TISSUES LECTURE NOTES Francis J. Liuzzi, Ph.D.

EPITHELIUM(Chapter 5)

I. BASIC FUNCTIONS OF EPITHELIA -RELATIONSHIP TO STRUCTURE

A. protection -protective epithelia are multilayered or stratified examples:

epidermis of skinlining of esophagus

B. absorption - absorptive epithelia are single layered or simple and have surface specializations to increase the absorptive surface areaexample: intestinal epithelium

C. secretion -secretory epithelia typically form glands which can range in complexity from a single secretory cell (e.g. goblet cell) to a complex secretory organ (pancreas)

D. sensation - very specialized epithelia are found in the taste buds, olfactory epithelium and inner ear

E. contraction -myoepithelial cells - specialized epithelial cells found in the acini of glands - these cells help to squeeze the secretory product from the gland

II. BASIC CHARACTERISTICS OF EPITHELIAL TISSUES

A. AVASCULAR - one does not typically find blood vessels in epithelial tissues - they derive their nutrients from vessels located in underlying connective tissues

B. CELLS ARE TIGHTLY PACKED - there is little extracellular space in epithelial tissues

C. EPITHELIAL TISSUES FORM COVERINGS OR LININGS OF ORGANS AND FORM GLANDS

D. ALL EPITHELIAL TISSUES HAVE A BASAL LAMINA - this extracellular structure separates the basal epithelial cells from the adjacent connective tissue - BASAL LAMINA CAN ONLY BE SEEN WITH THE ELECTRON MICROSCOPE

Basal lamina should not be confused with BASEMENT MEMBRANE which is visible with the light microscope

the major components of BASAL LAMINA are:1. type IV collagen2. laminin - a large glycoprotein

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3. proteoglycan - heparan sulfate

THE IMPORTANCE OF BASAL LAMINA: All cells that rest upon a basal lamina have receptors for laminin. In part, basal lamina helps determine the polarity of cells that rest upon it. Moreover, basal lamina acts in some ways as a boundary between tissues. It restricts epithelial cells from entering the underlying connective tissue. In the case of malignant tumor cells, i.e. cancer, the cells produce proteolytic enzymes to digest their way through the basal lamina. Among these enzymes are type IV collagenase and urokinase-type plasminogen activator. Benign tumors, such as those of breast, colon and stomach exhibit very little type IV collagenase activity while malignant tumors of these organs overexpress these enzymes. In experimental animals, inhibitors of type IV collagenase have been shown to significantly decrease metastases.

BASEMENT MEMBRANE is composed of basal lamina and an underlying RETICULAR LAMINA - the basal lamina is contributed by the epithelial cells while the reticular lamina is contributed by the underlying connective tissue

basement membranes such as those found in the renal glomeruli are made up of two basal laminae

E. ALL EPITHELIAL TISSUES ARE CHARACTERIZED BY INTERCELLULAR JUNCTIONS OR SPECIALIZATIONS (Note: These junctions are not confined to epithelial tissues)

1. DESMOSOMES - also known as MACULA ADHERENS - these are patch-like connections between adjacent epithelial cells which serve to hold the cells tightly together. Desmosomes have intermediate filaments associated with them - in the case of epithelium the intermediate filaments are made of cytokeratin and are called tonofilaments. Desmosomes are particularly abundant in epithelia that are exposed to physical strees, i.e. epidermis.

2. GAP JUNCTIONS - also referred to as NEXUS JUNCTIONS - these are very close appositions of adjacent cellular membranes - gap junctions are characterized by actual open channels (connexons) between cells. These junctions have been shown to allow for cell-to-cell communication. Materials smaller than 1500 Daltons can pass thru the channels.

In some epithelial tissue, such as that of the intestines, there is a very specialized, characteristic junctional complex joining the apical ends of the cells. This junctional complex can often be seen at the light microscopic level and was termed the TERMINAL BAR by the early histologists.

With the electron microscope, the terminal bar can be seen to be made up three structures, the following two specialized junctions plus macula adherens..

3. ZONULA OCCLUDENS - also known as a TIGHT JUNCTION - this structure completely surrounds the cell like a belt or band. It is a tight fusion of the adjacent membranes that serves to prevent luminal materials from diffusing into the intercellular space between adjacent epithelial cells membranes (paracellular route). At tight junctions the membranes are fused into a pentalaminar (five-layered) structure.

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4. ZONULA ADHERENS - this junction, like the zonula occludens, circles the entire cell. It has morphological similarities to the desmosome in section. Its function is to hold adjacent cells together. It has actin microfilaments associated with it.

F. MANY EPITHELIAL CELLS HAVE SURFACE SPECIALIZATIONS

1. MICROVILLI - specialized for absorption - microvilli contain a core of microfilaments (actin) - these microfilaments are anchored to a web of microfilaments and intermediatefilaments (terminal web) in the apical end of the cell. The terminal web is believed to form a structural framework for the apex of the cell.

2. CILIA - longer than microvilli - have an organized cytoskeleton made up of nine doublets of microtubules and a central pair of microtubules - the microtubules are anchored to the basal bodies in the apical cytoplasm -function is movement - tracheal cilia move mucous toward the pharynx

3. STEREOCILIA - very long cellular processes - not involved in movement - internal structure similar to microvillus (core of microfilaments) - found in the epididymis and the inner ear

III. CLASSIFICATION OF EPITHELIAL TISSUES

A. by the number of cell layers -

1. simple - one layer

2. stratified - more than one layer

3. pseudostratified - looks like more than one layer

B. by cell shape

1. squamous - flattened

2. cuboidal - height=width

3. columnar - height>width

CLASSIFICATION IS ALWAYS BY THE SHAPE OF THE TOP LAYER

A specialized epithelium that does not fit into the classification scheme is TRANSITIONAL EPITHELIUM - characteristic of the urinary system. Transitional epithelium is named such because of its ability to adapt to changes in bladder and urinary system distension. Characterized by dome-shaped cells in top layer (undistended) with specialized, thickened apical membranes.

EXAMPLES OF CLASSIFICATIONS

epidermis of the skin - stratified squamous keratinized

epithelium of the esophagus - stratified squamous nonkeratinized

epithelium of the intestines - simple columnar with brush or striated border (microvilli)

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epithelium of the terminal bronchioles in the lung - simple columnar, ciliated

epithelium of the epididymis - pseudostratified columnar with stereocilia

IV. GLANDULAR EPITHELIUM

ALL GLANDS DEVELOP AS INGROWTHS OF EPITHELIAL CELLS INTO THE UNDERLYING CONNECTIVE TISSUE.

There are two major types of glands

A. EXOCRINE glands - secrete substances onto an epithelial surface via a duct system. They can be classified as simple or compound

1. SIMPLE - one unbranched ducta. tubularb. coiled tubularc. branched tubular d. acinar

2. COMPOUND - ducts branch repeatedlya. tubularb. branched tubularc. acinar

MODES OF SECRETION FOR EXOCRINE GLANDS

1. HOLOCRINE - the entire cell fills with secretory product and then the cell is shed - sebaceous and tarsal glands

2. APOCRINE - the apical end of the cell is filled with secretory product and that part of the cell is released - mammary glands

3. MEROCRINE - secretory cells are formed, accumulate near the apical end of the cell and are released by exocytosis - pancreatic acinar cells

B. ENDOCRINE GLANDS - during development, the endocrine glands lose their ducts and the glandular cells secrete their products (hormones) into the bloodstream

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CONNECTIVE TISSUE(Chapter 6)

Connective tissue is one of the four basic tissues. Typically, connective tissue is derived from the middle embryonic germ layer, the mesoderm. Today’s lecture is about connective tissue proper as opposed to the specialized connective tissues such as blood, cartilage and bone, which will be the subjects of future lectures. Connective tissue proper does pretty much what its name implies, i.e. it hold tissues together and forms the framework of organs. More importantly, connective tissue underlies epithelium. It contains blood vessels, nerves, and lymphatics. It is extremely important in defense against foreign bodies and repair of injuries. In addition, connective tissue forms tendons and ligaments. Adipose tissue (FAT), serves as insulation, a storage depot for energy and provides the physical contours that distinguish men and women.

