Histology Final Practical Exam PDF

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Final Practical Exam Summary ...

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EPITHELIUM  Coverings on external surfaces (skin)  Linings on internal surfaces (lumen of tubes)  Glands originate from invaginated epithelial cells Function  Protection from abrasion and injury  Transcellular transport of molecules  Secretion of mucus and hormones  Absorption of material Characteristics  Closely opposed and bound by junctional complexes  Have morphological and functional polarity (apical, lateral and basal domains)  Separated from the underlaying connective tissue layer by basement membrane  Avascular Classification  Number of layers of cells o One = Simple o More than one = Stratified  Morphology of the surface layer o Flat = Squamous o Squarish/Cube like = Cuboidal o Tall = Columnar  Surface specialisations o Microvilli (brush border) o Cilia o Keratin Membrane specialisations Interceullular junctions  tight or occluding junctions: luminal contents cannot penetrate between the lining cells e.g. intestines (zonala occludens)  adhering junctions: anchorage sites for cytoskeletal elements, band like (zonala adherens), desmosomes (macula adherens)  communicating junctions: gap junctions, allow passage of small molecules between adjacent cells

luminal (apical) surface

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cilia: relatively long motile structures, structured microtubular arrangement, movement along the surface (respiratory epithelium) microvilli: short extremely numerous projections of the plasma membrane, actin filaments provide some stability, contraction, increase surface area for absorption cells (intestines) stereocilia: merely long microvilli (epididymis), in single or small clumps

basal surface  basement membrane (basal lamina): between cells and underlaying CT, non cellular- ECM proteins, provides structural support, acts as a selective barrier  folds in BM: in cells active in ion transport (kidney), enhance surface area  hemidesmosomes: anchorage of cell cytoskeleton, plasma membrane and BM Epitheloid tissue exceptions  Close apposition  Basement membrane  NO free (apical) surface  Most endocrine glands, including….. o Interstitial (Leydig) cells of the testis o Lutein cells of the ovary o Islets of Langerhans in pancreas o Anterior lobe of the pituitary gland

MUSCLES

Function: essential for contraction Muscle is made up from 75% water, 20% protein, 5% organic and inorganic compounds Muscle cells are elongated= muscle fibres Contains myofilaments which allows the muscle to contract Skeletal muscle  Provides voluntary movement of body  Voluntary control, can be reflexive too  Very large, multinucleated cells  Nucleus located at periphery  Cross striations Microscopic anatomy of muscle  Nuclei are just beneath the sarcolemma o Specialised plasma membrane of muscle cells o Invaginations = t tubules  Cytoplasm filled with myofibrils o Perfectly aligned, ribbon like organelles, gives muscle fibre its striped appearance  Sarcomere o Contractile unit of the myofibril o Z to Z disc  Myofilaments o Thick filament= myosin o Thin filament= actin, tropomysin and troponin  Sarcoplasmic reticulum o Smooth ER, stores calcium, released when the muscle is stimulated to contract Longitudinal plane of skeletal muscle

Connective tissues

transverse/cross section

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Endomysium: around single muscle fibre Perimysium= around a fascicle (bundle) or fibres Epimysium= covers the entire skeletal muscle Fascia= on the outside of epimysium Tendon= dense CT attaching muscle to bone

Contraction and relaxation  Neuromuscular junction: motor neurons have a threadlike axon that extends from the brain or SC to a group of muscle fibres. Action potentials arise at the interface of the motor neuron and muscle fiber  Synapse: where communication occurs between a somatic motor neuron and a muscle fibre, the cleft is the gap that separates the 2 cells  Acetylcholine is released from synaptic vesicles into this cleft and is directed to the motor end plate  Telodendrion= where the axon loses its myelin sheath as its on the myocyte surface

Cardiac muscle  Involuntary (no conscious control)  Cardiac muscle tissue contracts when stimulated by its own autorhythmic fibres o This is initiated by the sinoatrial node (pacemaker of the heart)  Striated, single cell, nucleus centrally located  Branching cells connected by intercalated disc= allow muscle action potentials to spread to each fibre  Small amounts of endomysium between cells  Only found in the walls of the heart  Have the same arrangement of actin and myosin in skeletal muscle Cardiac muscle cell adaption  Compensatory hypertrophy  Increased work load  Loss of surrounding muscle cells  Cardiac fibers are arranged in spiral or 8 shaped bundles Longitudinal plane

Smooth muscle

cross section/transverse plane

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Involuntary (no conscious control) Spindle shaped (fusiform) with central nuclei Non striated (but contains actin and myosin) Small amount of endomysium Slow, sustained, tireless movement

