ArtExaminingInterpreting hystology

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THE ART OF EXAMINING AND INTERPRETING HISTOLOGICPREPARATIONS: A LABORATORY MANUAL AND STUDY GUIDE FOR HISTOLOGY

Second Edition

William J. KrauseDepartment of Pathology and Anatomical Sciences

School of MedicineColumbia, Missouri


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TABLE OF CONTENTS

Introduction 3Chapter 1 Getting started 4Chapter 2 Epithelium 8Chapter 3 General connective tissue 15Chapter 4 Specialized connective tissue 19Chapter 5 Muscle tissue 23

Chapter 6 Nerve tissue 25Chapter 7 Peripheral blood & bone marrow 30Chapter 8 Cardiovascular system 37Chapter 9 Lymphatic organs 42Chapter 10 Integument 47Chapter 11 Digestive system 50Chapter 12 Respiratory system 61Chapter 13 Urinary system 66Chapter 14 Male reproductive system 70Chapter 15 Female reproductive system 75Chapter 16 Classic endocrine glands 81Chapter 17 Organs of special sense 85

Appendix Tables 91Index 98


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INTRODUCTION

The examination and interpretation of tissue sectionsseen under the light microscope in a laboratory settingis an example of student-directed, independentproblem solving. The proper reading of a histologicsection is an acquired art that can only be developedthrough practice, close observation and repetition.

This laboratory manual was designed as a guide forstudentsto aid them in this endeavor. The laboratorystudy guide/manual was designed to be used as asupplement to any current textbook and/or atlas ofHistology.Learning objectivesprovide the overallgoals for each chapter. The narrative of the studyguide explains how to systematically breakdown,examine and interpret each tissue and/or organencountered, without regard to a given histologic slidefrom a specific slide collection. Thus, this systematicmethod can be used to examine and interprethistologic preparations from any collection or of any

species.

Thestudent is encouraged to sketch, label andcreate a personalized atlaswhile using thislaboratory manual as a guide. Thevocabulary thatshould be developed and used during the laboratorycan be found quickly by going to thebold facetype inthe appropriate segment of the text. Each chapter

contains one or moretablesin which key structuresused in the identification of a tissue/organ arepresented, offering the briefest possible summary ofimportant histologic features. As a final short review,anappendixprovides summary tables that compareand contrasts the basic differences of severalstructures that are somewhat similar in generalarchitecture.

WilliamJ. KrauseDepartment of Pathologyand

Anatomical Sciences, School of Medicine,Universityof Missouri-Columbia, Columbia, MO

August 2004


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CHAPTER 1. GETTING STARTED

Have an appreciation of how a histologicpreparation is made

The human body consists of two basic components:cellsandproducts ofcells (extracellular materials).The discipline of histology is concerned primarily withthe microscopic examination of these two

components and how they are organized into thevarious tissues and organs of the body.

Obviously, if the liver, or a similar organ, wereto be examined, it would be impractical to place theentire organ under a routine light microscope forstudy. It is not only much too large, but also opaque,therefore an examination of its microcomponentswould be impossible. For this reason, and severalothers, a small portion of a specific tissue or organmust beexcisedfrom a given organ and processedfor microscopic analysis. The excised tissue is placed,as soon as possible after removal, into a reagent

known as a fixative.Fixativesact to preserve the cellsand extracellular substances of tissues/organs andprevent autolytic (degenerative) changes. Althoughthere are numerous fixatives developed for a variety ofpurposes, 10% buffered formalin is one of the mostcommonly used, routine fixatives in biology, medicine(surgical and general pathology) and biomedicalresearch. The collected tissues, once fixed, are thendehydratedin graded solutions of alcohol or otherdehydrating agents. Following removal of the majorityof water from the collected specimens duringdehydration, the tissues are cleared. Clearingis theprocess of removing the dehydrating agent andreplacing it with a fluid that is miscible both with thedehydrating agent used and with the type ofembedding medium chosen to make the tissue samplefirm throughout. As with dehydrating agents, there area large number of clearing reagents, the selection ofwhich is dependent largely on the embedding mediumchosen. Xylene and toluene are in common use forparaffin embedding; propylene oxide for embedding inseveral of the plastic embedding media. The tissuesample is next infiltrated withandembedded inthechosen embedding medium so that a firmhomogeneous mass of material containing the tissue

sample is obtained. Paraffin is the most commonlyused embedding medium for routine preparations.The formed paraffin block, together with thecontained tissue, is then sectioned (cut) into very thinslices calledsectionsthat normally range between 4and 7 microns (m) in thickness. The instrument usedin cutting histologic sections is called amicrotome.The cut sections are then transferred (mounted) ontothe surface of clean glass microscope slides.

In order to prepare themounted sectionsforstaining, the paraffin embedding medium must beremoved. This is accomplished by passing the slidestogether with their mounted sections through xyleneor toluene to remove the paraffin and then throughdescending strengths of alcohol solutions to water, asmost dyes used are in aqueous solutions. Stainingis a

process of increasing the visibility of cells by theapplication of dyes or by the reaction of chemicalreagents with the tissue components to form visiblesubstances. A large number of stains are available butgenerally only two stains are used together to providecontrasting color: one to stain the cytoplasm of cells,the other to stain the nuclei. The most common anduniversally used combination is thehematoxylin andeosin (H&E) stain. When this stain is applied to asection of tissue thenuclei of component cells appearblue; thecytoplasmand most extracellularmaterialsa light pink-orange. Staining is necessary

because the vast majority of cells and theirextracellular materials are transparent and lack color.Only naturally occurring pigment granules such asmelanin and lipofuscin would be visible onexamination. The color of the dyes used duringstaining markedly increases the contrast of cells, theirsub-components, and the associated extracellularmaterials. Without staining, the examination of cellsand tissues with a routine light microscope would beextremely difficult. Following staining in aqueous dyes,the slides, together with their mounted, stainedsections, are passed back through ascendingconcentrations of alcohol for dehydration, clearedwith some solvent (usually xylene or toluene), andthen a permanent mounting medium is put on thetissue section. A thin glass cover slip is then placed onthe covering mounting medium and underlying tissuesection and allowed to dry. As the histologicalpreparation dries, the solvent evaporates from themounting medium, which hardens, permanentlycementing and sealing the tissue preparation betweenthe glass slide and overlying cover slip. The mountingmedium (balsam, damar, Permount) when dried hasnearly the same refractive index as glass. After drying,the histologic section is well protected and if stored

properly will usually remain unchanged for severalyears.


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Identification of the luminal surface (the lining of theinternal space) as well as the external surface will be ofimportance when this preparation is examined further.Does the preparation have a uniform consistency andappear as a solid mass of tissue cut in the shape of asquare, rectangle or wedge? If so, such a preparationusually indicates that the sample of tissue was takenfrom a large compact (solid) organ such as the liver,spleen, kidney or pancreas to list just a few. Once a

determination has been made with regard to the shapeand consistency of the tissue mounted on the slide,then examine it more carefully with regard to itsstaining characteristics. Of particular importance is tonote if one surface (external edge or luminal surface ofthe doughnut-shaped configuration) stains morebasophilic (light blue) than any other region in thesample of tissue being examined. Such basophilicstaining usually indicates a high concentration ofnuclei per unit area (nuclei stain blue with hematoxylindye). In this way, one of the basic tissues, epithelium,can usually be located even before the histologic slide

is ever viewed under the microscope. Are additionalsmall tubular or round structures present within thetissue sample? These may indicate small blood vessels,ducts or glandular structures.

Always begin the initial examinationof ahistologic preparation with the low-power(scanning) objectiveand carefullyview the entiresection. This opportunity should be used to confirmor deny the observations and speculations made bydirect observation with the naked eye. Add moredetails to the mental image being developed withregard to the preparation under examination. Note thepresence or absence of more than one tissue type,patches of deeper staining, other structures present,their locations and relationships to one another and tosurfaces. Only after a thorough examination with thelow-power objective should the intermediate- andhigh-power objectives be used. Of these, the medium-power objective is the more useful for study, althoughmore detail can be seen with the high-power objective.The disadvantage of the high-power objective is thesmaller field of view and, because of this, relationshipsbetween tissues are often lost. The oil objective, ifused at all, should be used only in the examination ofperipheral blood and bone marrow preparations.

Since the tissues and organs of the bodyconsist only of two elements, cells and cell products(extracellular materials) both deserve careful andthorough study. The initial exercise should be toexamine themorphologyof a cell. The followingobservations should be made during the examinationof various cell types.

1.Shape of the cell.2.Size of the cell (determine the position of the cell

membrane).3.Shape, size and position of the nucleus.4. Identify the nucleolus if present in the nucleus.

It is absolutely essential that the boundary ofthe cell and that of the nucleus be clearly defined.Examine several cell types of various sizes and shapes

to make these observations.