CONNECTIVE TISSUES CAN BE THOUGHT OF AS BEING MADE UP OF THREE DISTINCT COMPONENTS:

I. GROUND SUBSTANCE1. characteristics

a. amorphousb. colorlessc. homogeneous

2. compositiona. proteoglycans - synthesized and secreted by the resident cells of connective tissue have a protein backbone with glycosaminoglycans covalently bound to it. Proteoglycans are very hydrophilic. They are typically surrounded by a thick layer of solvation water.Table 6.3 on page 162 in your text lists the major glycosaminoglycans.

b. glycoproteins 1. laminin - a large (approx. 900kD) heterotrimeric, cruciform molecule made up of three subunits B1, B2 and A. Laminin is a major component of basal lamina. 2. fibronectin

c. tissue fluid - similar to blood plasma. Low molecular weight plasma proteins can leave capillaries and enter tissue fluid. Ionic composition of the tissue fluid is similar to that of plasma. Normally, there is very little tissue fluid present in connective tissue. Accumulation of tissue fluid leads to edema (see below), which can have a number of causes.

II. FIBERS1. collagen fibers

a. Over 20 types of collagen have been identified - 5 collagen types are the most abundant types (types I-V: please refer to TABLE 6.2, page 153 in Ross)b. type I forms classically described collagen fibers and can form collagen bundles.c. acidophilic (pink) with H&E stain

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2. elastic fibers a. composed of elastin, a relatively amorphous substance, and microfibrils composed in part by fibrillin, a protein. Marfan’s syndrome, an autosomal dominent connective tissue disorder, is characterized by abnormal elastic tissue. It has been shown that the fibrillin gene is defective in this disorder.b. unlike collagen fibers, elastic fibers stain very weakly, if at all with hematoxylin and eosin. Therefore, to visualize elastic fibers, one must use special stains such as orcein or resorcin-fuchsin. These stains stain elastic fibers purple, dark blue or blue-black.c. your text describes oxytalan, elaunin and elastic fibers as part of an elastic fiber “system”. These members of the elastic fiber system are differentially distributed in the body and are functionally different. Oxytalan fibers form the suspensory ligament of the lens and are found in the dermis. They are composed of microfibrils with little or no elastin. Elaunin fibers are composed of microfibrils with small amounts of elastin. These two fiber types are not as elastic as elastic fibers, which are composed of microfibrils and elastin.d. found in walls of blood vessels (usually forming fenestrated membranes in larger vessels)

3. reticular fibers a. formed by collagen protein (composed of type III collagen) b. the fibers are very thin and stain with silver salts and therefore are called argyrophilic (i.e. silver-liking)c. reticular fibers form the framework of organs such as lymph nodes and the spleen. Reticular fibrils compose the reticular lamina, which underlies the basal lamina of epithelium.

III. Cells

A. resident cells - those born and residing permanently in the tissue1. fibroblasts - most abundant cell type in CT - synthesize extracellular matrix (ground substance and fibers)

some texts describe a myofibroblast. This cell type cannot be morphologically distinguished from other fibroblasts at the light microscopic level. However, they contain bundles of actin microfilaments and dense bodies similar to those of smooth muscle cells are contractile cells and are particularly abundant at sites of woound healing.2. adipose cells (adipocytes) - specialized connective tissue cells for storing triglycerides (fat)

3. other resident cells found in special connective tissues - e.g. chondrocytes in cartilage and osteocytes in bone (discussed in a later lecture)

B. immigrant cells - those born elsewhere and transiently present in the tissue, i.e. migrating through

1. macrophages - called histiocytes by pathologist - derived from bone marrow - migrate into CT and take up permanent residence. They are part of MONONUCLEAR PHAGOCYTE SYSTEM (formerly called the reticuloendothelial system)

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2. mast cells - once believed to be derived from CT precursor cell (mesenchymal cell) -BUT NOW recent evidence suggest derived from bone marrow - precursor cells migrate via blood to tissues where they proliferate and differentiate- cytoplasm filled with basophilic granules, but appear reddish rather than blue because they are metachromaticgranules contain glycosaminoglycans (heparin sulfate or chondroitin sulfate), neutral proteases, eosinophil chemotactic factor (ECF) and HISTAMINE

function: plasmalemma has IgE receptors - IgE produced by plasma cells, bound to mast cells - causes degranulation histamine - causes contraction of smooth muscle cells in bronchioles, dilation of capillaries

3. plasma cells - derived from B-lymphocytes - plasma cells have very basophilic cytoplasm due to high content of RER - large eccentric nucleus - chromatin pattern gives it a "clock face" appearance -sometimes light juxtanuclear region where Golgi resides - produce antibodies (immunoglobulins) to foreign substances

4. leukocytes - white blood cells - derived from bone marrow - enter connective tissue by process called diapedesis

a. neutrophils - these cells are the first cells to arrive at a site of inflammation - characterize acute inflammatory reaction - some pathologists call these cell polymorphs because their nuclei are variably lobulated - they are phagocytic and are specialized for killing and removing bacteria

b. eosinphils - eosinophilic (bright pink-red granules) - bilobed nucleustypically increase during allergic response and parasitic infectionsphagocytize antigen-antibody complexes

c. basophils - have granules similar to mast cells - but important to remember that basophils are not the same as mast cells - granules contain histamine

d. lymphocytes - very small cells (6-8µm in diameter. for comparison, RBCs are 7µm)- have large dark nucleus surrounded by thin rim of basophilic cytoplasm - typically find lymphocytes in the connective tissue underlying the epithelium of the GI tract, also can find them in the CT of the respiratory systemlymphocytic accumulations typically characterize chronic inflammation

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CLASSIFICATION OF CONNECTIVE TISSUE

I. loose connective tissue - also called areolar connective tissue - has lots of cells compared to fibers

usually very vascular -typically lies immediately beneath epithelia. Loose CT in this location sometimes

referred to as lamina propria

II. dense connective tissue - more fibers (collagen) per unit volume of tissue than cells

A. dense regular - abundant fibers arranged in regular bundles and/or sheets of predominantly collagen fibers - examples tendons, aponeuroses and ligaments

B. dense irregular - abundant fibers arranged in very irregular fashion - random bundles of collagen fibers - best example is the dermis of the skin

ADIPOSE TISSUE (Chapter 9)

Two types of adipose tissue

I. white fat - made up of unilocular fat (adipose) cells - one large fat droplet pushes nucleus and cytoplasm off to sidewidespread distribution in adults - 22% body weight in normal adult females - 15% body weight in normal adult males

II. brown fat - made up of fat cells with numerous small fat droplets (multilocular) - not very common in adult humans

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CARTILAGE AND BONE(Chapters 7 and 8)

I. Cartilage

A. General characteristics

1. special type of connective tissue that is able to withstand severe mechanical stress

2. provides support for soft tissues such as in the respiratory tract3. forms articular surfaces where it provides shock absorption and a smooth surface for joint movements3. essential for the development, growth and repair of long bones4. avascular and contains no nerves

B. Components

1. extracellular matrix: a. fibers: predominantly type II collagen fibrilsb. ground substance: mainly proteoglycans: chondroitin-4-sulphate,

chondroitin-6-sulfate and dermatan sulfate. These proteoglycans are made up of a core protein with attached glycosaminoglycans (bottle brush appearance). The glycosaminoglycans act as polyanions, bind sodium by electrostatic bonds. A thick layer of solvation water surrounds these glycosaminoglycans. The hydrated matrix resists compression and permits diffusion of nutrients to the cells within the matrix.

2. cell typesa. chondroblasts: located in inner layer of the perichondrium. They resemble fibroblasts and can differentiate into chondrocytes.b. chondrocytes: These are maturing cartilage cells. They secrete the matrix components (collagen and proteoglycans) and become surrounded by it. They are located in lacunae (lakes).

II. Types of Cartilage

A. Hyaline cartilage

1. found in articular surfaces, respiratory tract and ventral ends of ribs. Hyaline cartilage also forms temporary skeleton during endochondral bone development.

