A Manual of HUMAN HISTOLOGY THROUGH LIGHT MICROSCOPY (1995) OSCAR M. TANGCO, M.D., M.S. Professor of Anatomy College of Medicine University of the Philippines Manila‘TABLE OF CONTENTS INTRODUCTION Figure 1. Parts of the Microscope ‘THE COMPOUND MICROSCOPE Procedure for the Routine Use of the Microscope Table 1, Total Magnification and Diameter of Microscopic Field Estimating Size TISSUE STAINING crtoLocy General Noture of Tissues Fundamental Tissues EPITHELIAL TISSUE Lining or Covering Epitheliue Glendular Epithelium, Exocrine Glands Endocrine Glands CONNECTIVE TISSUE Connective Tissue Proper Cartilage Bone Osteogenesis MUSCLE TISSUE L000 Red Blood Coll (Erythrocyte) Leukocytes: Blood Platelets Field Meandering Method for Examining Blood Smears Table 2. Adult leukocytes in dry blood smoar stained with Wright's Stein [BONE MARROW Developeont of the Eosinophil Leukocyte " * % Bosophil " * " Mogakaryocyte " "Monocyte " "Lymphocyte Tobie 3, The Erythrocyte Series Table 4, Tho Noutrophil Series SKIN RESPIRATORY SYSTEM [BLOOD VASCULAR SYSTEM Capillery end Sinusoid Lorger Blood Vessels Transitions Arteries Special Types of Arteries Table 5. Comparison of Artoriole and Venule Table 6. Conparison of Muscular Artery and Mediu Sized Vein Table 7. Comparison of Elastic Artery end Large Vein Heart Impulse Conduct ing Syston vist Vist vit. vant Vin. Kt xa xa XA x2 x3 xa xt X12 XL.2 X12 XI3 x13 xI4 x5 xIL XII xiv. XIV. XIV.3 xIV.3 XIV.3 xaved XIV.5 xIV.6 XIV.7 xav.8LYMPHATIC SYSTEM Palatine Tonsil Lymph tiode Sploon Thymus DIGESTIVE SYSTEM Oral Cavity and Pharynx Tongue Accassory Glands of the Oral Cavity Parotid Gland Submaxillery Gland Sublingual Gland Esophagus. Stoaach Small Intostines Large Intestines Liver Gell Bladder Pencroas ‘URINARY SYSTEM Kidney Urotor and Urinary Bladder Fonale Urethra MALE REPRODUCTIVE SYSTEM Tostis, Seminiforous Tubules, ond Interstitial Tissue Tubult Rect Rete Testis Ductull Efterentes Ductus Epididymis Ductus Deferens Seminal Vasicle Prostate Mole Urethra and Bulbourethral Glands FEMALE REPRODUCTIVE SYSTEM Ovary Oviduet Uterus Carvix Uteri Vogine Placenta Fonale Manaary Gland ENDOCRINE GLANDS: Hypophysis Thyroid Gland Parathyroid Glend ‘Adrenel (Supraronal) Gland Epiphysis Corebri (Pineal Glend) =-0000000-- xvA1 Xv.2 XV.3 xVe4 WAS XVI xVL2 XVL.2 XVL.3 XVLS XV XvLA VLA XVL,S XVL7 XVL.8 NVI,9 ELI XVIE.12 XVI. XVILT XVIL. XVII. XvITEAA XVEEEAT XVILL.2 XVII, XVIEL.S XVLLL,3 XVEIL.S XVIILA XVILA WIL, XIX XIX XIX3 XIK4 XIX.5 XIX5 XIK.6 XIX.7 xxi XK XK.3 KA 4INTRODUCTION This manual is a guide for the beginning histologist in his study of human tissues with the use of the student laboratory micro- scope. This is not intended to replace the textbook or the class- room lectures. Information contained here concerns only those struc- tures that can be seen with the optical microscope, using mostly routine staining techniques. Some additional information are mention- ed here merely for the purpose of correlating between certain aspects of structure that are seen under the optical microscope but are better explained or demonstrated through special means like the elec- tron microscope (EM) and histochemistry. Presentation of subject matter in this manual follows a sequence designed to progressively develop the student's acumen in histology. I recotmend that you study thoroughly the sections on the microscope, the estimation of size, and the notes on tissue staining before pro- ceeding to the rest of the work. To gain maximum benefits from your laboratory work, you should have attended the lecture and read your textbook on the assigned topic before studying the same in the laboratory. You should develop as early as possible the skill in using and caring for your microscope and class slides so that your work will not be hampered by the struggle between you and your instrument. You would be better able to explain what you see under the microscope if you are always alert to the limit- ations of your instrument and the staining technique that was used in preparing the specimen. You will find it easier to interpret the var- fous features of structures in the course of your study if you culti- vate the skill of visualizing objects in three dimensions. Above all, do not hesitate to consult your professors. omr.FIGURE 1, Parts of the compound microscope that you have to be most femiliar with.I. THE COMPOUND MICROSCOPE ‘The compound microscope is a precision optical instrument that magnifies minute structures in properly prepared specimens. You can take full advantage of its potentials if you use it properly. ‘The following are the working parts of most compound microscopes (Refer to Figure 1.). Identify them in your own microscope. 1. Eye piece, ocular lens or “ocular* 2. Body tube or Viewing tube 3. Revolving nose piece 4. Objective lenses (or “objectives") set in the nosepiece 5. Body or Stand 6. Mechanical stage or Mobile & adjustable specimen holder 7. Object stage or Specimen stage 8. Coaxial knobs for moving the mechanical stage 9. Knob for focusing the condenser lens 10. Substage condenser housing, containing: a. Condenser lens b. Filter holder c. Condenser diaphragm with lever for regulating its opening. 11, Coarse focusing knob 12. Fine focusing knob 13. Base of stand, or Microscope base 14. Mirror OCULAR LENS ‘The commonly used ocular has a magnification of ten diameters (10X). It should be provided with a pointer so that you can conveniently refer to a structure that you want to discuss with your professor or classmate. If it has no pointer, ask the laboratory technician to attach one for you. When removed from the body tube, it may be used as a magnifying lens by viewing the specimen through its other end. Take care that you do not drop the lens. OBJECTIVE LENSES There are usually three objective lenses screwed into the revolving nose piece, namely, low power, high power (or “high-dry"), and oil inmersion lenses. Low Power Lens - This usually has a 10X magnification. High Power Lens - This has a magnification of 40X, but depending on the brand of the microscope, the magnification may be as high as 45x. Oil Immersion Lens - This has a magnification of 100X. Some microscopes may have 95X or 97X. When using this lens, a drop of immersion oil bet- ween the slide and the lens is needed to prevent light dispersion outside the area of the lens.SUBSTAGE CONDENSER ‘This is located below the object stage and consists of a system of lenses designed to bring parallel rays of light to the specimen. It is focused by means of the condenser focusing knob. CONDENSER DIAPHRAGM This is located below the condenser. You can regulate the amount of light passing through the condenser by varying the opening of the dia- phragm. This opening must be adjusted to the particular objective lens being used to obtain optimum illumination. With a very low power object~ ive (e.g., 4X) and with the oil inmersion lens, the diaphragm should be wide open. With the low power objective (10x) and high power objective it should be only partially open. Excessive light will cause undue fatigue. When working with unstained or low-contrast materials, it may be possible to increase contrast, and hence see more parts, by markedly decreasing the diaphragm opening or by lowering the condenser slightly. CARE OF THE MICROSCOPE Keep the microscope clean at all times. Be especially careful when using liquids on the slide. Wipe up spills immediately. If it is necessary to remove the eye piece when aligning or adjust- ing the microscope, do not leave the body tube open for a long time to minimize problems with dust. Use lens tissue to clean the optical parts. If this is not available, use soft clean cotton and wipe gently. To avoid breakage of the high power or oil inmersion objectives, do not focus down on the slide by using the coarse focusing knob while viewing through the microscope. Always carry the microscope vertically by supporting its base with one hand while holding the body with the other. Never carry the micro- scope with one hand. PROCEDURE FOR THE ROUTINE USE OF THE MICROSCOPE 1. Place the microscope in front of the light source. Seat comfortably behind your microscope. Look through the microscope by focusing your eyes at infinity and keeping both of thet open. Do not squint or strain. 2. Focusing the Condenser - With the low power objective in position, tilt the plane mirror until half the microscope field is bright and half is dark. Carefully focus the condenser until the metal rim of the mirror is in focus. When both the tissue on the slide and the rim of the mirror are in focus, the condenser is in focus for both the low power and high power objectives. Having focused the condenser, move the mirror while looking through the microscope until the.entire microscopic field is uniformly filled with bright illumination. 3. Illumination - Get proper illumination by so adjusting the mirror that a uniform bright light is obtained over the entire field. The diaphragm should be wide open while the light is being adjusted. The concave mirror is used with a very low power (4X) objective, while the plane mirror is used for the higher magnifications. However, when there are obstructionsacross the light source that confuse the microscopic image, the concave mirror may be used with the condenser racked up. This will provide a workable illumination but at the risk of introducing some optical dis~ tortions. 4. Iris Diaphragm - optimal setting of the diaphragm is determined by removing the ocular and looking through the body tube. When the diaphragm is closed, only the central portion of the objective is illuminated. As the diaphragm is opened, the diameter of the illuminated area increases up to a critical point beyond which it will no longer increase although the intensity of the light will. The optimal setting of the diaphragm has been attained when the upper lens of the objective is just complete- ly €illed with light. However, when working with unstained specimens, it may be desirable to have less than the optimal illumination. 5. Using the Low Power Objective - With the low power in position, decrease the distance between the objective and the specimen by turning the coarse focusing knob so that the lens is closer to the specimen than when in focus. Then, while looking through the ocular, slowly increase the objective- specimen distance, using the coarse focusing knob, until an approximate focus is attained. Get a sharp focus by using the fine focusing knob. ‘The condenser diaphragm should be approximately 2/3 open. 6. Using the High Power Objective - Check whether the low power and high power objectives are parfocal. A lens is parfocal with another when both are in focus at the same distance from the specimen. If the lenses are not parfocal, switching to the high power after having attained a focus with the low power lens may result in breakage of the cover slip of your slide and/or damage to the highpower lens. ‘The following is a method for checking the parfocality of your high power lens: With the high power lens in position, decrease the lens-to- specimen distance with the use of the coarse focusing knob so that the lens coomes very close to, but not touching, the top of the cover slip. DO THIS PROCEDURE WHILE WATCHING FROM THE SIDE WITH YOUR EYES LEVEL WITH THE OBJECT STAGE TO ENSURE THAT YOU WILL NOT BUMP THE LENS AGAINST THE COVER SLIP. Then while observing the specimen through the microscope, in- crease the specimen distance with the coarse focusing knob until an ap- proximate focus is attained. Sharpen the focus with the use of the fine focusing knob. Having focused the high power lens, switch to the low power and note if the specimen is still in focus. If the focus is considerably below that of the high power the lenses are not parfocal. If this is the case, always increase the specimen distance before shifting from low to high power. The condenser diaphragm should be approximately 2/3 open. 7. Using the Oil Immersion Objective - Rack the condenser all the way up. ‘The oil immersion lens has a very short focal length and has to be po- sitioned very close to the specimen. The clearance of the lens is reduced further by the cover slip and its mounting medium. Some cover slips are so thick that the lens cannot be focused on the specimen at all. Place the slide on the object stage. Put a small drop of immersion oil on the cover slip or uncovered specimen. With your eyes level with the slide, raise the object stage (or lower the body tube, as the case may be) carefully with the coarse focusing knob until the lens enters the oil, droplet and almost touches the cover slip or specimen, - Look through the microscope and in- crease the specimen distance by turning the fine adjustment until the1.4 specimen comes into focus. If you pass the plane of focus, carefully de- crease the distance a fraction of a turn with the fine adjustment. NEVER DECREASE THE SPECIMEN DISTANCE WITH THE FINE ADJUSTMENT MORE THAN A QUARTER TURN WHILE LOOKING THROUGH THE MICROSCOPE TO AVOID BREAKING THE COVER SLIP AND DAMAGING THE LENS. If the specimen does not come into focus after following these steps, the structure is probably below the focal point of the lens, Repeat the above procedure. If you still cannot get a focus on the specimen, the cover slip may be very thick or the speciten may be on the other side of the slide. A€ter using the oil inmersion lens, wipe the oil from the slide and the lens with lens paper or a piece of clean soft cotton. A piece of lens paper or cottonmoistened with xylene should be used to retiove the last traces of oil. Finally, the slide and the objective should be gently wiped dry with clean cotton or lens paper. MAGNIFICATION AND CHOICE OF A LENS COMBINATION ‘The magnifying power of a compound microscope depends on the ocular~ objective lens combination. It is the product of the magnifying power of the two lenses (total magnification). When the length of the body tube is 160 mm, and the ocular has a 10X magnification, the approximate total mag- nification and corresponding diameter of the field when using the follow~ ing objective lenses are given below in Table 1. TABLE 1: Total magnifications and respective diameter of fields when ocular lens has a 10X magnification and body tube is approximately 160 mm. long. Magnification of ocular lens 10x 10x 10x 10x Magnification of objective lens 4x 10K 40K 95=100%. Total Magnification 40x 100X400 ~—950-1000x Approximate diameter of Field (nm.) 5 2L 04 0.2 Got a panoramic view of the specimen before making a detailed exatt- ination at higher magnifications. The most important part of a tissue may be missed if you fail to survey the entire field. First examine the mount- ed specimen with the unaided eye. You may next examine it with a magnify- ing lens. Then examine the slide through the microscope using the low power objective. Select the area that you wish to examine at higher magnification. Place that area at the center of the field before switching to the high-dry objective. Place the same area at the center of the high power field before changing to the oil inmersion lens. The oil inmersion lens is used for studying finer cytological detail. It is not routinely used for the purpos of this course. The cotton should not be soaking wet but should only be barely moist. With the immersion oi] supplied by the department USE 95% ETHANOL INSTEAD OF XYEERE.ESTIMATING THE SIZE OF A STRUCTURE It is useful to know the size of a structure under the microscope. Using a micrometer eye piece is the best way for accurately determining the Linear dimensions of a structure. However, this is not very necessary in ordinary microscopy and what you need most frequently is the skill to make a rapid estimate of the said dimensions. Train yourself to make rapid estimates of the size of objects that you are examining under the micro- scope. You can estimate the size of an object by comparing it with fixed reference points such as the diameter of the microscopic field or the average diameter of a normal erythrocyte which is frequently found in the various tissues that you are going to study. Example 1. The length of an object is about 0.2 mm. if it is half the diameter of the high power field which is 0.4 tm. Length of the cell ts about half of the dianoter of the high pover field, It is, therefore, about 0,2 m1, long. eK 0.4m. — al HIGH POWER FIELD Example 2. The diameter of a cell is about 15 micrometers if it has twice the diameter of a normal erythrocyte which is about 7.74 micro- meters. Gell 1s about twice the dioneter of the ‘erythrocyte. It 1s, therefore, approx () imately 15 micrometers in diameter, be micRosooPIc FIELDII. SOME NOTES ON TISSUE STAINING Tissue preparations most conmonly used in this course are stained by a combination of two dyes, hematoxylin and eosin (H & E). Hematoxylin is blue while eosin is red. For simplicity, hematoxylin may be considered a basic dye while eosin is an acidic dye. An acidic structure will be stained by a basic dye. Tt is said to ex- hibit basophilia, or that it is basophilic. A basic structure will be stained by an acidic dye. It shows acidophilia - it is acido- philic. In routine H & E preparations, therefore, acidophilic struc- tures will take up eosin and will be colored red; basophilic struc- tures will take up hematoxylin and will be colored blue. STAINING OF PROTEINS Proteins can be stained by basic or acidic dyes if they have reactive secondary carboxyl or amino groups that are available for staining. The number and type of the reactive groups as well as the PH of the staining solution will determine whether the substance will be basophilic, acidophilic, or unstainable. At a given pH, if the reactive carboxyl group is the predominant component, the structure will be stained by the basic dye. If the reactive amino group is pre- dominant, that structure will be stained by the acidic dye. Special staining is required to demonstrate some protein subs tances. STAINING OF NUCLEIC ACIDS Nucleic acids are strongly acidic compounds because of their phosphoric acid component. Although the pH of the staining medium will affect their acidity, they will be negatively charged (baso- philic) at alkaliine, neutral and moderately acid pl. They will only be non-ionized (unstainable with basic dyes) at very acid pH. STAINING OF MUCOPOLYSACCHARIDES AND MUCOPROTELNS These compounds contain sulfate groups which are very acidic. Like nucleic acids, they are basophilic at alkaline, neutral and moderately acid pH, and are unstained with the basic dye at very acid ph.11.2 STAINING OF FATS AND CARBOHYDRATES Special staining is needed to demonstrate these substances. With routine H & E procedure, they are either dissolved out of the tissue or remain unstained. In certain instances where the unstainable substance is large enough to be within the resolving power of the optical microscope, its presence tay be intimated by an unstained spot in the cytoplasm - a form of “negative image”a1I.1 10 IIT. cyroLocy There are many types of cells. Their variety is reflected in differences of size, shape, structure and staining. ‘This section emphasizes those features that are common among different cells when seen under the light microscope (LM) and demon- strated by routine staining and some special staining procedures that may be found in your slide collection. For the sake of having a more complete picture, certain structures that are undemonstrable by rou- tine techniques will be mentioned. When advantageous, some features that are found only in some cells may be mentioned also. THE HYPOTHETICAL CELL Description of this non-existent cell refers to those features that are commonly found in different types of real cells. ‘The cell has two main parts, the cytoplasm and the nucleus. The only exception is the mature red blood cell which lost its nucleus during the late stage of its devlopment. You will see the cytoplasm as the body of the cell. Tt is bound- ed by the cell membrane or plasmalenma. the Nucleus is contained with- in the cytoplasm. The nuclear membrane or envelope covers the nucleus and apparently segregates it from the cytoplasm. PLASHALEHMA Compare the plasmalenma of the various cell types. Note that it is thinner in some than in others. Examine its texture. In some it is smooth and sharply demarcated while in others it may be fuzzy. Regional specialization on the surface of some cells may be re- Presented by the presence of a cilium or flagellum, or many cilia. There tay be tiny cytoplasmic evaginations or invaginations which in both instances are covered by plasmalenma. The evaginations may be microvilli. While these are too small to be resolved by the LM, if they are densely packed at a particular area they would contribute to the fuzziness of the cell boundary. If these microvilli stand very closely and parallel to each other, their presence may be appreciated under the LM as a brush or striated border, or as Filamentous structures called stereocilia. CYTOPLASM Note the color of the cytoplasm. Based on the color of the acidic or basic stain that was used, identify its staining reaction (aciao- philic or basophilic). Note the intensity of the color, and decide whether the reaction is faint, moderate, or intense. See if the staining is uniform throughout the cytoplasm or if there are color variations in certain areas. Is the cytoplasm homo- geneous or heterogeneous in texture ?14 IIE.2 Identify cytoplasmic structures that have been revealed by the staining. Cytoplasmic organelles and inclusions are the two general types of formed elements in the cytoplasm. Not all of them are de- monstrable routinely. They are listed below and each is indicated if visible under routine or special staining. Organelles ? 