Large cells

Examine a section of ovary for ova. These are locatedin the ovarian cortex at the periphery of the ovary.The ovum represents a very large, round cell. Becauseof their large size, these light-staining cells can befound, by using the low-power objective, near theperiphery of the ovary. After examining an ovum atlow power, study it further using increasedmagnification. Note again the large size of the ovum,

the abundant light-staining cytoplasm, and the centralround nucleus separated from the cytoplasm by a well-defined nuclear membrane (envelope). Identify thenucleolus. It is usually round in profile and stainsintensely. The nuclei of several ova may have to beexamined, as these cells are so large that the plane ofsection may not pass through the nuclear regioncontaining the nucleolus in all ova. Examine theremainder of the ovary and note the differences in thesize and shape of the different cell types. Note that thenuclear shapes most often assume the shape of thecells being examined and that the cell membrane ofmost cells cannot be resolved with the lightmicroscope. Therefore, when examining a number oftissues and organs the nuclei of component cells areoften relied on in determining the orientation andshape of the cellular component and the cytoplasmicboundaries of a give cell type are estimated. The shapeof the ovum is generally spherical, that of surroundingcells cube-shaped, whereas more distant cells in theovary are spindle in shape. Examine the nuclear profileof each group of cells, noting how the shape of thenucleus reflects the shape of the cell. In addition, notethe dark-staining, clumped nature of the chromatin issome nuclei. This material is referred to as

heterochromatinand often lies adjacent to thenuclear envelope. The lighter-staining nuclear materialis referred to aseuchromatin.

The next histologic preparation that should beexamined at this time is a transverse section throughthe spinal cord. Visual examination of the preparationwill reveal an H-shaped area (gray matter) near thecenter of the preparation that surrounds a smallcentral canal.


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Examine the gray matter with the low-power objectiveand locate numerous, large neurons (nerve cells) foundin the ventral horn. These too are exceptionally largecells with several irregularly shaped, elongatedprocesses. Make a clear distinction between thecytoplasm, nucleus and nucleolus.

Makea small labeled sketch of several cells fromthesepreparations illustratingthecytoplasm, thesizeand shapeof the

nucleus and theposition of thenucleolus.

Cytology: structural components of a cell that canbe examined with the light microscope

This laboratory guide briefly presentsa method forexamining the cytologic and histologicdetails ofhuman morphologyutilizing the routine H&Epreparation. This preparation primarily demonstratesthe nucleus and the surrounding cytoplasm of a givencell. It must be understood, however, that specialstaining methods can be used to demonstrate the

majority of organelles, inclusions and components ofthe cytoskeleton within a given cell, as well as a varietyof cell products and extracellular materials.Thesespecial staining methods include a variety of dyes,antibodies and other probes.

Techniques such as immunohistochemistry, insituhybridizationand autoradiographyare powerfultools in demonstrating structure, secretory products,and/or messages (mRNA)withincells, as well as cellreceptors not seen with routine preparations. Thestudy guide focuses primarily on what can bevisualized using the routine H&E preparation unlessotherwise stated.

Know the basics: the basic tissues

The termtissue[French tissu, woven cloth] is definedas a collection of similar cells and surroundingextracellular substances that perform related functions.Fourbasic tissuetypesoccur and these are woventogether to form the fabric of all organs.

The four basic tissues are: epithelium,connective tissue,muscle tissueandnervoustissue. A thorough understanding of each of thesefour basic tissues is necessary before beginning an

examination of individual organs or systems.


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CHAPTER 2. EPITHELIUM

Epithelia consist of closely aggregated cells separatedby only minimal amounts of intervening intercellularsubstances. Two general categories are recognized:alining, barrier or covering type of epitheliumorganized into sheets of cells that form barriers andglandular epitheliummodified for secretion. Thesheet (barrier) form of epithelium covers the external

body surface as the epidermis, lines the body cavities(pleural, pericardial, peritoneal) as well as the lumina ofthe cardiovascular, digestive, respiratory and urogenitalsystems. Thus, the majority of, if not all, substancesentering or exiting the "substance of the body" mustfirst cross an epithelial barrier. All epithelia lie on abasement membrane and are avascular. Thebasementmembraneappears as a thin interphase between theepithelium and underlying connective tissue. Itconsists of glycoproteins, proteoglycans rich inheparin sulfate and type IV collagen. Usually thebasement membrane appears only as an interphase in

H&E preparations but can be demonstrated clearlyusing special staining techniques. As different types ofepithelium are examined, a mental record should beestablished, keeping track of not only where a specifictype of epithelium is found in a given organ but alsoits functional ramifications. Later, in the examinationand identification of organs under the microscope, theidentity and location of a specific epitheliumis oftenakey featurein the identification.

Lining/covering (barrier) form of epithelium

Learning objectives for lining or covering

epithelium:

1. Be able to locate, identify and classify the varioustypes of epithelia in a given section of histologicmaterial.

2. Be able to identify the various specializations of thecell membrane associated with specific epithelia.

Classification

If the covering or lining form of epithelium consistsonly of a single layer of cells it is termedsimple. If

two or more layers of cells are present, of which thesuperficial most-cells do not reach the basementmembrane, the epithelium is classified asstratified.Once this determination has been made the next stepis to determine thegeometricshapeof thesuperficial-most cellsto complete the classification.Epithelial cells can be divided into three typesaccording to their geometric shape:squamous(thin,flat, plate-like cells),cuboidal (height and width of thecell are approximately equal with the nucleus nearly

touching all surfaces), andcolumnar(height of cell isgreater than its width). Cells intermediate in heightbetween cuboidal and columnar also occur and arereferred to as low columnar. Thus, epitheliumconsisting of a single layer of cells can be classified as:simple squamous, simple cuboidal, or simplecolumnar. A fourth type of simple epithelium,

pseudostratified columnar, consists of more thanone cell type whose nuclei occur at different levelsfalsely suggesting that the epithelium is made up oftwo or more layers. All cells of this type of epitheliumreach the basement membrane but not all reach theluminal surface.

The termendotheliumis the specific namegiven to the simple squamous epithelium that lines thecardiovascular and lymph vascular systems. Examinethe luminal (interior) surface of several blood vesselsfor this type of epithelium. The namemesotheliumisgiven to the simple squamous epithelium that lines the

pleural, pericardial and peritoneal cavities. Examinethe external surface of a region of the stomach,jejunum or ileum for mesothelium. Examine theluminal surface of each of these organs for simplecolumnar epithelium. Note:Prior to examining theseorgans under the microscope, each should beexamined with the naked eye. As an epithelium mustlie on onesurfaceor the other, and in the case of thegut, both, examine each of these surfaces under themicroscope initially with low power for orientationprior to moving to higher power objectives for a moredetailed examination.

Simple squamous epithelium

Examine the external surface of the stomach or smallintestine. Note that the cells making up this form ofsimple squamous epithelium appear extremelyattenuated, with their flattened, dense nuclei separatedby considerable distances. The cytoplasm oftenappears only as a thin interphase between nuclei.

Simple columnar epithelium

Examine the luminal surface of the stomach for typical

simple columnar epithelium. Note the basal positionof the oval-shaped nuclei and that the apical cytoplasmis filled with unstained secretory granules. These canbe visualized more clearly by lowering the intensity ofthe light and/or by dropping the condenser of themicroscope.Terminal barsalso can be seen in mostpreparations between the apices of adjacent cells.


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They appear as minute, dense-staining short bars andrepresent the light microscopic appearance of thethree part junctional complex (zonula occludens,zonula adherens, macula adherens) seen with theelectron microscope. Examine a textbook illustration(electron micrograph) of this important junctionalspecialization. Examine the luminal surface of a regionof small intestine. Note that it, like the stomach, islined by a simple columnar epithelium. Carefully

examine the intestinal lining epithelium and identifythestriated(microvillus)borderon the apicalsurface. Terminal bars also may be seen between theapices of cells forming this epithelium, as well as mostother simple columnar or cuboidal epithelia.

Simple cuboidal epithelium

This type of epithelium is best seen in a section ofkidney medulla, which also contains numerous tubuleslined by either a simple columnar or simple squamousepithelium. Tubules lined by either simple squamous,

simple columnar or simple cuboidal should beidentified and compared. The nuclei of cells in a goodsimple cuboidal epithelium should nearly touch apical,basal and lateral cell membranes. As different tubulesare examined, several examples of "low columnar" willalso be encountered.

Sketch and comparetubules formed byclassicexamples of thethreetypes of simpleepithelia observed.

Stratified squamous epithelium

Stratified squamous is the most common of thestratified epithelia. A good example of stratifiedsquamous is the epidermis of skin. Once again,examine the slide visually, noting the surface on whichthe epithelium rests, then examine it under low power.Note the presence of additional components of theskin but do not examine them at this time. Select anarea of epidermis and examine it carefully underincreased magnification, beginning at the basal surface.It should be quite obvious that this form of epitheliumis multilayered and consists of cells that vary in theirgeometric shape. Cells resting on the basementmembrane are columnar in shape whereas those above

assume a more spindle-shaped configuration. In theoutermost (superficial) layers, the cells become flat andplate-like, ie. squamous. Despite the large number ofcells with different shapes, recall that the classificationscheme remains based on two questions:

1.Are two or more layers of cells present, theoutermost of which is not in contact with thebasement membrane?

2.What is the geometric shape of the superficial-mostcells?