2. perichondrium a. dense C.T. covering, except on articular surfaceb. abundant type I collagenc. outer fibrous layer: fibroblastsd. inner cellular layer: chondroblasts

3. chondrocytesa. located in lacunaeb. immature chondrocytes at the periphery of the cartilage are ellipticalc. deeper within the cartilage they are roundedd. mitotic division of a single chondrocytes can result in groups of up to eight chondrocytes called isogenous groups

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4. matrixa. territorial matrix (capsular matrix): newly formed matrix immediately surrounding chondrocytes. It is rich in sulfated proteoglycans which are negatively charged. Therefore, the territorial matrix is basophilic.b. interterritorial matrix: as collagen type II accumulates in matrix, becomes less basophilic or more acidophilic. (remember, collagen fibers are acidophilic)

5. growth - two modesa. interstitial growth: mitosis of chondrocytes within cartilage (expansion of cartilage from within). As isogenous groups form, individual daughter chondrocytes begin secreting matrix that separates the cells.b. appositional growth: differentiation of chondroblasts from the perichondrium (addition of new layers of cartilage upon older layers)

B. Elastic cartilage

1. found in external ear (auricle and external auditory meatus), auditory (eustacian) tube, epiglottis and cuneiform cartilage of the larynx

2. it looks very similar to hyaline cartilage histologically, especially in H and E preparations. It requires an elastic stain (orcein or resorcein-fuchsin) to more easily differentiate elastic cartilage from hyaline cartilage.

3. contains abundant elastic fibers in addition to collagen type II fibrils

4. contains chondrocytes in lacunae

5. surrounded by perichondrium

C. Fibrocartilage1. can be viewed as a hybrid (mixture) between dense connective tissue and cartilage.2. found in intervertebral disks (annulus fibrosa), symphysis pubis, and attachments of some ligaments to bone (we have examples of all three in lab)3. contains numerous collagen type I fibers, often forming parallel bundles in direction of stress5. contains chondrocytes in lacunae, often arranged in long rows between the collagen bundles6. unlike hyaline and elastic cartilage, fibrocartilage is not surrounded byperichondrium

III. Bone

A. General characteristics of bone tissue

1. hard tissue, provides support and protection2. forms system of levers for musculoskeletal movement3. serves as calcium reservoir4. unlike cartilage, bone is well-vascularized

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5. red marrow is site of hematopoiesis

B. Components of bone tissue

1. bone matrix

a. organic (osteoid): collagen type I fibers, proteoglycans and some glycoproteins (sialoprotein and osteocalcin)

b. inorganic: calcium and phosphorus in the form of hydroxyapatite crystals and amorphous calcium phosphate

2. bone cells

a. osteoblasts: secrete osteoid (organic matrix); they are located at bone surfaces; they are cuboidal or columnar shaped cellsThey have a basophilic cytoplasm and develop from osteoprogenitor cells which have fibroblast-like appearance

b. osteocytes: are mature bone cells that maintain the organic matrix; they are located in lacunae and have cytoplasmic processes in canaliculi. The processes are connected by gap junctions. Osteocytes develop from osteoblasts.

c. osteoclasts: derived from the fusion of blood-derived monocytes and are, therefore, members of the mononuclear phagocyte system. They resorb bone matrix and are located in Howship's lacunae. Because they are derived from fused monocytic cells, they are multinucleate giant cells. Like many phagocytic cells, they are characterized by a ruffled border. Around the ruffled border is a region devoid of organelles, but rich in actin microfilaments. It is believed that this clear zone allows attachment of the osteoclast to the bone matrix and creates a microenvironment into which the cell secretes lysosomal enzymes.

Osteoclasts are hormonally regulated:

parathyroid hormone - increases number of osteoclasts and activates them. It increases bone resorption and thereby serum calcium

calcitonin from the parafollicular cells of the thyroid - inhibits osteoclasts, decreases bone resorption

C. Types of bone tissue

1. on the basis of morphological, gross appearance (naked eye) can identify two types of adult bone

a. compact (cortical): very dense area without cavities, provides strength to the outer portions of bones

b. spongy (cancellous, trabecular): numerous trabeculae (bridges) and spicules separated by marrow cavities.

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Under the microscope, compact and spongy bone are structurally similar. Both compact bone and the thicker trabeculae of spongy bone have Haversian systems (see below).

2. During development and during bone repair following a fracture, two stages of bone formation are observed.

a. primary (woven) bone: immature bone. characterized by an irregular array of collagen fibers, a low mineral content and a greater number of osteocytes. In adults, primary bone is found in the tooth sockets, near the sutures of the flat bones of the skull and at the insertions of some tendons.

b. secondary (lamellar) bone: more typical of adults. Collagen fibers are numerous and organized into parallel lamellae or concentric lamellae around blood vessels.

D. Bones as Organs

1. Gross structure of long bones such as the humerus

a. diaphysis (shaft): outer compact bone surrounding inner spongy bone around the marrow cavity. Compact bone gives the bone strength for weight bearing and movement while the spongy bone in the central shaft allows the bones to be lightweight.

b. epiphysis (bulbous end): mainly inner spongy bone covered by a relatively thin layer of compact bone

Short bones (e.g. metacarpals) are structurally similar to long bones. Core of spongy bone is completely surrounded by compact bone.

Flat bones, such as those of the skull, have outer layers of compact bone called plates and a thin inner layer of spongy bone called diploe

2. Connective tissue linings of bone

a. periosteum: covers external surfaces of bones; has two parts; (1) an outer fibrous layer of dense C.T. and (2) an inner cellular layer of osteoprogenitor. The cells of the inner layer can differentiate into osteoblasts.

b. endosteum: lines internal surfaces; thin layer of osteoprogenitor cells

3. Microscopic structure of secondary (lamellar) bone

a. Haversian systems (osteons):

1. concentric lamellae of collagen fibers and bone matrix parallel collagen fibers

2. lacunae containing osteocytes located between or in the lamellae

3. the center of a Haversian system is an endosteum-lined canal containing blood vessels, nerves and loose connective tissue.

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4. Haversian systems are longitudinally oriented, i.e. parallel to the long axis of the bone. They are not restricted to compact bone and can also be observed in the thicker trabeculae of spongy bone.

5. Volkmann's canals - provide communication between Haversian systems and between Haversian systems and marrow cavity. Essentially run perpendicular to Haversian systems and are not surrounded by concentric lamellae.

b. outer circumferential lamellae: located under periosteum

c. inner circumferential lamellae: located around marrow cavity

d. interstitial lamellae: remnants of remodeled Haversian systems

E. Bone development: There are two distinct ways in which bones can develop

1. Intramembranous ossification

a. mode of flat bone formation. Best example, flat bones of skull.

b. begins by a condensation of mesenchymal cells: This condensation is referred to as a membrane.

c. groups of mesenchymal cells within the membrane differentiate into steoblasts. This site of osteoblast formation becomes a primary ossification center.

d. osteoblast lay down bone matrix. Matrix encapsulates osteoblasts and calcifies. Cells become osteocytes located in lacunae. Spicules of woven bone form and these grow together forming trabeculae. Compact bone forms outer plates. Inner bone around the developing marrow cavity becomes spongy bone.

2. Endochondral ossification (within cartilage)

a. mode of development of long bones and most short bones

b. a hyaline cartilage model of bone forms in the embryo - replaced by bone during development. (NOTE: Hyaline cartilage does not become bone.)

c.. endochondral ossification consists of two phases:

1. hypertrophy and degeneration of chondrocytes2. osteogenic bud of osteoprogenitor cells and blood vessels penetrates the space left by the dead chondrocytes3. Sequence of Events: Development of long bones

a. Formation of the primary ossification center (prenatal)

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1. bone collar forms around diaphysis by differentiation of osteoblasts from the inner perichondrium, followed by deposition of bone matrix. This process, bone collar formation, is actually a form of intramembranous bone formation, i.e. bone formation within the perichondrium.

2. cartilage hypertrophy, degeneration, and calcification occur in area surrounded by the bone collar

3. osteogenic bud of blood vessels and osteoprogenitor cells penetrates bone collar and enters space left by degenerating cartilage

4. osteoblasts differentiate and secrete bone matrix on a scaffold of remaining calcified cartilage

5. primary ossification center expands to occupy the whole diaphysis

b. a secondary ossification centers arise later in the epiphyses (postnatal). This secondary center again forms by endochondral ossification

c. epiphyseal plate (connects epiphysis & diaphysis) – allows for long bone growth. It has 5 identifiable layers or zones

1. zone of reserve (resting) cartilage: typical hyaline cartilage

2. zone of proliferation: chondrocytes proliferate, form columns

3. zone of hypertrophy: chondrocytes enlarge, become vacuolated (in this area, chondrocytes begin synthesizing type X cartilage which appears to precede calcification)

4. zone of calcification: remaining cartlage matrix calcifies, the chondrocytes degenerate. This region usually appears basophilic.