1, Endolasmic Reticulum - Only the rough-surfaced reticulum (RER), if abundant, may be seen as basophilic areas. With Niss1 stain, it can be seen as deeply blue with a granular texture. The stooth-surfaced re- ticulum (SER) can only be seen with the EM, 2. Ribosome, and RNA/RNP - If abundant, the cytoplasm will show baso- philia, in some instances in the forni of fine basophilic granules, while in others in the form of a diffuse basophilia of the cytoplasm. 3. Golgi Complex - With H & E or Wright's stain, this may be seen as an unstained spot, often close to the nucleus (negative Golgi image) This can only be seen if the Golgi complex is large and "compact". 4. Mitochondrion - Dentonstrated by Janus Green stain or EM. 5. Fibrils - Some fibrils are demonstrable by H & E, like myofibrils. 6. Lysosome - This can be demonstrated by histochemical procedures that reveal the presence of lysosomal enzymes. However, in the case of the neutrophil and basophil leukocytes, staining with Wright's stain de- monstrates the azurophil granules in both cells which have been pre- viously identified histochemically as lysosomes. The specific granules of the eosinophil leukocyte is also a form of lysosome. 7. Cytoskeleton ~ Seen under the EM. 8. Centrosome - Usually demonstrated by special techniques. However, if it is large enough, it may be an unstained area close to the center of the cell and adjacent to the nucleus. It is often close to the Golgi complex, so that it may contribute to the appearance of the so-called negative Golgi image. It is best seen under the EM. 9. Microvesicles - Seen under the EM. 10. Peroxisome - Seen under the EM and confirmed as such thro ugh histo- chemical analysis. 11. Plasmalenma - Seen under the LM, as described earlier, but it is not satisfactorily resolved to show its components and modifications clearly. 12, Vacuole or Vesicle - This is usually seen in the living specimen. Inclusions 1. Secretory *granules* or droplets - Not demonstrable in all instances. Tt may be coarsely granular. 2. Pigment granules - Detonstrable under the LM, like melanin and Jipofuscin. 3. Fat "vacuole" or droplet - Seen as the unstained portion of the fat cell with H&E.12 XIE.3 4, Glycogen granules - Not demonstrated by routine H & E sstaining/ 5. Phagocytosed matter ~ May be seen in phagocytic cells. ‘THE INTERPHASE NUCLEUS ‘The following are seen under the LM: Nuclear Envelope - Compare the nucleus of the various cells in the List. Note that the nuclear envelope in one appears thinner than in another. In others you may notice that its thickness is not uniform. Note also if its cytoplasmic surface is smooth or granular. Nucleolus - This is a globular or ovate body which may or may not be Present in the nucleus. It is usually deeply basophilic but in some may show acidophilia. It varies in size and number in the various cell types. Heterochromatin - This is intensely basophilic. Varies in size and form, from fine and "powdery" to coarse discrete granules, clumps or masses. Look for these differences. Nucleoplasm - This is the structureless portion of the nucleus. Tt is supposed to be the fluid portion. Do not confuse this with parachromatin which under the LM apparently occupies the same area. They are not the same. The parachromatin is cotiposed of the nucleoplasm plus the other nuclear structures that are not seen under the LM. Number of the Nucleus - Examine the various cells listed. Note that there is usually one nucleus in a cell. But in some there may be mores Size of the Nucleus - Note that the size of the nucleus varies not only in absolute terms but also relatively in proportion to the size of the cell. Position of the Nucleus - Compare the position of the nucleus in the various cells. If the nucleus is not in the center of the cell, it is said to be eccentric in position. Shape of the Nucleus - Each cell type usually has a nucleus of a char- acteristic shape. It is described as rounded or spherical, oval or ovate, flattened, elongated, fusiform, indented, kidney-shaped, C- or U-shaped, lobated, bilobed or multilobed. Look for examples of the:13 IIT.4 CELLS SUGGESTED FOR CYTOLOGY PLASMALEMMUA ‘Smooth & sharply demarcated: Erythrocyte - blood ssear or any tissue containing blood vessels Epithelium of renal collecting tubule, 41a Epidermal colls, 560 Fuzzy: Epithelium of renal proxinal convoluted tubule, 41a or b Cli tated: Epithelium of trachea, 39 Flagol lated: Sperm coll, testis, 52 a or b Brush or striated border Mucosal epitholium of small intestines, 3la, bs or c Epitholium of renal proximal convoluted tubule, 41 a or b Storocilia: Lining epithelius of ductus epididyins, 52 CYTOPLASM Bosophilic: Zymogonic coll, gastric fundus, 300 Norve coll, cerebral cortex, 7a Aetdophi lic Parietal coll, gastric fundus, 300 . Paneth coll, snall intestines, 31a, b. or c Muscle coll, 6b ORGANELLES, RER, Ribosomes - soo basophilic cytoplasm Golgl complex - Plasma cell, bone marrow saoar Fibrils - myofibrils of sekeltel muscle, 6b INCLUSIONS Secretory "granules" or droplets - acinar cells, pancreas, 36 Ponoth coll, small intestines, Sia, b, or © Goblet coll, tracheal epithelium 39, micosal opithelium, intestines, 31a,b, or © Pigment granules ~ melanin pignent, epidermis, 56a Fat "vacuole" - fat coll, osentum, 1 or 23 Phagocytosed matter - dust or heart failure cells, lung, 40 NUCLEAR ENVELOPE - all cells that you encounter NUCLEOLUS - Nerve coll, 7a, ganglion coll, 9, ovum, 45 NETEROGHROMATIN - 011 cells that you encounter NUCLEOPLASM ~ all colls tht you encounter NUCLEAR NUWBER - Liver cell, Say by or ey Osteocl NUCLEAR SIZE - Mogakaryocyte, bone marrow smear or section,14, Ovum, 45, Lymphocyte, blood smear, Plasma coll, bone marrow snear te 3d POSITION OF NUCLEUS - Skeletal muscle, 6a or by cardiac misc coll, 36, plosma coll, bone marrow smear 6c, pancreatic acinar NUCLEAR SHAPE - Plesna coll, bone marrow smear, Noutrophil leukocyte, blood sneer, Megakaryocyte, bono marrow smear or section, 14, Skeletal muscle, 6a ab Lymphocyte, blood sneerWw 14 IV. GENERAL NATURE OF TISSUES A tissue is composed of cells (living components) and inter- cellular substance (non-living) surrounding the former. Intercellular material is composed of formed elements (intercellular fibers and fibrils) and amorphous substance composed of water, protein - carbo- hydrate complexes, amino acids, sugars, glycerides, minerals, elec- trolytes, hormones, vitamines, and cellular catabolic products. In any given tissue, there is a typical proportion of cells to intercellular substance, a preponderance of a single type of cell or mix of several types, with characteristic groupings that form recog- nizable structures. The intercellular material may also show predo- minance of certain formed elements which may be organized into struc- tures. All these contribute to the varied but distinctive appearance of the different tissues. FUNDAMENTAL TISSUES All the tissues comprising the various organs of the body are made up of four classifiable types - the so-called fundamental, primary, or basic tissues. They are the following: 1. Epithelial tissue 2. connective tissue 3, muscle tissue 4. nervous tissue Each fundamental tissue possesses structural and functional char- acteristics that reflect its role in an organ. Integrated function of fundamental tissues is important in the normal physiology of an organ. A degree of specialization (or differentiation) occurs during the development of fundamental tissues. This makes it necessary to classify each tissue into subtypes as you will see in the following sections. In general, there is a pervasive association of connective tissue with the other primary tissues. An important characteristic of this is the presence of a basal lamina that is interposed between the connec- tive tissue and the other fundamental tissues. The basal lamina is not constantly resolved by the LM.15 V. EPITHELIAL TISSUE Epithelial tissue is derived from any of the three germ layers. ‘Thus you will find it in the organs that developed from the particular layer. Regardless of its origin, epithelial tissue is characterized by scanty intercellular substance. Thus you will notice that an epithelium is highly cellular. Because it covers a surface or lines a cavity, at least early in its development, it possesses a free surface and an at- tached basal surface that is closely associated with connective tissue between which is interposed a basal lamina. Bach cell, therefore, is polarized along its perpendicular axis, and in many instances there are structural and functional differences between the two poles of the cell. Epithelium is generally classified into lining or covering epi- thelium, and glandular epithelium. Bach type may be further classified as follows: LINING OR COVERING EPITHELIUM The general morphologic classification of lining or covering epithelia is based the combination of two features: 1. Shape of epithelial cell. This refers to the shape of the cell when viewed from the side (see Figure 2.) ~ a. squamous - width is greater than height b. cuboidal - width is approximately equal to height c. columnar - width is very much less than height 2. Number of cell layers (see Figure 3.) a. simple - one layer of cells only b. stratified - two or more layers of cells in which the basal cells are the only ones that touch the basal lamina. When classifying lining or covering epithelia, therefore, a com- bination of the two features is used. For example, a simple squamous epithelium has one layer of flat cells, a simple columnar epithelium has one layer of tall cells, and stratified colummar epithelium has more than one layer of tall cells (see Figure 3). FIGURE 2. Lateral profile of epithelial cells: EF Lo] Squamous cuboidal columnar 1 basal lamina16 v.2 FIGURE 3. Morphological types of lining and covering epithelia: SIMPLE Eermietrn fa U eee. ofoletgl Jojelore basal lamina Simple squamous Simple cuboidal Simple columnar STRATIFIED EPITHELIA 2 lof elo} PEED aos Stratified squamous* Stratified cuboidal stratified columnar Note: * In any stratified epithelium, the basal cells are either cuboidal or columnar. When classifying epithelium, however, the cell shape referred to is derived from the side view of the most superficial layer of cells. Pased on appearance under the LM, two epithelia have been given fames that are not directly referrable to the preceding method of Classification, as follows: 1. Transitional Epithelium This lines the hollow organs that are part of the conducting Portion of the urinary system that periodically become distended with urine. When the organ is relatively empty, the epithelium is similar in appearance to stratified squamous epithelium except that its most Superficial cells are rather thick and have convex free surfaces. When the organ is distended, the epithelium is laterally stretched and the cell layers appear to decrease in number, and all the cells decrease in height. 2. Psoudostratified Columnar Epithelium This is called pseudostratitifed because even the most superficial cells touch the base. The epithelium appears stratified because the cells vary greatly in height and their nuclei consequently are at different levels. Other Structural Features That May Be Used To Differentiate Epithelia: A. Presence of Cilia - Cilia may be found in some columar epithelia. Thus the term "ciliated* is added to the name of the epithelium. Example: Simple columnar ciliated epithelium 2. Presence of Goblet cells - Simple columnar ciliated epithelium with goblet cells, or simple columnar epithelium with goblet cells.17 3. Presence of Keratinization - The terms ‘keratinized* or "keratinizing" and *non-keratinized" or "non-keratinizing™ are confined to stratified squamous epithelia in which the super- ficial cells are either impregnated or not with keratin, res- pectively. ‘The terns "secretory epithelium* and "neuro-epithelium™ refer more to function than to structure. "Secretory" because an import- ant function of that epithelium is secretion, “neuro-" because the function is reception of stimuli. Exercise: Lining or Covering Epithelium. Simple squamous epithelium - Slide 1 or 2, Omentum. Look for the lining epithelium of the omentum. This will be a surface view of the structure. Because it is very low in height and wide in lateral dimension with ir- regular borders, it is also referred to as "pavement" epithelium. You can often locate this epithelium by first looking for the thicker portions of the tissue where there are blood vessels (longitudinal tubular struc- tures). Center the blood vessel in the microscopic field under low power, decrease the light by partly closing the diaphragm, or lowering the con- denser so that the illumination appears’ to darken slightly. Then care- fully and slowly turn the fine focusing knob counterclockwise until the epithelium appears or comes into sharp focus. Slide 34 a or c, Liver. There are many blood vessels in the periportal connective tissue. The epithelium lining the blood vessel wall is on the luminal side and it will be seen in profile. Sometimes the cytoplasm of the individual epithelial cells are very thin and difficult to see, but the flat nucleus which occupies the thickest part of the cell will be Simple cuboidal epithelium - Slide 20, Thyroid gland. The cells forming the wall of the thyroid follicle are usually cuboidal in profile in the normal gland. In the relatively inactive gland the cells become low cuboid al or even squamous. When the gland is highly active the cells may become columar. Slide 45a, b, or c, Ovary. Look for the surface epithelium. The cells will be in profile. The epithelium is usually low cuboidal. Slide 41 a or b, Kidney. Look for the initial portion of the collecting tubule in the medullary ray. The tubules will usually be cut longitudi- nally so that the epithelium will be seen in profile. The distal convol- uted tubule in the granular cortex, which will usually be cut in crose- or obligue section, is lined by cuboidal cells. Simple columnar epithelium - Slide 31a, b, or c, Duodenum, jejunum or ileum. Look for the epithelium of the mucosa. The epithelium may be easier to appreciate in the Crypts of Lieberkuhn. Note also the presence of goblet cells. In routine H & E preparations it is easily spotted because the mucigen content of the cytoplasm has been washed away leav- ing an empty space in the apical, portion of the cell. This space will appear as a gap between epitheial cells. Stratified squamous epithelium ~ Slide 56c, Thick skin. The epithelium18 ved is in side view and is keratinized. Verify the columar or cuboidal profile of the cells at the base of the epithelium. Next examine the succeeding layers above the basal cells. Note the progressive flat- tening of the cells as the surface is approached. The superficial cells are definitely flat. Note that the epithelial surface is covered by layers of material that have lost their cellular appearance. The kerat~ inized portion of the epithelium is mostly located here. Slide 48a or b, Vagina. This epithelium is non-keratinizing. Note that the most superficial cells have not lost their cellular boundary and sotte nuclei may still be seen. Slide 28a or b, Esophagus. The iucosal epithelium is non-keratinizing. Compare the superficial cells with those of the epdernis. Stratified colurmar or cuboidal epithelium - Slide 25, Parotid; Slide 26, Mandibular gland - The tain duct and larger interlobar ducts are usually lined by stratified columnar or cuboidal epithelium. Verify if the lining is really stratified and not pseudostratified epithelium. Slide 56a, b, or c, Skin. Look for the duct of sweat gland. This is stratified low cuboidal epithelium. ‘Transitional epithelium - Slide 43a or b, Urinary Bladder; Slide 42, Ureter. Compare the lining epithelium of the two specimens. It is thick- er in the urinary bladder. Look for differences in appearance between stratified squamous epithelium (skin, vagina, esophagus, etc.) and transitional epithelium of the urinary bladder. Pseudostratified columnar epithelium - Slide 39, Trachea. The mucosal epithelium lining the lumen is nortially ciliated and with goblet cells. Slide 52c, Ductus epididymis. The cilia-like structures are sometimes called stereocilia. GLANDULAR EPITHELIUM Glands may be classified into two general types on the basis of the route taken by the secretion from the cell, nately: 1. Exocrine gland - ompties its secretion onto a body surface, and 2. Endocrine gland ~ empties its secretion via the intercellular portion of the connective tissue tg the blood or lymph capillaries of the latter. Exocrine Glands 1. Unicellular Gland- The secretory unit is composed of a single cell which is distributed attong the more numerous “ordinary” epithelial cells. It is usually found in columar types of epithelia. The goblet cell is the only example in man. Slide 3la, b, or c, stall intestines; Slide 39, trachea. 2, Multicellular Gland - This is composed of many cells arranged in various degrees of complexity, as follows: a. Secretory Epithelial Sheet - Some lining epithelia are also secretory in function. The cells are laid out as in lining epi- thelia. They do not form invaginations into the underlying con~v.58 19 nective tissue. Some examples of this type are the epithelium of the seminal vesicle, interfoveolar epithelium of the stomach, epithelium of the choroid plexus, and the uterine endometrial Lining epithelium at a certain stage of the menstrual cycle. Slide 29, Gastroesophageal junction, Slide 29; Stomach, Slide 30a, b, Or c; Seminal vesicle, Slide 55. b. Intraepithelial Gland - The secretory cells are aggregated in small groups within the lining epithelium. There is a recogniz- able attempt at formation of a lumen within the gland but there 4s no definitive invagination into the underlying connective tissue. This type is rather rare in man. It has been seen in the nasal mucosa, among the mucosal lining epithelium of the lacuna of Morgagni in the male, and the conducting portion of the respir~ atory tract. ©. Specialized invagination of the lining epithelium into the underlying connective tissue, as follows: (1) Simple glands - The lumen of the gland (and its duct, if present) are either unbranched or form only one generation of branches. (a) Simple tubular gland - This usually has no duct and may be likened in shape to a test tube. Example: Crypt of Lieberkuhn, Slide 31a, b, and cy (b) Simple coilea tubular gland - A duct is often present. ‘The secretory portion is coiled. Example: Sweat gland, Slide 56a, b, c, or e. (c) Simple branched tubular gland - There is only one gener- ation of tube-shaped branches arising from a short duct or the stem of the secretory portion. Example: Gastric glands, Slide 30a or b; some glands of Brunner, Slide 31a. (4) Simple branched alveolar gland - Sac-like secretory portions open into a single duct, or a single large sac is divided into smaller sacs. Example: Sebaceous gland, Slide S6e. (2) Compound glands - There are several generations of branches. Bach generation is surrounded by connective tissue which has characteristic thickness and arrangement associated with the degree of that particular generation. Thus a compound gland is composed of several lobes separated by connective tissue part itions that send thinner partitions into the interior of each lobe to form smaller lobes, etc. Depending on the predominant shape of the terminal secretory portion, there are two types of compound glands: (a) Compound tubular gland - The secretory portion is tubular in shape. Example: Kidney, Slide 41; Bulbourethral gland; some glands of Brunner, Slide 31a; Gastric cardiac glands, Slide 29, and Pure mucous glands of the oral cavity. (b) Compound alveolar (or acinar) gland - The secretory20 portion, or at least the terminal portion of the secretory part is in the form of a sac. Example: The bigger salivary glands, Slides 25, 26, & 27) Glands of the respiratory tract producing purely serous or mixed mucous and serous secretions; Exocrine pancreas, Slide 36; Prostate gland, Slide S4a or b; and Mammary gland. Slide 5ic. EXERCISE Examine the glands mentioned in the various types of exocrine glands. Look at several examples of each gland, not only from your slide collection but also from your clasamate's. Look for the ducts or evidence of direct opening of the secretory portion to a body surface. The types of ducts found in compound glands will be dis- cussed in the class lecture. Endocrine Glands Endocrine glands are derived from epithelium. Since their secretion oes not pass directly onto a body surface, no functional duct can be found. The variously arranged clusters or columns of secretory cells usually do not show any lumen in their midst. In those endocrine organs that arose througha process of invagination of a lining epi- thelium, some vestiges of a lumen may remain including occasionally a vestigial non-functional duct. The details on endocrine glands will be taken up later in the course. EXERCISE Examine the slides of endocrine organs to familiarize yourself with the following features: 1. Cellularity of the gland in keeping with its epithelial natur 2, Absence of duct, or at most presence of vestigial ducts only. 3. Presence of many blood vessels among the secretory cells. Slides: 19a or b, Hypophysis; 20, Thyroid; 21a ox b, Parathyroid; 22, Adrenal gland; and 23a or b, Epiphysis cerebri.Viel at VI. CONNECTIVE TISSUE AND CONNECTIVE TISSUE PROPER Connective tissue is derived from mesenchyme or mesoderm. Derivatives of ectoderm, endoderm, as well as mesoderm itself, grow into or develop with= fn mesenchyme. I definitive tissues, therefore, the parenchyma or the characé teristic functional cells of a tissue or organ are always physically and functionally associated with connective tissu connective tissue is characterized by the abundance of intercellular sub- stance. However, intercellular substance is its most variable component. There are two general types of connective tissue: 1. Connective Tissue Proper - composed of all the different connective tissues except the specialized connective tissues, and 3 Specialized Connective Tissue - composed of cartilage, bone, dentine, blood, and lymph. CONNECTIVE TISSUE PROPER Based on the relative density or number of intercellular fibers, connec~ tive tissue proper can be classified into loose and dense types. A third type, smedified loose connective tissues" is proposed for loose connective tissues with additional distinctive characteristics. For the sake of clarity, the following outline is presented to help explain the classification of connective tissue proper: 1. Ordinary Loose Connective Tissue (LCT) - Interncellular fibers are far apart and consist mainly of collagenous fibers. Areolar tissue is a good exan- ple of this. | Omentum, Slides 2a & b. 2. Modified Loose Connective Tissue - The fibers are far apart, but the tissu have varying histologic characteristics due one or a combination of the follow- ing: predominance of a certain cell type, preponderance or prominence of a type of intercellular fiber, or a consistency that is quite distinct from the ordinary loose connective tissue. a. Mucous Connective Tissue - In the fresh specimen, appearance of this tissue suggests mucus. Also, its ECM gives a positive test for mucin. Mesen- chymal connective tissue may be included here. Connective tissue of the umbi- lical cord is another example and is perhaps easier to obtain. umbilical Cord, Slide 50. b. Reticular Comective tissue- There axe few collagenous and elastic fibers, Reticular fibers constitute the bulk of the fibrous elements that support the large number of cells. Bone Marrow, Slide 14a. Note that marrow appears devoid of fibers due to the fact that the reticular fibers are un- Stained. The same is true for the H & E- or Masson-stained specimens of the jymph node, spleen, and liver. Lymph Node Stroma, Slide 15; Splenic Strom, Slide 16a & b; Liver, Slide 34a, and Liver, Silver Stain, Slide 34b- c. Adipose Tissue - Fat cells predominate in the tissue. Omentum, Slides 2a & b; Subcutaneous tissue of the skin, Slides 56a, b, & e+ d. Pigment Tissue - Pigment cells are present in some loose connective tissue, Eyeball, look for the tunica suprachoroidea and lamina fusca, Slide 58.vE.2 22 ©. Lamina Propria - This is the connective tissue supporting the micosal epithelium of the gastrointestinal and respiratory tracts. Its general char- acteristic is the presence of many cells, especially lymphocytes. Intestines, Slides 31, 32, & 33; Lung, Slide 40. 