Biomedical Histology Quiz 2

what is purpose of connective tissue (CT)

• physical and chemical support of other tissue and organs

  • around/between other tissue types

  • cells do not form junctions with each other

  • Extracellular matrix (ECM) is usually the most abundant component

  • ECM usually gives the tissue its characteristic properties

  • conduit for blood vessels and nerves

what are the components of ECM

1. Fibres

• Proteins that polymerize into long or short fibrils and fibres

• Primarily collagen and elastin

2. Ground substance

• ‘hydrated space’ (binds to water) with many proteins, multiadhesive glycoproteins (bind more then 1 thing) and proteoglycans (have more sugars then protein)

• Glycosaminoglycans =GAGs (hyluronan): polymers of modified sugar residues

  • sequesters water bind to hematoxylin

what are the 2 types of cells in Connective tissue

1. Resident (fixed) cells

• Synthesize and maintain ECM

• Fibroblasts and members of the fibroblast family

2. Transient Cells

• Cells of the immune system: leukocytes

• Secondarily take up residents in CT (macrophages) and/or traffic between CT and immune organs (lymphocyte)

Examples of transient CT cells

1. Macrophages

• Derived from blood monocytes

• Phagocytose microorganisms and damaged cells

• Long-term or short term residents

2. Mast Cells

• Similar to blood basophils

• Prominent granules contain vasoactive substances

• involved in allergic reactions

How do we classify fibrous CT

Loose (areolar) CT

• Abundant ground substance, few and relatively sparse fibres

• thin collagen 1 fibre bundles (may not be obvious in LM)

Reticular Connective tissue

• Fibres form 3D network of Type III collagen in a loose disposition

• Inside loose CT or in lymphoid organs where it forms scaffold to support cells

Dense CT

• Abundant collagen 1 fibres in large/prominent fibre bundles, comparatively less ground substance, few cells

  • Dense irregular CT = collagen bundles randomly oriented

  • Dense Regular CT = collagen bundles in regular orientation (aligned with fibroblasts)

How does Reticular Connective tissue stain

H and E

• collagen type III doesn’t stain

• Reticular cells are fibroblasts that make this collagen

• other cells are immune system cells

Silver stain - for reticular fibres

which is dense irregular and regular

left = dense irregular

Right = dense regular

what does loose and dense mean how is it shown

loose is the lighter pink, dense is darker pink (thinker fibres)

• this is because it is rich in collagen type 1 which is + so binds to eosin

What are ECM fibres made up of

Collagen

• approx 90% CT fibers are collagen

• protein polymers that make long straight fibres and meshworks

• Tensile strength (can withstand being stretched and pulled)

• very eosinophilic

Elastin

• Approx 10% CT fibers

• stretch and recoil

• poorly eosinophilic, light refractile, binds special stains

NOTE: most fibers see in LM will be collagen

what collagen made of/ collagen polymerization

25 different types

• all are made up of 3 polypeptides, arranged as triple helix

• outside cell, these collagen molecules polymerize into long fibrils

1. molecules polymerize into striped fibrils

2. fibrils grouped into larger fibres

3. fibres may further aggregate into fibre bundles

what each pointing at

Fibril

Fibre

Fibre bundle

porcollagen molecule

what are main types of collagen

Collagen Type I

• whole procollagen molecule is all triple helix

• makes lingest/largest and strongest fibers of body

• very strong in tensile strength but not stretchy

Collagen Type IV

• Procollagen molecules has globular/non helical regions at either end

• Form short segments of lateral association with multiple other type IV molecules (meshes)

• meshwork of collagen type IV interacts with laminin, fibronectin and other basal lamina proteins, collagens and ground substances in ECM

Collagen Diseases

Proper collagen arrangement in ECM requires

• many steps and enzymes both inside and outside cells to form correct fibrils

• several collagens and other molecules to form fibers and fiber bundles and other arangements