5. zone of ossification: osteoblasts differentiate, deposit bone matrix on remnants of calcified cartilage. This region usually appears more acidophilic (pink) in H and E.

F. Bone growth and remodeling

1. growth in length

a. due to growth of epiphyseal plate, i.e., interstitial growth of cartilage followed by endochondral ossificationb. epiphyseal plate maintains a constant thickness during growth phase

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2. growth in width (appositional growth)

a. due to bone formation by osteoprogenitor cells within the innerperiosteum around the diaphysis. This is actually a form of intramembranous ossification.

b. the marrow cavity increases in diameter due to the resorption of bone from internal surfaces by osteoclasts

3. internal remodeling

a. continuous resorption and deposition of bone results in remodeling

b. remodeling maintains shape of bone during growth

c. in adult bone, Haversian systems undergo continuous remodelling leaving interstitial lamellaer as a result

G. Fracture Repair

1. blood clot forms at site of fracture2. connective tissue of periosteum and endosteum proliferate3. hyaline cartilage forms in this connective tissue4. primary bone forms at site by both endochondral and intramembranous bone formation5. bone callus temporarily unites fracture6. remodelling occurs with healing

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NERVOUS TISSUE(Chapter 12)

Nervous tissue is highly specialized for the transmission of information over long distances. Nervous tissue comprises the CNS: central nervous system (the brain and the spinal cord), the PNS: peripheral nervous system (sensory ganglia and peripheral nerves) and the ANS: autonomic nervous system (sympathetic and parasympathetic ganglia). Nervous tissue can be found in most organs of the human body.

THERE ARE TWO CLASSES OF CELLS IN THE NERVOUS SYSTEM

I. nerve cells or neurons - are very specialized cells of neuroectodermal or neural crest origin which have evolved the ectodermal cell characteristics of excitability or irritability and cell-to-cell communication to their highest levels.

A. the human central nervous system (CNS, brain and spinal cord) has a large variety of different neuronal types (200 or more) which have specific morphological characteristics depending upon the particular part of the CNS in which they reside. In laboratory, you will examine the large multipolar neurons of the ventral horn of the spinal cord, motoneurons, which innervate the skeletal muscle of the body. You will also examine the pyramidal cells of the cerebral cortex which are named for the shape of their cell bodies and you will examine the Purkinje cells of the cerebellum which have a large rounded cell bodies and in silver preparations can be seen to have a very elaborate dendritic arborization. These are but three of the thousands of neuronal types in the mammalian nervous system. A group of neurons in the CNS that are found together and are functionally related is called a nucleus.

B. The peripheral nervous system (PNS) also has a number of different types of neurons all of which are derived from neural crest. Groups of functionally related neurons in the PNS are typically encapsulated within a ganglion. Sensory neurons (primary afferent neurons), those that carries sensations such as touch, position, pain and temperature from the skin, muscles, joints and viscera are found in dorsal or posterior root ganglia along the spinal cord and in cranial nerve ganglia in the head and neck. The cells of the dorsal root and the cranial nerve sensory ganglia are called pseudounipolar. There are also autonomic ganglia, sympathetic and parasympathetic, that give rise to postganglionic efferents.

C. Characteristics of neurons.

1. Typically, neurons have cytologically distinct regions.a. cell body or soma - contains the nucleus of the neuron and most of the

protein synthetic organelles. The neuron is an incredibly active cell. Its nucleus is characterized by a prominent nucleolus and preponderance of euchromatin. The cytoplasm is characterized by basophilia which can be seen to occur in clumps. These basophilic clumps are called Nissl bodies or Nissl substance and when viewed with the electron microscope, can be seen to be composed of RER and polysomes.

b. most neurons have one or more dendrites - the dendrites branch and form what is called a dendritic arborization or dendritic tree. Neurons with more than one dendrite are called multipolar. Some neurons such as those of the dorsal root ganglia and the cranial nerve sensory ganglia have no dendrites, these particular cells

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are called pseudounipolar. The proximal portions of dendrites can contain Nissl substance and therefore, stain with basic dyes.

Dendrites and the somata are typically the receptive parts of the neuron. They receive inputs from other neurons. Functionally, dendrites typically do not display an action potential. Instead, they exhibit a graded potential.

c. Most neurons have one axon. Depending on the type of neuron, the axons can be as short as a few microns or can reach lengths of more than a meter. Axons can have branches that come off at right angles. These are called axon collaterals. Or neurons can, toward their ends, branch into axonal arborizations called telodendria. Axons cannot be seen with routine stains (H and E or cresyl violet) because they contain no Nissl substance. Axons are the effector part of the neuron. Information typically travels along the axon as an action potential.

d. Neurons communicate by specialized junctions called synapses. There are basically two types of synapses within the nervous systems of mammals.

1. Chemical synapses most common type. The presynaptic ending contains synaptic vesicles which fuse with the presynaptic membrane and release transmitter into the synaptic cleft. Chemical synapses can be excitatory or inhibitory. The transmitter effects receptors in the postsynaptic membrane. Excitatory synapses cause in an influx of Na+ ions into the postsynaptic element and hence a change in the intracellular potential, typically a depolarization, i.e. going more positive. Inhibitory synapses cause a hyperpolarization, i.e. the intracellular potential goes more negative (K+ ions flows out). Postsynaptic elements such as dendrites usually exhibit a graded potential rather than an action potential. There can be thousands of synapses on the dendrites and soma of a neuron. The graded potentials from a number of inputs can summate resulting in an action potential in the axon.

2. Electrotonic synapses are present in some regions of the CNS of mammals. These are gap junctions between cells and hence when the potential in the presynaptic element changes, that in the postsynaptic element also changes. These types of synapses occur where neurons need to act coordinately.

II. Neuroglial cells or glia (glia means glue). These cells are the supportive cells of the nervous system.

A. In the CNS there are 5 different types of glia cell. Four derived from neuroectoderm and 1 derived from the bone marrow.

1. ependymal cells - these are simple cuboidal or columnar cells that line the ventricular system of the brain and the central canal of the spinal cord. They look very much like an epithelium, a result of their ectodermal origin. Some ependymal cells have cilia and they often are characterized by tight junctions (zonulae occludens).

2. Related to the ependymal cells are the cells of the choroid plexus. These cells are responsible for the elaboration of cerebral spinal fluid (CSF). CSF is produced by a combination of the ultrafiltration of the blood as well as active secretion. The composition of CSF is similar to plasma. Lumbar punctures are used to collect CSF for clinical examination. A high content of certain proteins or leukocytes can be indicative of CNS injury or infection.

3. astrocytes - also called astroglia - there are two morphologically distinct types of astrocytes in the CNS.

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a. fibrous astrocytes - typically found in the white matter. Thes cells have long slender processes packed with 10nm intermediate filaments composed of the protein glial fibrillary acidic protein (GFAP).

b. protoplasmic astrocytes - typically found in the gray matter of the CNS - have short, thick sort of bushy processes. They have relatively fewer intermediate filaments as compared to the fibrous astrocytes and hence stain relatively less intensely with antibodies to GFAP.

c. recent biochemical and immunohistochemical studies have shown that the two morphologically distinct astrocytic types. These two types may actually be made up of many subtypes.

d. astrocytes form barriers between the CNS and non-CNS. They form the glia limitans a barrier between the connective tissue of the meninges and the CNS parenchyma. They are also now known to be responsible for inducing CNS endothelial cell to form the blood-brain barrier.

e. astrocytes can be thought of as playing the role of connective tissue in the CNS where there are virtually no fibroblasts and relatively little collagen. They provide structural support to the CNS and are involved in the exchange of materials between the vasculature and the neural parenchyma. Moreover, astrocytes, like fibroblasts respond to tissue injury by dividing and hypertrophying to form a glial scar. These reactive astrocytes, which stain intensely with GFAP antibodies, are called gemistocytic astrocytes by pathologists.