3. Dense Connective Tissue - The intercellular fibers are abundant and closely packed, hence the cells have limited areas to occupy, and consist mostly of fibroblasts. a. Dense Regular Connective Tissue - There is a definitive pattern or orientation in the disposition of the fibers. Tendon, Slide 3. b. Dense Irregular Connective Tissue - There is no discernible pattern in the orientation of the fibers. Dermis of the Skin, Slides 56a to g. COMPONENTS OF CONNECTIVE TISSUE PROPER Listed below are the elements that are generally found in connective tissue Proper. There are differences in the number of each element among the different members of connective tissue proper. In some, a particular cell or intercellular fiber may be absent or rarely encountered. In others, an intercellular fiber or @ particular cell or several cell types may predominate in the tissue. 1, Intercellular Fibers @, Collagenous fiber - This has an acidophilic staining reaction. It is pink in routione Hl & E preparations. It is blue with Masson's trichrome stain. Skin, Slides 56a to g, look for the fibers in the dermis; Omentum, Slides 2a & b; Tendon, Slide 3; Adventitia of the aorta, Slide 13a. b. Elastic fiber - This is usually unstained in H & E and Masson sections. However, it may be seen as a refractile and short wavy fiber contrasting from the acidophilic collagenous fibers or smooth muscle fibers adjoining it. It is black with orcein and Weigert stains. Aorta, Slide 13a, Examine the tunica media. Look al so for the internal elastic lamina of arteries in other tissues. c. Reticular fiber - This can be seen in the section that has been stained with silver. Liver, Slide 34b, The fibers are black and anastomosing among the hepatic cells. 2. Cells - The following are either inhabitants (fixed) or are visitors (wander- ing cells) of the tissue: a. Fibroblast - This is found in all members of connective tissue proper. Its identity is easier to confirm when it is attached to or is closely associated with a collagenous fiber. Omentum, Slides 2a 6 b; Tendon, Slide 3; Perichondrium of cartilage, Slides 4a & b; Periosteum of bone, Slides 5b, c, & d; Perimysium of muscle, Slides 6a & b. b. Adipocyte (fat cell) - The slide of the omentum shows the fat cells in their characteristic "signet ring" appearance. Mesothelium, Slide 1; Omentum, Slides 2a & b. ¢. Mesenchymal cell - Under the LM, this is very difficult to distinguish from the fibroblast in adult tisues. However, it is usually smaller than the jatter and is often associated with, or are close to capillaries in contrast to collagenous fibers. Umbilical Cord, Slide 50. d. Macrophage - Like the fibroblast, the cell is found in all the members of connective tissue proper. It is relatively more abundant in the lymph node, spleen, lymphatic tonsils, and lamina propria of the digestive tract. The von Kupffer cells of the liver sinusoid and the dust or heart failure cells of thevr.3 23 lungs are macrophages also. Liver, Slide 34a; Lung, Slide 40; Lymph Node, Slide 15; Spleen, Slides 16a & b; Palatine tonsil, Slides 18a & b; Intestines, Slides 31, 32, 6 33. @. Plasma Cell - This is easier to find in lymphatic organs, lamina propria mucosae of the digestive and respiratory tracts, and bone marrow. I suggest that you postpone studying this cell until you reach hemopoiesis when you will be studying bone marrow smears. Lymph Node, Slide 15; Spleen, Slides 16a & b; Palatine Tonsil, Slides 18a & b; Lamina Propria Mucosae of the Intestines, Slides 31, 32, 6 33. £. Mast Cell - This cannot be identified in routine sections. g. Neutrophil Leukocyte - This is encountered in large numbers during bact~ erial infections. Otherwise, it is absent or very rare in connective tissue proper. Palatine Tonsil, Slides 18a & b; Appendix, Slide 32; Intestines, Slides 31b & c. h. Lymphocyte - This is most abundantly found in lymphatic organs and the lamina propria mucosae of the digestive and respiratory tracts. It is easy to find in peripheral blood and in bone marrow. But I suggest that you postpone looking for it in the last two examples until you are already studying.those tissues. Lymph Node, Slide 15; Spleen, Slides 16a @ b; Palatine Tonsil, Slides 18a & b; Lamina Propria Mucosae of the intestines, Slides 31, 32, & 33. i. Monocyte - This blood cell might be somewhat difficult to find in connect- ive tissue proper because it transforms into macrophage when it leaves the blood vessel. Lymph Node, Slide 15; Spleen, Slides 16a & b; Palatine Tonsil, Slides 18a & b;Lamina Propria Mucosae of the intestines, Slides 31, 32, & 33. 4. Eosinophil Leukocyte - In routine H & E preparations, you can easily spot this cell even under the low power objective because of its large and brilliant red cytoplasmic granules. The best preparations for locating this cell is thelamina propria of the digestive tract and the lymphatic tonsil. You will study this cell in more detail when you reach peripheral blood and hemo- poiesis in your studies. Palatine Tonsil, Slides 18a & b; Intestines, Slides 31, 32, & 33; Lung, Slide 40. k. Basophil Leukocyte - Like the mast cell this cannot be identified in H&E sections. You will be able to see it in the blood smear preparation. 1. Pigment Cell (Melanocyte) - This cell is found only in some members of connective tissue proper, It contains melanin pigment. Skin, Slides 56a, b, e, £, & 7 Eyebal, Slide 58.vine. 2h VII. CARTILAGE There are three types of cartilage, namely, hyaline, elastic, and fibrous. ‘These vary in gross and microscopic appearance due mainly to differences in the content and proportion of their intercellular material. Look for the following components of cartilage in your slides: 1. Chondrocyte (cartilage cell) - It is embedded in the intercellular subs- ance. The space that it fully occupies during life is the chondrocyte lacuna. In preparations, the dead chondrocyte is often shrivelled to a small hyper- chromatic mass, leaving a vacant space around it which is the part of the jacuna that has been exposed by cell shrinkage. Be careful when identifying the chondrocyte in this situation because you may mistakenly identify the shrunken cell as the nucleus and lacunar space as the cytoplasm. In adult hyaline and elastic cartilage, the chondrocytes are roundish and appear to be distributed randomly in the center of the tissue. They come closer together and progressively flatter towards the periphery or surface. In fibro- cartilage, many ofthe chondrocytes in the center of the tissue may be elongated rather than rounded. Increase in the number of chondrocytes in growing cartilage is by two means: a. Interstitial Growth - The tissue increases in bulk through the addition of material from the within the tissue itself. The condrocyte within the cart- ilage divide, the daughter cells separate from each other through their indiv- idual secretion of matrix. Immediately after cell division, and in the early stages of laying down matrix, the daughter cells arising from one chondrocyte are clustered together (isogenous group). If the tissue preparation was done at the time when the daughter cells were actively secreting matrix, you will be able to see amore tasophilic matrix surrounding each chondrocyte (territor- ial matrix) in the isogenous group. In growing long bones, interstitial growth of the cartilage at the epiphys- eal plate is responsible for the elongation of the shaft. The direction of pro- liferation of the chondrocytes is only along one plane, i.e., the longitudinal axis of the bone, so that, instead of finding isogenous groups you will see the chondrocytes arranged in longitudinal columns. b. Appositional Growth - The cartilage increases in size through the devel opment of new chondrocytes by the chondrogenic layer (deeper layer) of the perichondrium followed by the secretion of matrix by these new cells. Increase in bulk in this case is through the addition of new substance on the surface. ‘The chondroblasts (cartilage-forming cells) have a basophilic cytoplasm. They are in the perichondrial layer next to the newly laid cartilage. 2. Cartilage Matrix - This is the intercellular substance of the cartilage. It is of "amorphous" ground substance and intercellular fibers. Collagen- ous fibers are always found in thematrix of the three types of cartilage. You will be able to distinguish between the three types of cartilage mainly through the appearance of their respective matrices. In hyaline cartilage, the matrix appears homogeneous in routine preparations with staining tending towards baso-25 VEE.2 philia. The fibers, which are mainly collagenous, are not seen. In elastic cartilage, there are many elastic fibers. Although elastic fibers are not usually stained in routine H & E preparations, “fibrous” streaks, often seem- ingly forming networks, often basophilically staining, are seen’ in the matrix. This is the feature that easily differentiates elastic cartilage from hyaline cartilage at a glance. In fibrocartilage, there is an abundance of collagenous fibers to the extent that the fibrous texture of the tissue is very evident. ‘The amorphous portion of the matrix is mostly obscured by the fibers and my only be seen clearly around the chondrocytes. 3. Perichondrium - This is a type of dense fibrous connective tissue in which the fibers are arranged more or less parallel to each other, although not as well ordered as in the other types of fibrous membranes. It is composed mainly Of collagenous fibers and fibroblasts and is the result of the "condensation" ofthe mesenchyme surrounding the cartilage during its formation. It surrounds cartilage everywhere except over articular surfaces. Its outer surface blends with the surrounding connective tissue so that you may be able to trace some Of its fibers leaving the perichondrium and joining the surrounding connective tissue. There is a gradual transition froma dense fibrous membrane to that of a cartilage as you examine the part of the perichondrium next to the cartilage. It is believed that most of the undifferentiated cells of the perichondrium are found in its innermost part. These cells are the ones that transform into cartilage-forming cells or chondroblasts which in turn are responsible for ap~ positional growth in developing cartilage. In the perichondrium of mature cart— ilage, neither the undifferentiated cell nor the chondroblast may be identified in routine preparations. The cells in the deepest part are similar in appear- ance to fibroblasts. However, in cartilage undergoing repair, the deep layer (chondrogenic layer) of the perichondrium returns somewhat to its youthful character so that the chondroblasts may be easily identified. EXERCISES: Compare the three types of cartilage. Hyaline Cartilage, Slide 4a; Fibro- cartilage, Slide 4b; Elastic Cartilage, Slide 38a. Look for differences in appearance of the cartilage matrix, the shape of the chondrocyte or its lacuna jn the various areas of each cartilage, as well as in the three types of cartilage. Identity the perichondrium if its is found in the preparation. Study its texture in relation to the surrounding ordinary connective tissue and the cartilage it covers. Look for evidence of cartilage growth in the slide that you axe examining. If present, identify if its is interstitial or appositional in type. Look for areas of interstitial and appositional growth in the slide show- in intracartilagenous ossification. If present, interstitial growth will be seen in the epihyseal plate, while appositional growth will be found in the shaft of the bone and in the widest part of the cartilagenous epiphysis. Intracartilagenous Ossification, Slide Sd.26 VIII.1 VIIT. BONE ‘There are two general types of formed bone, as follows: 1. Spongy Bone - Gross examination reveals a structure that suggests a sponge. There are many holes or spaces between partitions of bony tissue. 2. Compact Bone - Grossly, this type appears solid, with occasional holes that will allow partial passage of an ordinary pin (Volkmann's canals). The following are the principal components of bone: 1. Cells 1.1. Osteocyte - This is the only cell type embedded in the matrix of formed bone. Each osteocyte is contained in an osteocyte LACUNA. Both cell andlacuna are described as a flattened oval, somewhat similar to the shape of a melon seed. The cell has short cytoplasmic processes that are inserted into the openings of CANALICULI in the lacunar wall. If the cell is well preserved it will show a faintly basophilic cytoplasm. Its nucleus is usually oval and has one or more nucleoli. 1.2. Osteoblast - This is mostly found on the surface of developing bone, butmay also be encountered occasionally on the surface of formed bone. It is somewhat bigger than the osteocyte, has a large nucleus and a big nucleolus. The cytoplasm is strongly basophilic. A negative Golgi image is often encountered. This cell has cytoplasmic projections that may "inter- connect” with neighboring osteoblasts. In many instances, osteoblasts are arranged on the surface of bony spicules in a manner suggesting a simple cuboidal epithelium. 1.3. Osteoclast - This is a multinucleated giant cell found on the surface of bone that is undergoing resorption. It may be found in a depression of ‘the bone called HOWSHIP'S LACUNA. Its size increases with the number of nuclei. It has a pale and “foamy* cytoplasm which often shows branching processes with rough edges at sites next to the bony surface. Staining re- action of the cytoplasm ranges from faint basophilia to acidophilia. In the class slides the cytoplasm is usually acidophilic. The nucleus is vesicular and frequently has a small dark nucleolus. 2. Bone Matrix (intercellular substance) - Regardless of the type of defini- tive bone, this is always arranged in the form of plates or lamellae. Its components are: 1.1, Amorphous Substance - This is composed of acid mucopolysaccharides and inorganic salts, mostly crystals of calcium, phosphates, and small amounts of sodium, magnesium, and bound water. 1.2, Fibers - Collagenous fibers are abundant but "masked", i.e., they are not seen in ordinary H & E preparations but can be seen with silver staining. Collagenous fiber bundles penetrate the bone from the periosteum (Sharpey's fibers) but these should not be considered as part of the in- trinsic intercellular elements of bone. Elastic fibers may also penetrate the bone from the periosteum, either with Sharpey's fibers or separatély. Like Sharpey's fibers, they are not considerd intrinsic elements of bone matrix.27 VIII.2 Architecture of Spongy Bone Under the LM, spongy bone shows irregular, often branching and anastomos- ing plates or bars of bony substance called bony TRABECULAE or bony SPICULES. Bach trabecula is composed of bony lamellae containing osteocytes. Canaliculi interconnect neighboring lacunae. There are usually no Haversian systems and blood vessels in the trabeculae. Marrow is found in the spaces between trab- eculae. Connective tissue of the marrow is condensed on the surface of the trabecula. This is called ENDOSTEUM. It contains stromal cells that can trans- form into osteoblasts or osteoclasts. The outer surface of spongy bone often has a thin covering of compact bone so that one cannot always say that spongy bone is covered by periosteum. Architecture of Compact Bone ‘The presence of HAVERSIAN SYSTEMS or OSTEONS is the one feature that histo- logically distinguishes compact bone from spongy bone. The haversian system is the unit structure of compact bone and can be described in a simplified way as a cylinder with a thick wall and a very narrow lumen. The cylinder is not a simple straight tube but a branching structure that connects with neigh- boring osteons and may end at the central marrow cavity or outer surface of long bones. The lumen is the haversian CANAL. It contains loose connective tissue, and usually two small blood vessels (capillary or postcapillary venule). It may join haversian canals of adjacent systems and may open into the central marrow cavity. The wall of the osteon is composed of concentric layers or jamellae of bone. Each lamella has osteocyte lacunae which are interconnected with neighboring lacunae by canaliculi. Canaliculi near the haversian canal open into that canal, while those in the outermost lamella xecurve into the system. CEMENT LINES define the outer limits of definitive osteons. They usu- ally do not have canaliculi and stain differently from the lamellae of the: osteons. VOLKMANN'S CANALS, approximately perpendicular or oblique in direction with respect to the long axis of the bone, are connected to the haversian can- als. They may open at the central marrow cavity or the outer surface of the bone. They carry relatively larger blood vessels that are directly continuous with those in the haversian canals, the marrow, as well as on the outer surface of the bone. Volkmann's canal pierces successive layers of haversian lamellae to reach the haversian canal. Hence, orientation of lamellae around the Volk- mann's canal is not concentric. The diaphysis of long bones is a cylinder in which the central or axial cavity is the CENTRAL MARROW CAVITY. Inmediately surrounding this are several bony lamellae that are circumferentially arranged. These are the INNER CIRCUMFERENTIAL LAMELLAE of long bones. Spongy bone, continuous with the inner surface of the innermost circumferential lamella may be found especially towards the epiphysis which is essentially spongy in character. The internal surface of the innermost circumferential lamella is lined by endosteum. Endos- teum also lines both haversian and volkmann's canals. Forming the outer sur- face of the diaphysis are layers of circumferentially oriented bony lamellae called the OUTER CIRCUMFERENTIAL LAMELLAE. Between the inner and outer circum- ferential lamellae are the osteons that are closely packed and arranged more ox less parallel to the long axis of the diaphysis. The angular areas formed by adjoining osteons are occupied by the remnants of older haversian systems that have been partially resorbed during bone remodeling. These are called INTERSTITIAL LAMELLAE. Sharpey's fibers may be found in the interstitialas vitT.3 lamellae, especially near the more superficial parts of the bone, and in the outer circumferential lamellae. ‘he architecture of flat bones is similar to that of long bonés. Since these are not cylindrical, the lamellae lining their internal and outer surfaces are called INNER and OUTER BASIC LAMELLAE, respectivley. Periosteum Periosteum covers the outer surface of bone except where it is covered by cartilage, at insertions of tendons or ligaments, and in sesamoid bones. It dis dense connective tissue and may be divided into two zones. The outer is dense, relatively acellular, and contains blood vessels and nerves that supply the bone. The inner zone is looser in quality and has relatively more cells. Collagenous fibers and bundles from the outer zone pass through the inner zone and penetrate the more superficial parts of the bone as Sharpey's fibers, bringing with them blood vessels and nerves. The periosteum also contains net~ works of elastic fibers which may penetrate the outer parts of the bone. Bigger vessels passing through the periosteum enter the Volkmann's canals. The inner zone of the periosteum has spindle shaped cells that are hard to distinguish from fibroblasts but possess the potentiality to transform into osteogenic cells. Firm attachment of the periosteum to bone is evidenced by the density of collagenous fibers entering the bone from the periosteum. The periosteum is firmly attached to most of the surface of short bones. In long bones, the the points of firmer attachments are in the regions where Shazpey's fibers penetrate the bone, around points of attachment of muscle and tendon, and also at the epiphyses. EXERCISES 1, Using the low power objective of your microscope, differentiate between spongy and compact bone. Slide Sa, Ground Bone; Slide Sb, Decalcified Bone; Slide Sd, Intracartilag- enous Ossification. 