affinity IgG, longer-lasting. 9. Any four: neutralisation, agglutination, precipitation, complement activation, opsonisation, ADCC. 10. MHC I: all nucleated cells. MHC II: antigen-presenting cells (dendritic cells, macrophages, B cells) and thymic epithelium. 11. Cytosolic proteins are degraded by the proteasome, peptides enter the RER via TAP, load onto MHC I, and the complex goes to the surface for CD8 T cells. 12. Signal 1: TCR binding peptide-MHC. Signal 2: co-stimulation (CD28 binding B7). 13. Perforin and granzymes, and Fas ligand, which trigger apoptosis. 14. Type I: anaphylaxis or hay fever. Type II: transfusion reaction. Type III: SLE. Type IV: contact dermatitis or tuberculin test. 15. It destroys CD4 helper T cells, which coordinate both humoral and cellular immunity. 16. Overlapping endothelial cells act as flap valves, no continuous basement membrane, anchoring filaments pull them open when tissue pressure rises. 17. The left venous angle (junction of left internal jugular and subclavian veins). 18. Concentric whorls of keratinising epithelial cells in the thymic medulla; they identify the thymus. 19. Positive selection (cortex): thymocytes that bind self-MHC survive. Negative selection (medulla): those that bind self-antigen strongly are deleted. 20. B cells in the follicles of the outer cortex; T cells in the paracortex (deep cortex). 21. Venules with cuboidal endothelium in the paracortex through which blood lymphocytes enter the node. 22. White pulp: lymphoid tissue (PALS and follicles) around central arterioles. Red pulp: cords and sinusoids that filter and remove old red cells. 23. The spleen's marginal zone is key to clearing encapsulated bacteria from blood; without it, the risk of overwhelming sepsis rises. 24. Specialised epithelial cells over Peyer's patches that transport antigens from the gut lumen to the lymphoid tissue. 25. Stratified squamous epithelium dipping into deep crypts, with many lymphoid follicles along them and a capsule on one side.

Glial cells of the CNS 1. Astrocytes - The most abundant and largest glial cells; star-shaped with many processes; contain glial fibrillary acidic protein (GFAP, an intermediate filament used as a marker in immunohistochemistry). - Types: protoplasmic (grey matter, short thick branched processes) and fibrous (white matter, long thin processes). - Functions: - structural support and scaffold for neurons - their end-feet wrap brain capillaries and induce the blood-brain barrier (BBB) - take up K⁺ and neurotransmitters (glutamate) from synapses, recycling glutamate into glutamine - store glycogen and supply lactate to neurons - form the glia limitans under the pia mater - are part of the tripartite synapse (modulate synaptic transmission) - react to injury (reactive astrocytosis, gliosis) and form glial scars - Blood-brain barrier: formed mainly by continuous capillary endothelial cells joined by tight junctions (with few vesicles), plus the basement membrane, pericytes and astrocyte end-feet. Lets through water, gases and lipid-soluble substances; glucose and amino acids need transporters. Protects the brain but blocks many drugs (a challenge in treating brain diseases). Absent in circumventricular organs (such as the area postrema, which detects toxins and triggers vomiting). 2. Oligodendrocytes - Small cells with few processes. Each cell myelinates segments of several (up to 50) axons in the CNS. - Also form satellite cells around neuron cell bodies in grey matter. - Clinical: destroyed in multiple sclerosis. 3. Microglia - Small, elongated cells with a dense elongated nucleus and short spiny processes. - The resident macrophages of the CNS, derived from yolk-sac precursors (mesodermal origin, not neuroectoderm). - Constantly survey the tissue; when activated they become phagocytic, remove debris and dead cells, prune synapses, and present antigens. - Clinical: HIV infects microglia (HIV-associated neurocognitive disorder); involved in Alzheimer disease. 4. Ependymal cells - Cuboidal to columnar cells with cilia and microvilli, lining the ventricles of the brain and the central canal of the spinal cord. No basement membrane; joined by gap junctions and desmosomes. - Cilia help circulate cerebrospinal fluid (CSF). - Specialised ependymal cells with capillaries form the choroid plexus, which secretes CSF (about 500 mL/day). - Tanycytes: specialised ependymal cells in the third ventricle floor. Glial cells of the PNS 1. Schwann cells (neurolemmocytes): form myelin around a single segment of one axon in the PNS; also surround unmyelinated axons (several per Schwann cell); essential for regeneration of peripheral nerves. 2. Satellite cells: flattened cells surrounding neuron cell bodies in ganglia; regulate the chemical environment (like astrocytes). Clinical - Gliomas: tumours of glial cells (glioblastoma, the most aggressive, astrocytic; oligodendroglioma; ependymoma). Mature neurons rarely give tumours. - Schwannoma (acoustic neuroma of cranial nerve VIII); neurofibromatosis. Exam focus: list the four CNS glial cell types with their functions, and describe the blood-brain barrier.

Parts of a neuron 1. Cell body (soma, perikaryon) - The metabolic centre; 5-150 µm. - Large, round, pale (euchromatic) nucleus with a prominent nucleolus ("owl's eye"): high transcription activity. - Nissl bodies (chromatophilic substance): clumps of RER and free ribosomes, strongly basophilic, seen with Nissl stains (cresyl violet, toluidine blue). Make proteins for the whole neuron. They are present in the soma and dendrites but absent from the axon hillock and axon. After axonal injury they disperse (chromatolysis). - Well-developed Golgi, many mitochondria, lysosomes. - Lipofuscin: brown "wear and tear" pigment (residual bodies) that accumulates with age. Neuromelanin in the substantia nigra (lost in Parkinson disease). - Cytoskeleton: - neurofilaments: the intermediate filaments of neurons, forming neurofibrils on silver staining; maintain axon calibre - microtubules: tracks for axonal transport; stabilised by the microtubule-associated protein tau in the axon (MAP2 in dendrites) - microfilaments (actin): in growth cones and dendritic spines - clinical: hyperphosphorylated tau forms neurofibrillary tangles in Alzheimer disease 2. Dendrites - Short, tapering, highly branched processes that receive signals; they contain Nissl bodies and conduct graded potentials toward the soma. - Dendritic spines: small protrusions that are the sites of excitatory synapses; their number and shape change with learning (synaptic plasticity). Reduced in some intellectual disabilities. 3. Axon - A single, long process (up to 1 m, from spinal cord to foot) of uniform diameter that conducts action potentials away from the soma. - Axon hillock: the cone-shaped region where the axon leaves the soma; lacks Nissl bodies. - Initial segment: the trigger zone, rich in voltage-gated Na⁺ channels, where action potentials start. - The axon may give branches (collaterals) and ends in many terminal branches (telodendria) with axon terminals (synaptic boutons, knobs). - Axolemma (membrane) and axoplasm; no ribosomes, so all proteins come from the soma. Axonal transport - Anterograde (from soma to terminal): - fast: about 100-400 mm/day; carries membrane-bound vesicles and organelles (synaptic vesicle components, mitochondria) along microtubules by the motor protein kinesin (moving to the plus end) - slow: about 0.2-8 mm/day; carries cytoskeleton proteins and soluble enzymes - Retrograde (from terminal to soma): about 100-200 mm/day; by dynein (to the minus end); returns worn-out components for degradation, and carries signals from the target (nerve growth factor) to the nucleus. - Clinical: some pathogens and toxins travel retrogradely to reach the CNS: rabies virus, herpes simplex and varicella-zoster (latent in ganglia), tetanus toxin, poliovirus. Axonal transport is also used for neural tract tracing in research. Vincristine (a microtubule inhibitor) causes peripheral neuropathy. Classification of neurons - Structural (by number of processes from the soma): 1. multipolar: one axon and many dendrites. The most common type: motor neurons and interneurons in the CNS, autonomic ganglion cells, pyramidal and Purkinje cells. 2. bipolar: one axon and one dendrite at opposite poles. Rare: retina, olfactory epithelium, inner ear (spiral and vestibular ganglia). 3. unipolar (pseudounipolar): one short process that splits into a peripheral branch (from the receptor) and a central branch (to the CNS); the soma is off to one side. Sensory neurons of dorsal root ganglia and cranial nerve ganglia. The peripheral branch functions and is structured as an axon. - anaxonic neurons (no clear axon): some in the retina and brain - Functional: 1. sensory (afferent) neurons: carry impulses from receptors to the CNS; mostly unipolar 2. motor (efferent) neurons: carry impulses from the CNS to muscles and glands; multipolar 3. interneurons (association neurons): between sensory and motor neurons within the CNS; about 99% of all neurons; multipolar - Also by size (Golgi type I, long axons; Golgi type II, short axons). Exam focus: describe Nissl bodies and where they are absent, list the types of axonal transport with their motors and speeds, and classify neurons structurally and functionally.