4. Oligodendroglial cells or oligodendrocytes - these cells form myelin in the CNS. Myelin is formed by the plasma membranes of the oligodendroglia which wrap the axons in a jelly role fashion. Myelin is part of the living oligodendrocyte. It is not extracellular and is not secreted by the oligodendrocyte. A single oligodendrocyte can myelinate many axons.

5. MICROGLIAL CELLS - these cells are not derived from neuroectoderm. They are resting macrophages within the CNS derived from bone marrow precursor cells (monocytes). They migrate into the CNS during the development of CNS vasculature. They respond to CNS injury by dividing and phagocytosing injured neurons and glia. When the CNS is injured, the microglial nuclei enlarge and elongate. These cells are then called rod cells by pathologist. Increased damage causes the microglia to become phagocytic. They are then called gitter cells by pathologists.

B. In the PNS there are two types of supportive cells (many texts , both of which are derived from the neural crest.

1. Satellite cells - These small cells with rounded nuclei form a unicellular layer around ganglion neurons. After peripheral nerve injury, these cells divide and begin to synthesize increased amounts of neurotrophic factors that may serve to support the neurons during the time they are regenerating an axon.

2. Schwann cells are the cells of the PNS that form myelin or ensheath groups of unmyelinated axons. A single Schwann cell can myelinate only one axon.

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Everything said above about CNS myelin holds for PNS myelin. The protein compositions and the ultrastructural appearances of the two myelins are different.Peripheral nerve myelin, but not CNS myelin, is characterized by Schcmidt-Lanterman clefts or incisures. These are areas where the myelin is distended by Schwann cell cytoplasm (refer to Figure 12.10 on page 330).

Like satellite cells, Schwann cells respond to injury of the peripheral nerve. They divide, become phagocytic and secrete cytokines and neurotrophic factors.

III. PERIPHERAL NERVE AS AN ORGAN.

Peripheral nerve is made up of a number of tissues and hence can be described as an organ.

EPINEURIUM The thick connective tissue that surrounds the entire peripheral nerve.

PERINEURIUM The sheath of flattened multilayered epithelioid cells that surround individual fascicles of axons. Is a component of the blood-nerve barrier.

ENDONEURIUM The connective tissue located between individual axons within a fascicle. Endoneurial capillaries are part of the blood-nerve barrier.

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MUSCLE TISSUE(Chapter 11)

Muscle is one of the four basic tissues.

There are three histologically and distinguishable types of muscle tissue:1. SKELETAL MUSCLE2. CARDIAC MUSCLE3. SMOOTH MUSCLE

The first two, skeletal and cardiac muscle, are called striated muscle because they can be seen at the light microscopic level to have a regular, repeating band structure.

I. SKELETAL MUSCLE

A. skeletal muscle as the name implies, is typically associated with movements of the skeleton.

B. skeletal muscle is functionally termed voluntary muscleC. histologically, skeletal muscle cells are long, cylindrical cells (a muscle cell is

called a fiber) having multiple, peripherally located nuclei. Each muscle cell is surrounded by a basal lamina, which can only be seen at the EM level.

D. The parts of muscle cells are specially named:1. Sarcolemma - the muscle fiber (cell) plasmalemma2. Sarcoplasm - the muscle fiber cytoplasm3. Sarcoplasmic reticulum - specialized muscle fiber smooth endoplasmic reticulum

E. As stated above, skeletal muscle is called striated because it has a regular banded appearance in the light microscope. In the light microscope, one can see alternating light and dark bands. These bands were named by their appearance in polarized light. In polarized light the A band appears bright (anisotropic). The I band appears dark (isotropic). With H&E staining, the stain routinely used in histology and pathology, the A band stains darkly and the I band stains lightly. The Z line is a dark line that bisects the I band and the H band is a light band that bisects the A band. Finally, the H band has a darker M line in its center. A sarcomere is the region between two successive Z lines. The sarcomere is the functional unit of striated muscle.

F. Ultrastructural significance of banding seen at the light microscopic level.

1. EM reveals that the bulk of skeletal muscle cytoplasm (sarcoplasm) is made up of myofibrils.

2. Myofibrils are made up of very regularly organized myofilaments. These

myofilaments are of two types:a. thin filaments made up of:

1. actin -found in filamentous form, F-actin polymer which is made up of globular, G-actin monomers2. tropomyosin long thin molecule wraps the actin filaments3. troponin complex of three subunits, a TnT subunit which binds to tropomyosin, a TnC subunit which binds to calcium ions and a TnI subunit which inhibits the actin-myosin interaction

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b. thick filaments made up of myosin - myosin molecule made up of two heavy and two light chains. The heavy chains have globular projections at one end which appear as cross-bridges at the EM level. These globular heads have ATP binding sites and with actin as a co-factor are capable of hydrolyzing ATP (i.e. act as an ATP-ase).

3. EM shows that the I band contains thin filaments only

4. the A band contains thick and thin filaments

5. the H band contains only thick filaments

6. the Z line is where thin filaments are bound together by the protein actininin

7. the M line is where thick filaments are bound by sidearms to adjacent thick filaments major protein in the M line is creatine kinase

G. MUSCLE CONTRACTION

The accepted hypothesis of muscle contraction is the sliding filament hypothesis. This hypothesis is based upon light microscopic observations of living frog muscle during contraction. It was seen that the A-band remains the same length while the I band becomes shorter with contraction. This basic, but important observation, suggests that the thin filaments are sliding past the thick filaments. Muscle contraction involves binding of ATP to the ATP-binding sites on the myosin globular heads. But myosin requires actin as a co-factor for the rapid hydrolysis of ATP. At rest, myosin cannot associate with actin because the troponin-tropomyosin complex associated with the F-actin molecule is blocking the actin-myosin binding site. However, on activation of the muscle, CA2+ ions are released into the sarcoplasm and bind to the TnC subunits. The binding causes a change in the spatial configuration of the molecules, thereby exposing the myosin binding site on the actin molecule. Release of energy from the ATP-myosin complex results in a bending of the myosin head and a sort of ratcheting of the thin filament past the thick filament. ATP is also essential for detachment of the actin-myosin bond.

H. SARCOPLASMIC RETICULUM (SR) - specialized network of endoplasmic reticulum around the myofibrils. Acts as a Ca2+ sink (i.e. actively transports Ca2+ back into cisternae after contraction.

I. TRIAD Associated with sarcoplasmic reticulum is a system of deep infoldings of the sarcolemma called transverse tubules (T-tubules) In skeletal muscle, form a unit at the A-I junction called a triad. On either side of a T-tubule is a terminal cistern of SR. (Triad = SR-TT-SR at the A-I junction)

Functionally the T-tubules act to rapidly spread depolarization throughout the muscle fiber so that there is widespread release of Ca2+ from the SR allowing for uniform contraction of the muscle fiber.

J. SKELETAL MUSCLE- THE ORGAN One can speak of skeletal muscle, the tissue and skeletal muscles, the organs.

1. Organization of the organ Skeletal muscles have a dense connective tissue sheath, the epimysium which surrounds the entire muscle. Around each muscle

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fascicle is a connective tissue sheath called perimysium. Between muscle fibers, there is a very thin endomysium.

2. There are three types of muscle fibers:

a. red fibers (slow twitch- resistant to fatigue) - found in the muscles of mammalian limbs and some avian (bird) limbs as well as in the shoulder and breast muscles of flighted birds. Red muscle fibers are endurance fibers in that they fatigue slowly. Physiologically, they are termed slow twitch fibers. They appear red because of their high content of myoglobin. They contain large numbers of mitochondria. Red muscle fibers are smaller than in diameter than white fibers. Energy derived from the phosphorylation of fatty acids.

b. white fibers (fast twitch – fatigue rapidly) - found in the breast muscles of flightless birds. They are paler in appearance and larger in diameter than red fibers. Physiologically called fast twitch. Extraocular muscles are fast twitch. Glycolosis is source of energy.

c. intermediate fibers

INTERESTINGLY, FIBER TYPE SEEMS TO BE CONTROLLED BY THE INNERVATION OF THE MUSCLE. IF A NERVE THAT NORMALLY INNERVATED A PREDOMINANTLY FAST TWITCH MUSCLE IS ROUTED TO A DENNERVATED MUSCLE MADE UP OF PREDOMINANTLY SLOW TWITCH FIBERS, THEN THE FIBER TYPE WILL CHANGE TO FAST TWITCH.

