BMS100_ATH1-01_ConnEpithel_W23.pptx

General Histology – Epithelial and Connective Tissue BMS 100 Week 1 Overview In-class: Introduction to Histology Characteristics of Epithelial Tissue Morphology & Function Junctions & Interactions with Connective Tissue Characteristics of Connective Tissue Morphology – Connective Tissue proper Fu...

General Histology – Epithelial and Connective Tissue BMS 100 Week 1 Overview In-class: Introduction to Histology Characteristics of Epithelial Tissue Morphology & Function Junctions & Interactions with Connective Tissue Characteristics of Connective Tissue Morphology – Connective Tissue proper Functions Characteristics of Ground Substance Epithelial-connective interfaces - membranes Skin GI Tract Learning Outcomes For the following commonly used stains, list the molecules, cells, &/or organelles that will be highlighted. Hematoxylin, Eosin, Periodic acid-Schiff stain (PAS), Masson’s trichrome. Epithelial tissue: Review the common function of epithelial tissue Review the morphology, function, and common locations of the following epithelial cell types: Simple squamous, simple cuboidal, simple columnar, pseudostratified columnar, stratified squamous, and transitional epithelium For the following cell junctions, describe their key functions, locations, and the proteins involved (& their functions). Tight junctions, adherens junctions, desmosomes, hemidesmosomes Compare and contrast structure and function of the cell junctions listed above. Describe the structure and function of motile cilia and the primary cilia. Learning Outcomes Connective tissue proper: Describe the composition (cell types, fiber types, ground substance), location, and function of the following types of connective tissue proper: Loose connective tissue, dense irregular connective tissue, and dense regular connective tissue. Compare types I, II, III, and IV collagen in terms of structure and function. Describe two major components of ground substance in terms of structure and function. Explain how dysfunction in the epithelial lining, epithelial cells, or connective tissue can contribute to common sources of pathology. The skin: Describe the structure and composition of the epidermis and dermis of the skin. Briefly describe the epidemiology, etiology, common symptoms, and pathophysiology of atopic dermatitis. The Intestinal mucosa: Describe the structure of the intestinal mucosa and describe the two ”route” of transport through the epithelium. Briefly describe the epidemiology, etiology, and pathophysiology, and common symptoms of celiac disease. Introduction to Histology • Study of tissues under a microscope, usually after the tissues have been prepared in some way • Light microscopy – can visualize structures as small as 0.2 microns (µm, 1/1,000,000 of a metre)  Anything smaller needs to use a beam of electrons as the radiation source  Types of light microscopy include fluorescence microscopy and confocal microscopy • Confocal microscopy can view a cell or tissue in a particular plane (i.e. doesn’t see the plane above or the plane below, so it’s a thin, almost “2-D” image) • Fluorescence microscopy involves loading a cell with a fluorescent probe  Cells can be living or dead, depending on preparations, and allows for visualization of cells and larger organelles Introduction to Histology • Electron microscopy can visualize structures that are as small as 3 nm (i.e. molecular level of resolution)  Tissue sections are often frozen in liquid nitrogen and sliced into thin sections  Tissue or cell being imaged can also be coated in a thin layer of gold (scanning electron microscope)  Tissues/cells are always dead, but this is the only method that allows good visualization of organelles and large molecules Exotic Microscopes Confocal microscope – University of Saskatchewan Electron Microscope https://commons.wikimedia.org/wiki/ Histological Tissue Prep – Light Microscopy In general, tissues are prepared for examination by: • Fixation – chemicals cross-link proteins and inactivate enzymes that degrade cells/cellular components  However, chemical characteristics of molecules are mostly retained so that staining the tissue still occurs • Dehydration & clearing – tissues are passed through alcohol solutions (replaces the water) and then the alcohol is removed • Infiltration and embedding – the tissue is infiltrated with a substance (i.e. paraffin wax) and then allowed to harden • Trimming – tissue is sliced into thin, almost transparent slices using a microtome Histological Tissue Prep – Light Microscopy • Cells and the thin slices of tissue that are used in microscopy are pretty much transparent  Living and dead cells can be “loaded” with dyes that improve visualization • The process of exposing a cell to a dye or molecule that improves visualization is known as staining  Usually done with dead, fixed cells, but some stains can be done with viable cells  The process of staining often highlights certain molecules that have particular chemical properties  Cells can also be stained with fluorescent antibodies that bind to a very specific