Resting membrane potential (RMP) - About -70 mV in neurons (inside negative); -90 mV in skeletal muscle. - Caused by: 1. unequal distribution of ions: more K⁺ inside; more Na⁺ and Cl⁻ outside; large negative proteins trapped inside 2. different permeability: at rest the membrane is much more permeable to K⁺ (leak channels) than to Na⁺, so K⁺ diffuses out along its concentration gradient, leaving the inside negative, until the electrical gradient balances it. The RMP is close to the K⁺ equilibrium potential (about -90 mV). 3. the Na⁺/K⁺-ATPase pumps 3 Na⁺ out and 2 K⁺ in, maintaining the gradients (and contributing a few mV directly). - Clinical: hyperkalaemia depolarises cells (dangerous arrhythmias), hypokalaemia hyperpolarises them. Graded potentials - Small, local changes in membrane potential (at dendrites and soma, at receptors) whose size depends on the stimulus strength. - Depolarisation (less negative) or hyperpolarisation (more negative). They fade with distance (decremental) and can be summed (temporal and spatial summation). - If the sum reaches threshold (about -55 mV) at the axon initial segment, an action potential starts. Action potential (AP) 1. Resting state: voltage-gated Na⁺ and K⁺ channels closed. 2. Depolarisation: at threshold, voltage-gated Na⁺ channels open quickly; Na⁺ rushes in (positive feedback: more depolarisation opens more channels); the membrane potential rises to about +30 to +40 mV (overshoot). 3. Repolarisation: Na⁺ channels inactivate (within about 1 ms); slower voltage-gated K⁺ channels open, and K⁺ flows out, restoring the negative potential. 4. Hyperpolarisation (after-hyperpolarisation, undershoot): K⁺ channels close slowly, so the potential dips below rest briefly. 5. The Na⁺/K⁺ pump and leak channels restore the resting state (few ions actually move, so the gradients hardly change during one AP). - Duration about 1-2 ms in neurons. All-or-none principle - A stimulus below threshold gives no AP; any stimulus at or above threshold gives a full-size AP. Its amplitude does not depend on the stimulus strength. - Stimulus intensity is coded by the frequency of APs (and by the number of neurons recruited), not by their size. Refractory periods - Absolute refractory period: from the opening of Na⁺ channels until they are reset (inactivated); no stimulus can trigger a new AP. It limits the maximum firing frequency and ensures one-way propagation. - Relative refractory period: during hyperpolarisation; only a stronger than normal stimulus triggers an AP. Propagation - The AP at one segment depolarises the next segment to threshold (local currents), and so on along the axon; it does not decrease with distance. The segment behind is refractory, so the AP travels in one direction. - Conduction velocity increases with axon diameter (less resistance) and with myelination (saltatory conduction; see the nerve fibre note). Clinical - Local anaesthetics (lidocaine, bupivacaine) block voltage-gated Na⁺ channels from inside the axon, stopping AP generation and conduction; small, unmyelinated pain fibres (C fibres) are blocked first, so pain goes before touch and motor function. - Tetrodotoxin (puffer fish) and saxitoxin block Na⁺ channels. - Some antiepileptic drugs (phenytoin, carbamazepine) stabilise Na⁺ channels in the inactivated state. - Class I antiarrhythmics are Na⁺ channel blockers. Exam focus: explain the ionic basis of the resting potential and the phases of the action potential, and explain how local anaesthetics work.

Types by location - Axodendritic (most common), axosomatic, axoaxonic (presynaptic modulation), dendrodendritic. Electrical synapses - Gap junctions (connexons) connect the cytoplasm of two cells; ions flow directly. - Very fast, synchronous, usually bidirectional. - Found in some brain regions (synchronising neuron groups), the retina, and extensively in cardiac and smooth muscle. Chemical synapses - Structure: 1. presynaptic terminal (bouton): synaptic vesicles containing neurotransmitter, mitochondria, and the active zone (where vesicles dock), with voltage-gated Ca²⁺ channels 2. synaptic cleft: about 20-30 nm wide, with extracellular matrix 3. postsynaptic membrane: receptors and a postsynaptic density (thickened, especially in excitatory synapses) - Slower (synaptic delay about 0.5 ms) but one-way and highly modifiable. Steps of neurotransmitter release 1. Synthesis of the transmitter and loading into vesicles (by vesicular transporters); peptides are made in the soma and carried by axonal transport. 2. Mobilisation: vesicles held on the cytoskeleton (by synapsin, linking them to actin) are released from the cytoskeleton by phosphorylation of synapsin. 3. Docking of vesicles at the active zone (SNARE proteins: synaptobrevin on the vesicle, syntaxin and SNAP-25 on the membrane). 4. Priming: the SNARE complex partly zips. 5. Arrival of the action potential opens voltage-gated Ca²⁺ channels; Ca²⁺ enters. 6. Ca²⁺ binds synaptotagmin, causing fusion of the vesicle with the membrane (exocytosis) and release of the transmitter into the cleft. 7. The transmitter diffuses and binds receptors on the postsynaptic membrane. 8. Recovery of the vesicle membrane by clathrin-mediated endocytosis (or kiss-and-run), and refilling of vesicles. - Clinical: botulinum toxin cleaves SNARE proteins, blocking acetylcholine release (flaccid paralysis; used therapeutically for spasticity, dystonia and cosmetically). Tetanus toxin cleaves synaptobrevin in inhibitory interneurons (spastic paralysis). Lambert-Eaton syndrome: antibodies against presynaptic Ca²⁺ channels. Postsynaptic effects - Ionotropic receptors (ligand-gated channels): fast responses (e.g. nicotinic ACh receptor, AMPA and NMDA glutamate receptors, GABA-A receptors). - Metabotropic receptors (G-protein-coupled): slower, longer-lasting, modulatory (e.g. muscarinic ACh, dopamine, most noradrenaline and serotonin receptors, GABA-B). - Excitatory postsynaptic potential (EPSP): depolarisation (Na⁺ entry), bringing the neuron toward threshold (main transmitter glutamate). - Inhibitory postsynaptic potential (IPSP): hyperpolarisation (Cl⁻ entry or K⁺ exit) (main transmitters GABA in the brain, glycine in the spinal cord). - Each neuron integrates thousands of EPSPs and IPSPs (summation) at the axon hillock. Main neurotransmitters - Acetylcholine: neuromuscular junction, autonomic ganglia, parasympathetic endings, brain (memory; lost in Alzheimer disease). - Amino acids: glutamate (main excitatory), GABA and glycine (main inhibitory). - Monoamines: noradrenaline (sympathetic endings), dopamine (movement, reward; Parkinson disease, schizophrenia), serotonin (mood, sleep; depression), histamine. - Neuropeptides: substance P (pain), endorphins and enkephalins (analgesia), neuropeptide Y. - Others: ATP, nitric oxide (a gas, diffuses freely). Tripartite synapse - The synapse includes three elements: the presynaptic terminal, the postsynaptic element, and the perisynaptic astrocyte process. - Astrocytes take up transmitters (glutamate), release gliotransmitters (glutamate, ATP, D-serine) and modulate synaptic strength. Inactivation of neurotransmitters - Termination is needed for precise signalling. Three mechanisms: 1. enzymatic degradation in the cleft: acetylcholinesterase breaks acetylcholine into acetate and choline (choline is taken back up) 2. reuptake into the presynaptic terminal or astrocytes by transporters (noradrenaline, dopamine, serotonin, glutamate, GABA), then reloading or degradation by monoamine oxidase (MAO) and COMT 3. diffusion away from the cleft - Clinical: SSRIs (fluoxetine) block serotonin reuptake (depression); cocaine blocks dopamine and noradrenaline reuptake; acetylcholinesterase inhibitors treat myasthenia gravis (neostigmine, pyridostigmine) and Alzheimer disease (donepezil); organophosphate poisoning (insecticides, nerve agents) irreversibly inhibits acetylcholinesterase (cholinergic crisis; treat with atropine and pralidoxime); MAO inhibitors are antidepressants. Exam focus: list the steps of neurotransmitter release, compare electrical and chemical synapses, and give three ways transmitters are inactivated with a drug example for each.