Thus, the classification must be stratified squamous.In the case of the epidermis the superficial most cellsalso undergo a transformation, known askeratinization. As a result of this process, cells loosetheir nuclei and the cytoplasm becomes filled with aproteinaceous material calledkeratin. These

transformed dead cells lie immediately above the intactlayer of squamous cells. When this type of surfacefeature is present, it is usually incorporated into theterminology of the classification scheme of theepithelium. Thus, in the case of the epidermis, thecomplete classification of the epithelium would bekeratinizedstratified squamous epithelium.Repeat this exercise examining the epithelium liningthe lumen of the esophagus. This epithelium lacks thelayer of keratin on its luminal surface. Therefore, it isclassified as anon-keratinized (wet) stratifiedsquamous epithelium. The term wet is often used

when this type of epithelium makes up a portion of amucous membrane or lines a moist environment. Notethe presence of intact nuclei in cells comprising thesuperficial most layer of this form of stratifiedsquamous epithelium.

Comparetheoverall thickness of this epitheliumwith that of theepidermis and makea labeled sketch of each.

Stratified cuboidal

This type of epithelium is limited in distribution toregions of ducts from larger glands where there is atransition from a simple epithelium into a stratifiedepithelium. Stratified cuboidal epithelium also lines theducts of sweat glands. Sweat glands are coiled tubescomprised of epithelial cells that extend from the baseof the epidermis into the tissue of the underlyingdermis and hypodermis. Because of their coiled nature,a profile of an entire sweat gland is rarely if everencountered in sectioned material. Using the low-power objective, scan the tissue beneath the epidermisin a section of skin looking for groups of small circularand oval profiles. These cellular profiles are oftenencountered surprisingly deep within the underlying

tissue. Two profiles will be encountered: one lightstaining, the other subtlety darker staining. The moredarkly stained portion of the tubule is the duct regionof the sweat gland. Confirm that the duct regionconsists of a stratified cuboidal epithelium, two cellsthick, which surrounds a minute central lumen.

Sketch theduct region of a sweat gland toillustratea stratifiedcuboidal epithelium.


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Stratified columnar

Like stratified cuboidal, stratified columnar isrestricted in its distribution confined primarily to thecavernous urethra, fornix of the conjunctiva and largeexcretory ducts of major glands. This epithelium ismost conveniently observed in the large ducts of theparotid or submandibular gland. Examine one of theseglands for the ducts only. They can be identified

initially with the low-power objective by scanning thegland and looking for tubules with a very large luminaldiameter. Once located, examine the wall of the ductand determine the nature of the lining epithelium. Is itstratified? Is the epithelium stratified cuboidal orstratified columnar? Both types will be encountered.Search the preparation until an example of each isfound.

Makesketches of twolargeducts: onelined bystratifiedcuboidal, theother lined bystratified columnar.

Pseudostratified columnar

This type of epithelium is a simple form of epitheliumas all component cells are in contact with theunderlying basement membrane, thereby satisfying thedefinition of a simple epithelium. The cells varyconsiderably in height and not all reach the luminalsurface. As a result, the respective nuclei are found atdifferent levels within this epithelium and form whatappears to be two or three layers of cells, falselysuggesting stratification, hence its name.Pseudostratified columnar epithelium is somewhatrestricted in its distribution, being confined primarily,but not exclusively, to the conducting portion of therespiratory system and the excurrent ducts of the malereproductive system.

Examine the trachea and epididymis forexamples of this type of epithelium. Examine theluminal surface of the trachea and note the height ofthe epithelium and the stratified appearance due to theposition of component nuclei. With the high-powerobjective examine the epithelium in detail beginning atthe base noting the different sizes and shapes of cellsforming this epithelium. Scattered within this epitheliallayer are unicellular exocrine glands known as goblet

cells. Goblet cellsare sandwiched among the otherepithelial cells and usually have a drinking goblet(wineglass) shape. The base of this cell is usually verynarrow and contains the nucleus. The apical region isexpanded due to the presence of numerous mucingranules. The latter are unstained in H&E preparationsand appear as clear vacuoles. With special mucin stainsthey appear solid and stain brilliantly. Examine theapical surface of the pseudostratified columnarepithelium for apical specializations called cilia. Cilia

appear as small tufts or hair-like structures protrudingfrom the apical cell surface. When cilia areencountered they are usually included as a prefix in thename of the epithelium. Hence, the epithelium liningthe trachea would be termed aciliatedpseudostratified columnar epithelium.

Examination of the epididymis with the low-power objective reveals an organ that consists ofseveral profiles of small tubules. In actual fact, the

epididymis consists of one extensively coiled,elongated tubule. An appreciation of this fact at thestart is of importance in developing three-dimensionalmental reconstructions of images from two-dimensional images examined under the microscope.Examine the pseudostratified columnar epitheliumlining the epididymal tubule at increasedmagnification. Note that it consists of two distinct celltypes: a small basal cell and a tall columnar cell knownas the principal cell. The latter have elongate, branchedmicrovilli extending from their apical surface, whichare calledstereocilia.

Makea sketch of theciliated pseudostratified columnarepitheliumliningthetrachea and compareit with an additionalsketch illustratingthepseudostratified columnar epitheliumliningtheductus epididymidis.

Transitional epithelium

Transitional epithelium is a stratified cuboidal type ofepithelium found only in the urinary system. Examinethis epithelium lining the interior of either the urinarybladder or ureter. Note the thickness of thisepithelium and the large dome-shaped cells at theluminal surface. The latter may be binucleate.

Makea sketch of transitional epitheliumfromtheliningof theureter.

Specializations associated with the cell membraneof epithelial cells

Microvilli

Closely re-examine the apices of cells forming thesimple columnar epithelium lining the small intestine.

Note thestriated (microvillus)borderfound at thislocation. Next, find and examine the proximalconvoluted tubule of the kidney. This tubule is foundonly in the renal cortex (the outer region of thekidney), is the longest tubule in the cortex (thereforeexhibits the most numerous tubular profiles) and is themost granular and darkly stained tubule. The apices ofcells forming the proximal tubule exhibit numerousmicrovilli closely packed together to form thebrushborderof light microscopy.


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Re-examine thestereociliaon the apices of theepithelial cells lining the tubule of the epididymis.Stereocilia are a thin, highly branched form ofmicrovillus.

Cilia

Re-examine the ciliated pseudostratified columnarepithelium lining the trachea. Note that individual cilia

can be seen. If the preparation is good (particularly ifthe embedding medium is a plastic resin of some type)numerousbasal bodiescan be visualized in the apicalcytoplasm immediately beneath the cilia and appear asa dense beaded line. Examine the ciliated simplecolumnar epithelium lining the oviduct for the samefeatures.

Makesketches comparingtheapical specializations found on theepithelial surfaces examined above. In addition, examineseveraltextbook illustrations of each apical specialization for theirultrastructural features.

Basal striations

Two organs that contain cells that show excellentexamples of this specialization of the cell membraneare the distal convoluted tubules of the kidney cortexand the striated ducts of the submandibular gland. Re-examine the cortical region of the kidney with the low-power objective for light-staining tubules. Whenexamined at higher magnification note that theselighter-staining epithelial cells show distinct nuclearprofiles and lack the microvillus border observed incells forming the proximal convoluted tubules.

Careful examination of the basal cytoplasm of cellsforming the distal tubule will reveal faint striations.The intensity of light in the microscope may have tobe decreased and/or the condenser lowered to makethe basal striations more visible. With special stainingtechniques (iron hematoxylin - which actuallydemonstrates mitochondria) the basal striations aredramatic. Basal striations represent a complexinfolding of the basolateral cell membrane plus a

parallel arrangement of associated mitochondria.Examine a section of submandibular gland for asmaller caliber duct within the lobules of the gland.The striated ducts are intralobular ducts and appear asnumerous circular profiles with wide lumina within theglandular tissue. The epithelium lining these ducts issimple cuboidal to columnar in type, is light stainingand agranular. Close observation of the basalcytoplasm using the same conditions as whenexamining the distal convoluted tubule of the kidneywill demonstrate faint basal striations within cellsmaking up the striated ducts. Note: these ducts were

so named because of the basal striations. Examine atextbook electron micrograph of cells from the distalconvoluted tubule of the kidney for basolateralinfoldings. Compare these features with those seenwith the light microscope.

Makea sketch illustratingthis morphological feature.

Table 1. Location of epithelia.

Type of Epithelium Location Specialization

Simple squamous Endothelium, mesothelium, thin segment of loop of Henle, retetestis, pulmonary alveoli, parietal layer of Bowmans capsule

Simple cuboidal Thyroid, choroid plexus, ducts of many glands, lens epithelialcells, covering surface of ovary, corneal endothelium

Surface lining epithelium of stomach, gallbladder, ducts of

several glands

Surface lining epithelium of small and large intestines Striated border

Proximal convoluted tubule of kidney Brush border

Distal convoluted tubule of kidney Basal striations

Simple columnar

Oviducts, uterus, small bronchi and bronchioles CiliaTrachea, major bronchi, eustachian tube Cilia

Large excretory ducts of glands, portions of male urethra

Pseudostratifiedcolumnar

Epididymis Stereocilia

Esophagus, epiglottis, corneal epithelium, vaginaStratified squamous

Epidermis of skin Keratin

Stratified cuboidal Ducts of sweat glands, large ducts of salivary glands

Stratified columnar Large ducts of glands, cavernous urethra

Transitional Restricted to urinary system: renal calyces to urethra


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Glandular forms of epithelium

Glands are comprised of epithelial cells specialized tosynthesize and secrete a product of some type. Avariety of different criteria can be used in theclassification of glands. The simplest classificationscheme is to divide the glands into endocrine(secretion into the lymph/ vascular system) and

exocrine(secretion onto an epithelial surface or into aduct) glands. A consideration of the endocrine glandswill be presented later as a specific topic.