Collagenopathies due to mutations in collagen genes

• osteogenesis imperfecta =fragile bones

Collagen damage by imflammation

• rheumatoid arthritis

Defective collagen production/processing

• scurvy (vit C deficiency) = collagen goes to RER, vit C is a cofactor for collagen to be hydroxylated to help form proper connective tissue

What is elastin, what made of

• random coiled elastin molecules attached covalently together

• when fibre is stretched the coiled straighten but molecules dont pull apart; recoil upon release of stretch

• produced by fibroblasts and vascular smooth muscle cells

• two proteins in elastic fibres: core of elastin, surface network of fibrillin

• Fibrillin network acts as scaffold for assembly of elastic fibres

• mutations in fibrillin lead to Marfan Syndrome

  • general CT defects, elongated ones, hyperflexible joints, aortic aneurysm

More ground substance details

• water (60-90%) - can sequester

• Carbohydrate polymers

  • Glycosaminoglycans (GAG) - neg charge

  • Proteoglycans

• Multiple other soluble proteins and glycoproteins, including fibronectin, laminin and growth factors

• stains poorly with H and E

• provides turgor pressure (resistance to compression)

  • occupies large volume and resists compression because of bound water

• Can be watery (loose CT) to very thick gel (cartilage) depending on GAG concentration

  • gels inhibit cell movement depending on amounts of GAGs

  • water in gels is the medium of diffusion through CT

GAGs and PGs characteristics

GAGs: long, straight, polymers of sulphated or carboxylated sugars

PGs: multiple GAG molecules attached to a protein core

• Both carry net negative charge (bind water and basic dyes)

what adipose tissue made up of and found?

• Made up of adipocytes (fat cells)- found as single cells or in small groups in loose CT

• differentiate from mesenchymal/fibroblast like precursor

• Adipose tissue highly cellular (unlike most CT)

Adipose tissue types

1. Unilocular (white)

• triglycerides stored as single lipid droplet

• rim of cytoplasm with flattened nucleus

• energy homeostasis and hormone secretion; padding and insulation when large numbers

2. Multilocular (brown)

• triglycerides stored in multiple droplets

• round nucleus

• obvious cytoplasm with lots of mitochondria

• lipid metabolized in cell for heat production

  • animals that hibernate, babies as they grow disappear slowly

EXTENSIVE BLOOD SUPPLY

which is which

left = unilocular

Right = multilocular

Blood details

• plasma - tissue fluid (like ground substance minus fibres)

• Fibres of blood are soluble; proteins become fibrous upon activation of coagulation

• View blood using cell smear

  • eosin and 2 basic dyes but basophilic still blue even though hematoxylin not used

what are components of blood

1. “Formed elements” = Erythrocytes and platelets

• No nuclei or organelles

• Fragments of cells

2. Leukocytes (white blood cells)

• Granulocytes

  • Nucleated blood cells with specific granules or storage vesicles

• Agranulocytes

  • Nucleated blood cells without specific granules

What is the origin of blood cells

Hemopoiesis

• Red bone marrow = makes blood cells

• yellow bone marrow as get older red bone marrow replaced by fat

RBC lose their nucleus as they mature

what are Erythrocytes

• red blood cells

• O2/CO2 exchange and pH balance

• most abundant “formed element” in blood (40% volume)

• Homogeneous eosinophilic cytoplasm - 30% hemoglobin

• present in most tissue sections

erythrocyte morphology

• anuclear bi-conclave discs for maximim surface area

• very eosinophilic due to high hemoglobin content

• single mutation in hemoglobin gene leads to sickle cell generation

What is a leukocyte

• Nucleated cells or blood are larger than erythrocytes

• not really involved in functionality of blood directly

• Travel from site to site and surveillance (defence)

• Associated with responses to tissue homeostasis, damage, infection, immunity, allergy

cells in blood vs CT vs both

Granulocytic Leukocytes

1. Eosinophils - stain pink because granules are eosinophilic

2. basophils - stain hematoxylin purple

3. Neutrophils - little granuals don’t have significant colour

common features:

• from common bone marrow precursor

• Multilobed nucleus

• azurophilic granules (1degree granules; lysosomes)