Histological Tissue Prep – Light Microscopy • Hematoxylin and eosin – usually both done together in a tissue preparation  Hematoxylin is a dark blue basic dye, and will bind to negatively-charged molecules (DNA in particular) • Molecules that bind to basic dyes are known as basophilic molecules  Eosin is a pink acidic dye – it binds to positively-charged molecules (i.e. cytosolic proteins) • Eosin is acidophilic • Periodic acid-Schiff stain – great at showing glycogen and many glycoproteins Common histologic staining procedures Stain Molecules highlighted Hematoxyli DNA, negatively-charged n molecules Eosin Proteins, positivelycharged molecules DAPI AO/EB stain DNA DNA & cell membranes Masson’s trichrome Keratin, collagen, DNA, cytoplasmic proteins Methylene blue PAS DNA Glycoproteins, glycogen Acid Fuchsin Collagen, mitochondrial elements Cells/organelles highlighted Nucleus Example: cartilage Proteins (cytosolic or extracellular) Example: keratin (contains many +vely charged AAs) Nucleus Apoptotic cells (programmed cell death) Muscle fibres, nuclei, collagen – complicated stain Nuclei Cytosol, mucous, some ECM Mitochondria Small intestine stained with H&E Small intestine stained with PAS basophilic cell nuclei are stained purple while cytoplasm stains pink. See how clearly the mucus-rich goblet cells and the apex of the intestine stain  glycoprotein mucus oligosaccharides on glycoproteins, such as the ends of the cells at the lumen (L) or mucus-secreting goblet cells (G), are poorly stained Better resolution/visualization Membrane stains more intensely (can visualize glycoproteins in cell membranes) Trichrome stains Masson's trichrome stain of rat airway. Connective tissue is stained blue, nuclei are stained dark red/purple, and cytoplasm is stained red/pink. Mouse skin stained with Masson's trichrome stain https://en.wikipedia.org/wiki/Masson Why is histology useful? • At the junction of anatomy and physiology  Much of the function of a cell or tissue can be deduced by its microscopic structure  Look back at the trichrome stain of the airway – what are those structures for? • Cells/tissues that are sick often look sick under the microscope  Can you pick out the fatty liver? Epithelial Functions Review • Protection  Function of all mucous membranes and the skin  Bladder is an interesting case – chemical protection from urine and can stretch for storage • Transport  Absorption - water, nutrients, electrolytes… almost anything your GI tract chooses  Secretion or removal of wastes – GI tract, kidney, lung  Exchange epithelium - Optimizes diffusion • thin cells that reduce diffusion distanced • endothelial cells, alveolar cells • Secretion of useful substances  Glands that secrete substances into ducts (exocrine) or hormones into the blood (endocrine) Epithelial Form and Function - Review Exchange epithelium Transport epithelium Microvilli in the GI tract are specialized for absorption and secretion Cilia – main function is motility (i.e. mucous) Microvilli – main function is increasing surface area Note: • Epithelium is avascular – no blood vessels • Nutrients, gases, wastes need to be exchanged via blood vessels in underlying connective Epithelial Form and Function - Review skin Chemical protection from urine Transport through epithelial linings: • Paracellular  between epithelial cells, movement across junctions • Transcellula r  through epithelial cells, movement across apical and Epithelial Form and Nomenclature • Epithelial cells/tissues are named according to their shapes  If flattened  squamous  If “square”  cuboidal  If “tall”  columnar • If there’s only a single layer of epithelial cells  simple • If multiple layers  stratified (we mostly have stratified squamous epithelial cells)  Stratified epithelia (i.e. skin) is named based on the shape of the cell farthest from the base  i.e. – even though there are cuboidal cells at the base of the epidermis, the cells at the “top” are flattened, they are still stratified squamous epithelium • Therefore stratified squamous epithelium • If there are cilia… then it’s a ciliated epithelium The Cytoskeleton in an Idealized Epithelial cell - review • Actin filaments – shape and motility of the cell • Intermediate filaments – structural “strength” to the cell • Desmin, keratin • Microtubules – determine polarity, cell division, movement of cilia (if present) Molecular Biology of the Cell, 6th ed. p. 893, fig. 16-4 Epithelial Cells and Junctions • Tight junctions • Adherens junctions • Desmosomes • Gap Junctions • Hemidesmosome s Junqueira’s Basic Histology, p. 74, fig. 4-4 Tight Junctions • Located at the apical aspect of almost all epithelial cells  Found in the gut, brain, skin, respiratory tract  Closest to the lumen of all the junctions • Key functions:  Barrier that prevents movement of undesirable substances to the tissues below  Regulates the movement of a variety of molecules between cells, through the barrier  Helps establish polarity – TJs seem to help direct membrane proteins to the apical vs. basolateral sides Tight Junctions Components Key proteins: • Claudins – trans-membrane