Exocrine glands can be classified further as towhether they are unicellular or multicellular. A classicexample of aunicellular exocrine glandis the gobletcell. Re-examine the epithelium lining the intestine andtrachea for goblet cells. Carefully study this cell, payingparticular attention to its overall shape, position of thenucleus, and the apical accumulation of secretory(mucin) granules. Recall that the mucin granules,although unstained with the H&E preparation, can

usually be visualized by lowering the intensity of thelight and by lowering the condenser.The majority of glands aremulticellular

exocrine glands. Multicellular glands can assume awide variety of morphologies. They may occur as asmall group of secretory cells that lie wholly within anepithelial layer, clustered about a small lumen. Theseare calledintraepithelial glands. An example of thisglandular organization can be found in the non-keratinized stratified squamous epithelium lining thepenile urethrae of the male reproductive system.Examine this lining epithelium. The intraepithelialglands (glands of Littr) appear as clusters of clear or

light-staining cells within the more darkly stainedlining epithelium. The nuclei of component secretorycells are generally compressed to the base and theapical portion of the cell is filled with unstained mucinsecretory granules. A somewhat similar glandularorganization is thesecretory sheet. In this case, thecells form a continuous epithelial layer. An example ofthis type of multicellular gland is the gastric liningepithelium of the stomach. This glandular formconsists of a simple columnar, mucous secretingepithelium that secretes directly into the lumen of thestomach. Examine this epithelial form again.

The majority of multicellular, exocrine glandssecrete into a ductal system. These are classifiedaccording to the morphology of the ducts and howtheir secretory cells are arranged to form the secretoryportion of the gland. If theduct branchesthe glandis classified ascompound; if theduct does notbranchthe gland is classified assimple. The secretorycells of the gland may be arranged into tubulesand/ oracini (alveoli) (berry-like end pieces).Subsequent classification depends on the shape and

configuration of the secretory unit and whether theseportions also branch. Thus, simple glandscan beclassed as simple tubular, simple coiled tubular, simplebranched tubular, or simple branched acinar (alveolar).Compoundglandsare subdivided into compoundtubular, compound acinar and compound tubuloacinar(compound tubuloalveolar).

Learning objectives for glandular epithelia andexocrine glands:

1. Be able to classify glands according to theirhistologic organization, type of material secreted andmanner in which material is secreted.

Simple glands

Simple tubular glands

Examine a section of colon with the low-power

objective. Find and examine the luminal surface of thisorgan and note that it is lined by a simple columnarepithelium. Observe the large number of goblet cells.Identify tubular invaginations that extend from thisepithelium into the underlying tissue. These are thesimple tubular glands (intestinal glands) of the colon.The epithelium forming the walls of these glands is thesame as that lining the surface. If the plane of sectionthrough the wall of the colon is at an oblique angle,the glands may appear as isolated oval or circularcollections of columnar cells surrounding a tiny lumen.If the glands are cut parallel to their long axis, thelumen of the gland can be traced to that of the colon.

Next, examine a section of small intestine. In the smallintestine, the simple tubular intestinal glands openbetween the bases of fingerlike extensions of tissuecovered by simple columnar (intestinal) epitheliumcalled villi. Compare the glands at this location withthose of colon. Note the numerous mitotic figureswithin the epithelium forming these glands.

Drawand label a sketch of thesestructures, includingthelocation of thesimpletubular intestinal glands in a section ofcolon and small intestine.

Simple coiled tubular glands

Re-examine the section of skin for eccrine sweatglands as an example of a simple coiled tubular gland.Using the low power objective examine the deepsubcutaneous tissue far beneath the overlyingkeratinized stratified squamous epithelium for theseglands. The duct is long, extending from the surfaceepithelium to deep within the underlying tissue.


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The secretory portion ends as a highly coiled structuresimilar to that of a coiled snail shell. A section throughsuch a unit results in several circular cross-sectionalprofiles. These are lined by lightly stained simplecolumnar epithelial cells. The ducts are more darklystained and lined by stratified cuboidal epithelium.

Simple branched alveolar glands

Continue to examine the preparation of skin forsebaceous glands. These glands are classified as simplebranched alveolar glands and are almost alwaysassociated with hair follicles. The latter areinvaginations of the epithelium into underlying tissuethat produce and contain hair shafts. Study thesebaceous glands carefully. Note the presence of largeflask-shaped secretory units, the alveoli. Alveoli ofsebaceous glands consist of large cells filled with smalllipid droplets, which gives them a light-staining,vacuolated appearance. Note that two or three alveolidrain into a single, short unbranched duct lined by

stratified squamous epithelium. The duct empties intothe lumen of an adjacent hair follicle. The overallthree-dimensional shape of these glands is similar to athree or four leaf clover.

Sketch, label and comparea simplecoiled tubular sweat glandwith a simplebranched alveolar (sebaceous) gland of theskin(integument).

Simple branched tubular glands

Study a section taken from the pyloric region of thestomach. Identify the gastric pits. These are tubularinvaginations of the gastric surface lined by simplecolumnar epithelium that extend into the underlyingtissue. Emptying into the bottoms of the gastric pitsare the pyloric glands, an example of simple branchedtubular glands. Cells forming these glands have basalnuclei and a light/clear supranuclear cytoplasm thatcontains mucin granules. Note that in this glandularform, the duct remains unbranched (simple) and that itis the secretory tubule that branches.

Sketch and label thesubcomponents of a simplebranchedtubular gland fromthepyloricregion of thestomach.

Compound glands

Compound tubular glands

Examine a slide of the duodenum and scan it carefully.Find the location of numerous light-staining, secretorytubules of the duodenal (Brunners) glands within theintestinal wall. These glands are found in the tissuebeneath the bottoms of the simple tubular intestinal

glands examined earlier in other regions of theintestinal tract. Now examine the duodenal glands atincreased magnification. The terminal portions(secretory units) of the duodenal glands are branched,coiled and of uniform diameter. Component cells arelight staining with basally positioned nuclei. Thebranching ducts are lined by a similar appearingepithelium to that forming the secretory units. Theducts of the duodenal glands unite with the bottoms

of the overlying intestinal glands. Note the differencesin the lining epithelium between glands where thistransition occurs.

Sketch and label a duodenal gland includingits association withan overlyingsimpletubular intestinal gland.

Compound tubuloacinar (alveolar) glands

This type of gland represents the most commonglandular organization of the compound glands. As anexample, study a section of the submandibular

(submaxillary) gland with the low-power objectiveidentifying several important features. The gland isorganized into lobes and lobules. These glandularsubdivisions are limited by fibers of the surroundingconnective tissue. Next identify the duct system. Theducts can be recognized by their round profiles, widelumina, and the light-staining cytoplasm of componentcells. The epithelial lining is usually simple cuboidal orsimple columnar although stratified forms of bothtypes can be found on occasion lining the very largeducts. Note that two categories of ducts can berecognized: intralobular ducts and interlobular ducts.The former occurswithinthe lobules and in thesubmandibular gland are the most numerous. Theinterlobular ducts are larger and occur betweenlobules. The lining epithelium is usually simplecuboidal or simple columnar. Can basal striations beobserved with increased magnification? Because thebranched ductal system of the submandibular is welldeveloped, numerous profiles of the ductal system areobserved. Carefully examine the apices of cellsforming the ductal epithelium. Note the minute, densestaining points between cell apices. These are terminalbars. Next, examine the secretory units of thesubmandibular gland and observe that two markedly

different cell types makeup the tubules and acini(alveoli) of this gland. The most numerous areserouscells. Serous cells are characterized by a dark stainingcytoplasm filled with numerous, distinct secretorygranules. The basally positioned nuclei usually exhibita round or oval profile. The less numerous cell typemaking up scattered tubules is themucous cell. Thesecells are characterized by a white (clear) cytoplasm offrothy appearance. The latter is filled with unstainedmucin granules.


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Nuclei appear as darkly stained, compressed profilespositioned adjacent to the basal cell membrane. Someserous cells are organized into small units calleddemilunesthat cap terminal regions of the scatteredmucous tubules.

When both serous and mucous cell types make up thesecretory units of a gland, the gland is often referred toas amixed gland.

Sketch and label thesubcomponents of a lobefromthesubmandibular gland.


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CHAPTER 3. GENERAL CONNECTIVE TISSUE

It is important to realize from the onset that theconnective tissues are classified according to the typeandarrangementof theextracellularmaterialsrather than features of the cellular components, as istrue of epithelium. General connective tissues areclassified as loose or dense according to whether theextracellular materials are loosely or tightly packed.

Looseconnective tissue can be subdivided further onthe basis of special constituents such as adipose (fatty)tissue or a concentration of specific extracellularfibers.Denseconnective tissue can be subdividedaccording to whether the extracellular fibers arerandomly distributed (dense irregular connectivetissue) or orderly arranged (dense regular connectivetissue).

