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LECTURE 1 – Epithelium 25/02  

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One of the four tissue types (epi, connective, muscle, nervous) Can be: - Covering (outermost) - Lining (innermost) - Gland – invagination of epithelial cells Separated from underlying connective tissue by basal lamina Between lateral surfaces – junctional complex Morphological/functional polarity (b/w lateral and basal) Avascular – no blood supply  obtains O by diffusion

Function  Protection  Transcellular transport  Secretion  Absorption  Selective permeability  Sensation Classifying 1. No. layers  simple/stratified 2. Morphology of surface layer  flat (squamous), cuboidal, columnar, (also psuedostratified – squamous, transitional - stratified) 3. Surface specializations  microvilli, cilia, keratin  Only squamous can be keratinized SIMPLE Simple Squamous  Flat and irregularly shaped  Passive diffusion  May have surface receptors for locally acting chemicals  Endothelium – lining blood/lymph vessel  Mesothelium – lining cavities (pleura, peritoneal, pericardial)  Endocardium – lining inside heart Simple Cuboidal  Typically lines ducts  Height ~ width  Excretory, secretory, absorptive Simple Columnar  Nuclei often elongated and usu at basal end  Absorptive (intestine), secretory (stomach)  Often have microvilli/cilia Psuedostratified Columnar

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Simple Every cell is attached to basal membrane, not all cells reach apical surface Nuclei located at diff levels – gives appearance of stratification Respiratory & male reproductive system Usu have cilia and often goblet cells

STRATIFIED Stratified Squamous  Outermost cells are squamous  Surface of skin, inside of oesophagus  Basal layer tends to be cuboidal, constantly dividing – may see mitotic layers  Middle layer is polymorphic  Moistened by glandular secretions  Keratinized – e.g. for epidermis under soles of feet Stratified Cuboidal  Lines larger ducts – sweat/salivary glands  Basal layer usu very cuboidal  Outermost layer may be more columnar Stratified Columnar  In largest ducts – pancreas/salivary gland Transitional Epithelium  Surface cells are large, pale and round  scalloped outline  Exclusively lining urinary system  Specialised to withstand toxicity of urine and be able to stretch Membrane Specialisations Intercellular Junctions  Tight/occluding  luminal contents cannot penetrate  Adhering  structural junctions that bind cells together  Communication  small junctions can cross between (gap junction) Apical Surface  Cilia – beat in rhythmic pattern; structured arrangement; e.g. resp system  Microvilli – short, numerous; projection of plasma membrane; non-motile; increase SA for absorption  Stereocilia – long microvilli; found in male reproductive tract Basal Surface  Non-cellular – made of matrix proteins; acts as a selective barrier  Folds in membrane – increase SA (cells in kidney)  Hemidesmosomes – anchors LECTURE 2 – Histological Techniques 04/03

Types of Specimens  Cytological – smear, scrape, washing, etc  Biopsy – piece of tissue  Whole organ  Post mortem sample Tissue Preparation Modes  Routine paraffin blocks  tissue embedded in paraffin wax  Urgent surgical cases  done while patient is still under anaesthetic to check if all cancer has been removed during the operation  Enzyme/lipid studies  Staining w/ antibodies  Special tissue studies  In situ hybridization (DNA)  Electron microscopy  Cytology Cell Death and Changes  Putrefaction  microorganism destruction  Autolysis  cells lyse  Post-mortem changes should be minimised  fixation of tissue Fixation of Tissue  Benefits: increases firmness, retains morphology, increase permeability for future chemical processing, prevent tissue breakdown  Store in a cool place, do autopsy ASAP  lowers autolysis/putrefaction  Organs removed at autopsy  Described and weighed  Placed in bucket of 10% PHO buffered formalin for a period of time depending on tissue type and size  Important factors for chemical preservation: - pH - T - Penetration - Osmolarity - Conc (10x more fixative than tissue, usually) - Duration - Size of tissue  affects the time taken to fix (e.g. biopsies are v small, so they can be tested very quickly after fixing) Processing tissue  Remove water from tissue so it can be embedded in wax by replacing the water with alcohol  Place in xylene (solvent) which is miscible with wax and alcohol  Vacuum infiltration with molten wax  Orientation of tissue during embedding is important