2. Identify periosteum and endosteum. Slide 5d, Intracartilagenous Ossification. 3. Using your slides of compact bone, identify the following: a. Osteon and its parts, namely, lamellae, lacunae, canaliculi, and haversian canal b, Interstitial lamellae c. Cement lines (if demonstrable) . Volkmann's canals e. Sharpey's fibers Slide 5a, Ground Bone, Slide 5b, Decalcified Bone 4. Using your slide of developing bone, identify osteoblasts and osteoclast: Slide Sd, Intracartilagenous Ossification. OSTEOGENESIS The following are the two general types of bone formation: 1.tntramembranous Ossification - This is bone formation through transformation of precursor cells into osteoblasts and the laying down of bony matrix result- ing in the development of bone without a preceding cartilage model.viIr.4 29 EXERCISE: Look for areas of deposition of bony matrix by the periosteum Gver the cartilagenous diaphysis of long bone. Why is this a type of intramembranous ossification 7 Slide 5d, Intracartilagenous Ossification. 2, Imtracartilagenous or Endechondral Ossification - This is bone formation ja which there is a previous cartilage model which is replaced by bone- EXERCISE: Identify the areas of intracartilagenous ossification. What are the structural evidences of this process in your slide ? cartilage growth and bone formation in the epiphyseal cartilage plate is the asin mechanism by which long bones increase in length. Study this region qaty well because you can see here most of the stages of endochondral ossif- \eation. You can recognize five zones in the epiphyseal plate in most of your class slides of developing bone. These are enumerated below in the same sequence as in endochondral ossification: 1. tone of Resting Cartilage (Cells) - This is nearest to and continuous with the epiphysis. There are no special physical changes visible here. The chon” Gnocytee appear to be randomly distributed and are small compared to those in the succeeding zones. 2. tone of Proliferation or Proliferating Chondrocytes - The chondrocytes are Giviaieg mitotically in a longitudinal direction, parallel to the long axis of the diaphysis, so that the cells become aligned in longitudinal colums. The pee ccellelar matrix between suceeding cells in a column appears as very thin partitions so that the chondrocytes (of lacunae) appear to abut into each pther. the matrix between adjacent columns of chondrocytes is a little more substantial. 4. tone of Hypertrophy (or Hypertrophied Chondrocytes) - The cells have enlarged 2k the expense of the mtrix. Nearest the fourth zone, the cells have acquired phosphatase. A. tone of Calcification (or Calcified Cartilage Matrix) - The calcified matrix je deeply basophilic. Most of the chondrocytes here are either dead or dying From the marrow cavity, connective tissue with small blood vessels aod osteoblasts invade the colums of lacunae containing the dead or dying chopdro- Cytes. The thin matrix immediately surrouoding the lacunae dissolves so that the thin partitions separating adjacent lacunae in a column disappear. This fecults ip the confluence of lacunar spaces in each column and further reduct- fon in the thickness of the matrix separating the columns, 5. toue of Bone Deposition or Metaphysis - Osteoblasts brought by the advanc” {hg conoective tiissue from the central marrow cavity lay down bone matrix of the surface of the remnants of calcified cartilage, producing a type of spongy bone (woven bone). In the continuing process of bone elongation, the tips of the bony spicules of the newly formed bone are resorbed. You have a greater chance of finding osteoclasts at this site. EXERCISE: Identify the various stages of endochondral ossification in your glide. Slide Sc, Ossification, Long Bones Slide Sd, Intracarti- lagenous Ossification.30 1x1 IX, MUSCLE TISSUE ‘The muscle cell is advantageously elongated to serve its contractile function. The striated varieties are preferentially referred to as muscle FIBERS because of their shape. Histologically and functionally, there are three types of muscle cells, camely: 1. Smooth Muscle - has no cross banding (striation); is usually involuntary 2. Skeletal Muscle - has cross banding; is voluntary 3. Cardiac Muscle - has cross banding; is usually involuntary. SMOOTH MUSCLE ‘The cytoplasm usually appears homogeneous or faintly longitudinally stri- ated in routine preparations. Smooth muscle is an integral part of all sys~ tems of the body except the central nervous system. It is usually arranged in sheets and small bundles, although single cells may be found, e.g., in small arterioles. It isusually fusiform in shape. Its average diameter at its thick- est portion is about six micrometers. Its length varies widely from 15 to 500 micrometers. The smallest are found in small blood vessels while the big- gest sre found in the pregnant uterus. The sarcoplasmic matrix is obscured by well developed eosinophilic myofibrils except in the periouclear region where the myofibrils skirt around the oucleus. The light staining oucleus is elong- ated with tapering rounded ends; its heterochromatin is distributed prominent- ly at the periphery. It is usually located in the thickest portion of the cell (about midlength) and is slightly eccentric in position. It has a tenden- cy to fold or wrinkle with contraction of the cell. Faint longitudinal stria- tions, especially near the periphery, may be seen when the light entering the microscope is reduced. These striations are produced by the longitudinally oriented myofibrils. When arranged in sheets, the lateral boundaries of the overlapping cells may be hard to distinguish. Cellular boundaries are better seen in cross sections where the profile of the cell is usually round. Depend- ing on the level of the section, the diameter of the cell will vary and its nucleus may or may not be included. SKELETAL MUSCLE Longitudinal sections of the fiber show cress striations due to alternate Light and dark staining segments in each myofibril which are "in register™ with those of the other myofibrils in the cell.the cell is multinucleated. ‘The nuclei are generally distributed in the periphery, just beneath the sarc- lemma. The diameter of the muscle fiber ranges from 10 to 150 micrometers, while the length is extremely variable. The fiber is very long and may run the entire length of the muscle. Since a section of the muscle involves a small piece of the whole length, it would be extremely hard to demonstrate the end of the fiber. In a cross section, the myofibrils may be aggregated in groups called Cohoheim's fields. This feature is believed by many to be ao artifact. In well prepared specimens, you will be able to distinguish some of the cross striations (Figure 4), oamely, the light band (I band or isotropic band), dark bank (A band or anisotropic band), 2 line or disc, and H band. In the uncontracted muscle, the I and A bands are about equal in length. The 2% disc is a thin dark line running transversely at the middle of the I band. ‘The H band is lighter staining than the A band but oot as light as the I band; it runs transversely accross the middle of the A band.31 1X.2 Associated connective tissue of skeletal muscle is named according to its location. The ENDOMYSIUM, a loose type of connective tissue, surrounds each individual muscle fiber. It is thin and has reticular fibers, a few fibroblasts, blood capillaizes, and small nerves. In some fibers, it may be thicker because of some collagenous and elastic fibers. the SATELLITE CELL has a flat oucleus and scanty cytoplasm. It is closely associated with the sarcolemma of the muscle fiber and is often mistaken for the oucleus of the fiber. The endomysium is continuous with the PERINYSIUM which binds the muscle fibers into bundles and forms septa within the muscle. The perimysium is thick- er than the endomysium. It has collagenous and elastic fibers, aod more cells It carries bigger blood vessels, lymphatics and nerves. The EPIMYSIUM invests the entize muscle. It is thicker than the perimysium and its texture is coarser because its fibers are stouter. It also contains larger blood vessels and nerves than the former. Lymphatic vessels are also numerou: FIGURE 4. Cross banding in the myofibril of skeletal miscle that can be seen under the HP objective with routine H & E staining. CARDIAC MCLE, Cardiac muscle is found in the heart and sometimes in the root of the great vessels of the heart. Like skeletal muscle, it is cross striated. However, it Possesses usually only one oval nucleus and this is axially situated at the center of the fiber. More sarcoplasmic matrix is seen next to the poles of the pucleus. Lipofuscin pigment may be seen also at this region. The fiber is about 14 micrometers long in the adult. It branches and forms networks with neigh- boring fibers. The branches are thinner than the main part. Cardiac miscle fibers in the atria tend to be smaller or thinner than those in the ventricles. The fibers are arranged end to end. The junction between fibers is at the INTERCALATED DISC. The intercalated disc is a dark transverse line or structure occuring at the level of the Z disc or the I band; it may be steplike in its course instead of being a straight transverm line. Compared to skeletal muscle, the texm "fiber" in cardiac muscle has a slightly different meaning in the sense that one may be referring to a series of cardiac muscle cells connected end to end. The fiber has @ similar banding pattern as the skeletal muscle except for the presence of the intercalated disc. Associated connective tissues are in the form of endomysium and périmysiui Bodomysium consists of reticular and fine collagenous fibers surrounding individual1X3 32 muscle fibers. Perimysium consists of somewhat larger fibers with elastic fibers around bundles of muscle fibers. Compared to skeletal muscle, cardiac muscle has much more blood vessels in the endomysium and perimysium. Components of the impulse conducting system of the heart, sometimes referred to as the "Purkinje system", are modified cardiac muscle fibers. This portion will be included in the chapter on the cardiovascular system. EXERCIS! Examine the cross- and longitudinal sections of the different typ muscles, using at least the following slides. 1. Smooth Muscle: Slide 31a, b or c, Duodenum, Jejunum, or Ileum. The muscle fibers are found in the muscularis mucosae and muscularis externa. Slide 47c, Uterus, secretory phase. The muscle is found in the muscularis layer Of the uterus (myometrium). Compare the size of the smooth muscle here with those of the small intestines. 2. Skeletal Muscle: Slide 6a, Voluntary Muscle, cross section 6b, "| longitudinal section Identify the associated connective tissues in the specimens Slide 24a, ox b, Anterior or Posterior Tongue The cross striations illustrated in Figure 4 are more often seen better in these slides of your collection. Cardiac Muscle: Slide 6c, Cardiac Muscle; Slide 12a, Atrium, b, Ventricle Compare the muscle fibers in the atrium and ventricle. Look for the intercalated disc. EXERCISE: Make a table of the physical differences among the three types of muscle when examined under the LM.33 ha xX BLOOD Like other tissues, blood is composed of cells and intercellular substance. Plasma is the intercellular substance in blood. It is a fluid and as such the cellular components are free to float about so that no fixed spatial relation- ships can be established among them. Unlike the other connective tissues, blood does not have intercellular fibers except when it clots. Blood also contains functional fragments from a cell normally residing in the bone mar- row. In this chapter, we are interested in studying the formed elements (cells and cell fragments) of the blood of the adult human. You will study mainly the thin dried blood smear that has been stained with Wright's stain which colors acidophilic parts red and basophilic parts blu: RED BLOOD CELL (erythrocyte, normocyte, or rbc) This is the most plentiful cell type in the blood (about 5.5 million pe: cubic millimeter of blood in men, and slightly lower in women during the child-bearing period). Erythrocyte count may be slightly higher in people living in high altitudes. Filipino researchers in the 1960s observed that erythrocyte counts were lower among Filipinos. Current observations tend to show that the count has increased. The erythrocyte is a biconcave disc. In thin dry blood smears tbc measures about 7.5 micrometers in diameter and 1.9 micrometers thick at the edge (thickest part). It is slightly bigger in circulating blood and slightly smaller in tissue sections. In a thick blood smear or inside blood vessels where circulation has slowed down, erythrocytes tend to stick together by their concave surfaces producing an appearance likened to a stack of coins (rouleaux formation). It is not nucleated. Its cytoplasm is acidophilic although it may still be considered normal to find a few that are faintly basophilic staining. These are called polychromatophilic erythrocytes or reticulocytes*. The positive evidence that you are looking at a reticulocyte is when the specimen is stained with brilliant cresyl blue where the baso- philic elements in the cytoplasm are in the: form of a network (reticulum). LEUKOCYTES (white blood cells) Unlike the rbc, leukocytes are nucleated and are therefore complete cells. They possess ameboid motion which partially explains their comparatively le: regular cellular boundary compared to that of the former. There are about 5,000 to 9,000 leukocytes in the normal adult. Some authors go as high as 10,000 per cubic millimeter of blood as the upper limit. If the total leuko- cyte count is below 5,000 per cubic millimeter the condition is called LEUKOPENIA. Above the upper limit is called LEUKOCYTOSIS. Leukocytes are classified into two groups based on the presence of specific granules. Specific cytoplasmic granules are those that are seen in a given cell type after staining. They are specific for that cell type. The cells are the following: 1.Granulocytes - These are leukocytes with specific granules. They are com- posed of the neutrophil, eosinophil, and basophil leukocytes. * Perhaps you should not look for the reticulocyte in your class slides because of possible confusion when you find some rb that are basophilic because of deficiencies in the staining procedure.34 X.2 2. Agranulocytes - These are the leukocytes with no specific granules. ‘They may show some cytoplasmic granules but these are inconstant features of the Particular cell. They are composed of the lymphocytes and monocyte BLOOD PLATELETS fhese are tiny cytoplasmic fragments of a giant cell in the bone narrow. Theix mumber is extrenely variable, which {s probably why various suthoce Give widely differing figues. some give 150,000 per cubic millimeter es che aoner neznal; 400,000 to 500,000 per cubic millimeter is given as the nighor Boral. They are biconvex in shape and show a round or ovoid outline whan viewed flat, and spindle or rod shaped when seen in profile.Thetrdiensten sae, chon 210M Of one micrometer to a high of four micrometers. In blood nised. g eeatend te aggregate in small clumps. Two parts can be easily recog aizgt 2 Contzal thicker chromomere which cokntains small azurophilie gran- wiles, and a peripheral clear and light blue hyalomere. EXERCISE: Fresh blood smear. CAUTION: DO NOT USE THE OIL IMMERSION LENS WHEN STUDYING FRESH WET MATERIAL. Study the shape and color of the rbc in the fresh wet preparation. Look for rouleaux formation and any marked variation in size and shape. As water oe nro the specimen, observe its effect on the cell. How would you explain this phenomenon ? What is it called ? tiomoedtes may be seen as small rofractile elements in the fresh prep- seis aasprneiz visibility may be improved by reducing the aperture oF tne ixis diaphragm of your microscope. EXERCISE: Dry blood smear stained with Wright's Stain. hep locating the formed elements of the blood, it is advantageous to look SUust at @ wider field through the use of the low power objective, wren you and centan ize that you would want to study, shift to the high-dzy objective Gnd Center the object in the microscopic field. In a slide with a cove slip, seed esse othe, Bish Power objective sharply on the structure. In the ontop BF thie dona eousing with the high-ary objective is unsatisfactory so the aos acucnamine the red blood cells as to size, shape and staining. since you will the ae eetamining your own blood, you could start on the assumption chee the specimen is normal. If this were the case, erythrocytes lined edge to Sage, actoss the of] immersion field should number about 25 to 27 (server the firm that in general the shape is a xound biconcave dise, Whe, viewing the se eaauthe flat position, the boundary of the cell will tell you shecer ct SS Tound of not. Could you tell if the cell is biconcave ? If yea, Nery it no, why ? Hook at the staining of the erythrocytes. Since they are acidophilic, they Should be pinkish red. Be on the alert for some erythrecytes thac may have a basophilic (bluish) tinge. If the color variation is net an artifact, what beoooniai, rithtocyte be 7 If you see many erythrocytes with suspicions besophilia among the normally acidophilic ones, call the attention ae your Professor.35 2. Examine the leukocytes for the purpose of identifying the five types. Table 2 will help you in this. After you have mastered the identification of the different leukocytes, make a DIFFERENTIAL LEUKOCYTE COUNT by count- ing 200 of these cells. To avoid counting a cell more than once, the Field Meandering Method of scanning the blood smear may be used as a methodical way of accomplishing the count (see Figure 5). Report your findings to your professor. FIGURE 5. FIELD MEANDERING METHOD - The arrows indicate the movement of the slide when examining contiguous microscopic fields under the oil immersion lens. The circles indicate the microscopic fields to be scanned. Contiguous fields should not overlap. 3. Look for blood platelets during your identification of leukocyt Since these are often in clumps, you can easily see them under the low power objective. Identify the parts of the platelet under the oil immers- ion lens.TABLE 2: The adult leukocytes seen in dry smears and stained with Wright's stain. : GRANULOCYTE S AGRANULOCYES + NEUTROPHIL a EOSINOPHIL " BASOPHIL f LYMPHOCYTE MoNocyTE c Very smell relatively un- Red, round to oval.retrac-Blue to dark violet.Vari- Occasional, non-specific, Occasional non-specificy yy GRANULES stained specific granules tile,larcer than neutro- able nunber & staining azure, azure "dust". may give a pinkish haze phil secondary granules, in a cell. Round to engu- T in the cytoplase, Generally unitora stain- lor shope,variable size 6 Secondary granules ere inf of all granules. in a coll - larger ones lerger, fewer & reddish bigger than eosinophil Pp purple or azure in color granules; small ones are L majority, are intersedi- ‘ate botwoon eosinophil & A neutrophil secondary s granules. M coLoR Colorless to faint pink. Colorless Colorless to ton Blue, clear, may be pale Greyish blue blue VACUOLES Occasional 8 Usually 2-3 but may be Usually Bilobed Usually Uor J shaped, Often round.A slight ‘Oveid or kidney shoped, N SHAPE v as many as 6 lobes con rarely bilobed slight- indentation occasionally or horse shoe shaped. ‘ nected by thin strands. ly. Often obscure Rarely round L £_NucuoLus "None" "None" "None" Invisible Occasional u ‘i en Goorse clusps.violet to Goerse clumps, smaller Finer clumps than in Coarse cluaps,closely Finer clumps; lighter violet purplish red. than in neutrophil,Coler neutrophil & eosinophil. packed, smudged eppear- compared to lysphocyte's. tends to be bluer, Often pale staining. ance. Dark violet. Often has "raked" appearance aL NER 9-12 10-14 8-10 6 = 8 (small) 12 - 15. May be as large os (nicrosstera) Tends to be slightly Tends to be slightly 10 12 (mediua! 