proteins that can act as channels for small molecules (paracellular)  Some are permeable (Claudin-2), some are relatively impermeable (Claudin-1) • Occludin – trans-membrane protein, function not clear • Junctional adhesion molecules (JAM)  Trans-membrane protein that may mediate permeability to larger molecules • ZO-proteins  Important in tight junction formation, interact with the cytoskeleton https://commons.wikimedia.org/wiki/File:Life_cycle_and_protein_associations_of_connexin Tight Junctions Structure: • Claudins and JAMs are transmembrane proteins • extend into the extracellular space and bind to claudins and JAMs on the neighbouring cells • ZO proteins bind to the intracellular face of claudins and JAMs, linking them to the actin cytoskeleton underneath • Cell membrane proteins, even lipids seem unable to cross the “belt” of the TJs  Keeps basolateral and apical cell membrane components separated https://commons.wikimedia.org/wiki/File:Life_cycle_and_protein_associations_of_connexin Adherens junctions • Found immediately below tight junctions • Function:  Strengthens and stabilizes tight junctions  Participates in cellcell signaling that regulates cell division and proliferation Junqueira’s Basic Histology, p. 74, fig. 4-4 Adherens junctionsComponents Key proteins and their function • Cadherin – transmembrane protein that interacts with other cadherins on the neighbouring cell (similar to claudins) • Catenins – linker molecules that connect the intracellular face of claudins to the actin cytoskeleton  Beta catenin can also act as a signal  When cadherins connect across cells, beta-catenin remains associated with cadherins  When they don’t connect, beta-catenin can dissociate and signal cell division  How might this regulate wound healing? https://commons.wikimedia.org/wiki/File:Life_cycle_and_protein_associations_of_connexin Desmosomes • Both adherens junctions and tight junctions circle the entire apical aspect of a columnar of cuboidal epithelial cell  Desmosomes only attach to certain spots of the epithelial cell membrane Desmosomes and adherens junctions • Similarities:  Strong adhesion between cells  Desmosomes use cadherinlike molecules  Both have intracellular “plaques” that interact with proteins that can act as “signalers” and “linkers” (i.e. beta-catenin) • Differences:  Desmosomes connect to intracellular intermediate filaments (i.e. keratin)  Desmosomes provide more structural stability to the cell Hemidesmosomes • Significant differences in structure and function between hemidesmosomes and desmosomes  Transmembrane “linking” protein is an integrin, not a claudin-like molecule  Integrin binds to a component of the basement membrane known as laminin • Does not bind to a molecule on an adjacent cell  Hemidesmosomes do not seem to have important intracellular signaling functions • Hemidesmosomes do link to intracellular intermediate filaments • Function – adhesion of the epithelial cell to the basement membrane Cellular junctions and the Epithelium • Take-away – outline the structures that contribute to each role listed below (be specific):  Transport of substances from the apical side to the basal side of the epithelium (paracellular transport) • Tight junctions  Barrier that restricts movement of substances from the apical side to the basal side of the epithelium • Tight junctions  Strength of the epithelial lining • Adherens and desmosomes  Determination of polarity (apical vs. basal) across the epithelial cell • Tight junctions  Signaling and regulation of the activity of the epithelial cell • Desmosomes  Anchoring the epithelial cell to the underlying connective tissue • Hemidesmosomes Cilia and the Epithelial Cell • Some epithelial cells possess motile cilia (columnar)  The columnar epithelial cells of the uterine tubes and respiratory epithelium of the larger airways are prominent examples • Cilia have a 9 + 2 structure of microtubules, with a central doublet  Bound to a basal body-like structure at the apex of the membrane – also composed of microtubules (axoneme) • Whip-like movements that propel fluids (i.e. mucous) in a single direction Junqueira’s Basic Histology, p. 80, fig. 4-10 Motile Cilia – close-up Junqueira’s Basic Histology, p. 80, fig. 4-10 Cilia and the Epithelial Cell • Almost all cells – including epithelial cells - have one primary cilia  These are non-motile cilia that have a ring of 9 microtubular structures, but no central doublet  Very long – range from 1 – 10 microns (much longer than microvilli) • The primary cilia have a range of receptors and intracellular signaling mechanisms that communicate information from the external environment to the cell  Extremely important in development of the embryo, sensing fluid movements, and sensing the presence of growth factors Connective Tissue Proper Review Includes loose and dense connective tissue • Loose – often found beneath the epithelial lining of many tissues  Lamina propria of the intestine, respiratory tract  Can also be found as “packing” between muscle fibres, within nerves, etc. • Lots of ground substance, many cells, relatively