K. innervation of skeletal muscle

1. motoneurons in the ventral horn of the spinal cord innervate somatic skeletal muscle. Axons leave the spinal cord by the ventral (anterior) roots and join sensory axons to form spinal nerves and then peripheral nerves

2. myelinated motor axons approach muscle, lose their myelin sheath, and form dilated endings on muscle cells called motor endplates, myoneural junctions or neuromuscular junctions

3. motor unit - a single motor axon and all the muscle cells (fibers) it innervates. Motor units can be very large for muscles that require course movements, such as the quadiceps femoris muscle. By contrast, in muscles that require fine control such as the extraocular muscle, those that control eye movement, a motor unit is one axon innervating one muscle fiber.

4. skeletal muscle has receptors for acetylcholine (AcH) that is released by motor axons at the neuromuscular junction. Myasthenia gravis is an autoimmune disease in which antibodies are made to the Ach receptor. The antibodies bind to the receptors and the muscle cells continually internalize the bound receptors and synthesize new ones. Myasthenia gravis is characterized by progressive weakness. Treatment can involve thymectomy, immunosuppressive drugs or inhibitors of acetylcholinesterase, the enzyme that inactivates Ach within the neuromuscular junction cleft.

II. CARDIAC MUSCLE

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A. involuntary striated muscle which has some morphological similarities to skeletal muscle, i.e. regularly organized sarcomeres

B. But cardiac muscle has some major differences:1. each muscle fiber has one or occasionally two, centrally positioned nuclei

2. the fibers (cells) are branched

3. fibers joined by intercalated disks - specialized junctional complexes between cells that allow cells to act in synchrony

4. ultrastructurally, cardiac muscle has diads at the Z line rather than triads at the A-I junction5. 40% of cardiac muscle cell volume is occupied by mitochondria versus about 2% for skeletal muscle

III. SMOOTH MUSCLE involuntary, non-striated

A. elongated, spindle shaped cells with a single centrally located nucleus

B. typically found in walls of hollow organs and blood vessels

C. contain actin, tropomyosin and myosin, but no organized sarcomeres. Filaments are loosely organized in association with dense bodies. Dense bodies apparently serve same function as Z-line in striated muscle.

D. desmin (skeletin) is the major intermediate filament protein in all smooth muscle cells. Additionally, vimentin occurs in vascular smooth muscle cells

E. have no troponin - CALMODULIN IS Ca2+ binding protein. NOTE: Contrary to Junquiera, tropomyosin is present in smooth muscle. It is just NOT complexed with troponin.

F. smooth muscle cells can occur singly or in sheets

G. distinction made between visceral smooth muscles, which are poorly innervated and multiunit smooth muscle, like those of the iris of the eye, which have precise, graded contractions

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BLOOD(Chapter 10)

A. General features of blood

1. Components

a. cells (formed elements): red blood cells (RBCs) or erythrocytes, white blood cells or leukocytes and platelets

b. intercellular fluid

1. plasma is the fluid that suspends blood cells; it is an aqueous solution and contains proteins (7 -8% of volume), inorganic salts (0.9% of volume); The major proteins in plasma are albumin which is essential for the maintenance of blood osmotic pressure. Additionally, blood contains globulins including immunoglobulins (Igs), which are antibodies, and fibrinogen (made in the liver), which is essential for clotting (fibrin clot).

Blood also serves as a carrier for amino acids and hormones as well as vitamins.

2. serum is a clear yellowish fluid that remains after the blood clots.

2. Hematocrit After centrifugation, erythrocytes end up at the bottom of the tube, leukocytes

and platelets on top of them and plasma at the top. The hematocrit is useful in that it provides an estimate of the volume of these components. Red cells are around 43% of the total volume. White cells and platelets are around 1% and plasma is the rest. Low hematocrit is indicator of anemia.

3. Blood smears Blood smears are an important and one of the oldest clinical tools. They use

Romanovsky-type stains (Wright stain or Giemsa stain) that contain methylene blue, eosin and azures.

4. Functions

a. transport of O2 (mainly in RBCs) and CO2 (both in RBCs and in solution as

CO2 and HCO3), nutrients, metabolic waste products and hormones.

b. helps maintain homeostasis: body temperature, acid-base balance (pH) and osmotic balance

c. blood specimens are readily available for clinical testing: blood counts (eg. CBCs), blood chemistries and blood gases.

5. Hematopoiesis (blood cell production). Most blood cells have short lifespans. They are, therefore continuously produced by replication and differentiation of hematopoietic stem cells in the bone marrow.

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B. Erythrocytes (red blood cells, RBCs)

1. Normal count: around 4-5 million/mm3. Males normally have more RBCs than women as reflected in their higher hematocrits. The number of RBCs can decrease (hypocythemia) and such a decrease causes anemia. An increase in the number of RBCs is called polycythemia. Polycythemia can be an adaptation to living at a high altitude.

2. Normal hematocrit: normally around 43%. Males normally have a higher hematocrit than females. Significant increases (polycythemia) and decreases (hypocythemia) in the number of RBCs can be detected with the hematocrit.

3. Normal morphology:

a. shape: biconcave disk. This shape optimizes the surface to volume ratio and is important for gas exchange.

b. size: 7.5 µm diameter in an isotonic suspension. About 7µm in tissue sections. Abnormalities in size can be indicative of pathology. Macrocytes have diameters greater than 9µm. Microcytes have diameters less than 6µm.

c. mature, normal RBCs have no nuclei, organelles or cytoplasmic inclusions.

4. RBC membrane (plasmalemma) maintains its unique shape by its association with cytoskeletal components such as spectrin, actin and ankyrin. They permit flexibility as the cells pass through the narrow lumens of capillaries. Moreover, the flexibility of the RBCs allow the blood to maintain a relatively low viscosity.

5. Hemoglobin content: About 33% of the cytoplasmic volume.

a. reversibly combines with O2 to form oxyhemaglobin and CO2 to form

carbaminohemaglobin. Hemaglobin irreversibly with CO to form carboxyhemaglobin. This latter combination blocks oxygen and carbon dioxide transport and can consequently be fatal (carbon monoxide poisoning).

b. abnormal hemoglobin can impair gas transport. In the case of sickle cell disease, a point mutation causing a replacement of glutamic acid with valine. This seemingly innocuous variation actually causes serious impairment that affects gas transport, causes hemaglobin polymerization that results in rigid, inflexible RBCs. These rigid, sickle-shaped cells can impair bloodflow.

c. anemia is caused by a low blood hemoglobin concentration. It can be due to decreased number of RBCs (hypocythemia, see above) or to decreased hemoglobin per RBC (hypochromic anemia).

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6. Energy metabolism: main source of energy for RBCs is glucose.

a. glycolysis (anaerobic: 90% of glucose breakdown): utilizes glucose to generate ATP

b. hexose monophosphate shunt (aerobic: remaining 10% of glucose degradation): regulated by glucose-6-phosphate-dehydrogenase.

7. Reticulocytes: Younger RBCs. By special staining (cresyl blue), they can be seen to contain some residual ribosomal RNA. During normal maturation the cells lose mitochondria and ribosomes. They are released from the bone marrow at this stage and are then present in the peripheral blood for about one day. They are not usually visible on routine blood smears. Reticulocytes are normally about 1% of circulating RBCs; represents daily replacement of 1% of RBCs. Reticulocyte count is useful in diagnosis of anemia or in monitoring response to treatments that increase RBC production.

C. Leukocytes: White blood cells.

1. General features

a. Leukocytes are classified as either granulocytes (polymorphonuclear) or agranulocytes (mononuclear).

b. There are two classes of cytoplasmic granules:1. specific granules that are found only in granulocytes. They are

neutrophilic, eosinophilic (red), or basophilic (blue). They stain specifically because of their contents. Granulocytes are classified on the basis of the type of specific granules they contain.

2. azurophilic granules stain with the azures in the dye mixtures. They are lysosomes and are found in granulocytes and agranulocytes.

c. leukocytes are transient components of blood. They serve their functions in the tissues.

d. leukocytes function in defense against foreign materials and participate in inflammatory reactions and immune responses.