20 when stretched. _ ‘smaller than neutrophil, smaller than neutrophil, 12- 15 (large, usually fandin tissues only. PER CENT OF 50-75 5 1-45 05-15 20 - 35 5; Sone claim 3-88 TOTAL COUNT that 45 § 1s high normal. x37 XI, BONE MARROW ‘Two preparations are available for the stuly of the hone maxrow: 1, Slide 14, Bone marrow section, H & E stain 2. Bone marrow smear, Wright's stain Bone Marrow Section (Slide 14) You may be able to see parts of the bone maxxow framework plus remnants of bony spicules, and the more abundant developing and mature blood cells. The following elements may be considered as constitutingthe bone marzow frame- work: venous sinuses, reticular cells, fat cells, and reticular fibers. The venous sinuses and fat cells axe easiest to see. It is quite difficult to locate the xeticular cell. The reticular fibers cannot be seen in this prepazation. The venous sinus is a type of capillary with a wide lumen. It usually contains mostly matuxe blood cells. If you can see the wall of the vessel, this most prob- ably would be part of its lining endothelium. The nucleus of the endothelial cell should bulge towards the lumen of the vessel. The reticular cell is mainly a flat cell that is closely applied to the outer surface of the vessel. Its nucleus should bulge away from the vessel wall. Fat cells are usually close to the vessels. You will usually see only the more Prominent part of the cells containing the large fat vacuole. This would be a Jaxge circular unstained structure with a thin wall which is additionally demarc- ated by the surrounding blood cells. The predominating elements in the microscopic field are the abundant developing and matuxe blood cells that are located between the meshes of the bone marrow framework. The megakaryocyte is usually easier to locate in this preparation than in the bone marrow smear. Bone Marrow Smear (Unnumbered ) This is an aspirate of the free cells and fluid of the marrow which is spread on a glass slide, dried, and then stained. The elements of the marrow framework axe, therefore, not included in this preparation. ‘This preparation is easier to examine under the oil immersion lens than the bone marrow section. Consequently the finer cytological details that axe visible under the optical microscope may be easier to find here. ‘The following two tables, Table 3, the Erythrocyte Series, and Table 4, the Neutrophil Series, xespectively compare the stages in the development. of the erythrocyte and the neutrophil leukocyte through differential features that can be seen under the optical microscope. In general, developmental features of the eosinophil and basophil leukocytes axe similar to those of the neutrophil series. The features in their development that axe different axe described in separate paragraphs. Development. of the megakaryocyte, and agranulocytes axe also described in separate paragraphs.“a xI.2 SUGGESTIONS: ‘The student who is about to embark on a study of the various cell types in the marxow may be overwhelmed by the variety of cellular characteristics. It might help make your study easier if you note the following: 1, Identify only those cells that axe well presexved (intact) and stained. 2. Look for the cellular representative of each of the variuos stages of a cell series befoxe going to the next series. However, if you encounter a beautifully stained cell that obviously does not belong to the sezies you axe currently studying, tzy to identify and study that cell also so that, you may save time when you get to the series where it belongs. 3.bymphocytes constitute abouta fourth of nucleated marrow cells. In the exythro~ cyte series the developing and mature cells constitute also about a fouzth of the marrow population. The lymphocytes will always be moze than the total numbex of erythroblasts. 4. The number of cell of any series is always lowest at the earliest stage and increases progressively with each older stage. Development of the Eosinophil Leukocy Stages in the development. of the eosinophil leukocyte axe similar to those of the neutrophil with the following exceptions: Myelocyte Stage - They are less in number. The pexipheral clumps of heterochroma- tin show a coarser pattern. The specific granules are largex, easiex to stain, and are eosinophilic. Metamyelocyte Stage - No band forms develop. Nuclear lobations axe less, often only two in number. Heterochromatin is less compact. resulting in a less intense basophilia. Development. of the Basophil Leukocyte Stages in the development. of the basophil leukocyte are similar to those of the neutrophil with the following exceptions: Myelocyte Stage - Very much less in numbex than the other myelocytes. The nucleus is paler staining than the other myelocytes. Theze are relatively fewer specific granules which are also variable in siz Metamyelocyte Stage - The nucleus is usually mexely deeply indented with no distinct lobation, and with only occasional slight constzictions of not moxe than three. Thus it is difficult to differentiate this stage from the mature form. Theze axe no band forms. The developmental stages of this leukocyte is axticularly difficult to find because there are very few of this cell type and the specific granules are hard to presezve. Development. of the Megakaxyocyte ‘The MEGAKARYOBLAST is the youngest stage that may be identified in the cell line. This is a large cell, much laxger than any of the biggest cells in the other series. Ite cytoplasm is basophilic and homogeneous. Its nucleus may be round or oval and often indented. There may be two nuclei ox a large lobated nucleus. Nuclear heterochromatin is generally loosely arxanged but may appeax denser in the periphery. ‘The PROMEGAKARYOCYTE is larger than the preceding stage (30-45 micrometers). Its cytoplasm is less basophilic than the previous stage. The bigger cells of this stage show an even paler cytoplasm. You will encounter more cells with lobated nucleus in this stage. There are azurophilic cytoplasmic gxanules esp-39 xI.3 ecially towards the center of the cell. Nuclear heterochromatin is more dense than in the previous stag: ‘The MEGAKARYOCYTE is even larger than the previous stage (50-100 micrometers). During the initial part of this stage, the cytoplasm is similar to that of the promegakaryocyte, showing dispersed azurophil granules near the central part of the cell with the peripheral cytoplasm usually being free from them. In the lat part of this stage, the cytoplasm shows a patchy basophilia in which the azuro- phil granules are gathered in small groups each surrounded by a clear zone of cytoplasm. The cell border at this time has become indistinct or fuzzy. The nucleus is highly multilobed. The lobes may be clumped together, or in some parts, some of the lobes may be separated from the main mass revealing a slender nuclear stalk" connecting each one to the main mass of the nucleus. The heterochromatin is much denser and more clumped than in the preceding stage. Development of the Monocyte Stages in the development of the monocyte is difficult to identify in the bone marrow because there are very few cells of this line. Furthermore, the MONOBLAST is difficult to differentiate from the myeloblast. The PROMONOCYTE develops from the monoblast. It is about 15 micrometers in diameter which makes it slightly bigger than the average monocyte. Its nucleus varies from roundish to oval to indented. It show fine chromatin which are often dispersed in arrangement. There are usually two or more nucleoli. The basophilic cytoplasm often contains a few azurophilic granules nearer the center of the cell than in its periphery. ‘The average monocyte is smaller than the promonocyte (about 12 micrometers). ‘The shape of its nucleus is similar to the preceding stage. Its nucleoli have become indistinct and relatively smaller than in the preceding stage. Its cyto- plasm, which is less basophilic than than in the promonocyte, has acquired a greyish-blue cast and azurophilic granules usually are greater in number. Development ofthe Lymphocyte ‘The lymphoblast is a relatively large cell (about 15 micrometers). It has a large nucleus with prominent nucleoli. The cytoplasm is usually homogeneous and basophilic. It is practically impossible to distinguish it from the hemocytoblast. Further differentiation of the lymphoblast gives rise to a smaller cell, the PROLYMPHOCYTE. This is slightly larger than the mature lymphocyte. The cytoplasm is usually a thin rim around the nucleus and is more basphilic than in the previ- ous stage. In addition, azurophil granules are often seen in the cytoplasm. The nucleus shows fine heterochromatin which is more dense and compact than in the previous stage. There are quite a number of this stage in the marrow (about 20 per cent of the marrow lymphocytes). However, in many instances the nucleus shows clumping of chromatin due to mitosTABLE 3: THE ERYTHROCYTE SERIES (Bone Marrow Smear, Wright's Stain) 1 1 1 BASOPHILIC | POLYCHROMATOPHIL | ]POLYCHROMATOPHIL [__NemmcrtosuasT | PROERYTHROBLAST | eryrmoatast | ervmumoptast | MORPBLST | erviaocrre | dteseter 1 10-15 | 2-20 ! 10 \seatter than preceding |smalier than pre- |Saaiter than pre- Icatcroneter) | i I i | I stoge coding stagoe c I [Thin rim to moderate |Thin rim to moder | 5 1 1 © 1 setative [2nount eround nucleus Janount: relatively | yi 1 Gia oe \ ee | Ioreater than preced- | 1 i \ co L Ling stage * 1 i L L et t 1 1 ["waddy® blue thru gray [Reddish orange with | P| color | Pete blue | Medium biue | Deep biue Ito reddish violet [faint bluish tinge. | a! 1 | | | Iaimost sinter to | | 1 | i \ Inoraccyte.Slight | i L L L 1 Iaray areas rare | | Nuclear [Indistinct, delicate, | [Thicker than |Thicker than |Hard to see; nucleus| [*nembrane" [thin |__ distinct [preceding stage lpreceding stage [is usually pyknotic | 1 |Fine stipple or net. |Fine stipple or net, | |More condensed & more | 1 | Wotero- [There may be small or uniformly dispersed| Coarse network, [clumps than preceding | Coarse blocks: | oe [chromatin |condensed areas at |More distinct than | Some clumps |stage."Cartwheel" or | densely packed 1 ° 1 |peripnery [preceding stage 1 | "checkerboard" pattern | 1 L | 1 1. nay be seen 1 L Tipore- 1 I [Distinct, mainly oie | T 1 spereins [Distinets continuous [Distinct: less Icontinuous. May have |Indistinct or blurred | Blurred or absent | I i Jcontinuity [pinkish tinge L 1 i I t 1-2 1 1-4 1 1 i 1 | Nucleolus | [Blue & hyaline with | Usually not seen | None: | None 1 | I I 1 i eT indistinct border | + This is @ relative incresse because of the faster decrease in nuclear volume. vmxTABLE 4: THE NEUTROPHIL SERIES (Gone Merrow Smear, Wright's Stain) T T | WELOBLAST T T T BAND FOR MOCrTOBLAST PROMYELOCYTE ocrre -AUYELOCYTE L = AST |__ueuxoat ast \ u 1 wre herein Late Me onyelocyte T | | [Bigger than preceding | 1 1 cnet el 10-17 ‘stage, up to 24 micra, 10 (ero indistinct [size decreeses with each | Smaller than preceding| Initosis Approaching adult size sass atin ee a a lpetetive [Thin rim to moderate |Similar to preceding |More than preceding |More than preceding | More than preced- | More than preced- © I poune lanount around the |stage, but slightly | stage * Istage * | ing stage * | stage * y | Jnucieus more * 1 1 1 1 ¥ | | [Deeper blue(similer to |Varies from less to sore |Less blue than preced- |Less blue than | Faint lilac to 0 | Color | Pale blue [thet of mature small blue than preceding stegal ing stage. Some lilac | preceding stage. | colorless ep | 1 |1ymphocyte) or tan Joften with localize 1 |Some Lilec | cL 1 1 |pinkish areas 1 1 | al 1 1 |More azurophil. Some [Decreasing number of |Fewer ezurophil, |Few azurophil, many S| Granules |Occasicnal azurophil |Qccasional ezurophil Ispecific granules may —_|ezurophil, Increasing |more specific gran-|specitic granules wl 1 1 [be seen [specific granules Jules. CLOSE TO ADULT NO. OF GRANULES. 1 |Lerge, round to oval; |Large, round to oval ]Round to oval,occasional-|Ovoid, becoming irregu- ‘shaped; |"C* or "U" shaped: N | Shape |*undifferentiated” — | ly with slight indentation| er with each mitosis [deeply indented |unifors thickness of = wi 1 L i 1 1 Lisbs Tai Tart ee Tne | wasnt | ities ei i I Fine stipple or net, |Meinly granular,arr- \Granular, often arrenged | More dense & compact; 1 | | Hetero- |There may be small —_|anged in delicate [Like @ sieve; later be- | increased amount; |coarse, dark stain-|Mature configuration € [chromatin condensed areas at [pattern or like & Icoming denser around nuc-| coarser clumps: ling clumps 1 out Iperiphory |sieves Some clumps Iteolus & at periphery. | | | L 1 |More clumps 1 i i s | Distinet; continuous: | Distinct; continuous peas but discontin~ ! Less distinct | Uses distinct ! Less distinct Tweleolus | 1-2 | 1 oF more [Pale blue, becoming in- | 1 1 I | Blue & hyaline | Pale blue conspicuous in later part| Not seen | Not seen | Not seen 1 1 1 lof this stage 1 1 1 = This 1s a relative incresse because of faster decrease in nuclear volune. oxXIT.1 ka MII. THE SKIN The skin is the largest. organ of the body. Among its many functions are protection of the underlying tissues, heat. regulation, sensation, and express ion. In addition, it participates in the control of fluid and salt balance of the body. Many of the structural characteristics of its various components suggest the functions ascribed to it. The student should endeavor to cor- relate the degree of development of the skin, including size and frequency of occurrence of its various components with the region where they are found. The skin has two general layers, (1) the EPIDERMIS - a stratified squan- ous keratinizing type of epithelium, and (2) the DERMIS (corium, dermis, cutis vera) - situated beneath the preceding layer, and composed of dense irregular connective tissue. In a section perpendicular to the surface of the skin, the epidermis shows downward projections (epidermal ridges, former- ly called rete pegs) into the dermis. The dermis sends upward projections into the epidermis (dermal papillae). Many of the elements of the dermis continue into the underlying hypodermis (subcutaneous tissue). There is no clear demarcation between these two layers. In general, the hypodermis has many adipocytes and less connective tissue fibers compared to the dermis. However, these characteristics vary from region to region, i.e., being frankly loose in character in some areas, and being suggestive of dense connective tissue in another. SLIDE 56a, -b, -£, & g, SKIN: The following procedure may help the student to identify the various componenets of the skin, and verify the structural char- acteristics described in the textbook: 1, Identify the epidermis and dermis. Examine the junction between the two and verify the presence of the epidermal ridges and dermal papillae. 2. Identify the layers of the epidermis. Specimens of thick skin will show the maximum number of layers. 3. Confirm that the dermis is a dense irregular connective tissue. Identify the papillary and reticular layers of the dermis. The papillary layer is that portion where you will find the dermal papillae. The boundary between these two sublayers is located slightly lower than the crest or tip of the epidermal ridges. Note that the fibrous elements in the papillary layer are finer and seemingly sparser than in the reticular layer. 4, Identify the hypodermis. Note the presence of many adipocytes. Note also that the fibrous elements are sparser - fitting the general description of this part as a loose type of connective tissue. 5. Find out whether a particular specimen has hair follicles. If present, determine whether they are plentiful or sparse, and whether they are large or small. Look for evidence that the hair follicle is an evagination of the epidermis. Locate the tip (base) of the hair follicle, whether it is in the dermis ox hypodermis. Correlate this with the size of the follicle. Observe the oblique position of the hair follicle relative to the surface of the skin.aS XII.2 Identify the hair shaft including its root and matrix. 6. Look for ARRECTOR PILI muscles. Note their position relative to the hair follicle to which they are attached. 7. Look for sebaceous glands. Note their position relative to the hair follicle. Try to locate the junction of its duct with the hair follicle. 8. Look for sweat glands. Distinguish between the secretory portion and the duct as far as cytological appearance of the wall, location and frequency of their sections, and general direction or orientation of the tubules. 9. Two sensory nerve endings are easily identified in the H & E preparation. ‘These are Meissner's corpuscle located in the dexmal papilla, and the corpuscle of Vater-Pacini (Pacinian corpuscle) in the deeper portion of the dermis, sometimes very close to the hypodermis. ‘The Meissner's corpuscle is often ovate in appearance with its longitudinal axis perpendicular to the surface of the skin. It often looks transversely striated with some flattened nuclei also oriented transversely. ‘The Pacinian corpuscle is a large globular structure and appears circum- ferentially striated with scattered nuclei oriented in the same manner. 10.Sections of nerves are also seen in the dermis. The bigger ones are found in the basal part of the reticular layer, while the smallest ones are seen in the papillary layer. 11.Varying sizes of small vessels are seen in the dermis. The larger art- erioles and venules are found in the basal part of the reticular layer, while the smallest ones are seen more in the papillary layer. Capillaries are found throughout the dermis but are easier to locate in the superficial part of the dermis. 12.Finally, compare the appearance of the skin in the slides listed above. Note the structures that you did not see in a given specimen. Note also variations in size and frequency of occurrence of those that you found. Reconcile your findings with the descriptions in your textbook regarding the characteristics of the skin in the various regions of the body.Ah XIIT.1 XIII. THE RESPIRATORY SYSTEM ‘The respiratory system consists of the nose, pharynx, larynx, trachea, bronchi. bronchioles, alveolar ducts, and alveoli. Bach lung includes intrapulmonary bronchi, bronchioles, alveolar ducts and alveoli, nerves and blood vessels with their associated connective tissues, and the visceral pleura. ‘The system may be divided into two parts, namely, (1) the conducting portien, concerned principally with the conveyeance of air to and from the second portion, including the additional functions of warming and humidify- ing the air, its filtration, and elimination of trapped materials out. of the system through ciliary action, and (2) the respiratory portion, con- cerned with the exchange of gases between alveolar air and the blood, and the phagocytosis of particulate matter that has been inhaled or has origin- ated from the tissue itself. ‘The conducting portion is composed of the nasal passages, the nasopharynx, larynx, and tracheobronchial tree as far as the terminal bronchioles. The respiratory portion is composed of the respiratory bronchioles, alveolar ducts, and alveoli. Actual exchange of gases occurs at the level of the interalveolar septa. Each respiratory bronchiole together with the alveolar ducts branching from it, and the alveoli arising directly from it or from the alveolar ducts constitute a PULMONARY LOBULE. ‘The epithelium of the respiratory tract is generally of two basic type: namely, (1) a pseudostratified columnar ciliated epithelium with goblet cell: which lines the conducting portion in general, and sometimes referred to as a "respiratory type of epithelium", and (2) a simple squamous epithelium which lines the areas where actual exchange of gases occur. There is a gradual epithelial transition in the conducting portion respiratory portion is approached. The epithelium becomes progressively thinner, becoming simple columnar ciliated, then low cuboidal ciliated, and finally flattening out into the simple squamous epithelium lining the aly olar ducts and alveoli. In certain areas, a stratified squamous epithelium may be found instead of the pseudostratified epithelium. Goblet cells in the epithelium and small mixed serous and mucous glands in the supporting connect~ ive tissue keep the epithelium moist. The goblet cells are always found in the pseudostratified epithelium and also in the parts lined by simple colun- nar and cuboidal epithelium so long as the epithelium is still ciliated. Goblet cells become scanty and finally disappear before the cilia are gone. ‘The bigger glands, either mixed or purely mucous, are found only in the lamina propria of either the pseudostratified or the stratified epithelium. Initially the lamina propria is well developed in the conducting portion but becomes thinner as the diameter of the passageway decreases and the epithelium thins out. In the nasal cavity, particularly in the nasal conchae, the lamina propria is extremely vascular and contains plenty of venules. The lamina propria is fibroelastic in nature; the elastic elements are condensed into a prominent layer in the pharynx, trachea, and extrapulmonary bronchi. This elastic layer can be said to separate the lamina propria from the submucosa in the lateral portions of the nasopharynx, in the part of the pharynx that is directly con- tinuous with the esophagus, in the trachea, and extrapulmonary bronchi. In theAS XIII.2 other regions, this layer is not very prominent and is directly continuous with the connective tissue supporting the underlying structures of muscle, bone, or cartilagi In general, there is a rigid structure (cartilage, bone, or fibroelastic connective tissue) that supports the wall of the conducting portion. Thes are absent in the respiratory portion. In the bronchioles, elastic fibers form an interlacing network around the tube and literally anchor the wall to the surrounding connective tissue of the respiratory portion. ‘The hilus of each lung receives a primary bronchus together with accompany- ing blood vessels, nerves, lymphatics, and associated connective tissue. Immediately upon reaching the hilus, the