little collagen  Collagen is randomly distributed Connective Tissue Proper Dense irregular connective tissue • Fewer cells, less ground substance than loose connective tissue • Much more collagen  Collagen is arrayed in bundles that are not parallel, but arranged in many different directions  Resists stresses from multiple different directions • Found in capsules that surround organs and in the dermis (located beneath epidermis of skin) Junqueira’s Basic Histology, p. 117, fig. 5-19 Connective Tissue Proper Dense irregular connective tissue • In this picture, both dense irregular (D) and loose connective tissue (L) are visible  Eosin stains the collagen pink  The nuclei are a darker purple  Note the difference in both across the two tissues Junqueira’s Basic Histology, p. 117, fig. 5-19 Connective Tissue Proper Dense regular connective tissue • Lots of collagen (type I) with less ground substance and cells than loose connective tissue • Collagen is oriented in one particular direction  Resists stresses along one line or plane • Typical examples – tendons, ligaments, aponeuroses • This image shows the orderly orientation of collagen (top) and the fibroblast that is surrounded by type I collagen fibres it has built Junqueira’s Basic Histology, p. 118, fig. 5-20 Connective Tissue Proper Fibres Collagen • Synthesized by fibroblasts • Different types of collagen have different functions  Type 1 – resists tension, multiple triple helices bound together to form fibrils, and fibrils are organized to form fibres  Major collagen type in dense CT and bone Junqueira’s Basic Histology, Text and Atlas – 13th ed Page 108, fig 5-11 Connective Tissue Proper Fibres Collagen cont… • Type II collagen – smaller fibrils with less organized orientation than dense regular tissue  Major component of cartilage – mainly resists pressure and absorbs shock • Type III collagen – reticular fibres  Major component of loose connective tissue • Type I, II, and III collagens are known as fibrillar collagens • Type IV collagen – forms the basement membrane that connects epithelial and connective tissue layers  Forms a sort of cross-linked “net” with laminin (glycoprotein) and proteoglycans interspersed within it Type IV Collagen in the Basement Membrane • The basement membrane is formed from an organized meshwork of type IV collagen, proteoglycans, and laminin  Note that integrins (hemidesmosomes) bind to the laminin in the basement membrane Pompili et. al., Front. Med. 8:610189 2021 doi: 10.3389/fmed.2021.610189 Connective Tissue Proper – Ground Substance Two major components: • Multi-adhesive glycoproteins  These bind to a wide variety of components of the extracellular matrix • i.e. laminin binds to type IV collagen and the integrins of hemidesmosomes • Fibronectin binds to collagen, glycosaminoglycans (GAGs) on proteoglycans, and some integrins • Proteoglycans – 3-part structure:  A very long, linear polymer of hyaluronic acid (a GAG)  Linking proteins attached to the hyaluronic acid polymer  Shorter GAG chains attached to the linking proteins Proteoglycans • Highly hydrated – “collect” water in the ECM due to the OH-groups on the carbohydrate GAGs • Link between collagens and glycoproteins – help bring structural integrity to the ECM • An ECM rich in proteoglycans is difficult for most bacteria to penetrate • “Store” of growth factors  Messengers can be “stored” within the ECM – associated with proteoglycans  When the ECM is broken down, these factors are liberated  replacement of the ECM Interactions – Epithelial and Connective Tissue • The skin and the mucosal membranes are involved in a wide range of conditions relevant to naturopathic medicine  Understanding these conditions hinges on understanding the normal microscopic anatomy of the epithelial and connective tissue interfaces and functions • Common sources of pathology:  Disrupted barrier or protective function of the epithelial lining  Disrupted transport across the epithelial lining  Inflammation in the connective tissue below the epithelium • Due to autoimmune or allergic conditions The Skin – General Structure • Stratified squamous epithelium  Typical functions of a stratified squamous epithelium? • Protection against abrasian • Apical layers – cells that accumulate keratin, “compact” it, and eventually die  Stratum granulosum, lucidum, and corneum  Keratin = main intermediate filament in keratinocytes  Keratin is strong and forms bundles – a barrier that prevents water loss from deeper layers and microbe invasion  It complexes with another protein – filaggrin – that helps compact keratin and attracts water, aiding in skin moisturization The Skin – General Structure • As skin matures from deeper layers, junctions are modified  Loss of hemidesmosomes (no contact with the basement membrane)  Modification of desmosomes  Tight junctions remain • Net result – the “outside” surface of the skin is flattened layers of dead “bags” of keratin and filaggrin linked by tight junctions The Skin – General Structure • The dermal layer lies under the epidermis  Dense irregular connective tissue  Capillary loops extend