Loose (areolar) connective tissue

Areolar connective tissue is a loosely arranged

connective tissue that is widely distributed throughoutthe body. It consists of three extracellular fibers(collagen, reticular, elastic) in a thin, almost fluid-like,ground substance. The latter is not preserved inroutine preparations and accounts for some, but notall, of the spacing observed between the fibrous andcellular components. Areolar connective tissue formsthestromathat binds organs and the components oforgans together. It forms helices about the long axesof expandable tubular structures, such as thegastrointestinal tract and other visceral organs, theducts of glands, and blood vessels.

Fibers of connective tissue

Learning objectives for connective tissue fibers:

1. Be able to identify and distinguish between the threetypes of connective tissue fibers.

2. Be able to classify general connective tissuesaccording to the arrangement of their extracellularfibers.

Collagen fibersare present in all connective tissues,vary in thickness from 1 to 10 m and are of

undefined length. In H&E preparations they stain apink or pink-orange color. Because of theproteinaceous subcomponents these fibers, dependenton the dye used in staining, they can also be stainedblue, green, yellow or red. Examine the dermis of skin(that region underlying the keratinized stratifiedsquamous epithelium [epidermis]) for collagen fibers.Note the variation in size and the wavy, homogeneousappearance of the pink-orange staining collagen fibers.The majority of oval, densely staining nuclei in the

field are those of associatedfibroblaststhat secreteand maintain the collagen fibers. The extent of thefibroblast cytoplasm usually cannot be seen in H&Epreparations and what is actually visualized arefibroblast nuclei. Examine the external surface of amedium-sized (named) vessel for collagen fibers andfibroblasts. If available, examine a spread preparation

of loose areolar connective tissue for collagen fibersand fibroblasts. The advantage of this type ofpreparation (usually a portion of a mesentery) is that itis not a section of tissue but rather an intact tissue,which is thin enough to allow the transmission oflight. Examine the fibroblasts carefully, first notingtheir oval-shaped nuclei and then their associatedcytoplasm. In these preparations the extent of thefibroblast cytoplasm can often be traced forconsiderable distances. Note that the fibroblasts lieimmediately adjacent to, or on, collagen fibers, whichstain lightly (a light pink in most preparations). Close

observation of some fibroblast nuclei will reveal a lightappearing strip crossing the blue-stained fibroblastnuclei. These strips are collagen fibers as seen againstthe stained chromatin background of the fibroblastnuclei. Return to a section of skin and re-examine thedermis with low power. Note, once again, that it formsa thick interwoven layer beneath the overlyingepithelium. At increased levels of magnification notethat the abundant thick, collagenous fibers areinterwoven to form a compact network. The dermis isa classic example ofdense irregular connectivetissue. Next, a longitudinal section of tendon orligament should be examined in detail. Note theregular, precise arrangement of collagen fibers intobundles that run parallel to one another. Fibroblastsare the primary cell type present and occur in rowsparallel to the bundles of collagen fibers. Fibroblastnuclei usually are the only feature of these cellsvisualized and appear elongate and densely basophilic.Tendons and ligaments are classic examples ofdenseregular connective tissue.

Sketch and label thesubcomponents of a region of dermis andtendon (or ligament). Illustratethearrangement of collagen fibersand their association with fibroblasts. Examinean electron

micrograph froma textbook illustratingthefact that eachcollagen fiber (typeI) consists of banded unit fibrils, thesmallestmorphologicallydefined unit of collagen.

Elastic fibersappear as thin, homogeneousstrands that are smaller and of more uniform size thancollagen fibers. Usually elastic fibers cannot bedistinguished easily in routine H&E preparations andrequire special stains (orcein or Verhoeff's elastic stain)to make them visible.


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If a spread preparation of loose areolar connectivetissue stained to demonstrate elastic fibers is available,examine it carefully and note darkly stained, variouslysized, thin cylindrical fibers coursing across the field.These are elastic fibers. Look for an elastic fiber thathas been broken. Because of their elastic properties,broken fibers will form a highly undulated snarl muchlike broken elastic fibers of clothing (eg. stockings). Ifa specifically stained preparation is not available,

elastic fibers can be visualized to some degree usingthe same morphological criteria as demonstrated byspecial staining. However, in this case the elastic fibersstain the same color as collagen fibers but arenarrower (thread-like), smooth and homogeneous inappearance, and of more uniform diameter thancollagen fibers.

Examine a section of a named (muscular)artery for elastic tissue. Locate the lumen of the vesseland examine the region immediately beneath the liningendothelium. Note the highly scalloped, homogeneouslayer of elastic tissue (the internal elastic lamina) at this

location. If the vessel is specifically stained for elastin,move through the vessel wall and examine it for otherdark-staining elastic fibers of various sizes. Return tothe vessel interior. The internal elastic lamina is not afiber per sebut a thick homogeneous sheet of elastin.Now, and in the future, when additional arteries areencountered in routine H&E preparations, examinethese vessels for the internal elastic lamina. Inroutine preparations this highly scalloped appearing

membrane, although stained similar to collagen, has aslightly different refractive index. The appearance ofthe elastin can be made more visible by dropping thecondenser of the microscope. Use the known positionto locate and examine the negative image of theinternal elastic lamina using this technique. Examinesections of any other tissue specially stained todemonstrate elastic fibers. Examine them in bothlongitudinal and transverse profiles. Note again the

smooth, homogeneous nature of these darkly stainedfibers. They often give a "copper wire-like" appearancewhen seen in sections of tissue.

Reticular fibers, like elastic fibers, are notseen in routinely prepared sections but can bedemonstrated with silver stains or by the periodic acid-Schiff's (PAS) procedure. These are small fibers thatform delicate networks and are a major component ofthestromathat binds the cells of tissues and organstogether. The most commonly used organs todemonstrate reticular fibers are the liver, kidney,spleen and lymph node where these fibers are

especially prominent. With silver-stained preparationsthe reticular fibers stain black. Note the fine delicatenetwork of fibers supporting the cellular components(parenchyma) of these organs.

Makea sketch of reticular fibers and their association withparenchymal elements. Comparethesefibers with a sketch ofelasticfibers.

Table 2.Key histologic features of connective tissue fibers.

Fiber type Light microscopic appearance Primary locations

Collagen fibers (typeI collagen) Coarse fibers 0.5-10.0m indiameter, indefinite length, stainwith protein dyes

Tendon, ligament, dermis, fascia,capsules, sclera, bone, dentin

Reticular fibers (typeIII collagen) Delicate network of fine fibers,must be stained specifically to bedemonstrated, usually by areduction of silver or the periodicacid Schiffs (PAS) stainingreaction

Stroma of lymphatic organs, bonemarrow, glands, and adipose tissue

Elasticfibers Smooth, homogeneous fibers ofvarying diameter, must be stained

specifically to demonstrate well(orcein or Verhoeffs stain)

Dermis, lung, arteries, organs thatexpand

Cells of connective tissue

General connective tissue may contain a wide varietyof cell types. Some are indigenous (residents) ofconnective tissues; others are transients and migrate toand from the general connective tissue from thevasculature.

Learning objectives for connective tissue cells:

1. Be able to distinguish and identify the following celltypes (both indigenous and transient cells) foundwithin connective tissues: fibroblasts, macrophages,plasma cells, fat cells, mast cells, neutrophils,eosinophils and lymphocytes.


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Indigenous cells

Fibroblastsare the most common of the connectivetissue cell types. They are large, spindle-shaped cellswith elliptical nuclei. The boundaries of the cell are notseen in most routine preparations and the morphologyand staining intensity of the nuclei vary with the stateof activity. Active fibroblasts exhibit plump, light-staining nuclei; nuclei of inactive fibroblasts appear

narrow and densely stained. Re-examine preparationsof dermis, tendon (ligament) and areolar connectivetissue and compare fibroblast nuclei.

Macrophagesare abundant in general areolarconnective tissue. They are commonly described asirregularly shaped cells with blunt cytoplasmicprocesses and ovoid or indented nuclei that are smallerand stain more deeply than those of fibroblasts. Inactual fact, unless macrophages show evidence ofphagocytosis, they are difficult to distinguish fromfibroblasts. If special preparations are available,utilizing tissues from animals injected with India ink or

trypan blue, examine the areolar connective tissue orliver preparations for cells that have phagocytized thematerials injected. These will be macrophages. Onelocation in which to examine macrophages in a"natural" setting is the center (medulla) of lymphnodes. Examine a routinely prepared lymph nodeunder low power. Note that its central region is lighterstaining and consists of anastomosing cords of cellsseparated by wide spaces. Examine the cords of cellsand the adjacent spaces carefully for large roundedcells with brown-gold colored particulate materialwithin their cytoplasm. These are macrophages.

Continue to look carefully within the interiorof the medullary cords of the lymph node and notenumerousplasma cells. These cells appear somewhat"pear-shaped" with small eccentrically placed nuclei inwhich the heterochromatin is arranged into coarseblocks forming aclock face pattern. The cytoplasmis basophilic and a weakly stained or light area ofcytoplasm often appears adjacent to the nucleus on theside facing the greatest amount of cytoplasm. Thislight-staining area is referred to as anegative Golgiimage. I f available, examine a section of lactatingbreast. The connective tissue surrounding thesecretory units of this gland often contains numerous

plasma cells. Plasma cells also are present in largenumbers in the connective tissue (lamina propria) ofthe intestinal tract. Examine the connective tissue thatlies between adjacent intestinal glands for both plasmacells and small lymphocytes. The latter show a round,dense nucleus and only a scant rim of cytoplasm.