Cutting and Section Preparation  Sectioned on a microtome (not automated), usu cut at 5um  Floated on a waterbath to remove compression wrinkles.  Picked up individually on glass slide  Baked on Deparaffinization of Sections  Heat to melt wax  Place in xylene to dissolve away wax  Rehydrated by decreasing graded alcohol  Dye  Haematoxylin and Eosin is the most common, dyes usually stain nucleic acid OR cytoplasm and surrounding connective tissue  H stains nucleic acid blue, E stains cyto, membranes and proteins pink  Manual staining is done when urgent or when using expensive reagents; usu done with a slide staining machine instead Mounting  Coverslip glued on with DPX  Requires dehydration through increasing alcohol, putting in xylene, then gluing with DPX  Legal requirement that tissue sample is kept for 20 years Alternatives to Paraffin Embedding  Resin  for semi thin sections (1-2um) light microscopy; or v thin (100200nm) electron microscopy  Paraffin – histological sections 5-10um thick for light microscopy  Frozen – 10-50um for urgent diagnosis or enzyme research  Agar for thick sections for research Lipid Loss  Lipids, esp neutral lipids are lost during solvent processing  Fat is dissolved, therefore cannot be stained – you will see large white spaces LECTURE 3 – Glands and Skin 4/3 Glands  Originate from epithelia  Penetrate connective tissue  Manufacture basal lamina and then become secretory  Usually consists of simple cuboidal epithelium  Exocrine - Outgrowths of epithelium into connective - Have secretory segment and excretory segment = secretory unit - Connected by duct to epithelial origin - Can be defined as a specialized collection of secretory cells Exocrine Types

Simple:  Unicellular  goblet cell (secretes mucus)  Tubular  simple (intestinal wall); coiled (sweat gland); branched (stomach wall)  Acinar  simple (urethra), branched Complex:  Compound tubular  duodenum  Compound acinar  pancreas  Compound tubuloacinar  salivary Modes of Secretion  Holocrine  whole cell disintegrates to release contents (sebaceous)  Eccrine  exocytosis  Apocrine  buds off cell in vesicles (mammary gland) Myoepithelial Cells  Muscle-like epithelial cell  actually a non-muscle contractile cell  Sits below epithelium in glands and contract to help excretion Endocrine Glands  Secrete hormones directly into connective tissue/blood stream  Pancreas  insulin  Thyroid  T3, T4  Adrenal  adrenalin Skin 

Function: - Barrier - Immunological information - Homeostasis - Sensory information - Part of endocrine system - Excretory

Epidermis  Stratified, keratinized squamous, avascular  5 stratum: - Basale  undergo mitosis, 1 layer - Spinosum  bulk of epidermis, several layers, polymorphic, have spines - Granulosum  1-3 cells thick, contain granules of precursor to keratin - Lucida  only in thick skin, 1-2 cells thick, contains keratin intermediate - Corneum  keratin layer, nucleus and organelles gone, dead  Cells in epidermis  - Keratinocytes (main) - Melanocytes  secrete melanin, protect against UV; also called halo cells - Langerhan’s  immune cells – first layer of defence, migrate into higher layers when a foreign particle enters the skin

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- Merkel  type of nerve, mechanic receptor for sensory perception Dermo-Epithelial Junction - Dermal papillae (up) and rete ridges (down) create an interlocking interface - Increased presence in areas of high-mechanical stress  aka fingerprints Dermis - Papillary layer – loose connective, thin collagen & elastic, many nerves and blood vessels - Reticular layer – thicker, less-cellular, thick collagen and elastic, oriented in regular lines Hypodermis - Loose connective - Fine collagen/reticulin/elastin fibres - Energy storage and insulation