Unique features

• specific granuals (2 degree granules)

• unique functionality for each cell type

Neutrophils (PMNs) details and function

• most abundant granulocyte

• multi lobed nucleus, immature has band like nucleus

• pale, poorly stained cytoplasm

• specific granules not abundant or large, so best seen in TEM

Function

• usually first immune cell to enter site of inflammation/infection

• recruited to specific sites by mediators and cytokines in underlying CT

• kill microorganisms by phagocytosis and secretion of antibacterial enzymes/peptides/ROS

• Most die in process; accumulated dead and dying PNMs create pus

Eosinophils details and function

• mid-sized and fairly abundant leukocytes in blood

• large bi-lobed nucleus

• pale, poorly stained cytoplasm

• large prominent eosinophilic specific granules

• granules contain rod shaped inclusions visible in TEM

function:

• Kill endo-parasites

• contents of specific granules able to perforate parasite membranes

• eosinophil products modulate mast cell activity

  • trigger histamine release

  • promote survival

  • intensify allergic inflammation

• Play important role in Asthma

Basophils details and function

• very rare <0.5% of all leukocytes

• large bi-lobed nucleus

• pale, poorly stained cytoplasm

• large, deeply stained basophilic granules, may obscure nucleus

• granules stain metachromatic (cause colour shift) with Toluidine Blue

Function

• cells releared from bone marrow and circulate for short time

• modulate allergic reactions: trigger rxn

  • immediate reaction to second stimulus by antigen

• Degranulation causes vasodilation, edema, mucus secretion, bronchiole constriction

• Can be life-threatening: systemic

• Mast cells are related cell type found in tissue- usually a more localized response but can also be systemic response

Agranulocytic Leukocytes

1. Lymphocytes

2. Monocytes

Directly related to immune responses

• Adaptive immunity (lymphocytes)

• Intrinsic immunity (monocyte/macrophage)

General Features

• no obvious specific granules

• azurophilic granules as all leukocytes; may or may not be obvious in LM

• nucleus multilobed

• relatively less cytoplasm

Lymphocytes details and function

• Smallest nucleated cell in blood; not much larger than RBC

• very little cytoplasm; usually basophilic

• Nucleus with dense heterochromatin; nucleus is often only prominent feature, especially in sections

Function

• involved in targeted immunity; antigen specific, highly regulated and part of complex system

• 3 major types

  • B lymphocytes (B-cell): Ig (antibody) production

  • T-lymphocytes (Thymus): recognition and regulation, cytotoxicity

  • NK cells (Natural Killer Cells): killing of abnormal cells

• All look alike so need immunostaining to differentiate based on surface molecules

• Freq transit between blood, CT, lymph/lymphoid organs

Plasma cells

• Heterochromatic nucleus with obvious heterochromatin clumps

• nucleus eccentric; more extensive cytoplasm than lymphocyte

• Cytoplasm basophilic with light staining Golgi zone

• Differentiated daughters of B lymphocytes; only see in CT and lymphoid organs

• Clonally selected and specialized for secreting antibody

• limited lifespan

Monocytes

• largest blood cells

• indented nucleus, often kidney, ‘U’ or horseshoe-shaped

• No specific granules but azurophilic granules often prominent

Macrophages

• Differentiate from monocytes after exit blood

• ones that exit as a response to inflam/tissue damage look similar to monocytes but with more phagocytic vesicles

• ones that become permanent specialized residents look very different and unique for each organ of residence

• best way to identify macrophages is via presence of phagosomes

• LM sometimes can see engulfed pigments (especially with haemorrhage)

Paraffin sections

• only dependable ID is through presence of phagosomes eg. uptake of particulate dyes or blood pigment

TEM

• abundance of lysosomes, phagocytic vesicles and residual bodies

what is function of macrophage

Inflammation

• major cell types in sites of inflammation after PMN/neutrophils

• kill bacteria via cytotoxic secretions

Phagocytosis

• engulf debris, old cells, foreign cells/molecules tagged by antobodies

Immune response

• Coordinate immune activities via production of lymphokines and cytokines

• antigen presenting cell

Platelets (thrombocytes)