Fat cellsare specialized for synthesis andstorage of lipid. Individual fat cells may beencountered throughout the loose areolar connectivetissue or may accumulate in large numbers to form fat

(adipose) tissue. In routine sections, fat cells appearlarge, round and empty due to the loss of a storedcentral lipid droplet during tissue preparation. Theremaining cytoplasm appears only as a thin rim arounda large empty central space and if the nucleus isencountered in these large cells, it lies flattened on oneside of the cell. Groups of fat cells have theappearance of chicken wire or a honeycomb. Fat cellsare abundant in the hypodermis of skin - that region

of tissue lying beneath the dermis. Fat cells are alsocommon in the loose areolar connective tissue aroundthe perimeter of a lymph node. If the lymph nodepreparation has been stained to demonstrate reticularfibers, examine the delicate network of reticular fibersenveloping each fat cell.

Mast cellsare large, ovoid cells 20 - 30 m indiameter with large granules that fill the cytoplasm.The nucleus is oval or round in shape and centrallylocated. Mast cells are present in variable numbers inloose connective tissue and often accumulate alongsmall blood vessels. Examine a mesentery or areolar

spread preparation for these large granulated cells. Thegranules can be stained different colors depending onthe dye used. In tissues embedded in a plastic resinand stained with H&E, mast cell granules stain a lightred color. If sections of this type are available, examinesections of the stomach and intestinal tract. In eitherorgan the outer supporting muscular wall and adjacentconnective tissue components should be examined formast cells. They will appear as oval-shaped cells orcytoplasmic fragments of cells packed with coarse red-orange granules.

Transient cells derived from blood

Variable numbers ofleukocytesconstantly migrateinto the connective tissues from the blood to carry outtheir specialized functions. Neutrophilsare one typeof leukocyte characterized by a multilobed nucleus anda faintly pink-staining cytoplasm. They are generallyround in shape. Most often, the multilobed nucleus isthe only prominent feature recognized when thesecells are encountered in a section of generalconnective tissue. Eosinophil leukocytes also exhibitmultilobed nuclei and are characterized by bright red(eosinophilic)-staining granules within the cytoplasm.

Lymphocytesare smaller leukocytes (5 - 7 m indiameter) characterized by a central, round nucleussurrounded by a thin rim of cytoplasm. In tissue,groups of lymphocytes appear only as collections ofround, dark staining nuclei. Examine the outer, moredarkly stained region (cortex), of a lymph node andthymus for these small darkly stained leukocytes.Examine the lighter-staining central region (medulla)of the thymus for mast cells and eosinophils.


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Sketch and comparethevarious cell types encountered in generalconnectivetissue.

Questions:

In sections of plastic embedded material stained withH&E, the cytoplasmic granules of both mast cells andeosinophil leukocytes stain red. How can these twocell types be distinguished from one another? Clue:

consider their size and the nuclear profiles.

As plasma cells and lymphocytes are closely related,how do they differ morphologically?

Table 3.Key cytologic features of cells found in general areolar connective tissue.

Cell types Nuclear characteristics Cytoplasmic characteristics

Indigenous cells

Fibroblasts Oval, centrally placed, stainingintensity variable depending onactivity

Elongate, spindle- or stellate-shaped cell; usually not clearlydistinguished is sectioned material

Unilocular fat cells (whitefat) Usually compressed at edge of cell,

staining variable

Forms a thin rim around a single,

large central lipid droplet

Multilocular fat cells Central, spheroid, light staining Numerous lipid droplets, abundantmitochondria

Mast cells Central, spheroid to ovoid, mayshow abundant heterochromatin

Filled with secretory granules

Macrophages Large, ovoid, most frequentlyindented

Light staining, containsphagocytosed material

Plasma cells Usually eccentric, spheroid;heterochromatin clumps may formclock face

Basophilic, slate gray in color; mayshow negative Golgi image

Transient cells

Neutrophils Polymorphonuclear, 3-5 lobescommon, chromatin dense

Light lilac staining granules

Eosinophils Polymorphonuclear, 2-4 lobescommon, chromatin dense

Bright red-orange granules fill thecytoplasm

Lymphocytes Single, spheroid, abundantheterochromatin

Thin rim, light transparent bluestaining


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CHAPTER 4. SPECIALIZED CONNECTIVE TISSUE

Cartilage

The classification of cartilage into hyaline, elastic orfibrous is based on the differences in the abundanceand type of fiber within the matrix. Fibersand theground substanceconstitute thematrixof cartilage.

Learning objectives for cartilage:

1.Be able to identify the three types of cartilage(hyaline, elastic and fibrous (fibro) cartilage) andtheir subcomponents.

Hyaline cartilageis the most common typeof cartilage and forms the cartilages of the nose,larynx, trachea, bronchi, costal cartilages and thearticular cartilages of joints. Examine the trachea oranother tissue that contains hyaline cartilage. In thetrachea, hyaline cartilage appears as a large

homogeneous mass of tissue with a glassy appearance.The matrix appears homogeneous because the groundsubstance and the collagen fibers (type II) embeddedwithin it have the same refractive index. Scatteredwithin the light-staining, homogeneous cartilage matrixare small spaces called lacunae. These spaces containthe cells of cartilage known aschondrocytes.Chondrocytes generally conform to the shape of thelacunae in which they are housed. Note that deepwithin the interior of cartilage the cells and theirlacunae usually exhibit a rounded profile whereas nearthe surface (edge) they are elliptical and flattened withthe long axis oriented parallel to the surface. Near thecenter, chondrocytes often occur in small clusterscalledisogenousgroups. The more intensely stainedmatrix immediately around chondrocytes is termed theterritorial matrix. The less densely stainedintervening matrix is called theinterterritorial matrix.Except for the free surfaces of articular cartilages,hyaline cartilage is enclosed in a specialized connectivetissue membrane (sheath) called theperichondrium.The outer region of the perichondrium is formed by awell-vascularized, dense irregular connective tissue.The region adjacent to the cartilage matrix is morecellular and the transition into cartilage is

imperceptible. The perichondrial cells adjacent to thecartilage retain the capacity to form new cartilage.

Elastic cartilageis more flexible than hyalinecartilage and is found in the epiglottis, external ear,auditory tube and some of the small laryngealcartilages. Elastic cartilage differs from hyalinecartilage chiefly in that the matrix contains anabundance of elastic fibers. Elastic fibers form adense, closely packed mesh that obscures the groundsubstance deep within the cartilage, but, beneath the

perichondrium, the fibers form a loose network andare continuous with those of the perichondrium. Theelastic fibers of elastic cartilage, like those elsewhere,need to be specifically stained to be welldemonstrated. Identify the perichondrium, matrix,lacunae and chondrocytes in elastic cartilage andcompare them with similar structures found in hyaline

cartilage.Fibrous (fibro-) cartilageoccurs in the

symphysis pubis, intervertebral discs, in some articularcartilages and at sites of attachment of major tendonsto bone. Fibrous cartilage lacks aperichondriumandmerges into bone, hyaline cartilage or dense fibrousconnective tissue. Fibrous cartilage represents atransition between cartilage and dense connectivetissue. Typical chondrocytes enclosed in lacunae arefound but only a small amount of ground substance ispresent in the immediate vicinity of the cartilage cells.Chondrocytesmayoccursingly, in pairs or in

short rowsbetweenwell defined bundles ofdensecollagen fibers (type I).

Makea sketch of thethreetypes of cartilageand label theirsubcomponents.

Bone

Two forms of bone can be recognized by visualinspection: compact (dense) bone and cancellous(spongy) bone.

Compact (dense) boneforms the solid,continuous mass that forms the perimeter of thenamed bones of the skeleton. Cancellous (spongybone) is formed by an interlacing network of bonyrods calledtrabeculae. These branch and unite toform a three-dimensional system of bony rodsseparated by small communicating spaces that formthe marrow cavity. Bone is covered, except overarticular surfaces and where tendons and ligamentsattach, by a fibroelastic connective tissue membranecalled theperiosteum. A similar, less fibrousmembrane, theendosteum, lines the marrow cavity.

Learning objectives for bone:

1. Be able to identify bone tissue and itssubcomponents.

Compact bone

The initial examination of compact bone should bedone using a ground preparation rather than ahistological section of bone as the microscopic detailof thematrix is much more pronounced.