Nerve Endings  Called corpuscles  Sense touch and temperature  Meissners  in dermal papillae  Pacinian  in deep dermis/hypodermis; vibration/touch; looks like onion ring Glands  Sweat glands  eccrine, secrete to surface; secretory segment - simple cuboidal; duct – stratified cuboidal LECTURE 4 – Connective Tissue (Extracellular Matrix) 11/03 Extracellular matrix provides support to cells and organs CT  

Originates from mesoderm (middle embryonic germ layer) Mesoderm develops into mesenchyme  loose spongy, packing material - Supportive, provides strength, assists differentiation, permits diffusion - Forms CT, cartilage, bone, blood, lymphatic/haemopoietic tissue

Ground Substance of CT  Transparent, hydrated viscous gel  Lost during fixation – not seen in H&E stains  Contains glycosaminoglycans (GAG’s) and glycoproteins  Need to know Collagen Type !, II, III & IV GAG’s of GS  Linear polymers of repeating disacc units  Covalently linked as side chains to protein backbone – form proteoglycans  PG’s aggregate to form larger molecules that bind cations (w/ water) and regulate movement of molecules through GS

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GAG’s synthesized by CT cells: hyaluronic acid, chondroitin, dermatan, keratin, heparan sulfates

Glycoproteins of ECM  Structural proteins (can be seen under microscope)  Characterized by their individual AA sequence  GPs vs Proteoglycans - both join sugar chains to protein backbone - GP have shorter sugar chains w/ wider variety - protein determines main function o f GP - sugar chain determines main function of proteoglycans GP Fibres in CT  Provide tensile strength and deformability  3 types: Reticulin, Collagen, Elastin, Fibronectin  Produced by fibroblasts in CT  Reticulin - Type I - Very fine – found in areas that lack space – provides strength without bulk - Important in basement membrane, marrow, lymph nodes, supports fat, small blood vessels, nerves and muscles  Collagen - Type I & II - V thick  thickness & strength varies with type (38 combinations) - Mostly composed of fine fibres of 3 peptide chains helically twisted - Type II is finer than I  first tissue laid down when healing a wound; Type I forms the scar - Banding is caused by staggered arrangement of collagen molecules when they assemble into fibrils  Fibronectin - Aids cell adhesion, growth, migration, differentiation, healing & embryonic development - Has PG, fibrin & collagen binding regions - Involved with signalling of cells  Elastin - Found in loose connective tissue and blood vessels - Cylindrical threads or flat ribbons - High refractile (appears to shimmer) under a microscope - GP containing desmosine and isodesmosine - Randomly coiled molecule – not structured or patterned Basal Membrane  At basal surface of all epithelia  Molecular filter  All connective tissues are limited by BM  EM shows it is comprised of 3 zones

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Laminin - 4 arms  3 short bind other laminin (sheet forming); long arm binds to cells

Cells of CT  Fibrocytes - Retain ability to divide (stem cell) - Synth extracellular matrix - Pale, fusiform (cigar-shape) - Squashed amongst CT that they make – may be mistaken for smooth muscle cells - Healing wound  myofibrils form to help contract the tissue ends - Can form int, smooth muscle (blood vessels), & osteocytes/chondrocytes (bone) when healing a wound  Adipocytes - Synth and store triglycerides - Unilocular (one vacuole)  crowded signet ring appearance - Multilocular  in brown fat (E generating fat)  Macrophages - Ability to phagocytose - Contains lytic enzymes - Different name depending on tissue: Kupffer (liver), microglial (brain), Langerhans (skin), osteoclast, chondroclast  Mast cells, similar to basophils (from blood) - Contain large granules that can produce a stain colour different to original stain - 2 types: near blood vessels (contain anticoagulant and vassodilater); in mesentery (contain chondroitin sulfate) - Secrete molecules involved in inflammation and allergic response LECTURE 5 – Blood and Lymphatic Vessels 11/03 Tunica Intima  Endothelium  Basement membrane  Subendothelial layer of CT  made of III collagen & elastin  Internal elastic lamina (membrane) Tunica Externa  Vasa vasorum  blood vessels feeding the blood vessel