primary bronchus divides into second- ary bronchi, the number of divisions usually being three on the right and two on the left, corresponding to the respective number of lobes in the right and left lungs. Each secondary bronchus divides dichotomously to form tertiary bronchi. Regardless of the position of the bronchus in the system of branchings, we speak of extra- and intrapulmonary bronchi when we refer to those that are outside or inside the lung, respectively. Usually, the intrapulmonary portion begins in the terminal part of the primary bronchus. The C-shaped cartilage of the extrapulmonary bronchus becomes an irregular cartilage plate in the intrapulmonary portion. This may encircle the entire circumference of the bronchus in the form of a spiral. These cartilage plates become smaller as the diameter of the bronchus decreases. The smooth muscle fibers that are prim- arily located only in the interval between the ends of the cartilage in the extrapulmonary bronchus become distributed around the circumference of the intrapulmonary bronchus. The term "bronchiole" is used in this manual to mean that segment of the con- ducting portion that is devoid of cartilage. In its terminal portion, glands and goblet cells are absent and its epithelium has changed into a simple cil- jated columnar or cuboidal type. The bronchiole that gives rise directly to alveoli along its wall and to alveolar ducts from its distal end is called a respiratory bronchiole. It is lined by simple low cuboidal epithelium which is ciliated proximally and becomes non-ciliated distally. ‘The alveolar duct is a long passageway with a wall that is closely studded by alveoli and alveolar sacs. In longitudinal sections, the duct appears as an elongated space with a wall that is defined by the "knobbed" ends of sections of the interalveolar septa. The "knobs" are encountered in this location only and are not met in the portions showing alveoli that are not directly given off from the wall of the alveolar duct. These "knobs" are cross sections of smooth muscle and elastic fibers that encircle the opening of the alveoli and alveolar sacs directly arising from the duct. Muscle fibers are absent in structures beyond the alveolar duct. Hence sections of alveoli that are not. directly connected to an alveolar duct will not show knobbed ends in their interalveolar septa. Study the following slides of the respiratory sytem: SLIDE 37, OLFACTORY EPITHELIUM - Look for the epithelium and determine its type. Note that there are no goblet cells. Identify the three cellular compo- nents of the epithelium, namely, the supporting cell, olfactory cell, and basal cell. The supporting cells are tall and slender. Its nucleus is elong- ated or oval with its long axis perpendicular to the plane of the epithelium. ‘The nuclei usually occupy the highest level compared to those of the other cell components. The olfactory cell is also a tall cell. Its nucleus is usual- ly round and dark staining, with a prominent nucleolus. Although the nuclei occur at various levels, they are generally found between the level of the nuclei of the supporting cells and those of the basal cells. The axon and46 xIII.3 dendrite of this cell is not well demonstrated in the class slide. The basal cells are small and are truncated in shape. They are at the basal part of the epithelium. Their round nuclei form the lowest level of nuclei in the epith- elium. Nucleoli can also be seen but note that the basal cell nuclei appear the paiest staining compared to those of the other two cell typ identify the following features of the lamina propria: 1. There are many lymphoid cells. 2. It is very vascular - showing sections of capillaries in the superficial portions, and many venules in the middle and deep part. There are also a number of arterioles. 3. Lymphatic capillaries are seen especially near the deeper part. 4. Nerve bundles mainly belonging to the fila olfactoria, are numerous. 5. The serous secreting glands (glands of Bowman) are branched tubuloacinous structures which are distributed mainly in the superficial part of the lamina propria. Note that their secretory portions are mainly disposed horizontally whicle the ductal segments are vertical to the plane of the epithelium. There is a tendency for the terminal portion of the ducts to have a wide lumen. The cell containing pigment are often hard to demonstrate in the class slide. SLIDE 36a, EPIGLOTTIS - This is a longitudinal section of the entire thick- ness of the tissue. Identify the elastic cartilage at the core of the tissue. Examine the epithelial covering of the surfaces of the epiglottis. The strat- ified squamous epithelium covering one surface, from the base to the tip, and around the tip of the epiglottis should be easy to identify. at the other side, you will see a transition from stratified squamous to pseudo- stratified columar epithelium. The side covered by pseudostratified epith- elium is the posterior surface of the epiglottis. Examine the supporting jamina propria which should contain many lymphocytes and small blood vessel: You will usually see mixed mucous-serous glands in the deeper portions of the lamina propria close to the cartilage. There are usually more glands toward the base of the epiglottis than at the tip. SLIDE 36b, LARYNX - Confirm the presence of the true and false vocal cords in your class slide. Both of these structures are medial shelf-like project- ions with a recess (laryngeal ventricle) separating them. These are lined by stratified squamous epithelium. The supporting lamina propria has mixed mucous and serous secreting glands which are more abundant at the regions of the ventricular fold, laryngeal ventricle, and in those areas covered by pseudo- stratified epithelium. ‘The submucosa, which is loose connective tissue, is poorly demarcated from the lamina propria, but you will notice that there is a change in texture here, i.e., it has become less cellula while the connective tissue fibers have become more prominent. Deep to the submucosa are skeletal muscle fibers, usually cut in cross: section. These are fibers of the vocalis muscle. The hyaline cartilage found jateral to or close to the muscle is part of the thyroid cartilage. SLIDE 39, TRACHEA - Scan your slide using the LP objective. Identify the *c* shaped hyaline cartilage and the lumenal surface of the trachea which is cov- ered by respiratory epithelium.47 xIII.4 Confirm the presence of respiratory epithelium in the tracheal mucosa. Observe that there are many goblet cells in the epithelium. A prominent epi- thelial basement membrane can usually be seen. The supporting lamina propria has many elastic fibers which are basally "condensed" to form an elastic membrane in many areas. The submucosa is loose connective tissue. It contains adipocytes, and mixed mucous-serous secreting glands. ‘The perichondrium of the tracheal cartilage is fibroelastic in character. The trachealis muscle consists of smooth muscle fibers found mostly in the connective tissue continuation of the submucosa and fibroelastic compo- nents of the perichondrium at the gap between the ends of the cartilage. They are usually transversely oriented but some also run longitudinally and obliquely. SLIDE 40, LUNG - Locate the side of the tissue where the visceral pleura is attached. The pleura is fibroelastic in character with a covering of meso- thelium. This is where the base of the pulmonary lobule is situated. Examine the junction of the pleura with the parenchyma of the lung to look for ar where there are accumulations of connective tissue. These sites are usually the interlobular or intersegmental areas. Expect to find small pulmonary veins here. Next, scan the substance of the lung itself. You will notice that generally the tissue presents a lacework of pulmonary alveoli, alveolar sacs, and alveo- lar ducts. Scattered among these are islands or accumulations of connective tissue containing either a pulmonary vein, or a combination of bronchioles or intrapulmonary bronchi with pulmonary artery and bronchial arteries. The size of these connective tissue areas is related to the size of the pulmon- ary vein or bronchiole-pulmonary artery combination they contain. Next, Locate specific segments of the bronchial tree including the resp- iratory portions of the lung. Switch to the HP objective to identify and confirm the components of these segments. Examine the interalveolar septa and identify all that you can clearly see under the LP objective, e.g., the alveolar capillary, the alveolar macro- phages, and the interstitium between capillaries and its cellular components. ‘Try to identify the alveolar epithelium with the use of the HP objective. »xIV'y 48 XIV. BLOOD VASCULAR SYSTEM ‘he blood vascular system may be considered as a closed system of ves Sele composed of two circuits connected to each other through the heart. These aze the pulmonary and systenic circulations. The heart 1s the punp Ehat Propels blood through the various vessels of the system, aided by the Fecoil of elastic components of the wall of large arteries, and the con, traction of skeletal muscles particularly in the extremities. Tisted in consecutive order are the types of blood vessels through which blood passes, beginning from and ending in the hearts Heart (ventricle)- Elastic arteries (large size or conducting arteries) - Transitional arteries ~ Muscular arteries (medium qize or distributing arteries) - small arteries and/or Artarioles - Rapillaries* ~ Vonules and/or Small veins ~ Medium-size veins Large veins - Heart (atrium). Mieh the exception of the capillary which has only one basic layer in its wall, the other vessels, including the heart, are composed of thice gen- gia} Ravers which usually increase in thickness and complexity with thee increase in diameter. Since one basis for classifying blood vessels is the size or dianater Cr the structure, you should take this opportunity to estimate the sane fhrough the use of methods discussed at the beginning of this manual, CAPILLARY TCapillary" is the general term used to identify that part of the blood Vascular system which is the culmination of the system of branchings of the vessels. This is the part where the structural mechanism for materials and gasses between blood and tissue fluid resides, The various types of capillaries can hardly be differentiated from sr taser Under the LM beyond gross differences in size and uniformity Of tumenal shape in the case of “ordinary* capillaries versus sinusoid. consutt Your textbook while studying capillaries in the laboratory fo correlate the ultramicroscopic structure of these vessels with thelr appearance under the LM. "ORDINARY" CAPILLARY cee dade es mabtest type of vessel in the system. The EM can aisting- Tish two Kinds of ordinary capillaries, the CONTINUOUS and the FENESTaNte, pipes, No physical differences can be seen under the LM. The calibre of the jy'se2 Fanges from 8 to 12 micrometers, but in the vessel that 1s act active- t¥ conducting blood the diameter may ba as small as 7 micrometers. Shrinkage4g xIV.2 resulting from the processing of the tissue may contribute to the smaller measurement. The calibre of a capillary is more or less uniform throughout its length. You may verify this by noting that in the tissue you are exam- ining, the diameter of these vessels is generally identical. Note that in cross- and oblique sections of the capillary, the wall appears as a thin ring of uniform thickness. In some sections, a flattened nucleus may be seen. This nucleus has a tendency to bulge towards the lumen when the vessel is empty. This "ring" and nucleus may be interpreted simp- listically as the cytoplasm and nucleus, respectively, of the endothelial Lining cell. However, part of this structure is the basal lamina and asso- ciated reticular fibers that are not revealed in routine H & E sections. In a longitudinal section, you will see that the endothelial cell and its nucleus are elongated and aligned along the long axis of the vessel. A small cell closely associated with the abluminal surface of the endothelium may also be encountered. ‘This is the PERICYTE. Sometimes, only the nucleus of this cell may be seen and this may be mistaken for the endo- thelial cell nucleus. However, the pericyte nucleus will not bulge into the capillary lumen. Currently, "pericyte" is a name applied to several hard- to-identity cells closely associated with the capillary. ‘sInusorps Sinusoids have a relatively larger lumen than "ordinary" capillaries. ‘They are characterized by dilatations along their length making it difficult to categorically state their diameter. The EM has also shown that the sinu- soidal wall may be continuous or fenestrated, or may have both features. In one type there may be large intercellular gaps in the endothelium together with areas that have gaps in the basal lamina (discontinuous sinusoid). The lining cells of the sinusoid may be composed purely of endothelial cells ox may have both endothelial and phagocytic celle. Other cells may be associated with the endothelium, occupying the position of the “pericyte” as described previously in “ordinary” capillari: Note that unlike the "ordinary" capillary, the wall of the sinusoid follows closely the contour of the parenchymal elements in which it is located. The following slides are suggested for studying capillaries and sinusoids. Compare these vessels with each other and distinguish their dissimilarities in structure, ate. Contiquous Capillary: Skeletal Muscle, Slide 6a, -b; Cerebral Cortex, Slide 7a, Skin, Slide 56a to -g; Thymu by Slide 17 (study at cortical area) Fenestrated capillary: Kidney, Slide 41a, to-c (study peritubular capillaries & renal glomerulus) Choroid Plexus, Slide lla, & -b Intestinal Mucosa, Slide 3la, to -c (study at area of lamina. propria, and core of villus) Fenestrated Sinusoid: Hypophysis (anterior lobe), Slide 191, & -b; Adrenal Cortex, Slide 22; Pancreas (islet of Langerhans), Slide 36 Discontinuous Sinusoid: Liver (hepatic sinusoids), Slide 34a, to50 xIV.3 LARGER BLOOD VESSELS ‘There are two methods for classifying the larger vessels. The first method makes use of size, e.g., large-, medium-, and small-sized artery, and arteriole (which also means small artery). This method is also used for veins, thus, large-, medium-, and small-sized veins, and venules. ‘The second method takes into consideration the structural composition of the arterial wall but retains the terms used for classifying veins in the first method. Thus, we refer to an elastic type and a muscular type of artery, and arteriole. Other descriptive terms referring to the functional role of the larger arteries are also used in some instances, @.g., conduct- ing, and distributing arteries. While one could profitably and conveniently use the two methods of classification interchangeably, confusion could arise when terms are used without qualifying them. The most common source of misunderstanding is the term "small artery". The user of the term could be referring to the small type of artery with the characteristic structural components that differ- entiate it from the next type. However, the same person could be simply referring to the size regardless of the structure of the vessel wall. While he may have been talking about a small muscular type of artery, the listener could be interpreting his term to mean ‘arteriole* with its Amplied structural characteristics. It is suggested, therefore, that the student should use only the second method of classification as much as possible. The following Tables make use of this method. TRANSITIONAL ARTERIES ‘Transitional arteries occur in segments of the arterial tree where there is a transition of structural characteristics from the elastic to muscular type. Two types are generally recognized: 1. Mixed Type - Islands of smooth muscle interrupt elastic membranes in many places. Examples: external carotid, axillary, & common iliac arteries. 2. Hybrid Type - This is often found in places where a mixed type or elastic type abruptly gives rise to a muscular type with a markedly smaller diameter. ‘The media consists of an inner zone of smooth muscles and an outer zone of fenestrated elastic membranes. Examples: Coeliac & Mesenteric arteries at their point of origin. SPECIAL TYPES OF ARTERIES 1. Cerebral & Dural Arteries - These are thin walled, with a well developed elasticainterna but almost no elastic fibers in the media. The adventitia is poorly developed and mainly composed of collagenous fibers. 2. Pulmonary arteries - These are generally thin walled in comparison to other arteries of the same calibre elsewhere in the body. 3. Arteries of the Lower Limbs - These have more highly developed muscular tissue than those of the upper limbs. Note that the external iliac artery, which is a large artery, is muscular in type. 4. Penile and Pudic Arteries - Hyperplasia of the intima and media occur after puberty. The adventitia remains relatively thin. The intima is esp- ecially greatly thickened and has many longitudinal smooth muscle fibers in its outer part.5a xIVv.4 5. Umbilical Arteries - Elastica interna is indistinct and entirely missing in some places. The media has two muscle layers - an inner longitudinal, and an outer circular layer. True adventitia is lacking in the extra-abdominal portion and poorly developed in the intra-abdominal part. 6. Popliteal & Tibial Arteries - These are small arteries with elastic type of wall. Read also on the coronary artery. TABLE 5: Comparison of small arteries (arterioles) and smal} veins (venules). Note that in the nomenclature being used here the terms "arteriole" and “venule" refer to small-sized arteries and veins, respectively. ARTERIOLE VENULE Dinweter | Size Range: Dienoter | Size Range: (micro- (ntcro- geters) | 20-7 300 micrometers motors) | 20-7 2+000 micrometers 20 | Intima: 2 | Endothelial colls, basal lamina, a few = ditto reticular fibers. | Moai I A tew smooth muscle calls which may or = None = may not form 0 complete coat. | Adventitio ! Thin leyer of collagenous & reticular Longitudinally oriented collagenous fibers fibers, and sone c.t. cells. ond fibroblasts, 40 - 65 | Addition of olastica interna to intima: 45 | Partially differentiated esooth muscle sharply donarcates intima fron media fibers bogin to appear between endothol ive More collagenous, elastic & reticular and adventitia (beginning of a t. media), fibers in the adventitia, 1 100-200 | 5 - 4 Layers of smooth muscle in nodia, 200 | Continuous layer of smooth muscle fibers Advantitia 1s better denarcated from in media. modi 300 | Appearance of wall is similar to muscular | 2,000 | One of several leyers of smooth muscle artery, but lumen dianoter is often less then wall thicknoss, Tunica media 1s about equal to adventitia in thickness. colls in media, Sone fibroblasts, thin elastic end collagenous fibers in adventitia; fibers mostly longitudinally oriented, Lunen dissoter 1s always much greater then wall thickness.TABLE 6: sa XIV.5 Conparison of muscular (mediua-size) artery and medium sized voin, MUSCULAR ARTERY MEDIUM SIZED VEIN Intine: Endothelium - subondotholial elastic & collagenous fibrils & sone fibroblasts Elastica interne is well developed. Occasional smooth muscle calls, esp. at points of branching: lerger muscle bundles in larger arteries of this category, and in coronary artery. Mod = Thickost coat 25 = 40 layers of circularly orlented smooth muscle fibers Small anounts of fibroolastic connective tissue with some reticular fibers and fibroblasts, Elastic fibers increase in number & size with increase in size of artery, forming circunferenti- elly oriented networks In larger vessels and becoming fenestrated meabranes in the largest of this class. Advent: Mostly longitudinally oriented collagenous fibers. Increase in the nunber of elastic fibers which ere concentrated near the media forming coarse networks. Elastica oxtorna 1s soon in the bigger muscular arteries. Sono longitudinally oriented smooth muscle cells may be found in tho inner portion between elastic fibers. May have @ subendotheliel leyer of @ few elastic and collagenous fibers, It 1s denarcated occasionally from ‘the media by network of elastic fibers, Very thin Mainly circularly oriented smooth muscle fibers parated by longitudinal collagenous fibers end sone fibroblasts. Longitudinally oriented collagenous fiber bundl: and elastic networks. No elastica externa May have longitudinally oriented sooth muscle fibers In Inner portion close to media, Lunen diameter is greater then wall thickness Media is thicker than adventitia. (In the smaller varieties tho media aay be almost equal to the adventitie,) Lumen disseter is very much greater then well thickness, Media is thinner then adventitia. NOTE: The mein ertery supplying an organ is usually muscular in type and may reaein so through its main branches inside the orga, As @ general rule, the: voins draining the orgens are arteries aro still visible to the uneided eye. The so usually medium sized.53 XIV.6 TABLE 7; Comparison of elastic (large size) artery and large vein. ELASTIC ARTERY LARGE VEIN Intinas Endothelial cells are short & polygonal. Subondothel ial connective tissue consists of fine collagenous & elastic fibors & a fow fibroblasts, Mein part consists of coarser collagenous fibers, sone longitudinal smooth muscle fibers © many longitudinel elastic fiber: Internal elastic monbrane is celled INTERNAL FENESTRATED MEMBRANE, This Is often split into several seabranes, or it may fuse with similar fsonbranes of intina and media, Modto: Ribbons of concentrically arranged fenestrated ela: elastic monbranes anestonosing to form longitud~ inelly oriented networks. Fine elastic networks; circularly oriented smooth muscle cells betwoon fenestrated moxbranes; sone collagenous & reticuler fibers support smooth muscle colls, Adventiti Thin connective tissue composed of collagenous fibers with few elastic fibors, Occasional longitudinally oriented smooth muscle tiers. External Fenestrated Monbrana is not es distinct as internal fenestrated monbrano but clearly desercates adventitia from media, Cheracter is similar to medium sized veins. In lerger vessels connective tissue of intima may be very thick. Media is poorly developed & is similar to that of nodium sized vein. It 1s sometines absent. This layer makes up the majority of the wall. It is composed of loose connective tissue, mostly long- Atudinal collagenous fibers & thick elastic fibers. Prominont longitudinally oriented smooth muscle bundles & elastic networks adjacent to the sedi or if media is ebsent, to the intina. No equivalent In studying the different blood vessels, in addition to examining the structure of specitic vessels in the class slides, you should look at other tissues and organs and identity the type of blood vessels that you will encounter thers. In the larger blood vessels, look tor VASA VASORUM ‘and nerves in the adventitia. Skin, Slide 56a - Study the following slides: Kidney, Slide 41a - cy Spermatic Cord, Slide 53a, & torte, Slide 13a - cy -b, Testis, Slide 528 = c, andsh xIVv.7 THE HEART ‘The following are the three layers of the heart, with some comments on the components of each layer. Please note that the layers are presented consecutively. Endocardium 1. Endothelium - similar to those found in large arteris 2. Supporting fibro-elastic connective tissue - This contains nerves, blood vessels, and lymphatics. Occasional bundles of smooth muscles may also be found here. a, Subendothelial Jayer - a thin layer of fine collagenous and elastic Eibers. b. Main part - fibrilar elements are coarser in consistency. 