2. Neutrophils make up 55-60% of leukocytes in normal blood and thus are the most common leukocyte. The main function of neutrophils is the phagocytosis and killing of bacteria. They are very active phagocytes of small particles and as such are sometimes referred to as microphages. Particularly suited to survival in an anearobic environment since bacteria are often found in oxygen-poor regions.

a. The nucleus of neutrophils typically have 2-5 lobed nucleus. More immature cells have horseshoe-shaped nuclei (called stab or band cells). The appearance of large numbers of band or stab cells in the circulating blood is called a shift to the left and is indicative of a bacterial infection. Older neutrophils have more than 5 lobes

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(hypersegmented). In females, inactive X-chromosome (Barr body) forms a "drumstick" nuclear appendage in some cells.

b. specific granules: small (cannot be seen by LM). These granules are neutrophilic (salmon-pink). They contain alkaline phosphatase, collagenase,

lactoferrin, and lysozyme. Lactoferrin binds iron which is crucial to bacterial nutrition. Lysozyme specifically cleaves peptidoglycan that forms the cell wall of some gram+ bacteria. Collagenase is used by the neutrophils to move through the connective tissues.

c. azurophilic granules: larger, purple granules that contain acid phosphatase and other lysosomal enzymes. The azurophilic granules also contain myeloperoxidase.

d. During phagocytosis there is a production of superoxide (O2-), hydrogen

peroxide (H2O2) and nitric oxide (NO) which have a bacteriocidal action.

3. Eosinophils are much less numerous compared to neutrophils. They comprise 2-5% of leukocytes in blood. Their main functions are phagocytosis of antigen-antibody complexes, moderation of the allergic response (i.e. eosinophils make histaminase that neutralizes histamine) and participation in the killing of parasitic worms. Increased numbers of eosinophils (eosinophilia) are seen during allergic reactions and parasitic infections.

a. The nucleus of eosinophils is usually bilobed.

b. eosinophilic granules: large, eosinophilic, orange-red granules. At the EM level, the granules can be seen to have a crystalline core (internum). The internum contains major basic protein which has a large number of arginine residues. Major basic protein is responsible for eosinophilia of granules. Surrounding the internum is the matrix (externum) which contains acid phosphatase, arylsulfatase, RNAse, phospholipase, peroxidase, cathepsin and glucoronidase.

4. Basophils comprise the fewest number of white cells (0-1%). Although they arise from bone marrow stem cells different from mast cells, their function appears to be similar. They are involved in the mediation of inflammatory reactions and in some instances, may assist mast cells.

a. The nucleus typically has 2-3 lobes, but lobulation is difficult to see because basophilic granules in ther cytoplasm obscure the nucleus.

b. basophilic granules are large, purple and irregular in shape. They contain heparin, histamine and produce leukotrienes.

5. Lymphocytes comprise 30-35% of circulating white cells and are the most common agranulocyte. The circulating lymphocytes represent a mixed population of diverse cell types with similar morphologies. Lymphocytes can only be distinguished immunocytochemically based on cell surface markers, They function in immune responses and are common in C.T. and lymphoid tissue & organs.

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a. structure of lymphocytes1. small lymphocyte: spherical nucleus, condensed chromatin, thin rim of

basophilic cytoplasm and few azurophilic granules2. medium and large lymphocytes are larger in diameter, have more

cytoplasm and less heterochromatic nuclei. These cells are activated lymphocytes.

b. two main classes of lymphocytes1. B cells: Bursa- or bone marrow-derived cells. They have surface

immunoglobulins that recognize specific antigens. They differentiate into plasma cells and are responsible for humoral immunity. Some B cells differentiate into plasma cells and are then involved in antibody production; others form memory B cells. Memory cells are primed to respond rapidly to subsequent antigen exposures.

2. T cells: are thymus-derived. They have surface T cell receptors that are not immunoglobulins, but that recognize antigens on cells. They are responsible for cell-mediated immunity. T cells secrete lymphokines that modulate macrophage function. There are 4 types of T cells: helper, suppressor, cytotoxic, and memory T cells. Cytotoxic T lymphocytes (killer T lymphocytes) recognize foreign antigens on cell. surfaces. Virus-infected cells that display viral glycoproteins on their surfaces are classic targets of cytotoxic T lymphocytes. Helper T cells release lymphokines that stimulate B lymphocytes and other cells that participate in immune reactions. Suppressor T cells suppress the activity of B cells and may be important in suppressing the immune response to self molecules (i.e. preventing humoral autoimmunity).

c. null cells are lymphocytes with no T or B surface markers. It is believed that null cells may be circulating stem cells or natural killer cells.

6. Monocytes comprise 3-8% of circulating leukocytes. They are the largest leukocytes. Monoctytes are large cells with oval, horseshoe- or kidney-shaped nuclei with lightly stained chromatin. They have basophilic cytoplasm and small azurophilic granules. Monocytes differentiate into macrophages in connective tissue. They form all of the cells of the mononuclear phagocyte system (dust cells, Kuppfer cells, etc). Macrophages are responsible for phagocytosis, and cooperate with lymphocytes in antigen recognition and processing.

D. Platelets (thrombocytes). they are discoid cell fragments (non-nucleated). Platelets originate from megakaryocytes in bone marrow. Their primary function is to stop bleeding, form a temporary plug of an injured vessel and induce repair of the injury by endothelial and smooth muscle cells. They have a peripheral light zone (hyalomere), that has a bundle of microtubules to maintain cell shape and a central dense zone (granulomere) with 3 types of granules:

1. delta granules (dense bodies) contain calcium, ADP and ATP. They take up and store serotonin from the plasma. 2. alpha granules are larger than delta granules. They contain fibrinogen, platelet-derived growth factor and several other proteins.3. lambda granules are small vesicles and contain lysosomal enzymes.

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When blood vessels are damaged, discontinuities in the endothelial lining exposes the underlying connective tissue to platelets in the circulation. The platelets adhere immediately (primary aggregation) to this region forming a platelet plug. The platelets in the site release ADP, an inducer of platelet aggregation, and serotonin which causes contraction of vascular smooth muscle which reduces local blood flow. Platelets induce fibrinogen in the plasma to form a fibrin clot. The fibrin traps blood cells and forms a blood clot or thrombus. The resulting clot bulges into the vessel lumen, but contracts via the action of platelet actin and myosin in conjunction with ATP. Platelet-derived growth factor (PDGF) stimulates proliferation of fibroblasts and smooth muscle cells that contribute to repair of the injury site.

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HEMATOPOIESIS

A. Hematopoietic organs

Prenatally, primitive blood cells arise from yolk sac mesoderm. Later in embryogenesis, the liver and spleen develop hematopoietic abilities. Finally, the ossification of the fetal bones results in the formation of bone marrow, which becomes the primary hematopoietic tissue.

Postnatally, the bone marrow (myeloid or medullary tissue) produces most of the blood cells, except for the lymphocytes. Most of them are produced in lymphoid tissues and organs with some being produced in the bone marrow. If hematopoiesis in the bone marrow becomes inadequate, e.g., after excessive blood loss, the liver or spleen can reinitiate blood cell formation. This is called extramedullary hematopoiesis.

B. Bone marrow histology

Bone marrow is one of the largest organs of the body, comparable in total size to the liver (1500g). Under normal conditions, the bone marrow can rapidly increase its production of blood cells to adjust to the needs of the body. In the adult there are 2 types of marrow, yellow and red.

1. Yellow (inactive) bone marrow is found in long bones of adults. Under severe conditions, such as blood loss or hypoxia, yellow bone marrow can convert into red bone marrow. It contains numerous adipose cells and undifferentiated mesenchymal cells, which serve as a reserve of hematopoietic tissue.

2. Red (active) bone marrow is found mainly in the flat bones of adults. It functions in blood cell production, RBC destruction and iron storage. It has 3 main components:

a. hematopoietic cords: clusters of developing blood cells

b. stroma: network of reticular cells, reticular fibers and macrophages. The extracellular matrix molecules (laminin, fibronectin, hemonectin) are important for binding hematopoietic cells to the stroma. Hematopoietic growth factors (colony-stimulating factors) produced by the stromal cells are essential for hematopoiesis.

c. sinusoidal capillaries: large diameter capillaries with a continuous layer of endothelial cells. Some areas of the endothelium are thin and may be where mature cells enter the bloodstream. The endothelium is supported by an underlying reticulum formed by reticular cells and fibers. The mechanisms that regulate release of blood cells into the sinusoids are unclear.