Locations  Walls of veins, arteries, airways  Hollow organs= gut, gall and urinary bladder, utereus, ureter and fallopian tube  Fibromuscular stroma of the prostate  Muscles that attach to hair follicles Longitudinal plane

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cross section/transverse

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Ganglia= group of nerve cell bodies outside the CNS Nerves= collection of nerve fibres No lumen in nerves, solid structure Satellite cells= protects neuron cell bodies, surrounds a layer around the cell bodies of ganglia Bundles (fascicles) or nerve fibres are encapsulated by collagen with reticulum support layers o Endonerium= individual cells o Perinerium = nerve fascicles o Epinerium= whole nerve bundle Schwaan cell o Supports cell forms myelin o Made from glycolipid o Nodes of ranvier= exposed axon

BLOOD AND LYMPHATIC VESSELS

The circulatory system  Closed system consisting of 2 circuits: systemic and pulmonary  Arteries carry blood away from the heart  Veins carry blood to the heart General features of arteries and veins Intima  Endothelium= simple squamous epithelium lining the inside of lumen  Basal lamina= basement membrane of the endothelium (made up of collagen, proteoglycans and glycoproteins)  Sub endothelial layer= loose connective tissue  Internal elastic lamina= elastic tissue in arteries and arterioles Media  Smooth muscle cells= principal component in this layer  Other components= collagen, elastin and proteoglycans  External elastic lamina= in arteries, between the media and adventitia Adventitia  Collagen= principal component of connective tissue  Elastin= smaller component  Thicker in the vein relative to the respective artery o Veins require their own blood supply= vaso vasorum  Nervi vascularis= network of neurons that regulate contraction of SMC Arteries Large/ elastic arteries Medium/muscular arteries  E.g.  Most named arteries aorta and of the body primary branches,  Intima= thick, sub  Intima= thin, prominent internal endothelial layer of elastic intima (black collagen, elastin and line) SMC  Media= thick, whorls  Media= SMC and spiral layers of SMC, elastic layers, with with collagen but collagen and less elastin proteoglycans  Adventitia=  Adventitia= loose CT separated by with vessels and external elastic neurons lamina, thicker, same  Conducting vessels thickness as media  Resistance vessels

Small arteries & arterioles  Intima= Similar endothelium, internal elastic lamina is absent in arterioles  Media= thicker (8 layers) in small arteries, in arterioles its 1 or 2 layers, capillary end in arterioles forms into the precapillary sphincter  Adventitia= thin  Flow regulators

Capillaries  Networks of capillaries are the sites of exchange between the blood and surrounding interstitial fluid

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Narrowest vessels with a diameter comparable to a rbc (7um) Wall= single endothelial layer with a supporting basement membrane

Continuous  They have a continuous sheet of endothelial cells  Sealed together via zonula occludens  Pericytes surrounding the capillaries which are contractile  Found in the muscles, lungs and CNS

Fenestrated  Fenestrations which are 80100nm in diameter often with a thin membrane  Found in the GIT and glomerulus of nephrons

Sinusoids  Wider than other capillaries  Found in the liver, spleen and marrow

Venous system Large veins Medium veins Venules  e.g. vena cava  thin intima= no  Post capillary  no valves internal elastic venules with an  intima= SMC at the lamina endothelium intima media  media= thinner than surrounded by boundary artery, SMC with pericytes  media= thinner that collagen and elastin (inflammation) artery  adventitia= thicker  Muscular venules=  adventitia= thickest than the media posses thin media layer of longitudinal  possess valves to (no pericytes) and SMC prevent backflow adventitia LYMPHATIC SYSTEM  The lymphatic system is a network of vessels and associated organs that essentially runs in parallel to the circulatory system in most parts of the body  The fluid which travels through the system is called lymph  Terminal lymphatics or lymphatic capliaries make up this system o Found closely to capillary beds, except in the CNS bone o Blind ended tubes with a single endothelial layer and discontinuous basal lamina o Converge to form larger collecting vessels  Possess valves  Drainage o Lymph from right upper body drain into right lymphatic duct → subclavian vein o Lymph from other parts of body drains into thoracic duct → subclavian vein

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Without a pumping organ, lymph transport is very slow and relies on rhytmic contraction of smooth muscle, milking action of skeletal muscles and adjacent arteries and a pressure gradietn between thorax and abdominal cavities Lymph only carried in one direction: towards the heart