Myelin - A multilayered membrane wrapped around the axon: about 70-80% lipid (cholesterol, phospholipids, galactocerebroside, sphingomyelin) and about 20-30% protein (myelin basic protein, protein zero in PNS, proteolipid protein in CNS). - Insulates the axon, increases conduction speed up to 100 times. - Appears white in fresh tissue; dissolves in routine H&E processing, leaving a pale, net-like space (neurokeratin network); preserved with osmium tetroxide (black) or luxol fast blue. Myelination in the PNS by Schwann cells: steps 1. A Schwann cell surrounds a segment of a single axon. 2. The Schwann cell's plasma membrane forms a fold (mesaxon) that begins to wrap around the axon. 3. The Schwann cell rotates (the inner lip spirals) around the axon many times, laying down concentric layers of membrane. 4. The cytoplasm is squeezed out of the layers, so the membranes fuse into compact myelin: major dense lines (fused inner cytoplasmic faces) and intraperiod lines (outer faces). 5. The nucleus and most cytoplasm remain in the outermost layer: the neurilemma (sheath of Schwann), which is essential for regeneration. - One Schwann cell myelinates one internode (about 1 mm) of one axon. - Schmidt-Lanterman clefts (incisures): funnel-shaped areas of residual cytoplasm within the myelin. - Myelination starts in the fetus (month 4) and continues into adolescence (the brain until the mid-20s). This explains why infants have slower reflexes and uncoordinated movements. Unmyelinated fibres - Small axons (diameter under 1 µm) lie in grooves of a single Schwann cell, which surrounds several axons (up to 15-20) with one layer of membrane (Remak bundles). - Conduction is continuous and slow (0.5-2 m/s). - Examples: C fibres (slow pain, temperature), postganglionic autonomic fibres. Nodes of Ranvier - Gaps between adjacent Schwann cells (or oligodendrocyte segments), about 1 µm long, at regular intervals, where the axon membrane is exposed. - Very high density of voltage-gated Na⁺ channels at the nodes; K⁺ channels are under the myelin next to the nodes (juxtaparanodes). - The internode is the myelinated segment between two nodes. Saltatory conduction - In myelinated fibres the AP is generated only at the nodes. The myelin prevents ion leak, so current flows quickly inside the axon to the next node, where it triggers a new AP; the impulse "jumps" from node to node (saltare means to jump). - Advantages: much faster (up to 120 m/s in large Aα fibres) and more energy-efficient (fewer ions to pump back). - Classification of fibres: Aα (motor, proprioception, 70-120 m/s), Aβ (touch), Aδ (fast pain, cold), B (preganglionic autonomic), C (unmyelinated; slow pain, 0.5-2 m/s). Oligodendrocyte myelin in the CNS - One oligodendrocyte extends processes to myelinate internodes of many axons (up to 50). - No neurilemma; no Schmidt-Lanterman-like basement membrane; nodes are contacted by astrocyte processes. - Different proteins (proteolipid protein, myelin oligodendrocyte glycoprotein), making CNS myelin the target of distinct diseases. Multiple sclerosis (MS) - An autoimmune disease in which T cells and antibodies attack CNS myelin (oligodendrocytes), forming plaques of demyelination in the white matter (optic nerves, periventricular regions, brainstem, spinal cord). - Conduction slows or fails; symptoms depend on location: optic neuritis (blurred vision, painful eye movements), weakness, numbness, ataxia, bladder dysfunction; relapsing-remitting course in most. - Diagnosis: MRI (plaques disseminated in space and time), oligoclonal bands in CSF. - More common in women aged 20-40, in higher latitudes; linked to vitamin D and Epstein-Barr virus. - Peripheral nerves are spared (the PNS equivalent is Guillain-Barré syndrome, an acute demyelinating polyneuropathy after infection). Nerve sheaths: structure of a peripheral nerve - Endoneurium: delicate loose connective tissue (reticular fibres) around each individual nerve fibre. - Perineurium: several layers of flattened epithelioid cells joined by tight junctions, surrounding each fascicle (bundle of fibres). It forms the blood-nerve barrier. - Epineurium: dense irregular connective tissue with fat and blood vessels (vasa nervorum) surrounding the whole nerve and filling the space between fascicles. - Mnemonic: endo- (inside, each fibre), peri- (around each fascicle), epi- (upon the whole nerve). - A nerve is a bundle of axons in the PNS; the CNS equivalent is a tract. Ganglia are clusters of cell bodies in the PNS; the CNS equivalent is a nucleus. Nerve injury and regeneration - CNS: regeneration is minimal because oligodendrocytes do not form a regeneration tube, myelin debris and the glial scar (astrocytes) inhibit growth (Nogo, MAG), and microglia clear debris slowly. Spinal cord injuries are usually permanent. - PNS: regeneration is possible if the cell body survives and the ends are close. 1. Injury (the axon is cut). 2. Wallerian degeneration: the axon and myelin distal to the injury degenerate within days; macrophages and Schwann cells remove debris. The proximal end degenerates a short distance, back to the nearest node. 3. Chromatolysis in the cell body: it swells, the nucleus moves to the periphery and Nissl bodies disperse, as the cell increases protein synthesis for repair. 4. Schwann cells proliferate within the endoneurial tube and form a column (bands of Büngner) that guides regrowth; they secrete growth factors (NGF). 5. The proximal stump sprouts many new axon branches; one enters the tube and grows at about 1-3 mm per day (about 1 inch per month). 6. Schwann cells remyelinate the new axon, and connection to the target is restored. - If the gap is too large or the tube is blocked by scar, sprouts form a tangled mass: a neuroma (painful). - Surgical repair: aligning the fascicles (epineural or fascicular suture) or a nerve graft improves recovery. - Clinical: Tinel sign (tingling on tapping the regenerating nerve) tracks the growth front. Crush injuries (axonotmesis) recover better than cuts (neurotmesis). Exam focus: describe the steps of myelination by Schwann cells, explain saltatory conduction, compare the three nerve sheaths, and list the steps of peripheral nerve regeneration.