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Bone is characterized by the arrangement of its matrixinto layers called lamellae. Small, ovoid spaces, thelacunae, occur rather uniformly between and withinlamellae, each housing a single bone cell called anosteocyte. Minute tubules calledcanaliculi radiatefrom each lacuna, penetrate and cross lamellae to joinwith canaliculi from adjacent lacunae. In compactbone, lamellae show three configurations. Intransverse sections, most are arranged concentrically

into several cylindrical units, much like growth rings ofa tree. The concentrically arranged lamellae surround acentral space known as aHaversian canal. Each unit,consisting of 8 to 15 concentric lamellae that surroundthe central Haversian canal, is referred to as anHaversiansystemorosteon. Portions of additionallamellae can also be visualized filling in the regionsbetween osteon units. These are calledinterstitiallamellae. At the external surface, several lamellaecourse around the entire external circumference of thebone. These are called theoutercircumferentiallamellae. A similar but less well developed system of

lamellae (one or two lamellae in thickness) lines theinterior surface adjacent to the marrow cavity andthese are referred to as the inner circumferentiallamellae. Close inspection of individual osteons willreveal that they are outlined by a refractile line ofmodified matrix, thecement line. Note that thecement lines are not traversed by canaliculi. It must beemphasized that when viewing a preparation ofground bone (pieces of bone ground thin enough topermit the transmission of light) only the matrix isobservedand the lacunar spaces and canaliculi withinit. To visualize the cells of bone and the vascularizedconnective tissues associated with bone (periosteumand endosteum) a decalcified, histologically cut andstained section of bone must be used. Identify all thefeatures observed previously using the preparation ofground bone in a histologic section of bone. Note thatthe matrix, at initial inspection, appears smooth,homogeneous and stains a light pink-red. It appearssimilar to hyaline cartilage. On closer observation,however, note the organization of the matrix.Lacunaecontain osteocytesand are organized in acircular pattern around acentral Haversian canal.Using low power, scan the section and identify severalosteon units. By lowering the intensity of light and

lowering the condenser note that the lamellae arecrossed by fine, unstained canaliculi which can betraced to lacunae and give the latter a very irregularshape. Carefully examine these features under highpower. Use of the fine focus adjustment, focusingback and forth through the section, may be required tosee these structures. Carefully examine severalHaversian canals. Note that they contain a delicateconnective tissue that contains at least two small bloodvessels. In well-preserved specimens a layer of

flattened cells lines the limiting wall of the Haversiancanal. These cells have osteogenic potential, ie, theycan transform into bone-forming cells, osteoblasts,and can produce bone during the remodeling process.In addition, note the differences in diameter of theHaversian canals. Why is this? Is it related to theremodeling process? The delicate vascular connectivetissue found within the Haversian canals is anextension of the endosteum, which also lines the

marrow cavity. Compare theendosteumwith themuch thickerperiosteumcovering the externalsurface of bone. The outermost layer of theperiosteum is dense irregular connective tissue withabundant collagen fibers, some elastic fibers, scatteredfibroblasts and a network of blood vessels. The regionof periosteum closest to bone is more cellular andconsists of a loosely arranged connective tissue.Fibroblast-like cells immediately adjacent to the bonematrix are often called bone-lining cells orosteoprogenitor cells. I f stimulated, they assume acuboidal shape and synthesize and lay down new bone

matrix. If this occurs these cells are termedosteoblasts. Some blood vessels leave the periosteumand enter the bone throughVolkmanns canals.These are transverse canals that penetrate the bonefrom the endosteal and periosteal surfaces. Haversiancanals on the other hand follow a longitudinal courseparallel to the long axis of the bone and lie withinosteons. Note that Volkmanns canals are notsurrounded by concentric lamellae. Blood vesselspassing through Volkmanns canals unite with thosewithin the Haversian canals and link vessels in themarrow cavity and periosteum to the Haversiansystem. Large bundles of collagen fibers (Sharpeysfibers) enter the outer circumferential lamellae fromthe periosteum and firmly anchor the periosteum tobone. Re-examine both preparations of bone for bothVolkmanns canals and Sharpeys fibers.

Sketch and label both a ground bonepreparation and a sectionof decalcified bone. Compareand contrast theadvantages anddisadvantages of each preparation.

In addition to osteocytes, two other cell typesare directly related to bone: osteoblasts andosteoclasts. Both are most easily found in young or

developing bone and should be examined in detail.Developing bonecan form directly in a

primitive connective tissue (mesenchyme) by a processknown as intramembranous ossification or byreplacement of a pre-formed hyaline cartilage model,the process of which is called endochondralossification.


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Learning objectives for developing bone:

1. Be able to describe the process associated withmembrane bone formation and be able to identify themicroscopic detail associated with a developingmembrane bone.2. Be able to describe the process associated withendochondral bone formation and be able to identifythe microscopic detail associated with a developing

endochondral bone.

Intramembranous ossification

Flat bones of the cranium and part of the mandibledevelop by intramembranous ossification and, as aresult, are often referred to asmembrane bones.Examine a section of forming membrane bone at lowpower and note that much of the bone is of thecancellous, or spongy, type. Carefully examine severaltrabeculae of the cancellous bone at higher power.Note the layer of low cuboidal, basophilic cells

covering the external surface of each trabecula. Theseareosteoblasts. Examination of the interior of atrabecula will reveal lacunae with osteocytes. In thesame region note a thin layer of bone matriximmediately adjacent to the cuboidally shapedosteoblasts that stains lighter than the remainder ofthe trabecular bone matrix. This layer ofunmineralized matrix is calledosteoid. After a shortperiod it becomes mineralized to form true bone. Asnew osteoblasts are recruited from osteoprogenitorcells, which resemble fibroblasts, in adjacentmesenchymal tissue, they produce osteoid andeventually become enveloped in their own matrix.When this occurs the osteoblasts are termedosteocytes. With development, the trabeculae continueto thicken by the addition of new bone on theirexternal surface (appositional growth) and the spacesbetween some trabeculae are gradually obliterated. Asbone growth encroaches on vascular spaces within thesurrounding mesenchymal connective tissue, matrix islaid down in irregular, concentric layers around bloodvessels to formprimary osteons. The entrappedblood vessels and connective tissue form the contentsof the developing, primitive Haversian canals. Thesenewly formed osteon units then undergo remodeling.

Identifyand sketch several fields comparingonewith theotherand envision primaryosteon formation.

Endochondral bone formation

Bones of the extremities, pelvis, face, base of skull andvertebral column result from endochondral boneformation, a process that involves simultaneousremoval of a precursor hyaline cartilage model and

formation of bone matrix. Cartilage does notcontribute directly to the formation of bone but muchof the process is concerned with the removal of thecartilage precursor. Initial indications of ossification ofa long bone occur at the center of the cartilage modelin the shaft or diaphysis. In this area, the primaryossification center, chondrocytes hypertrophy, theirlacunae expand and the matrix between adjacentlacunae is reduced to thin, fenestrated partitions.

Simultaneously, the perichondrium becomes morevascular and assumes an osteogenic function. A thinlayer of bone called theperiosteal collarformsaround the perimeter of the altered cartilage and actsas a temporary splint. Blood vessels from the formervascularized perichondrium (now best termedperiosteum) invade the degenerating cartilage as aperiosteal bud. The connective tissue sheath thataccompanies the invading blood vessels contains cellswith osteogenic properties. As the cartilage matrixbreaks down, lacunar spaces are opened up, becomeconfluent, and narrow tunnels in the calcified cartilage

matrix are formed. Blood vessels grow into thesetunnels bringing with them osteogenic cells that alignthemselves on the surfaces of the calcified cartilage.The latter differentiate into osteoblasts and begin tolay down new bone matrix (osteoid). Thus, earlytrabeculae consist of a core of calcified cartilagecovered by a shell of bone. These are removedthrough the activity of osteoclasts that appear on thebony shell. In this way, an expanding cavity is formedin the developing shaft. Support is provided byexpansion of the periosteal collar, which becomesthicker and longer as the periosteum lays down newbone at the external surface. The process continues asan orderly progression towardboth endsof thecartilaginous precursor. Several zones of activity canbe distinguished in the remaining cartilage. Beginningat the ends furthest from the primary ossificationcenter, these are as follows:

Zone of reserve cartilage. This area consistsof typical hyaline cartilage with chondrocytes and theirlacunae randomly arranged throughout the matrix.

Zone of proliferation. In this regionchondrocytes actively proliferate and as a resultchondrocytes become aligned in rows or columns

separated only by a small amount of matrix.Zone of maturation and hypertrophy.

Here, cell division stops and chondrocytes mature andenlarge. Lacunae expand at the expense of theintervening matrix and the matrix between adjacentrows of chondrocytes becomes even thinner.

Zone of calcification and cell death. Thematrix between and around the rows of chondrocytesbecomes calcified and chondrocytes die, degenerateand leave empty lacunae.


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The thin regions of matrix between lacunae then breakdown resulting in irregularly shaped tunnels appearingin the matrix. The extent of this zone can berecognized by careful examination of the matrix,which stains slightly darker than that of the previouszones indicating the extent of calcium diffusion intothe matrix.

Zone of ossification. Vascular connectivetissue enters the tunnel-like spaces formed in the

adjacent zone and provides osteoprogenitor cells thatdifferentiate into osteoblasts. Osteoblastsgather onthe surface of the calcified cartilage and lay down bonematrix (osteoid). Examine these cells carefully.

Zone of resorption. The calcified cartilageand the bony covering are resorbed due to the actionof osteoclasts. In this way the marrow cavity increasesin size as the developing bone increases in length.Osteoclastsare large multinucleate giant cells with amoderately stained acidophilic cytoplasm. Some maycontain as many as 30 nuclei. Individual nuclei showno unusual features and usually are located in the part

of the cell furthest from the bone surface. The cellsurface adjacent to the bone may show a striated(ruffled) border. Osteoclasts reside in shallowdepressions on the surface of bone calledHowship'slacunae.