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Blood vessels leave and enter organ at hilum Larger are bundled together

Capillary  Forms meshwork in CT  every section is fed

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Inner surface is lined w/ endothelium Just tunica intima Elongated contractile pericytes wrap around capillary to squeeze if required 3 types: - Continuous  no gaps b/w epithelia, basement membrane is intact; substances enter and leave through vesicles - Fenestrated  has pores – not really a hole, just has a v thin membrane over the perforation - Sinusoidal  gaps b/w endothelial cells, basement membrane not intact, has larger holes  larger vessel (associated w/ macrophages)

LECTURE 6 – Muscles 18/03 Skeletal Muscle  Sarcolemma  membrane, invaginations = T tubules  Sarcomere  myofibrils linked in series  SR  muscle ER  Cells (w/ nuclei at periphery)  Fibre  Bundle  Fascicle  Connective tissue around muscle: Endomysium  Perimysium (type I collagen and fibroblasts)  Epimysium  Classification - Slow (Red)  type I, rich vascularization, abundant myoglobin - Fast (White)  Type IIA (no fatigue), Type IIB (fatigue), anerobic - Can sub-classify by location (or enzymes)  e.g. Type I prolific in postural muscles - 60% fast and 40% slow Enervation  Neuromuscular junction (motor) - Each muscle cell innervated by a peripheral motor neuron - Approaching junction  nerve loses myelin, branches into telodendria - Telodendria lie in shallow grooves on myocyte  Muscle spindles (sensory) - Consist of small groups of thin muscle fibres known as intrafusal fibres - Separated from extrafusal fibres by connective tissue capsule Smooth Muscle  Individual fusiform cells w/ central nucleus  Non-striated  Contain actin and myosin which criss-cross the cell (not nucleus)  whole cell contracts and cytoplasm is brought together around nucleus  Actin filaments anchored w/ dense bodies  Sparsely innervated  Can proliferate to replace lost smooth muscle cells Cardiac Muscle  Striated single cells, central nucleus

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Bound by junctions called intercalated discs  important for electrical coupling – contracts in unison Branched  interconnect to cells beside and across from it – one cell can have ~8 connections No ability to regenerate – can undergo hypertrophy  increased work load = cells will bulk up to do the work required Visceral – around organ, parietal – around cavity in which the organ sits Epicardium/Pericardium has a mesothelium layer  produces a lubricant

LECTURE 7 – Cartilage and Bone (18/03) Bone Types  Woven (spongy)  weight-bearing, mature  Lamellar (hard)  supportive, scaffolding of inner bone, precursor Cellular Composition  Osteoprogenitor  lining peri/endosteum; mesenchymal – stem cell  Osteoblast  bone forming, regulate mineralization  Osteocyte  quiescent/resting cells that regulate Ca & POH in serum  Osteoclasts  macrophage/remodelling; secrete collagenases; respond to mechanical stresses on bone to provide strength Chemical Composition  60-70% mineralized  hydroxyapatite, Na, Mg, F  Organic  Type I collagen (23%), GAG’s  10% water (bound to GAG’s) Growth  From Epiphyseal plate  Osteoid is mineralized and bone forms as it is vascularized

Lamellar Bone  From osteoid  Aligned into parallel sheets of fibrils  Composed of cylinders  osteons  Between each layer = osteocytes. Osteocytes penetrate the layers with canaliculae and can communicate w/ each other  Surrounded by circumferential lamellae and then periosteum  Endosteum lines marrow cavity  Central canal contains neurovascular components Woven Bone  Within core  Collagen runs in various directions  Oriented to provide strength against mechanical stress  Lined w/ osteoblasts Cartilage  Does not regenerate  Avascular  Supplied by perichondrium and synovial fluid  Form epiphyseal plate  So well-hydrated that it can be pressed and bounce back Cells   