EXTRACELLULAR MATRIX Connective tissues

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Originates in the mesoderm in the embryo- mesenchyme Mesenchyme is loose spongy tissue, serves as packing, penetrating the developing organ Support, strength, role in differentiation, communication between cels, diffusion of nutrients, oxygen and wastes The mesenchyme differentiates into connective tissue, cartilage, bone, blood, lymphaitc and hematopoietic tissues Scaffolding forms the inercellular matrix= ground substance = interstitium and fibres aka ECM or STROMA

ECM ground substance  Transparent, colourless and homogenous hydrated viscous gel at 37  Is lost during tissue prep for histo slides  Contains glycosminoglycans (GAGs) and glycoproteins (GP)  GAGs include o Linear polymers of repeating disaccharide units (hexosamine and uronic acid) o Covalently linked to a protein (becoming a proteoglycan) o Principally gags are hyaluronic acid (hyaluronan) and sulfates of chrondroitin, dermatan, keratan and heparan  Aggregate to form larger molecules binding na, k, ca and water  Cerebral cortex= background tissue= neuropil, pink in colour, contains ground substance as well as dendrites and axons coming from the nerves Glycoproteins  Structural proteins with one or more attached hetero saccharides connected to a polypeptide backbone  These can make up to form glycoprotein fibres that provide tensile strength within the body and resist deformability of the body  Examples:  Collagen, fibronectin, laminin and chondronectin (cartilage) Fibres in CT  Provide tensile strength & deformability  3 types: all are complex proteins in long chains of amino acids (AA) with peptide linkages o Reticulin o Collagen o Elastin Reticulin  Very fine collagenous fibres (type III collagen)  Single fibril  Forms a fine network of supportive fibres known as reticulin  Important in basement membranes, bone marrow, lymph nodes, support fat cells, small blood vessles, nerves and muscles  Forms fine partitions in the lung and supports liver sinusoids

Collagen  Extremely tough e.g. tendons  Thickness and appearance varies with type  Type of collagen depends on location and functional need  Composed of fine fibrils of 3 peptide chains helically twisted together (triple helix)  Made by fibroblasts Elastin    

Found mostly in loose connective tissue (skin) and other blood vessels Either cylindrical threads or flat ribbons which are highly refractile under light Glycoprotein containing amino acid desmosine and isodesmodine which crossink Elastic fibres include elastin, elaunin and oxytalan

Basement membrane  Basal lamina  At the basal surface of all epithelia  Flexible and firm support, role in differentiation, acts as molecular filter (sieve)  BM limit all CTS  Em shows 3 layers of basal lamina= lamina rara, densa, reticularis Cells of CT  Fibrocytes (resting, inactive) → fibroblasts (active)  Fat cells (adipocytes)  Macrophages and mast cells (from blood) Telocytes  Also known as interstitial cajal cells  Different from fibroblasts and pluripotent mesenchymal cells o Long extensions called telopodes  Controls microenvironment= communication, receive and send signals, stimulate cell differentiation Fibrocytes  Multipotent and retain mitotic activity  Synthesise the ECM when activated  Pale fusiform (cigar) shaped cells  Mistaken for smooth msucle cells

Fibroblasts  Derived from the fibrocytes  In wound healing acquire contractile filaments and become myofibroblasts and help contract wound size  Form pericytes around capillaries and can differentiate into smooth muscle

Adipocytes (fat cells)  Synthesise and store TAGS  Unilocular (one vacuole), signet ring appearance often polygonal because of their crowding o Nuceli are flat, pushed to the surface o During slide prep, fat is washed away by zylene  Multilocular (many vacuoles) seen in brown fat (heat production in young animals and hibernating ones) o Brown due to increased mitochondria o Nuclei are not as flat  Appear clear (empty) as fat is removed during slide preperation by solvents Macrophage  Tissue histiocyte  Has a phagocytic and surveillance role  Contains hydrolytic enzymes in lysosomes  Migrate out of tissues back into circulation through lymphatics  In liver = kupffer, brain= microglial, skin= langerhans Mast cells  Metachromatic granules in cytoplasm  Have IgE receptors on surface, responsible for allergic hypersensitivity reactions  Reside near blood vessels containing heparin (anticoag) and histamine (dilates capillaries)

BONE Lamellar Bone (80% skeletal weight)

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Aka cortical, compact or dense bone

Woven bone

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Aka cancellous, trabecular or spongy bone

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Weight bearing tissue of long bones, provides strength and movement Mature Is the transformation of osteoid= collagen aligned into sheets of parallel fibrils, composed of cylinders known as osteons or Haversian systems, this is surrounded by circumferential lamellae then periosteum, endosteum lines marrow cavity o Haversian canal and volksmann canal carries blood vessels