3, Subendocardial connective tissue - This is a loose type of connective tissue. Tt is absent in papillary muscles and chorda tendinae. Tt contains Purkinje fibers, blood vessels, nerves, and lymphatics. Myocardium This is composed of layers of cardiac muscles separated by loose con- nective tissue containing blood vessels, nerves, and lymphatics. Its connect- ive tissue is continuous with those of the subendocardial layer and epicardium. Epicardium 1. Supporting fibro-elastic connective tissue - The deeper part of this layer has fat and is somewhat loose in consistency, particularly in the portion immediately adjacent to the atrial myocardium. This deeper part is called the subepicardial connective tissu 2. Mesothelium - This is a simple squamous epithelium which covers the surface of the heart that is facing the pericardial cavity. CARDIAC VALVES The atrioventricular and semilunar valves consist of a core or plate of dense connective tissue which is "chondroid" in character, and covered on both sides by endocardium. A concentration of elastic fibers is found beneath the endothelium lining the surface of the valves facing the lumen. The free border of the atrioventricular valves is reinforced and tethered by chorda tendinae to the papillary muscles. The chorda are attached to the ventricular surface of the connective tissue plate. The endocardium is thicker on the atrial side of the atrioventricular valv ‘The semilunar valves have no supporting tendons but a fibrous nodule (Noduli arantii) at the middle of the free border of each valve leaflet aids in strengthening these valves.55 xIV. 8 IMPULSE CONDUCTING SYSTEM This is composed of modified cardiac muscle fibers. It is sometimes called the Purkinje System of the heart. It has the following components: 1. Sinuateial Node - This is found in the subepicardial connective tissue at the junction of the superior vena cava and the right atrium, Tts funct~ fonal components are more correctly referred to as nodal fibers rather than Purkinje Eibexs. These form a compact network. The fibers are much smaller than ordinary cardiac muscle fibers and may be mistaken for fibroblasts because of their size and shape. In well prepared specimens, cross striat- ions can be seen. 2. Atrioventricular Node - This is located in the subendocaridal connective tissue, in the lower portion of the interatrial septum close to the opening of the coronary sinus. Its cells are similar in appearance to those of the sinuatrial node. 3. Atrioventricular Bundle (Bundle of His) - This starts from the anterior portion of the atrioventricular node in the subendocardium and enters the fibrous portion of the interventricular septum where it divides into right and left branches. Its component fibers are similar to those in the AV node at the beginning but become larger than the ordinary cardiac muscle fiber lower down. These are now the typical Purkinje fibers. 4. Purkinje Fibers in the subendocardium of the ventricular wall and interventricular septum are also found in the papillary muscle. The fibers in the ventricle are the ones usually encountered by the student in the class slides of the ventricle. In your study of the heart, identify its layers and sublayers. Try to establish the structural differences between atrium and ventricle. Identify Purkinje fibers by looking for cytological differences between ordinary cardiac muscles and Purkinje fibers. Study Slides 12a, to ~d.56 Xv, THE LYMPHATIC SYSTEM ‘The lymphatic system is composed of the lymphocytes, their precursors and derivatives, lymphatic vessels and the tissues and organs whose cell population is distinctly lymphoid in character. It is believed that the precursors of a1} lymphocytes come from the bone marrow and that these are processed elsewhere for maturation into two types of cells. The T lymphocytes are processed in the thymus. The B lymph- ocytes are processed in the tissues or organs where they lodge after migra- tion from the bone marrow. These types of lymphocytes cannot be distinguish ed from each other by light microscopy with present technics. The plasma cell, which you have studied before, is derived from the B cell. In lymphatic tissues and organs, there is a basic structural framework composed of a network of reticular fibers and reticular cells. The reticular fibers are closely attached to the processes of the reticular cells. Except in the thymus, there are apparently two categories of reticularcells in a lymphatic tissue or organ: one is a type of endothelial cell, or fibroblast, and the other is a macrophage. The lymphoid cells are found between the meshes of the network. Lymphatic tissues and organs are classified and recognized ‘on the basis of the degree and manner of aggregation of the lymphocytes, plus other distinctive structural features that identify them. In addition to the tendency of lymphocytes to infiltrate epithelia and connective tissue proper, especially loose connective tissue, you will find that in the lamina propria of organs, particularly in the digestive and respiratory tracts, there are areas where the intense accumulation of lymph- ocytes leaves little doubt that you are looking at a lymphoid area, i a lymphatic tissue. You will discern that there is a gradation in the concentration of lymphocytes in these lymphatic tissues. You will encounter in various text- books and literature on the subject the use of the following descriptive terms, namely, diffuse, loose, dense, and nodular lymphatic tissue. The use of these terms is for the purpose of identifying and classifying the various degrees of accumulation and/or arrangement of the lymphocyt Your textbook (Bloom & Fawcett, A Textbook of Histology, 11th Fd.) uses only "diffuse" and "nodular" to classify lymphatic tissues. It lumps toge- ther both loose and dense lymphocyte accumulations as “diffuse* lymphatic tissues and recognizes "nodular" lymphatic tissue as that dense accumulation of lymphocytes with a discrete globular shape and which stands out distinct- Jy even amid a relatively compact area of “lymphocytes or dense lymphatic tissue. At least one author uses the term "diffuse" lymphatic tissue in a different context, i.e., that it is a lymphatic tissue that does not have a capsule. To avoid confusion, and at least for the duration of your stay in this course inhistology, I suggest that you use the classification in your text- book unless instructed otherwise by the professor in charge of this topic. Familiarize yourself with the following terms that are used to identify certain configurations of lymphatic nodule:57 XV.2 1. Solitary Nodule, or Solitary Follicle - The Nodule is alone in the connective tissue or lymphatic tissue, or other lymphatic nodules are relatively far from it. 2. Aggregated Nodules or Aggregated Follicles - Several lymph nodules lie close to each other within diffuse lymphatic tissue. The classical example of this is the Peyer's patch which is found in the ileum. 3. Primary Nodule - This is a nodule in which the lymphocytes do not mark- edly vary in size and staining from each other, and are distributed more or less uniformly within the structure. 4. Germinal Center - This is a nodule in which the center is composed of Jarger and paler staining cells while the peripheral portion has smaller and darker staining cells. Other authors call this nodule a "secondary nodule" and would distinguish the pale center as the “germinal center* (or reaction center) and the darker periphery as the "corona". The lymphatic tissues that are definitively recognized as lymphatic organs are the lymph nodes, the spleen, and thymus. You will notice that there are certain relatively constant characteristics in these organs: 1. They have a connective tissue capsule which makes them a discrete structure. 2. The lymphatic nodules are usually located either in a given region ox structure within the organ. 3. There is a recognized pattern of arrangement of the lymphatic or blood vessels that subserve the organ's function. 4. The organs are predictably constant in location. Some authors would classify the lymphatic tonsils as non-encapsulated. Others would claim that it is partially encapsulated. Perhaps this is why some would call it an organ. The vermiform appendix is also considered by some as a lymphatic organ because of its obvious lymphoid nature despite its being a part of the digestive system developmentally. Examine slides of the intestinal tract to identify diffuse lymphatic tissue, the solitary nodule, and aggregated follicles. Try to find the primary nodule and the germinal center. For diffuse lymphatic tissue, solitary nodule, primary nodule, and germinal center, examine the lamina propria of the following: Esophagus, Slide 28a, or -by Duodenum, Slide 31a; Jejunum, Slide 31b; and Rectum, Slide 33a, -b, or -c. For aggregated follicles, examine the ileum and the vermiform appendix. The aggregated follicle is principally located in the lamina propria but its basal portion may be in the submucosa. You may also find here some examples of the germinal center. Ileum, Slide 31c; Vermiform Appendix, Slide 32. PALATINE TONSIL, Slide 18a & -b. The organ is covered by stratified squamous epithelium which invag- inates into the interior at many places. These are the tonsillar crypts. The way the tissue was sectioned may isolate these crypts from their con-58 ceeeecn with the surface epithelium. Finding these crypts is an important Structural feature that would help you identify the preparation acs lymph- atic tonsil. The epithelium usually overlies a thin connective tissee which sends "septa" into the parenchyma. wn, no, ase Of the tissue, usually opposite the side covered by epi- fhelium, you my find dense connective tissue suggestive of » capsule. Look EGE Ate oxtromitios to soo its extant. Note the presence of conmbeting tissue “septa* coming from the basal connective ti. he parenchyma consists of an intense accumulation of lymphoid cells {iiteuse lymphatic tissue) in which lymphatic nodules are masy and often die close to the crypts. You may easilyb find germinal centers among these nodules. There are no lymphatic sinuses in the lymphatic toneile LYMPH NODE, Slide 15 Next, look for the afferent lymphatic vessels in the capsule. the fon sant lymphatic vessel in the hilue is harder to find but try to took for one. Sgontsty the subcapsular (marginal) sinus beneath the capsule and its coneanuations around the trabeculae. These are relatively cleus avene tee xoreed by fine extensions of £ibroelastic connective tissue of the capsule cecal wbectiae into the parenchymal reticulun. The sinuses acound the vee reeeate Ae called trabecular sinuses. They are part of the co-oalicn “intermediate" or *cortical* sinuses which arise trom the subcapsular sinus, Redullary cre Cortex, and becone continuous with the medullary sinuses, the Redullary sinuses and the subcapsular sinus converge on the nilec dymphatic vessels which, in turn, empty into the efferent lymphatic vessels. 1ozng, the LP objective lens, identify the cortex and medulla. The cortex the eae et Periphery of the organ. It is "separated" trom the capeaie by fhe subcapsular sinus. The medulla occupies the inner pact of the organ. wee Cortex appears denser than the medulla. There are two zones in the fonee "sth outer cortex containing most of the lymphatic nodules, aad che Aner or deep cortex (also called paracortical aren) usually with no or few Beales and appearing less dense than the former. Note that there is ce sharp boundary between the outer and inner cortices. the medulla has many wide sinuses (medullary sinuses) so that the Bocauece ae etizsue is Limited to areas between then as the medullary corde, tease OF these sinuses, the medulla haa many spaces which imparts upon eee nzes a Lighter appearance compared to the cortex. Note that the ah sity of the medullary cords is similar to that of the deapt sorter seceit, $Y to Adentity the vascular supply of the organ by usiag your EP objective lens to identify the landmarks and isolate bleed vessels, then, Sxitching to the HP objective ens, to confirm tha type of bloc vessels. The Aargest arteries and veins of the organ are found in the Maat connect~ Ave tissue, These usually continue through the hilar trabeceles oat event-59 ually leave the connective tissue to enter the medullary cords. You may find capillaries in the cords. The smallest arteries are in the deep cortex. these send capillaries peripherally towards the outer cortex. These capilla- ries loop around the lymph nodules and retura to the deep cortex where they empty into postcapillary venules. These venules empty into small veins in the medullary cord. The veins in the cord enter the hilar trabeculae to join the trabecular veins. SPLEEN, Slide 16a, or -b The spleen is a large lymphatic organ that filters blood instead of lymph. Tts afferent and efferent vessels are blood vessels instead of lymph- atics, Its lymphatic vessels serve to drain tissue fluid from the connect ive tissues of the capsule and trabeculae, Under the LP objective lens, examine the entire section. Identify the dense fibroelastic capsule at one of the edges of the preparation. The cap- sule sends trabeculae into the interior of the organ. Since the spleen is a Jarge organ, a histological section of it will seldom include its hilus. However, ite hilar connective tissue continues deep into the interior of the organ as branching trabeculae which are usually stouter than those coming from the capsule. The hilar trabeculae bring with them the branches of the splenic artery and the tributaries of the splenic vein. Look for those trabeculae in your slides. They will appear as islands of dense connective tissue of varying sizes, usually carrying an artery and/or a vein. With the LP lens, examine the splenic parenchyma. In the H & E prep- aration, you will see that the parenchyma surrounds the trabeculae and con- sists of two parts. One part is predominantly basophilic because it is mainly composed of lymphoid cells arranged as diffuse and nodular lymphatic tissues. This is the white pulp. It is distributed in the parenchyma as discontinuous areas. The other part is mainly acidophilic because its pre: dominant cellular component is red blood cells. It occupies a larger area of the parenchyma and is more continuous in its distribution. With the LP lens, distinguish between the diffuse and nodular lymphatic tissue of the white pulp. Look for a smalJ artery that is surrounded by diffuse lymphatic tissue. This is the central artery or artery of the white pulp. Notice that inside a lymph nodule this artery is not central in location. With the LP lens, examine the red pulp. Distinguish between the venous sinuses (sinusoids) and the splenic cords (Billroth cords). The sinuses wil) usually appear lighter because they have less cellular content. Look for the lining of the sinuses. The cords are usually thinner than the sinuses and appear denser because they have a greater concentration of cells. Look for sma}J arterioles in the cords. These are the penicillar arteries (arteries of the red pulp). Look also for the sheathed arteries (capilla~ ries) and "ordinary" capillaries in the cords. These are the branches of the penicillar arteries. The sheathed arteries are difficult to find because they are not very prominent in human spleen. The sheathed artery has a lumen diameter that should be similar to a capilJary's. Its wall will appear to be thick because of the connective tissue sheath around it. This sheath has a concentric pattern around the vessel, and there are cells in it including macrophages and the cellular constituents of the blood.60 XxV.5 guymus, Slide 17 ‘The thymus is a lymphoid organ whose primary function is apparently to provide and environment for nurturing a type of lymphocyte which is later identified as the thymus-dependent lymphocyte, or T lymphocyte. ‘It reaches its greatest development towards the end of fetal life. It attains its max- imum size at puberty. It rapidly regresses after puberty so that in adult life it is sigaificantly reduced in size, its substance largely replaced by adipocytes. Blood vessels are the important avenues for the functioning of this organ. There are no afferent lymphatiucs that have been demonstrated. BEf ent lymphatics have been seen in its connective tissue septa. Appearance of the organ will greatly depend on the degree of involution that it has undergone. In the postpubertal thymus, the gland may be seen as isolated groups of lobules separated by wide areas of adipose connective tissue. In the younger thymus you will see the organ as a more structurally integrated whole. With the LP lens, scan the preparation to identify the loose connective tissue "capsule", with maay adipocytes, which should cover the periphery of the parenchymal tissue. Look for connective tissue septa projecting inwards from the capsule. Secondary septa may be seen branching from the primary pta.These'may produce sublobular areas. At this time also, look for blood vessels in the septa. These are usually smal} arteries and veins. Next, distinguish between the cortex and medulla. The cortex is peri- pheral while the medulla is central in location. The cortex is darker stain- ing, appears more basophilic, and is denser than the medulla. There are more lymphocytes concentrated in the cortex. In addition, the supporting reticu- Jar cells have thia cytoplasmic processes so that only their nuclei can usually be seen under the light microscope. ‘Themedulla appears less dense, and often you will see that it is acido- philic. This is due to the fact that the lymphocytes here are farther apart and the reticular cells are larger and have more cytoplasm that is acidophil- ic. It is the acidophilia of the reticular cell cytoplasm that imparts the xeddish coloration to the medulla in an H & E preparation. Look for the Hassal's body (thymic corpuscle) in the medulla. It is usually a round acidophilic structure, frequently with a concentrically lamellated texture. You may see nuclei within its layers but often the structure is merely hyaline in appearance. Hassal's bodies are smaller and fewer in number in the fetal thymus. They tend to be larger and more in num- ber in the involuting organ. A lymph nodule may be found in the medulla but this is very rare. This is usually observed only when there is a pathology which perhaps involves an autoimmune reaction. Try to look for blood vessels in the parenchymal areas of the organ. ‘The vessels supplying the cortex are capillaries and are hard to find because of their small size. At the corticomedullary junction and in themedulla, many Vessels are postcapillary venules which are larger than capillaries and presumably are easier to locate.XVI.1 64 XVI. THE DIGESTIVE s¥sTEM The digestive system consists of the digestive tract and large associated glands. The tract may be divided into the following consecutive regions: oral cavity and pigryme, esophagus, stomach, small intestine (composed of duodenum, Jejunun cod dajims the appendix, and the large intestine (composed of cecum. colon, aad vect= um). he large associated glands are composed of the large salivary glands (parotid, Randibular, and sublingual glands), the liver with the gall bladder, end che pan= Seeedea nee yempty their secretion into the tract through ducta. In addition, con ablee the Lee gimeestine to the liver through the hepatic portal venous system one ables the latter to process substances absorbed by the tract. Structures in the system that subserve its function of digestion, absorption, and elimination of indigestible matter, can be seen to a large eetent mider seein with Fouting scaning. Cells that