• 2-3 um diameter formed elements

• are plasma membrane-enclosed cytoplasmic fragments of megakaryocytes

• no nuclei, but contain other organelles

• Function: clot formation (hemostasis) and other signalling cascades

• extensive network of membrane channels

• numerous granules that contain vasoactive substances, factors for clotting and clot dissolution, growth factors

• microtubule and actin network regulate platelet shape and adhesion

coagulation

• tissue damage or wounding triggers platelet activation

• adhesion characteristics are altered; platelets clump together adhere to damaged tissue and degranulate

• platelet factors activate clotting cascade leading to conversion of plasma fibrinogen to insoluble strands of fibrin

• fibrin network traps RBSs and forms clot to prevent further blood loss

• other platelet factors trigger repair/healing

Cartilage function and location

• specialized type of CT; still, pliable resilient and gel-like

• resists mechanical stresses, absorbs shock, slides on surfaces

• avascular: no blood supply, limit size, has poor capacity for repair, low, anaerobic metabolism

• Function: supports soft tissue, facilitates bone movement, guides bone development

Location

• articular surfaces of bones

• ears and nose

• airways and lungs

• growth plates of bones

components of cartilage

1. Extracellular Matrix

2. Cells

Extracellular matrix made up of

• accounts for ~95% volume; solid and firm but pliable

• resists compression

Ground substance (80%)

• 40%-80% glycosaminoglycans (GAGs, proteoglycans, glycoproteins)

• rest H2O molecularly bound to GAGs - gives nutrition and ox because tissue not vascularized

Fibers

• generally collagen Type II - limits compressive deformation, resists tension

• Elastin (in elastic cartilage) and collagen Type I (in fibrous cartilage)

Cells in cartilage made up of

Fibroblast-like cells

• edge of cartilage from a dense CT layer called perichondrium

• proliferate to produce chondroblasts

Chondroblasts

• acts as progenitors

• proliferate and differentiate into chondrocyte

Chondrocyte

• mature cartilage cells

• produce and are trapped in ECM

• reside in cavity called lacuna

overview classification of cartilage

Hyaline Cartilage

• Chondrocytes sit individually or in small clusters with viable amount of ECM

• usually considerable cell shrinking so lacuna seems only partly filled (artifact)

• ECM of hyaline cartilage either poorly stained or basophilic (sulfated GAGs)

• region of ‘territorial matrix’ (higher GAG, lower collagen) around each chondrocyte

• hyaline cartilage chondrocyte actively secreting ECM contains extensive Golgi and RER and mitochondria

• Nucleus mostly euchromatin

• region ECM immediately adjacent to cell rich in GAG and PGs with collagen type IV

• territorial matrix richer in collagen type II; less basophilic in LM

• these 2 regions always distinguishable in LM- use territorial matrix for the wall stained area adjacent to cell

Elastic Cartilage

• it is basically hyaline with elastin in ECM

• Elastin fibres form dense meshwork around chondrocytes

• looser network of elastin sheets throughout ECM

• elastin cartilage is yellowish when fresh; elastin is not readily stained with H and E (seed specual orcein stain)

• elastic cartilage very flexible; springs back into place

• unlike hyaline cartilage, it does not calcify

Fibrocartilage

• Essentially a mingling of hyaline cartilage and dense fibrous CT

• can be distinct layers of each or less organized

• Cartilaginous regions contain chondrocytes and sparse matrix; fibrous regions contain fibroblasts

• fibrous regions rich in collagen type I: tissue is much more eosinophilic than other cartilage

• Cushioning and tensile strength; resist tearing

• fibrocartilage chondrocytes contain extensive RER but not much Golgi; not producing much GAGs and PGs

• Nucleus is mostly euchromatin

• Region of ECM around chondrocytes fairly typical for cartilage

• also large prominent bundles of collagen in multiple direction - mostly collagen type I