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Supportive, scaffolding structure of inner bone In healing bone is the immature precursor Function: blood cell production Within the core of long bones Lined by osteoblasts

Cellular composition of bone  Osteoprogenitor cell= bone linining cells of peri and endosteiums, origin= mesenchymal and are fibroblast like  Osteoblast= bone forming cells, bone mineralisation, make up bone ECM,  Osteocyte= quiescent cells that regulate serum calcium and phosphate, maintain bone, sits in lacuna (23% collagen and 10% water)  Osteoclass= macrophage, remodels bone, collagenases Bone matrix composition  60-70% mineralised component= sodium, calcium phosphate  organic component= type 1 collagen and GAGS

Cartilage  doesn’t regenerate  is avascular (no blood supply)  nutrients by diffusion from perichondrium and synovial fluid  forms the epiphyseal plate (growth plate)  function: shock absorption (deformable gel)

structure  chrondroblasts= mesenchymal chondrogenic cells of the perichondrium  chondrocytes= sit in their own lacunae, arranged according to type of cartilage, secretes cartilage matrix o GAGS, hyaluronic acid, chondroitin and keratin sulphates, proteoglycans, collagen, hydrated (60-78% water) bound to neg charges GAGS= basophilic, picks up haematoxylin  perichondrium= capsule of mesenchymal cells, vascular supply that feeds the cartilage types Hyaline Elastic Fibrocartilage  glassy  type 2 collagen  type 1 collagen  type 2 collagen  contains elastin  withstands strong tensile  chondrocytes ordered at  chondrocytes are found in a forces the growth plate in threadlike network of elastin  opaque appearance, firm  chondrocytes are single and isogenic groups (of 2 or fibres, sits under 4) perichondrium, they are sparse single  found in the nose larynx  no perichondrium   located in intervertebral discs yellow colour and more trachea and bronchi opaque than hyaline  translucent blue grey solid colour  located in the pinna and epiglottis

TISSUE PREPERATION AND FIXATION Tissue preparation modes  Routine paraffin blocks  Frozen sections on a cryostat, a refrigerated microtome

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Enzyme or lipid studies Immune

Cell death  No oxygen leads to cell death (muscles 20 mins,, brain 2 mins)  Putrefaction – destruction by microorganisms  Autolysis- degeneration by lysosomal enzymes  Abnormal metabolism Fixation- chemical tissue preservation  An attempt to preserve the tissue in a lifelike condition  Body stored cool and autopsy performed ASAP to slow down autolysis and prevent putrefaction  Organs removed, described and weighed  Placed in 10% phosphate buffered formalin- st solution is 4%, prevents further autolysis and preserves tissue better  Reasoning: prevents tissue breakdown (a and p), increase firmness for handlin, retains tissue structure and increases permeability for future chemical processing  How: denatures protein, breaking down autolytic enzymes, unfolding molecules, disrupts internal bonds (hydrogen and disulphide), increasing permeability= allowing molecules to make new links with fixatives and dyes Types of tissue preservatives  10% phosphate buffered formalin  Aldehydes= formalin  oxidising agents= osmium tetroxide  protein denaturing= alcohols  physical- heat and microwave  formalin= 5-7 hrs  FAA- 4-5 hrs  Bouins 3-4 hrs Factors for good fixation method  volume of fixative should be ten times or greater than that of tissue volume= ensures no dilution effect by tissue fluids  failure of staining method usually is a result of inadequate fixation Processing of tissue  need to make the tissue hard and supported so it can be cut into thin sections, but the problem is that water and wax doesn’t mix  Dehydrate tissue in increasing graded alcohol= leads to the infiltration of paraffin wax  Clearing in solvent (xylene), intermediate chemical, miscible dehydrating agent and paraffin wax embedding agent  Infiltration and embedding: vaccum infiltration wil molten paraffin wax Embedding tissue  Support tissue for cutting

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Produce a solid block embedded in paraffin Tissue placed in moulds, and molten paraffin is poured over, set and cool

Cutting and section preparation  Tissue blocks are sectioned on a microtome (cryostat)  Cut at 5um thick  Floated out to remove compression wrinkles  Picked up individually on glass slide, then baked Deparaffinisation of sections  Problem= wax and water don’t mix  Tissue sections need wax removed, stains are water or alcohol based  Sections are preheated to melt off wax  Placed in solvent xylene to dissolve wax  Rehydrated through decreasing graded alcohol Staining  Unstained sections are opaque and tissue structure is obscured  Staining makes tissue visible and gives cellular elements contrast  Haematoxylin and eosin is the most common stain o Haematoxylin s...