1. How is the motor division of the PNS subdivided? 2. What is the difference between grey and white matter? 3. List the functions of astrocytes. 4. What forms the blood-brain barrier? 5. Where do microglia come from, and what do they do? 6. What do ependymal cells line, and what do they do? 7. Name the two glial cells of the PNS. 8. What are Nissl bodies, and where are they absent? 9. Compare fast anterograde and retrograde axonal transport. 10. Why can rabies and herpes viruses reach the CNS from the skin? 11. Classify neurons by structure and give an example of each. 12. What causes the resting membrane potential? 13. Describe the phases of the action potential. 14. What is the all-or-none principle? 15. How do local anaesthetics work? 16. List the steps of neurotransmitter release. 17. How does botulinum toxin cause paralysis? 18. What is a tripartite synapse? 19. How are neurotransmitters inactivated? 20. Describe the steps of Schwann cell myelination. 21. How do oligodendrocytes differ from Schwann cells? 22. What is saltatory conduction, and why is it faster? 23. What is multiple sclerosis? 24. Name the three connective tissue sheaths of a nerve. 25. Describe the steps of peripheral nerve regeneration.

1. Somatic (to skeletal muscle) and autonomic (to cardiac and smooth muscle and glands; sympathetic and parasympathetic). 2. Grey matter contains cell bodies, dendrites and synapses; white matter mainly myelinated axons. 3. Support, inducing the BBB, K⁺ and glutamate uptake, energy supply (lactate), tripartite synapse, glial scar formation. 4. Endothelial cells with tight junctions, basement membrane, pericytes and astrocyte end-feet. 5. From yolk-sac precursors (mesoderm). They are the resident macrophages of the CNS: surveillance, phagocytosis, synapse pruning. 6. The ventricles and central canal. They help circulate CSF, and choroid plexus ependyma secretes it. 7. Schwann cells and satellite cells. 8. Clumps of RER and ribosomes in the soma and dendrites; absent from the axon hillock and axon. 9. Fast anterograde: 100-400 mm/day, by kinesin, from soma to terminal. Retrograde: about 100-200 mm/day, by dynein, from terminal to soma. 10. They are carried by retrograde axonal transport from nerve terminals to the cell body. 11. Multipolar (motor neuron), bipolar (retina), unipolar (dorsal root ganglion sensory neuron). 12. Ion gradients maintained by the Na⁺/K⁺ pump, and a membrane much more permeable to K⁺, which leaks out. 13. Depolarisation (Na⁺ channels open, Na⁺ in), repolarisation (Na⁺ channels inactivate, K⁺ out), hyperpolarisation (slow K⁺ channel closing), return to rest. 14. A stimulus at or above threshold gives a full-size AP; below threshold none. Intensity is coded by frequency. 15. They block voltage-gated Na⁺ channels, preventing AP generation and conduction. 16. Synthesis and loading, release from the cytoskeleton, docking, priming, Ca²⁺ entry, fusion and exocytosis, receptor binding, recovery of vesicle membrane. 17. It cleaves SNARE proteins, blocking acetylcholine release at the NMJ (flaccid paralysis). 18. A synapse including the presynaptic terminal, the postsynaptic element and an astrocyte process that modulates it. 19. Enzymatic degradation (acetylcholinesterase), reuptake (transporters), diffusion. 20. A Schwann cell surrounds one axon segment, forms a mesaxon, spirals around it, squeezes out cytoplasm to form compact myelin, and leaves its nucleus and cytoplasm outside as the neurilemma. 21. Oligodendrocytes (CNS) myelinate segments of many axons and lack a neurilemma; Schwann cells (PNS) myelinate one segment of one axon and support regeneration. 22. The AP jumps from node to node because myelin insulates the internodes. Faster and more energy-efficient. 23. An autoimmune disease destroying CNS myelin, causing plaques and neurological deficits. 24. Endoneurium (each fibre), perineurium (each fascicle), epineurium (whole nerve). 25. Wallerian degeneration of the distal segment, chromatolysis, Schwann cells form bands of Büngner, proximal sprouting, growth of about 1-3 mm/day, remyelination.

The three types of muscle: overview - Skeletal: long cylindrical multinucleated fibres, nuclei at the periphery; striated; voluntary; attached to bones (also in the tongue, upper oesophagus, eye muscles). - Cardiac: branched cells with one or two central nuclei; striated; intercalated discs; involuntary; heart wall. - Smooth: spindle-shaped cells with a single central nucleus; no striations; involuntary; walls of hollow organs and vessels. Skeletal muscle architecture (from large to small) 1. Muscle (organ), wrapped by the epimysium: dense irregular connective tissue around the whole muscle, continuous with the deep fascia and tendon. 2. Fascicle: a bundle of fibres, wrapped by the perimysium (carries larger vessels and nerves). 3. Muscle fibre (cell), wrapped by the endomysium: delicate loose connective tissue with reticular fibres and capillaries around each fibre. - The three layers merge into the tendon, which transmits force to the bone (at the myotendinous junction). - Mnemonic: same as nerves: epi- (whole), peri- (bundle), endo- (each cell). Skeletal muscle fibre - A syncytium: formed by the fusion of many myoblasts during development, so it has hundreds of nuclei located at the periphery just under the sarcolemma. - Length up to several cm (up to 30 cm in sartorius), diameter 10-100 µm. - Satellite cells: muscle stem cells between the sarcolemma and the basal lamina; they allow limited regeneration and growth. Adult muscle grows mainly by hypertrophy (bigger fibres), not hyperplasia. - Sarcoplasm is packed with myofibrils, mitochondria, glycogen and myoglobin (an O₂-binding red pigment). Myofibril and the striations - Myofibrils (1-2 µm wide) run the length of the fibre; they are chains of sarcomeres. Their bands align across the fibre, producing cross-striations. - A band (anisotropic, dark in light microscopy, birefringent in polarised light): the full length of the thick filaments, including overlap with thin filaments. Its length does not change during contraction. - H zone: the paler centre of the A band where there are only thick filaments (no overlap). - M line: the centre of the H zone, where thick filaments are linked (myomesin, creatine kinase). - I band (isotropic, light): only thin filaments; bisected by the Z disc. - Z disc (Z line): a dense line where thin filaments of adjacent sarcomeres are anchored (α-actinin). - Sarcomere: the segment between two successive Z discs (about 2.5 µm at rest); the contractile unit of striated muscle. - Sequence: Z - I - A (with H and M in the middle) - I - Z. - Mnemonic: "dArk A band, lIght I band"; the H zone and I band shrink during contraction, the A band stays the same. Thick filament (myosin) - About 15 nm wide, 1.6 µm long; made of about 300 myosin II molecules. - Each myosin molecule: two heavy chains forming a long tail and two globular heads, and four light chains (two essential and two regulatory). - The heads (cross-bridges) project toward the thin filaments; each has an actin-binding site and an ATPase site. The middle of the thick filament is a bare zone without heads. - Titin: a giant elastic protein running from the Z disc to the M line; holds thick filaments in the centre and gives passive elasticity (a spring). Nebulin runs along thin filaments and sets their length. Thin filament (actin) - About 7 nm wide, 1 µm long, anchored to the Z disc. - Three proteins: 1. actin: two strands of globular G-actin monomers polymerised into filamentous F-actin twisted into a helix; each G-actin has a myosin-binding site 2. tropomyosin: a long, rod-shaped protein lying in the groove of the actin helix; at rest it covers the myosin-binding sites 3. troponin: a complex of three subunits bound to tropomyosin: - troponin T: binds tropomyosin - troponin I: inhibits actin-myosin binding - troponin C: binds Ca²⁺ - Clinical: cardiac troponins (I and T) are released into the blood when heart muscle is damaged, the key blood test for myocardial infarction. Other proteins - Dystrophin: links the actin cytoskeleton of the fibre to the dystroglycan complex in the sarcolemma, which binds laminin of the basal lamina. Protects the sarcolemma during contraction (see DMD in the NMJ note). - Desmin: intermediate filament linking myofibrils to each other at the Z discs and to the sarcolemma. Exam focus: draw a sarcomere and label its bands, describe the composition of thick and thin filaments, and list the connective tissue layers of a muscle.