C. Hematopoiesis

1. At one time there was a controversy over whether all blood cells, red and white, arose from a common stem cell (the monophyletic theory) or whether each blood cell type arose from a separate stem cell (the polyphyletic theory). The evidence, as

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outlined on the next page, supports the monophyletic theory, all blood cells arise from a common stem cell, a pluripotential stem cell (refer to Figure 10.4 on page 265).

a. in vivo studies in mice: bone marrow cells from donor mice are injected into lethally irradiated mice whose hematopoietic cells have been destroyed; the transplanted bone marrow cells form colonies of hematopoietic cells in the spleen. By using marker chromosomes, it can be shown that each colony is a clone of a single colony-forming cell or colony-forming unit (CFU).

b. in vitro studies using semisolid culture medium: hematopoietic colonies develop in vitro by placing stem cells in culture with specific colony-stimulating factors (CSFs). Genetic markers demonstrate that each colony is a clone of a single colony-forming cell.

c. pathophysiology of certain blood cell disorders: e.g., in chronic myeloid leukemia, a marker chromosome called the Philadelphia chromosome (representing a translocation) is found in all of the dividing granulocytic, erythrocytic, and megakaryocytic precursors (and occasionally in B cells), suggesting their origin from a common stem cell.

2. Hematopoietic lineage

a. pluripotential stem cell: single type of cell in bone marrow; produces all blood cell types. The pluripotential cell is self-renewing.

b. multipotential stem cells: self-renewing cell, consists of 2 types:1. lymphoid stem cell: forms lymphocytes (B, T, NK?)2. myeloid stem cell: forms all other blood cells: erythrocytes, granulocytes, monocytes, and megakaryocytes (platelets)

c. progenitor cells: self-renewing; several types of cells, each leading to production of a single blood cell type; called colony-forming cells or units (CFU); e.g., erythrocyte-colony-forming unit (CFU-E)

d. precursor cells: not self-renewing, i.e. cannot give rise to other cells like themselves; they form a single type of blood cell; can be morphologically distinguished; e.g., proerythroblast, myeloblast

4. Hematopoietic growth factors (colony-stimulating factors, CSFs, cytokines)

a. several CSFs have been identified and characterized:

1. interleukin 3 (IL-3): controls pluripotential and other stem cells. Produced by T lymphocytes.

2. GM-CSF: Controls myeloid stem cells. Produced by T lymphocytes, endothelium and fibroblasts.

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3. erythropoietin: Essential for RBC development. Erythropoietin is produced by kidneys when the kidneys are exposed to low O2. It is a glycoprotein that stimulates mRNA for globin synthesis.

b. CSFs regulate proliferation and differentiation of stem cells; they act at different stages of stem cell differentiation, and each can act at multiple steps. They also stimulate functions of mature cells.

c. recombinant CSFs are being used clinically to increase blood cell proliferation in various conditions; e.g., CSFs can enhance the success of bone marrow transplants; erythropoietin is used to treat anemia due to chronic renal failure; GM-CSF is used to treat neutropenia (decrease in neutrophils)

D. Erythrocyte Development

1. Erythrocytic lineagea. pluripotential stem cellb. myeloid stem cell - multipotential stem cell: CFU-GEMM (colony forming unit-

granulocyte, erythrocyte, monocyte, megakaryocyte)c. ECFU (erythrocyte colony forming unit)d. proerythroblast - precursor cellf. differentiation - described below

2. Cellular changes during erythrocyte development (from proerythroblast to erythrocyte)

a. decreased cell sizeb. decreased nuclear sizec. increased chromatin condensation: nucleus becomes pyknotic and finally

extrudedd. change in cytoplasmic color: basophilic to eosinophilic as rRNA decreases

and hemaglobin increases

3. Morphologically visible stages a. proerythroblast (pronormoblast) - relatively large cell (12-15µm) with

basophilic cytoplasm. The nucleus is large with one or two prominent nucleoli. Not easily identified in marrow smears. This cell can divide.

b. basophilic erythroblast (basophilic normoblast)* - this cell has a very basophilic cytoplasmic and a round nucleus with distinct, open spaces in the chromatin. This cell can divide.

c. polychromatophilic erythroblast (AKA polychromatophilic. normoblast)* - as hemoglobin accumulates in the cytoplasm and rRNA begins to "disappear", the cytoplasm takes on a grayish appearance due to a mixture of pink and blue. The nucleus is smaller with noticeably fewer spaces in chromatin. This cell can divide.

d. orthochromatophilic erythroblast (AKA normoblast)* - cytoplasm has nearly color of red cell cytoplasm. Nucleus is very small, eccentric and dark (pyknotic) with no spaces in chromatin

e. reticulocyte - retains some rRNA that can only be detected with cresyl blue stain

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f. erythrocyte(* indicates cells to be identified in lab)

E. Granulocyte development

1. Neutrophilic lineage: (separate lineages for neutrophils, eosinophils and basophils)

a. pluripotential stem cellb. myeloid stem cell - CFU-GEMM - multipotential cellsc. CFU-GM - (colony forming unit-granulocyte:monocyte) bipotential progenitor

cellsd. CFU-G - (colony forming unit-granulocyte) uni- or mono-potential progenitor

cellse. myeloblast (committed precursor cell)f. differentiation (see below)

2. Cellular changes during neutrophil development (from myeloblast to neutrophil)a. changes in cell and nuclear size and stainingb. formation of azurophilic (primary) granules (lysosomes)c. formation of specific (secondary) granules - appearance of specific granules is

first sign of maturation. Eventually fill cytoplasm

3. Morphologically visible stages

a. myeloblastb. promyelocyte* - characterized by basophilic cytoplasm and numerous

prominent, azurophilic granules, nucleus oval. This cell can divide.c. neutrophilic myelocyte* - first appearance of specific granules, nucleus

flattened on one side, eccentically positioned. This cell can divide.d. neutrophilic metamyelocyte* - more specific granules, indented nucleus.

This cell cannot divide.e. band (stab) cell* - u-shaped or horseshoe-shaped nucleus, can appear in

circulation. Large numbers called shift to left.f. mature or segmented neutrophil

4. Kinetics of Neutrophil Productiona. medullary formation compartment

1. mitotic compartment2. maturation compartment -

b. medullary storage compartment - capable of releasing large numbers of netrophils to circulation

c. circulating compartmentd. marginating compartment - adhere to endothelium - not in circulatione. marginating and circulating compartments about equal sizef. diapedesis (leaping through) - mechanism by which neutrophils enter

connective tissueg. neutrophils survive in connective tissue about 1-4 days regardless of whether

they have phagocytosed bacteria.

5. Changes in numbers of circulating neutrophils33

a. neutropenia - a decrease in the number of neutrophils. Can have a number of causes that include:

1. decreased productiona. irradiationb. drug-induced (anti-inflammatory drugs such as indomethacin)c. viral infections (AIDS, etc.)d. alcohol use

2. ineffective production such as in megaloblastic anemia3. increased destruction as occurs in overwhelming infections

b. neutrophilia - an increase in the number of circulating neutrophils can be caused by:

1. bacterial infections (shift to left)2. tissue injury such as burns, acute inflammation3. emotional stress4. physical stress - exercise releases neutrophils from the marginating

pool. Is a temporary increase.

F. Agranulocyte maturation - difficult to study histologically

1. lymphocytes - likely that all lymphocytes arise from bone marrow.a. T lymphocytes arise from undifferentiated lymphocytes that migrate from bone

marrow to the thymusb. other lymphocytes remain in the bone marrow - give rise to B lymphocytes

2. monocyte maturationa. monoblast - looks like a myeloblastb. promonocyte large cell, basophilic cytoplasm, neucleus is large and slightly

indentedc. monocytes enter circulation where they remain for about 8 hours. Then rthey

enter the connective tissue by diapedesis. Mature into macrophages in connective tissue where they can remain for months

G. Platelet Production

1. originate from mature megakaryocytes, giagantic cells in bone marrow

2. megakaryocytes arise from megakaryoblasts, large polyploidal cells (up to 30 times the amount of DNA found in normal cells)

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