Carefullyexamineand sketch thezones within thecartilageforthedetails described and then re-examinetheperiosteal collar.

Simultaneous with these events the periostealcollar increases in thickness and length, extendingtowards the ends of the developing bone. Its growthcontinues to provide a splint around the area ofweakened cartilage.

Near the time of birth, new centers ofossification (epiphyseal or secondary ossificationcenters) appear in the epiphyses. Hyaline cartilage ofthe epiphyses show the same sequence of events as

observed in the diaphysis, but growth and subsequentossification spreads simultaneously in all directions.Ultimately, all the cartilage will be is replaced by bone,except for the free end in the joint cavity, whichremains as articular cartilage. Hyaline cartilage alsopersists as a narrow plate between the diaphysis andepiphysis called theepiphysealplate. Its continuedgrowth permits further elongation of bone. Note thatthe epiphyseal plate continues to exhibit the variouszones associated with endochondral bone formation.When growth in the cartilage plate ceases, it is replacedby bone and further increase in length of the bone is

no longer possible. Thus, the rapid growth of a longbone is largely the result of cartilage growth and bonereplacement and not growth of boneper se. Examinethe external surface of the periosteal collar for a layerof osteoblasts and its associated osteoid.

Sketch this region, includingits relationship with thesurroundingperiosteum.

Table 4.Key histologic features used in identifying different types of compact connective tissues.

Type Arrangement of cells and matrix Additional features

Hyalinecartilage Glasslike matrix; lacunae with chondrocytesrandomly arranged, slitlike in appearance nearperichondrium

Isogenous groups, territorialmatrix

Elasticcartilage Matrix more fibrous in appearance; elastic fibers(need to be stained selectively); lacunae withchondrocytes randomly arranged

Large isogenous groups

Decalcified bone Lacunae with osteocytes show organizationwithin lamellae of osteons; Haversian canalslined by endosteum contain blood vessels

Tide marks, bone marrow,irregular shape of lacunae

Ground bone Matrix only arranged into distinct Haversiansystems; interstitial and circumferential lamellae

Canaliculi distinct betweenlacunae

Fibrous cartilage Dense fibrous appearance with a small amountof ground substance; round cells within lacunae

are arranged in short rows between collagenousfibers

Round-shaped chondrocytes

Tendon, ligaments,aponeuroses

Fibroblast nuclei dense staining and elongate, liein parallel rows between regularly arrangedcollagen fibers

Lacunae absent

Dermis, capsules of organs,periosteum, perichondrium

Fibroblast nuclei dense and elongate; randomlyscattered between dense interwoven, irregulararrangement of collagen fibers

Lacunae absent


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CHAPTER 5. MUSCLE TISSUE

Three types of muscle can be distinguished: skeletal,cardiac and smooth.

Learning objectives for muscle tissue:

1. Be able to distinguish between the three types ofmuscle.

Skeletal muscle

The skeletal muscle cell (fiber) is a giant cell thatranges between 10 and 100 m in diameter and isextremely variable in length. Skeletal muscle cells aremultinucleatedand may contain several hundrednuclei. All are peripheral in location, evenly spacedimmediately beneath the plasmalemma. The nuclei areelongated in the direction of the long axis of the cell.Chromatin tends to be distributed along the nuclearenvelope and one or two nucleoli are usually present.

The most outstanding structural feature of the skeletalmuscle cell is the presence of alternating light and darkbandsor cross-striationsthat are visible when thecell is viewed in longitudinal section. Thedarkbandsare calledA bandsand thelight bandsare called Ibands. Running transversely through the center of theI band is a narrow dense line, theZ line. Myofibrilsare elongated, thread-like structures that fill theskeletal muscle cell, compressing the nuclei to aperipheral location. Myofibrilsare the smallest unitsof contractile material that can be identified with thelight microscope and in transverse section appear assmall, solid dots within the muscle cell (fiber). Eachmyofibril shows the identical banding pattern to thatof the whole cell. Indeed, the banding of the skeletalmuscle cell results from the bands on the containedmyofibrils being in perfect alignment as theplasmalemma of the cell is transparent. Compare thebanding seen with the light microscope with thatvisible in an electron micrograph.

The interior of the tongue and lip areexcellent locations to carefully examine the details ofindividual skeletal muscle cells. Skeletal muscle isclosely associated with connective tissue at all levels oforganization. In examining a named muscle, the entire

muscle is surrounded by a connective tissue sheathcalled theepimysium. Septa pass from the deepsurface of the epimysium to envelop muscle fascicles(groups of muscle cells) as theperimysium. Adelicate connective tissue wraps each individual musclecell as anendomysium. Although given differentnames according to its association with differentstructural units of skeletal muscle, the connectivetissue forms a continuum and acts not only to bind thevarious muscle units together but also functions as aharness and aids in integrating and transmitting the

forces of contraction. It consists of collagenous,reticular and elastic fibers and contains severalconnective tissue cell types, the most common ofwhich are fibroblasts. The endomysium is delicate andconsists primarily of reticular and thin collagen fibers.It contains blood capillaries and small nerve branches.Larger nerves and blood vessels lie within the

perimysium.

Sketch and label longitudinal and transverseprofiles of skeletalmusclecells. In thetransverseprofileincludeits association withthesurroundingconnectivetissue.

Cardiac muscle

Cardiac muscleis associated with the heart andforms the majority of the heart wall or myocardium.Cardiac muscle cells are small cylindrical cells thatbranchand are linked to one another, end to end, by

specialized junctions known as intercalated discs.The latter appear as darkly stained transverse lines.Each cardiac muscle cell contains one or occasionallytwocentrally positioned nuclei. The same bandingpattern witnessed in skeletal muscle occurs in cardiacmuscle. Although A bands, I bands and Z lines arevisible they are not as conspicuous as those of skeletalmuscle. Myofibrils are fewer in number and oftengrouped into bundles that diverge around nuclei. As aresult, regions of cytoplasm appear structureless ateach nuclear pole. Examine both longitudinal andtransverse profiles of cardiac muscle cells to confirmthe position of their nuclei. A web of reticular and finecollagenous fibers is present between cardiac musclecells and corresponds to an endomysium. Numerouscapillaries are present in this layer of connective tissue.

Sketch and label both longitudinal and transverseprofiles ofcardiacmusclecells. Thelongitudinal profileshould includeanintercalated disc. Comparetheselight microscopicobservationswith thosefeatures seen in electron micrographs of cardiacmuscle.

Smooth muscle

Smooth muscleis widely distributed and plays an

essential role in the function of organs. It forms thecontractile portion of the walls of blood vessels,hollow viscera, such as the digestive, respiratory,urinary and reproductive systems, and is asubcomponent of most other organs.

Smooth muscle cells are shaped likeelongated spindleswith asingle,central nucleusoccupying the wide portion of the cell midway alongits length. The nucleus is elongated in the long axis ofthe cell. Smooth muscle cells lack the cross-striationsobserved in the two other forms of muscle.


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Smooth muscle cells may be present as smallisolated units or may from prominent sheets. In anyone sheet of smooth muscle, the cells tend to beoriented in the same directionbut are offset so thatthe wide portions of some cells lie adjacent to thetapering ends of neighboring cells. This is mostevident in transverse sections, where the outlines ofthe cells vary in diameter according to where alongtheir lengths the cells were cut. Nuclei are few in this

view and present only in muscle cells cut near theirlargest profiles (centers). A thin connective tissue offine collagenous, reticular and elastic fibers aids inbinding the smooth muscle cells into bundles orsheets. In routine preparations this fine reticuloelasticsheet is almost imperceptible and the field isdominated by profiles of smooth muscle cells.

Examineand sketch themusclewall of a transverselycutsegment of small intestineand notethat it consists of twosheetsor layers of smooth muscle. Thesmooth musclecells in theexternal layer will besectioned transversely, thoseof theadjacentinner layer will becut parallel totheir longaxis. Examinethesmooth musclecells carefullyat higher magnifications and notetheposition of their nuclei.

Next, examine the wall of any named artery

for its smooth muscle layer, which occupies the centerof the vessel wall. What is the orientation of cellswithin this smooth muscle layer? How can smoothmuscle cells be differentiated from adjacentfibroblasts?

Table 5.Key histologic features that distinguish muscle types.

Type Cell shape Nuclei Striations Other features

Skeletal Long cylinders Peripheral, multiple Present Endomysium, perimysium

Cardiac Short branching,anastomosing cylinders

Central, usuallysingle, occasionaldouble

Present Intercalated discs

Smooth Small spindles Central, single Absent Cells packed tightly together,occurs in sheets, layers orbundles with nuclei oriented inthe same direction


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CHAPTER 6. NERVE TISSUE

Neurons (nerve cells)

It should be emphasized that the majority of cellbodies (perikarya) of neurons are found in the graymatter of the central nervous system (CNS).Aggregates of perikarya occur in the gray matter of theCNS, which act as distinct functional units called

nuclei. Similar collections of individual nerve cellbodies are located outside the CNS and these arecalledganglia. Thus, the majority of nerve tissue (thenerves) seen in the peripheral nervous system are theprocesses of nerve cells and do not contain thesomaor cell body.

Learning objectives for nerve tissue:

1. Be able to find, ident

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