Sliding filament theory - During contraction, the thin filaments slide toward the M line, overlapping more with thick filaments. - Changes: - Z discs move closer together; the sarcomere shortens - I band and H zone shorten (may disappear) - A band stays the same length - the length of the filaments does not change - All sarcomeres in a myofibril shorten together, shortening the whole fibre. - Length-tension relationship: maximum force at optimal overlap (resting length about 2.0-2.2 µm); too stretched (little overlap) or too compressed (filaments collide) gives less force. The same principle underlies the Frank-Starling law of the heart. Cross-bridge cycle Precondition: Ca²⁺ binds troponin C, which moves tropomyosin away from the myosin-binding sites on actin. 1. Cross-bridge formation: the energised myosin head (holding ADP and Pi) binds to actin. 2. Power stroke: release of Pi triggers the head to pivot, pulling the thin filament toward the M line; ADP is released. The head is now in a low-energy state tightly bound to actin (rigor state). 3. Cross-bridge detachment: a new ATP binds to the myosin head, causing it to release actin. 4. Cocking of the myosin head: myosin ATPase hydrolyses ATP to ADP and Pi, returning the head to its high-energy "cocked" position, ready for the next cycle. - The cycle repeats as long as Ca²⁺ and ATP are present; each stroke moves the filament about 10 nm. Heads work asynchronously, so there is always some attachment. - Relaxation: Ca²⁺ is pumped back into the SR by SERCA (an ATP-dependent pump), tropomyosin blocks the sites again, and the filaments slide back passively (elasticity, titin). Rigor mortis - After death, ATP synthesis stops. Ca²⁺ leaks from the SR and cross-bridges form, but without ATP myosin cannot detach from actin, so muscles become rigid. - Begins about 2-4 hours after death, peaks at about 12 hours, and disappears after about 48-72 hours as muscle proteins degrade (autolysis). Used to estimate time of death. Energy for contraction: ATP regeneration - ATP stored in muscle lasts only about 2-4 seconds of maximal contraction. It is regenerated by: 1. Direct phosphorylation (creatine phosphate system): - creatine phosphate + ADP ⇌ creatine + ATP, catalysed by creatine kinase (CK) - very fast, no oxygen; lasts about 10-15 seconds (sprint, weightlifting) - clinical: raised CK in the blood indicates muscle damage (rhabdomyolysis, myocarditis, dystrophies) 2. Anaerobic glycolysis: - glucose (from blood or glycogen) to pyruvate gives 2 ATP per glucose; without enough O₂, pyruvate becomes lactate - fast, about 2.5 times faster than aerobic; lasts about 30-60 seconds; used in intense exercise (400 m run) - lactate goes to the liver (Cori cycle) or heart for use 3. Aerobic respiration: - glucose, fatty acids and amino acids are oxidised in mitochondria to CO₂ and water; about 30-32 ATP per glucose - slow but sustainable for hours; needs O₂ (delivered by blood and myoglobin); main source at rest and in endurance exercise (marathon) Oxygen debt (excess post-exercise oxygen consumption, EPOC) - After strenuous exercise, breathing stays rapid to take in the extra O₂ needed to: - replenish O₂ in myoglobin and haemoglobin - regenerate ATP and creatine phosphate - convert lactate back to pyruvate and glucose (liver) - restore glycogen - meet the higher metabolic rate from raised temperature Muscle fatigue - The physiological inability to contract despite continued stimulation. Causes: ionic imbalances (K⁺, Ca²⁺), accumulation of Pi (which interferes with Ca²⁺ release and cross-bridges), low glycogen, central (nervous) fatigue. Lactate itself is not the main cause. Heat production - Only about 40% of the energy released in contraction does work; the rest becomes heat (keeps body temperature; excess is removed by sweating and vasodilation). Shivering is involuntary contraction to generate heat. Effects of exercise - Aerobic (endurance) exercise: more capillaries, more mitochondria, more myoglobin; better endurance, strength and resistance to fatigue; also benefits heart, lungs, bones and metabolism. - Resistance (strength) exercise: muscle hypertrophy (more myofibrils and myofilaments per fibre), more glycogen and strength. - Disuse: atrophy (bed rest, immobilisation in a cast, denervation) with loss of mass and strength; after long denervation fibres are replaced by fibrous tissue. Skeletal muscle fibre types - Type I (slow oxidative, "red" fibres): - small diameter, many mitochondria, much myoglobin (red colour), many capillaries - slow myosin ATPase, aerobic metabolism, resistant to fatigue - postural muscles (back, soleus), endurance athletes (marathon runners) - Type IIa (fast oxidative-glycolytic, intermediate): fast, fairly fatigue-resistant; sprinters and middle distance. - Type IIb/IIx (fast glycolytic, "white" fibres): - large diameter, few mitochondria, little myoglobin (pale), much glycogen - fast ATPase, anaerobic glycolysis, powerful but fatigue quickly - short, powerful movements (arm muscles, sprinting, weightlifting) - Most muscles contain a mix; the proportion depends on genetics and training. All fibres in one motor unit are of the same type. - Example: chicken breast (white, flight muscle used briefly) versus leg (dark, used constantly). - Histochemistry (myosin ATPase or NADH-TR staining) shows a checkerboard of fibre types; lost in denervation (fibre type grouping). Exam focus: describe the four steps of the cross-bridge cycle and the role of ATP, explain rigor mortis, and compare red and white fibres.