from the papillary dermis, bringing nutrients and exchanging gases and wastes  Dermal vasculature allows immune cells to enter the epidermis • Fight infection • Heal wounds Atopic dermatitis • One of the most common skin conditions  Typically begins in childhood 10 – 30% of children affected  Becomes less common with age – 2 -10% of adults • Typical symptoms & signs:  Itchy papules and plaques that can become excoriated with scratching  Distributed over the extensor surfaces, face, and scalp  Worsen in response to allergen exposure https://commons.wikimedia.org/wiki/ Atopic Dermatitis Pathophysiology • Highly heritable – children of parents with atopic dermatitis have a 50% chance of developing the disorder  Subtle abnormalities in filaggrin impair the ability of the more apical strata to retain the moisture of the skin  Tight junction changes to more permeable types decrease the barrier function of the skin • Postulated sequence of events:  Impaired skin barrier  repeated introduction of antigens to immune cells that reside in the epidermis and the dermis  recruitment of other, particular immune cells into the dermis and epidermis from the blood stream  a specific type of inflammation (type 2) that causes excessive histamine release into the skin  chronic swelling and itch with further antigen exposure The Intestinal Mucosa – General Structure • Skin and intestinal mucosa have vastly different functions  Reflected in the structure of the epithelial and connective tissue layers • Simple columnar epithelium – prominent apical microvilli  Specialized for absorption of nutrients and water • Interspersed with cells that have glandular functions  Many of these secrete mucous (known as goblet cells)  Mucous has a protective and a digestive role Enterocytes and Goblet Cells M Small intestine stained with H&E basophilic cell nuclei are stained purple while cytoplasm stains pink. Small intestine stained with PAS Note again - mucus-rich goblet cells (G) and mucous covering over the microvilli (M) The Intestinal Mucosa – General Structure • The epithelial layer sits on a bed of highly vascularized loose connective tissue known as the lamina propria  Blood and lymphatic capillaries  absorption of nutrients and water from across the epithelial cell into blood  Immune cells are present in this layer  protection from hostile microbes and tolerance to healthy microbes  Layer of smooth muscle – the muscularis mucosa  helps maintain the shape of the structure • Finger-like projection of epithelium and lamina propria = the villus Anatomy and Physiology, 2e p. 1024, fig. 23.19 The Intestinal Mucosa – General Structure • As we zoom in we can see that there are two routes “through” the epithelium:  Paracellular route between adjacent enterocytes - mostly regulated by tight junctions  Transcellular route through enterocytes, across cell membranes – regulated by membrane proteins Anatomy and Physiology, 2e p. 1024, fig. 23.19 Celiac disease • A common immune-mediated disorder – individuals mount an immune response to a component of gluten (known as gliadin)  Gluten is present in wheat, barley, rye  Common – up to 1% of the population • The inflammatory response damages the delicate structure of the villus  Only small amounts of gluten are needed to perpetuate the migration and activation of massive numbers of immune cells into the lamina propria  The villus becomes flattened, enterocytes damaged  reduction of surface area for absorption (remember Fick’s law?)  diarrhea, poor absorption of nutrients, and varying degrees of malnutrition in many patients Celiac disease • How does gliadin get into the lamina propria?  Shouldn’t tight junctions keep it out? • Possible (likely) paracellular route 1:  Gliadin binds to a protein (FYI – chemokine receptor CXCR3)   signaling cascade that causes release of a signalling protein called zonulin   Zonulin release leads to phosphorylation of ZO proteins   disassembly of claudin and occludin proteins at the tight junction   leakage of gliadin into the immune cellcontaining lamina propria https://journals.physiology.org/doi/full/10.1152/ajpgi.00386.2020 https://commons.wikimedia.org/wiki/File:Life_cycle_and_protein_associations_of_connexins. Wrapping it up… • The interaction between epithelial tissue and connective tissue in a wide variety of organs is crucial to understanding many aspects of physiology and pathology  The interactions can get complicated (i.e. atopic dermatitis and celiac disease)  best understood within the context of the general structure and functional interaction between both sets of tissues  This interaction is at the cellular and molecular level FYI Articles for the curious: • Larazotide acetate: a pharmacological peptide approach to tight junction regulation  https://journals.physiology.org/doi/full/10.1152/aj pgi.00386.2020  Celiac disease article • Filaggrin in atopic dermatitis  https://www.jacionline.org/article/S0091-6749(09 )01123-3/fulltext • Claudin-1 mediated tight junction dysfunction as a contributor to atopic march  https://www.frontiersin.org/articles/10.3389/fim mu.2022.927465/full