Chondroblasts  mesenchymal, derived from perichondrium Condrocytes  quiescent, sit in own lacunae, secrete cartilage matrix Perichondrium  capsule of mesenchymal cells, vascular (feeds cartilage)

Matrix  Gel-like  Contains GAG’s, hyaluronic ac, chondroitin, collagen fibrils, proteoglycans, 80% water (bound to –ve GAG’s) Types  Hyaline (glassy)  Type II, grouped cells, perichondrium  Elastic (pliable)  Type II collagen, elastin single chondrocyte, perichondrium  Fibrocartilage  Type I, no perichondrium, structured, single chondrocytes LECTURE 8 – Nervous System Meninges  Dura mater - Outer - Fibrous - Epidural space periosteal side  attached to cranium, contains rich blood supply  Arachnoid mater - Leptomininge

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- Subarachnoid space contains vessels filled w/ CSF Pia mater - Leptomininge - Capsule of brain and SC - Delicate membrane covered w/ simple squamous epithelium

Brain  Grey matter (cortex)  nerve  White matter (medulla)  neuronal processes and neuroglia  CSF  outside and inside (subarachnoid space, ventricles, central canal of SC) Ependyma  Ciliated specialized epithelium w/ microvilli  No base membrane  Absorb and circulate CSF  Lines ventricle (brain) & central canal (SC)  Choroid plexus cells in ventricles make CSF Neuronal Cell Body  Up to 135 um  Uni-, bi-, and multi-polar  Varying shapes depending on location  Perikaryon - Around nucleus - Cell body - Remain in CNS (grey matter)  Nucleus - large & spherical - Fine chromatin  Nissl bodies - Evenly distributed - Clumps of rough ER and polysomes (ribosomes)  Neurofilaments - Throughout neuron and cell processes

Neuronal Processes  Axon  b/w mm and m in length - Transmit - No Nissl bodies - Most are myelinated (oligodendroglia, Schwann cells)  Dendrites  short, usu branched - Receive - Contain Nissl bodies Cerebellum  Many folds

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Thin layer of grey matter in cortex Granular layer of neurons (Purkinje cells) & neuroglia

Neuroglia (glue)  Support cells  metabolic exchange  Types: - Oligodendroglia  myelin - Astroglia  ground substance; attach to neurons, blood vessels, glia & pia mater; regulate [K+] - Microglia  phagocytosis Blood Brain Barrier  Capillaries have reduced permeability to macromolecules  Junctions b/w endothelial cells (zonulae occuldentes) prevent transport b/w them  Astrocytes provide a further covering Spinal Cord  Surrounded w pia mater  White mater of cortex  myelinated and unmyelinated  Grey matter of medulla  motor neurons and posterior horns (receives sensory information)  Note: matter of cortex and medulla is opposite to that of the brain PNS 

Ganglia - Group of nerve cells bodies outside CNS - Variable in size/no. of neurons  Nerve bundles in PNS - Axonal and dendritic processes - Encapsulated by collagen & w/ reticulin support  Epineurium  Perineurium  Endoneurium  Schwann cell  forms myelin; nodes of Ranvier (salutatory conduction) LECTURE 10 – Haematoxylin and Eosin Staining Chemistry  Natural/synthetic  Usu synthetic are organic - Aromatic  derived from HC benzene  Substitution of benzene is significant  known as quinone ring  O atoms w/ valency of 2 replace H in ortho or para positions simultaneously = quinoid/quinonoid cmpds Chromogens  Aromatic cmpds containing chromophore radicals  act as solvent dyes

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Alternating double bonds important to allow absorption of photons and thus colour Light transmitted or reflected f...