Neuromuscular junction (NMJ, motor end plate) - Structure: - the axon terminal of a motor neuron loses its myelin, branches and ends in boutons in shallow depressions (synaptic gutters) on the muscle fibre; covered by Schwann cell cytoplasm - boutons contain synaptic vesicles filled with acetylcholine (ACh) and many mitochondria - synaptic cleft (about 50 nm) filled with basal lamina containing acetylcholinesterase (AChE) - junctional folds of the sarcolemma (the motor end plate) increase the surface; their crests are packed with nicotinic ACh receptors - One NMJ per muscle fibre, usually in the middle of the fibre. - Events: 1. an action potential arrives at the axon terminal 2. voltage-gated Ca²⁺ channels open; Ca²⁺ enters the terminal 3. Ca²⁺ triggers exocytosis of ACh (each vesicle is a quantum of about 10,000 molecules) 4. ACh diffuses across the cleft and binds nicotinic receptors (ligand-gated cation channels) 5. Na⁺ enters (more than K⁺ leaves), producing an end-plate potential (local depolarisation), which is always large enough to trigger an AP in adjacent sarcolemma (a safety factor: one nerve AP gives one muscle AP) 6. the AP spreads along the sarcolemma and down the T tubules 7. AChE in the cleft breaks down ACh within milliseconds, ending the signal - Clinical: - myasthenia gravis: autoantibodies against nicotinic ACh receptors (type II hypersensitivity); fatigable weakness (ptosis, diplopia, then limbs and breathing); associated with thymoma or thymic hyperplasia. Treated with AChE inhibitors (pyridostigmine), immunosuppression, thymectomy. - Lambert-Eaton: antibodies to presynaptic Ca²⁺ channels (often with small-cell lung cancer); weakness improves with repeated use. - botulinum toxin blocks ACh release (flaccid paralysis). - curare and neuromuscular blocking drugs (rocuronium, vecuronium) block ACh receptors; used in anaesthesia. Succinylcholine is a depolarising blocker. - organophosphates inhibit AChE (fasciculations, then paralysis). T tubules and sarcoplasmic reticulum - Transverse (T) tubules: deep invaginations of the sarcolemma that penetrate the fibre and surround each myofibril. In mammalian skeletal muscle they lie at the A-I junctions (two per sarcomere). They carry the AP rapidly to the deep interior, so all myofibrils contract together. - Sarcoplasmic reticulum: a network of smooth ER surrounding each myofibril like a sleeve. It stores Ca²⁺ (bound to calsequestrin). - terminal cisternae: enlarged ends of the SR next to the T tubules - longitudinal SR with SERCA pumps - Triad: one T tubule flanked by two terminal cisternae (seen in EM at the A-I junction in skeletal muscle). In cardiac muscle, a dyad (one T tubule and one cisterna) at the Z disc. Excitation-contraction coupling 1. The AP travels along the sarcolemma and down the T tubules. 2. In the T tubule membrane, voltage-sensitive dihydropyridine receptors (DHPR, L-type Ca²⁺ channels) change shape. 3. In skeletal muscle, DHPRs are mechanically linked to ryanodine receptors (RyR1, Ca²⁺ release channels) in the terminal cisternae; the shape change opens RyR1. 4. Ca²⁺ floods from the SR into the sarcoplasm (concentration rises about 100-fold). 5. Ca²⁺ binds troponin C; tropomyosin moves, exposing myosin-binding sites on actin; the cross-bridge cycle begins. 6. When stimulation ends, SERCA pumps Ca²⁺ back into the SR (using ATP); Ca²⁺ falls, tropomyosin blocks actin again, and the muscle relaxes. - Clinical: malignant hyperthermia: a RyR1 mutation causes uncontrolled Ca²⁺ release after volatile anaesthetics or succinylcholine: rigidity, hyperthermia, acidosis, hyperkalaemia. Treated with dantrolene (blocks RyR1). Motor unit - A motor neuron and all the muscle fibres it innervates. The fibres of one unit are spread through the muscle and contract together. - Size: small units (a few fibres, e.g. 3-10 in eye muscles, fine control) versus large units (hundreds to over 1,000 fibres, e.g. in the thigh and back, power). - Force is graded by: - recruitment: activating more motor units (small units first, the size principle) - frequency of stimulation: summation, up to fused tetanus - Muscle tone: slight contraction at rest due to a low level of spinal reflex activity. - Clinical: EMG records motor unit potentials; denervation causes fibrillations; motor neuron disease (ALS) destroys motor neurons. Duchenne muscular dystrophy (DMD) - X-linked recessive; affects about 1 in 3,500-5,000 male births; a mutation (often a deletion) in the dystrophin gene (the largest human gene, on Xp21) with no functional dystrophin. - Without dystrophin, the sarcolemma tears during contraction; Ca²⁺ enters, fibres degenerate and are replaced by fat and fibrous tissue. - Onset at age 2-5 years: clumsiness, falls, difficulty climbing stairs; Gowers sign (using hands to climb up the legs to stand); calf pseudohypertrophy (fat and fibrous replacement); very high serum CK; wheelchair by about 12; death from respiratory or cardiac failure (cardiomyopathy), now often in the 20s-30s with care. - Diagnosis: genetic testing; muscle biopsy with immunohistochemistry (IHC) for dystrophin: normal muscle shows a continuous brown ring at the sarcolemma of each fibre; DMD muscle shows no staining. - Becker muscular dystrophy: in-frame mutation with a reduced, partly functional dystrophin; milder, later onset. - Treatment: corticosteroids, exon-skipping drugs (eteplirsen), gene therapy (micro-dystrophin), cardiac and respiratory care. Exam focus: list the events at the NMJ, describe the triad and excitation-contraction coupling, define a motor unit, and explain the defect in DMD and how it is shown by IHC.

Cardiac muscle cells (cardiomyocytes) - Short (about 100 µm), branched cylinders forming a three-dimensional network. - One (sometimes two) centrally placed nucleus, with a pale perinuclear zone containing lipofuscin, glycogen and the Golgi. - Striated: the same sarcomere arrangement as skeletal muscle. - Very many large mitochondria (about 35-40% of cell volume): the heart depends almost entirely on aerobic metabolism (fatty acids mainly); very rich capillary supply; much myoglobin. - Endomysium with capillaries between cells; no epimysium or fascicles as in skeletal muscle. - Atrial cells contain granules of atrial natriuretic peptide (ANP), released when the atria are stretched (promotes Na⁺ and water excretion). Intercalated discs - Dark-staining, step-like junctions where cells meet end to end, always at the level of Z discs. - Transverse portions (across the fibres), containing: - fasciae adherentes: anchor actin filaments of the terminal sarcomeres (like a Z disc) - desmosomes (maculae adherentes): prevent cells from pulling apart during contraction (anchor intermediate filaments, desmin) - Lateral portions (parallel to the fibres) containing gap junctions: allow ions to pass between cells, so the AP spreads rapidly and the myocardium contracts as a functional syncytium. - Clinical: arrhythmogenic cardiomyopathy involves desmosome gene mutations. T tubules and SR in cardiac muscle - T tubules are larger and located at the Z discs (one per sarcomere). - SR is less developed; forms dyads (one T tubule with one small terminal cisterna). - Excitation-contraction coupling uses calcium-induced calcium release: Ca²⁺ entering through L-type channels during the long plateau of the cardiac AP triggers RyR2 to release more Ca²⁺ from the SR. So cardiac contraction depends on extracellular Ca²⁺ (the target of calcium channel blockers and of digoxin, which raises intracellular Ca²⁺). - The cardiac AP has a long plateau and a long refractory period (about 250 ms), preventing tetanus so the heart can refill. Failure to regenerate - Adult cardiomyocytes are terminally differentiated and divide very rarely (renewal of about 1% per year in young adults, less with age). - After myocardial infarction, dead cells are replaced by a fibrous scar that cannot contract, leading to heart failure. The heart adapts to increased load by hypertrophy (bigger cells), not hyperplasia. - Research into stem cells and reprogramming aims to regenerate myocardium. Conduction system of the heart - Made of specialised cardiac muscle cells (not nerves) that generate and conduct impulses; they have few myofibrils. - Components (the order of conduction): 1. Sinoatrial (SA) node: in the wall of the right atrium near the entrance of the superior vena cava. The pacemaker: its cells depolarise spontaneously (the "funny" current, If) at about 60-100 times per minute, setting the sinus rhythm. 2. The impulse spreads through the atrial muscle (gap junctions and internodal pathways), making the atria contract. 3. Atrioventricular (AV) node: in the interatrial septum near the opening of the coronary sinus. Conducts slowly, delaying the impulse by about 0.1 s so the atria finish emptying before the ventricles contract. The only normal electrical connection between atria and ventricles (the fibrous skeleton insulates the rest). Intrinsic rate about 40-60/min. 4. Atrioventricular bundle (bundle of His): passes through the fibrous skeleton into the interventricular septum. 5. Right and left bundle branches: run down the septum toward the apex. 6. Purkinje fibres: large, pale cells (rich in glycogen, few myofibrils, few T tubules) under the endocardium; conduct very fast (about 2-4 m/s) and spread the impulse through the ventricular walls from the apex upward, so blood is squeezed toward the outflow valves. Intrinsic rate 20-40/min. - Autonomic control: the sympathetic system (noradrenaline) increases heart rate and force; the parasympathetic vagus (acetylcholine) slows the SA node and AV conduction. - Clinical: heart block (AV node disease); ectopic pacemakers; artificial pacemakers; Wolff-Parkinson-White syndrome (an accessory pathway bypassing the AV node). - Histology: Purkinje fibres are recognised in the subendocardium as large cells with pale, vacuolated cytoplasm and few peripheral myofibrils. Electrocardiography (ECG) - A recording of the electrical activity of the heart from electrodes on the skin (12 standard leads). - Waves: - P wave: atrial depolarisation (from the SA node) - PR interval (0.12-0.20 s): from atrial depolarisation to the start of ventricular depolarisation; mainly the AV nodal delay - QRS complex (under 0.12 s): ventricular depolarisation (larger than P because of the larger muscle mass); atrial repolarisation is hidden in it - ST segment: ventricles fully depolarised (plateau); raised in acute myocardial infarction (STEMI) - T wave: ventricular repolarisation - QT interval: total ventricular depolarisation and repolarisation; prolonged by some drugs and genetic channelopathies (torsades de pointes risk) - Uses: heart rate and rhythm (arrhythmias such as atrial fibrillation), conduction blocks (long PR interval in first-degree block), ischaemia and infarction, chamber hypertrophy, electrolyte disturbances (peaked T waves in hyperkalaemia). - Note: the detail of ECG interpretation belongs to Physiology and Cardiology; here the aim is to link the waves to the conduction system. Exam focus: describe the three components of the intercalated disc and their roles, list the conduction system in order with the role of the AV node, and link each ECG wave to cardiac events.

Locations - Walls of the digestive tract (from the middle oesophagus to the anus), airways (trachea to bronchioles), urinary tract (ureters, bladder), reproductive tract (uterus, vas deferens), blood vessels (tunica media) and lymphatics. - Also the iris (dilator and sphincter pupillae), ciliary muscle, arrector pili muscles of the skin, and capsules of some organs (spleen). Structure of the smooth muscle cell - Fusiform (spindle-shaped) cells, 20-200 µm long (up to 500 µm in the pregnant uterus), 3-8 µm wide at the centre. - One elongated, central nucleus; when the cell contracts the nucleus takes a corkscrew shape. - Cells are arranged in sheets or bundles, overlapping so the thick part of one cell lies next to the thin ends of others. In cross-section: circles of various sizes, only the largest showing a nucleus. - Each cell is surrounded by a basal (external) lamina and reticular fibres (endomysium), which transmit the force. - Often arranged in two layers at right angles: inner circular and outer longitudinal (in the gut, they produce peristalsis). - No T tubules; SR poorly developed. - Caveolae: flask-shaped invaginations of the sarcolemma that serve as T tubule equivalents, concentrating Ca²⁺ channels and signalling molecules near the SR. - Gap junctions (nexuses) connect cells electrically in single-unit smooth muscle. The contractile apparatus - Myofilaments are not organised into sarcomeres, so there are no striations: - thin filaments (actin with tropomyosin, but no troponin; associated with caldesmon and calponin) - thick filaments (myosin II), with heads along their whole length (side-polar), so they can pull actin over longer distances - ratio of thin to thick filaments is high (about 12:1 or more, compared with 2:1 in skeletal muscle) - intermediate filaments (desmin; vimentin in vascular muscle) - Dense bodies: contain α-actinin; the equivalent of Z discs. Scattered in the cytoplasm (cytoplasmic dense bodies) and attached to the sarcolemma (membrane-associated dense bodies, dense plaques). Actin filaments and intermediate filaments anchor to them. - The filament network is arranged diagonally, like a lattice in the cell, so when it contracts the cell shortens and twists, bulging between attachment points (a "corkscrew" appearance). Contraction of smooth muscle 1. Ca²⁺ rises in the cytoplasm: from outside (voltage-gated, ligand-gated and stretch-activated channels in the caveolae) and from the SR (IP₃ receptors when G-protein-coupled receptors are stimulated). 2. Ca²⁺ binds calmodulin (not troponin). 3. The Ca²⁺-calmodulin complex activates myosin light chain kinase (MLCK). 4. MLCK phosphorylates the regulatory myosin light chains, activating the myosin heads. 5. Cross-bridge cycling begins; actin filaments are pulled toward the centre of the cell. 6. Relaxation: Ca²⁺ is pumped out or into the SR; myosin light chain phosphatase dephosphorylates the myosin. - Caldesmon (and calponin) on the thin filaments inhibit actin-myosin interaction at rest; Ca²⁺-calmodulin binding or phosphorylation relieves the inhibition. - So smooth muscle regulation is thick filament (myosin)-based, while in skeletal and cardiac muscle it is thin filament (troponin)-based. Features of smooth muscle contraction - Slow, prolonged and resistant to fatigue: - myosin ATPase is 10-100 times slower - the latch state: dephosphorylated myosin heads stay attached to actin, maintaining tension (tone) with very little ATP use. Ideal for sphincters and blood vessel walls. - Stress-relaxation response: when stretched (filling of the bladder or stomach), smooth muscle first contracts and then relaxes, allowing the organ to fill without a big rise in pressure. - Length and tension: it can generate tension over a very wide range of lengths (up to 2.5 times the resting length), thanks to the absence of sarcomeres. - Hyperplasia: smooth muscle cells can divide (e.g. in the uterus in pregnancy; in the vascular wall in atherosclerosis and hypertension). They also secrete collagen, elastin and proteoglycans (the fibroblast-like role in vessels). Control of smooth muscle - Autonomic innervation: varicosities (swellings) along autonomic axons release neurotransmitters (noradrenaline, acetylcholine) into a wide synaptic cleft over many cells (diffuse junctions), not NMJs. The same transmitter can excite or inhibit depending on the receptor (e.g. adrenaline contracts vascular smooth muscle through α1 receptors but relaxes bronchial smooth muscle through β2 receptors). - Hormones and local factors: oxytocin (uterus), histamine, angiotensin II, nitric oxide (relaxation via cGMP), low O₂, CO₂, H⁺, stretch. - Types: - single-unit (visceral) smooth muscle: the most common; cells connected by gap junctions contract together as a unit; many are self-excitable (pacemaker cells, such as interstitial cells of Cajal in the gut) and respond to stretch. Examples: gut, uterus, bladder, small blood vessels. - multi-unit smooth muscle: each cell has its own nerve supply; few gap junctions; graded, precise contraction. Examples: large airways, large arteries, iris, ciliary muscle, arrector pili. Vascular smooth muscle - In the tunica media; circular or helical arrangement; controls vessel diameter, blood pressure and blood flow distribution. - Clinical: vasodilator drugs act here: nitrates (NO donors), calcium channel blockers (amlodipine), α1-blockers; β2-agonists (salbutamol) relax bronchial smooth muscle in asthma; phosphodiesterase-5 inhibitors (sildenafil) prolong cGMP. - Tumours: leiomyoma (fibroids of the uterus, the most common tumour in women); leiomyosarcoma. Comparison of the three muscle types - Cell shape: skeletal, long cylindrical; cardiac, short branched; smooth, spindle. - Nuclei: skeletal, many peripheral; cardiac, one or two central; smooth, one central. - Striations: skeletal and cardiac yes; smooth no. - Sarcomeres: skeletal and cardiac yes; smooth no (dense bodies instead). - T tubules: skeletal, at A-I junction (triads); cardiac, at Z disc (dyads); smooth, none (caveolae). - SR: skeletal, well developed; cardiac, less; smooth, poor. - Ca²⁺ source: skeletal, SR; cardiac, SR and extracellular; smooth, mainly extracellular and some SR. - Ca²⁺ regulatory protein: skeletal and cardiac, troponin; smooth, calmodulin (and MLCK). - Cell junctions: skeletal, none between fibres; cardiac, intercalated discs; smooth, gap junctions. - Control: skeletal, voluntary (somatic); cardiac, involuntary (autonomic, intrinsic pacemaker); smooth, involuntary (autonomic, hormones, local). - Speed: skeletal fast; cardiac intermediate; smooth slow and sustained. - Regeneration: skeletal, limited (satellite cells); cardiac, almost none; smooth, good (cells divide). - Tetanus: skeletal possible; cardiac impossible (long refractory period); smooth, sustained tone. Exam focus: describe the structure of a smooth muscle cell (dense bodies, caveolae, filament arrangement), explain the calmodulin-MLCK pathway, and fill in the comparison of the three muscle types.

1. List four functions of muscle tissue. 2. Name the three connective tissue layers of a skeletal muscle. 3. Why does a skeletal muscle fibre have many nuclei? 4. What are satellite cells? 5. Draw a sarcomere: which bands change length during contraction? 6. What proteins make up the thin filament, and what does each do? 7. What is the role of titin? 8. List the four steps of the cross-bridge cycle. 9. What is the role of ATP in contraction and relaxation? 10. Why does rigor mortis occur? 11. Name three ways muscle regenerates ATP. 12. What is oxygen debt? 13. Compare red (type I) and white (type IIb) fibres. 14. Describe the events at the neuromuscular junction. 15. What causes myasthenia gravis? 16. What is a triad? 17. Describe excitation-contraction coupling in skeletal muscle. 18. What is a motor unit, and how is muscle force graded? 19. What is the defect in DMD, and how does IHC show it? 20. What are the three components of the intercalated disc? 21. Why can cardiac muscle not heal after an infarction? 22. List the conduction system of the heart in order. 23. Why is there a delay at the AV node? 24. What do the P wave, QRS complex and T wave represent? 25. What are dense bodies and caveolae? 26. How is smooth muscle contraction regulated? 27. What is the latch state? 28. Compare single-unit and multi-unit smooth muscle.

1. Any four: movement, posture, joint stability, heat generation, moving substances, controlling openings. 2. Epimysium (muscle), perimysium (fascicle), endomysium (fibre). 3. It forms by fusion of many myoblasts (a syncytium). 4. Muscle stem cells under the basal lamina that allow repair and growth. 5. The I band and H zone shorten; the A band stays the same. 6. Actin (myosin-binding sites), tropomyosin (covers the sites at rest), troponin (T binds tropomyosin, I inhibits, C binds Ca²⁺). 7. A giant elastic protein from Z disc to M line that centres thick filaments and gives passive elasticity. 8. Cross-bridge formation, power stroke, detachment (ATP binds), cocking (ATP hydrolysis). 9. ATP detaches myosin from actin, energises (cocks) the head, and powers SERCA to pump Ca²⁺ back for relaxation. 10. Without ATP, myosin heads cannot detach from actin. 11. Creatine phosphate, anaerobic glycolysis, aerobic respiration. 12. The extra O₂ taken in after exercise to restore O₂ stores, ATP and creatine phosphate, convert lactate and restore glycogen. 13. Type I: small, many mitochondria and myoglobin, aerobic, slow, fatigue-resistant. Type IIb: large, glycolytic, fast, fatigue quickly. 14. AP reaches the terminal, Ca²⁺ enters, ACh is released, binds nicotinic receptors, end-plate potential triggers a muscle AP, AChE degrades ACh. 15. Autoantibodies against nicotinic ACh receptors. 16. One T tubule flanked by two terminal cisternae of the SR, at the A-I junction. 17. AP down the T tubule changes DHPR shape, which opens RyR1, Ca²⁺ is released from the SR, binds troponin C, and cross-bridge cycling starts. 18. A motor neuron and all the fibres it innervates. By recruitment of more motor units and by the frequency of stimulation. 19. Lack of dystrophin (X-linked). IHC shows no sarcolemmal staining, instead of the normal continuous ring. 20. Fasciae adherentes, desmosomes (transverse parts) and gap junctions (lateral parts). 21. Adult cardiomyocytes are terminally differentiated and hardly divide; dead muscle is replaced by scar. 22. SA node, atrial muscle, AV node, bundle of His, right and left bundle branches, Purkinje fibres. 23. So the atria finish contracting and emptying before the ventricles contract. 24. P: atrial depolarisation. QRS: ventricular depolarisation. T: ventricular repolarisation. 25. Dense bodies: α-actinin anchors for actin (Z disc equivalents). Caveolae: sarcolemmal invaginations acting like T tubules. 26. Ca²⁺ binds calmodulin, which activates MLCK, which phosphorylates myosin light chains, starting cross-bridge cycling. 27. Dephosphorylated myosin heads stay attached to actin, maintaining tone with little ATP. 28. Single-unit: gap junctions, contract together, often self-excitable (gut, uterus). Multi-unit: each cell innervated, precise (iris, large airways).