have endocrine function are usually not recemeiced in ing oe yprebarations. Their identity can only be established through special seatn- ing or ultramicroscopy. ray ne fatty embryo, the digestive tract is a relatively straight tube with a fairly uniform structure. Elongation and differential development of the tube result able Tee eeecializations that render the various segnents of the tract dencieee fple, Tn spite of these regional characteristics, you can appreciate « taste pattern an the architecture of the tract. Learning this basic structure te a key to easily understanding the histology of the same. The tract 18 essentially a tube of varying diameters at various regions. Ite wall is Composed of four basic layers. Some layers have sublayers. Beginning from the lu- minal side, the consecutive layers of the tract are the following: 1+ Tunica micosa or micous membrane, composed of the following sublayer: a. Mucosal epithelium - usually simple columnar b. Lamina propria €- Muscularis mucosae ~ usually a smooth muscle layer 2+ Tunica submucosa, a connective tissue of varying density 3+ Tunica muscularis or muscularis externa, composed of an inner circular and an nective egitudinal muscle layer (usually smooth muscle) supported by loose cun- nective tissue Te eany Mesothelium, and blends with the surrounding connective tlacees® Tf this layer is covered by mesothelium it is called a Tunica eerene the following characteristic variances are encountered in specific regions of the tract: qhe Mucosal epithelium is stratified squanous instead of simple columnar. The mucosa has folds, ridges, and/or grooves. The mucosa has villi. qhe lamina propria is intensely infiltrated with lymphoid cells Plastic fibers instead of smooth muscle are found at the level of the muscularis mucosae. ftiseularis externa 19 composed of skeletal instead of smooth muscle. Nuscularis externa has one or three layers instead of two.XVE.2 62 ORAL CAVITY AND PHARYNX Slides available: 24a - Anterior tongue, H&E, Masson, HAC b ~ Posterior tongue, H&E c ~ Posterior tongue, masson and H&E There are no specimens in your slide box that shows the wall of the oral cavity and pharynx. However, the slide of the palatine tonsil (18a & b) may enable you to see some of the components of this wall. The mucosa of the oral cavity and most of the pharynx are lined by stratified squamous non-keratinizing epithelium, The Tongue ~ This is a muscular organ. You can confirm this in your slide by see- ing the abundant striated muscles located beneath the mucosa and oriented in vari- ous directions. Note that there is no submucosa in the dorsum of the tongue. You may see a submucosa if the inferior or ventral surface of the organ is included in the specimen. The anterior two-third of the body of the tongue (oral part) is demarcated from the posterior third (pharyngeal part) by a "V" shaped groove, the sulcus terminalis. The apex of the "V" points posteriorly. The foramen cecum is located immediately behind this. Slide 24a, Anterior tongue: Note that the dorsum is covered by numerous filiform papillae. Fungiform papi- llae are scattered among the former and are very much fewer in number. The filiform papilla is narrower than the fungiform papilla. Its base is wider than its tip. The epithelium over it is thicker than that on the fungiform papillae. The superifical part of the epithelium has a thicker layer of flattened cells with degenerated nuclei. An upward projection of the lamina propria forms the connective tissue core (primary papilla) of the filiform papilla. This core, in turn, sends pointed branches into the epithelium (secondary papillae). ‘The fungiform papilla has a broad somewhat flattened superior surface. Its lat- eral side narrows toward its base. There are fewer layers of partially keratinized cells at the upper layers of the epithelium compared to that of the filiform pa- pilla. Note that the connective tissue core has many blood vessels. The core also sends secondary papillae into the epithelium. Occasional taste buds will be found in the epithelium, usually in the areas associated with the secondary papillae. The taste bud will be spotted as a pale, vertically oriented oval structure occupying almost the entire height of the epithelium. Study the details about the components of the taste bud in your textbook. Anterior lingual glands are located in the anterior tongue in the deeper portion of the lamina propria and in the interstitial connective tissue between the muscle strands and bundles. Those located at the tip of the anterior portion are predon- inantly serous secreting in appearance. Those located more posteriorly are predom- inantly mucous secreting with serous demilunes at the ends of some secretory tubules. Be alert to the ducts among the secretory elements and in the lamina pro- pria. Correlate the size of the duct with its lining epithelium. Slide 24b, and c, Posterior tongue: Unlike the anterior tongue, the dorsum of the posterior part will not have many Lingual papillae. Only the characteristic circumvallate papillae, which are quite few in number, will usually be seen.63 XVI.3 A section of the circumvallate papilla will show that it has a broad flat top and a narrow base. Its top is only slightly higher than the general Surface of the Gorsum. It is sunken, so that its lateral wall is surrounded by a groove which is also lined by stratified squamous epithelium. Identify the taste buds in the epi thelium of the papilla and the wall of its surrounding groove. Note that the lamina propria in the posterior tongue has many lymphocytes with a tendency to form lymphatic nodules. Posterior lingual glands, like those in the anterior tongue, these glands ate located in the deeper part of the lamina propria and in the subjacent connective tissue between the interstices of the muscular elements. Two types may be found here? 1, Gustatory glands (of von Ebner) which are closely associated with the circumval- late papillae. These are purely serous secreting. The ducts are usually very short and open into the circumvallate groove. 2. Mucous glands of the root of the tongue - These are purely mucous secreting. ACCESSORY GLANDS OF THE ORAL CAVITY Slides available: 25 - Parotid gland, H&E 26 - Submaxillary (Mandibular) gland, H&E 27 - Sublingual gland, H&E These are the large salivary glands that are classified as "salivary glands proper". They are the parotid, mandibular, and sublingual glands: All are paired glands. All are compound tubulo-alveolar glands. Therefore, expect to see lobular groupings of the secretory elelments. Look for them. A lobule will usually be de~ marcated by a narrow space around a group of secretory tubules. This space may appear empty or may be occupied by thin connective tissue elements. Expect also a duct system consisting of a main excretory duct, interlobar, interlobular, and Antralobular ducts. The names of these ducts suggest their location. Correlate these names with the type of lining epithelium that you expect to see. Two segments of intralobular ducts may be recognized. The length of either segment varies in the three glands and is related to the proportion of serous and mucous secretory ele~ ments. The first segment is a continuation of the interlobular duct as it enters the lobule. It is lined by simple columnar epithelium characterized by cytoplasmic acidophilia of the epithelial cells and vertical striations in the basal cytoplasm. This duct is called a striated duct. It is also called secretory duct because of certain secretory functions attributed to its lining epithelium. The second segment ‘is lined by simple cuboidal epithelium and has a very narrow diameter. It is direct- ly continuous with the serous alveoli. It is called intercalated duct because it 1s interposed between the secretory duct and the serous alveolus. Slide 25, Parotid Gland: Examine the capsule before looking at the parenchyma of the gland. ‘This is usually a purely serous secretory gland. Although it is classified as a compound tubulo alveolar gland, most of its secretory elements are in the form of alveoli. Gonfirm these two characteristics in your slides. Both striated and inter- calated ducts are long and branched so that you will frequently see sections of them within the lobules.64 XVI. Slide 26, Submaxillary (Mandibular) gland: Examine the capsule before looking at the parenchyma of the gland. this ie a mixed serous and mucous secreting gland. Host of the secretory ele~ mente cre serous alveoli. The mucous secretory componayss, te generally in the Tepalar segments so that you will hardly find se100e demilunes. Confirm these two tubtures in your slide. The secretory ducts are long ‘and branched but the inter~ fSiated ducts are much shorter compared to those $0 ‘the parotid gland. Therefore, Sou should expect to see sections of striated ‘ducts frequently but few intercalated ducts in the lobules. Slide 27, Sublingual gland: observe that the capsule of this gland is not well developed but interlobular septa are prominent in many instances. This $6 2 nixed serous and mucous secret~ seu gland. The major portions of the secretory, eleven fare mucous secreting tubu- see ecolar components. There are small groups of serous secreting alveoli, but in many areas you will see that the serous components £14 ‘at the ends of the mucous niveoli as serous demilunes. Confirm these characteristics in your slide. There are geually no intercalated ducts. The intralobulat ducts are few and short. They are ily 7e tetory in nature although patches of epithelia? cells occasionally Are gtriated in character. Most of the ducte that you will see are interlobular ones. ESOPHAGUS Slides available: 28a, Esophagus, H&E vy w "| Masson and H&E 29," Gastroesophageal junction, H&E Using the low power Lens, ascertain whether the spec inet is a cross- or a long- itudinel section. If it 4s a cross section, observe that the profile of its luminal a eeene ig undulant. This 1s due to longitudinal folds formed by the mucosa and saencosa resulting from contraction of the wall of the Gut Identify the layers of the esophagus, Note that the mucosal epithelium is stravified squamous. The outermost layer of the gut +8 an adventitia. In the lamina propria, the lymphocytes show a tendency £0 form lymph nodules. Opserve that the muscularis micosae is thick and is mainly composed of Long~ ttudinally oriented smooth muscles. There may be a fev cireumferentially orient~ sitetbers in the luminal side of this sublayer- Tf the specimen was taken from the part close to the pharynx, the muscularis mucosa may ‘also include some prominent elastic fibers. Examine the muscularis externa and confirm that the direction of the fibers is cireamferential in the inner and longitudinal in the oltel parts. Be alert to the type of muscle in the muscularis externa. 1f the ‘specimen was taken near the pha- tym, the Layer may be composed of skeletal muscles’ & mixture of smooth and rymeeal muscles is found in the middle segment, and purely smooth muscles in the lower third. there are two kinds of mucous secretory glands in the esophagu Esophageal cardiac glands, and Esophages} glands proper. Esophageal cardiac glands are located in the lanint propria at two levels. One group is located in the upper part of the esophaguss between the level of the65 XVL.S cricoid cartilage and the 5th tracheal cartilage. The second group is located at the lower end of the esophagus. These glands are simple branched tubular in type with some coiling at their basal ends. In some instances they may be small compound tub- ular glands. The lining epithelium is simple columnar showing a clear supranuclear cytoplasm. Smaller ducts join the larger excretory duct which may be cystically dilated. This always opens at the tip of the connective tissue papilla of the lamina propria. Esophageal glands proper are found in the submucosa and may be encountered at any level of the organ. They are small compound tubuloalveolar in type. The secro- tory epithelium is simple cuboidal to columnar. The main duct is usually lined vy stratified squamous epithelium. Examine the gastro-esophageal junction. Note that the stratified squamous epi- thelium of the esophageal mucosa is abruptly replaced by the simple columnar type of the stomach. If cardiac glands are present, note that they appear similar at the regions of the esophagus and stomach. STOMACH Slides available: 29, Gastroesophageal junction, H&E 30a, Gastric fundus, H&E 30b, Pyloric region, H&E 30c, Pyloroduodenal junction, H&E The stomach may be divied into three regions, namely, cardia, body and fundus, and the pyloric part. The cardiac area is approximately the initial inch or less of the stomach fol- lowing the gastroesophageal junction. The body is the next segment caudal to the cardia and constitutes the major part(about 2/3) of the organ. The fundus is that part of the body that projects upwards and at the left above the level of the gas- troesophageal junction. The pyloric part is caudal to the body of the stomach, and ends at the gastroduodenal junction. The gastric mucosa has ridges (rugae) mainly disposed longitudinally. They have a core of submucosa. They are close to each other when the stomach is empty but are far-apart and may flatten out when the organ is distended. In addition, the entire mucosal surface is divided by grooves into small irregularly shaped gastric areas (mamillated areas). The mucosal surface on each gastric area has many tubu- Yar invaginations called the gastric pits or foveolae. Glands in the lamina propria open into the bottom of the foveolae. With the LP lens, identify the layers of the stomach and confirm that their ar- rangement conforms to the general pattern of the digestive tract. Next, look for the characteristics that are unique to the stomacl Mucosa - The lining epithelium is simple columnar. It also lines the foveolae. The epithelial cells look uniformly alike, and are characterized by a large unstained supranuclear area. There are usually no goblet cells. The lamina propria is quite thick but a major area of it is occupied by the glands of the organ. It has many lymphoid cells. Solitary lymphatic nodules may be seen. The muscularis mucosae is well developed and consists of inner circumferential (circular) and outer longitu- dinal fibers. Some strands enter the lamina propria and may be found between the glands. An outer circular layer is sometimes seen at the pyloric area. Compare the three gastric regions. Differences reside mainly in the type of glands and dimensions of the foveolae. Observe that the foveolae are narrow and66 XVI.6 short in the cardia. Those in the body and fundus are wider and longer. The fov- eolae in the pyloric region have the widest diameter and are the longest, som times reaching as far down as half the thickness of the lamina propria. The cardiac glands are mucous secreting, composed of short simple branched tub~ ular, or small compound tubular elements. They are found in both the esophagus and cardia. Their lining cells have a pale cytoplasm. In those glands located in the part of the cardia contiguous with the body and fundus, some parietal cells may be found among the mucous secreting cells. ‘The gastric glands proper (or gastric glands or fundic glands) are found in the body and fundus. This type constitutes the majority of the glands of the stomach. They are simple branched tubular with some coiling towards the base. They are the longest glands in the stomach. They are at least twice the length of the foveolae to which they open. Several glands open into the bottom of each foveola. Because of these, the lamina propria of the body and fundus is reduced to narrow areas bet- ween the glands. Look for the three cell types that can be reliably identified in H&E slides. These are the mucous neck cells, parietal cells, and the zymogenic cells. Aside from. size and staining characteristics, these cells have a pattern of distribution in the gland. You may see the . ether cell types but accurate identification would be difficult. The mucous neck cell is largely confined to the neck of the gland. Its stain~ ing with HSE is similar to the lining cells of the foveolae although it is smaller and more slender than the latter. Its nucleus is often somewhat flattened towards the basal cytoplasm. ‘The parietal cell is also found in the neck but it is densely distributed in the body of the gland and decreases in number towards the bottom. Lt is usually pyra~ midal in shape. It is the largest cell of the gland, its base often indents the basement membrane and its apex projects into the glandular lumen beyond the level of the other glandular cells. You can easily find this cell even under the LP lens because of its size and red color imparted by coarse acidophilic cytoplasmic gra- nules. The zymogenic cell constitutes the majority of the cells in this gland. It is distributed in the body and bottom of the gland. It is mainly found in the bottom. It is interspersed among the predominat parietal cells in the body. Its cytoplasm is basophilic. It is usually cuboidal in shape. Pyloric glands are short tubular glands with greater coiling than the preceding. They are less in number. They are often shorter than the foveolae into which they open. They are lined by mucous secreting cells and have similar staining as foveo- lar cells in H&E preparations. Occasional parietal cells may be seen, especially in those glands that are close to the body of the stomach. Submucosa - The submucosa consists of coarser connective tissue fibers (collagen~ ous) and appear to be denser compared to the lamina propria. It has many lymphoid cells. Observe that this layer has larger blood vessels (arterioles and venules) compared to the lamina propria which mainly has capillaries. Muscularis externa - There are three layers of smooth muscles unlike in the gener~ al plan which has two. These layers are the the innermost oblique layer which does not form a complete coat, inner circular layer (middle in position) which is a complete coat, and is more developed in the pylorus to form the pyloric sphinc- ter, and the outer longitudinal layer which is lacking in some areas of the dorsal and ventral surfaces of the stomach, and in the pylorus.67 XVI.7 With the LP lens, look at the areas occupied by loose connective tissue between the muscle layers. Identify elements of the myenteric plexus of Auerbach consist ing of nerve fibers and ganglion cells. Serosa - The tunica miscularis is invested by a thin layer of loose connective tissue covered by mesothelium (visceral peritoneum) constituting the serosa. SMALL INTESTINE Slides available: 30c, Pyloroduodenal junction, H&E 31a, Duodenum, &E 31b, Jejunum, Masson, and H&E 3lc, Tleum, H&E The small intestine is composed of three segments, the duodenum, jejunum, and ileum, It commences at the pyloroduodenal junction and ends at the ileocecal junc- tion. Using the LP lens, examine the slides of the intestinal segments. Confirm that the basic layers are present. With the exception of the posterior surface of the retroperitoneal part of the duodenum, which is covered by adventitia, the rest of the small intestine is covered by serosa. Most of the important processes of digestion and absorption occur in the small intestine. To subserve these functions, the area of contact between intestinal contents and intestine is increased by the following structures which are mostly in the mucosa: 1, Plenty of microvilli (brush border or striated border) on the luminal surface of the mucosal and glandular epithelium. 2, Intestinal villi 3. Crypts (glands) of Lieberkuhn 4. Circular folds of the mucosa (plicae circulares, valves of Kerkring, or valvulae conniventes) The striated border is rarely seen in the class slides due to autolysis. Examine the profile of the free surface of the mucosa. You should be able to see intestinal villi projecting into the lumen. Each is composed of a core of lan- ina propria covered by mucosal epithelium. Compare the shape and size of the villi in the three segments of the intestine. Confirm that the villi are tallest and leaf-like in the duodenum, shorter and less leaf-like in the jejunum, and short est and finger-like in the ileum. Next, examine the core of the villi. Look for the central lacteal (a lymphatic capillary) closely accompanied by smooth muscle fibers. There are also a small arteriole with its capillary branches, and a venule. You have to look at several villi in order to have a complete picture of the struc~ ture of the intestinal villus. Look for the plicae circulares. These are shelf-like transverse projections of the mucosa. They are semicircular or spiral in shape. Each plica has a core of sub- mucosa, muscularis mucosae, and lamina propria. Its surface should be covered by intestinal villi. It is most developed in the lower part of the duodenum and prox- imal jejunum. It is usually absent in the first part of the duodenum and distal half of the ileum. Mucosal epithelium - This is simple columnar, composed of three general types of cells, namely, the absorptive cell, goblet cell, and enteroendocrine cell. The absorptive cells constitute the majority. These are the ones whose microvilli on their luminal surface are demonstrable under the LM, in well prepared specimens, as “brush” or "striated" or "cuticular" border. The goblet cells are the solitaryXVI.8 68 mucous-secreting cells which you have seen before when you were studying glands. Observe that they increase in number as you proceed caudally along the intestine. You should see more of them in the ileum compared to the duodenum. The entero- endocrine cells (basal granular cells, enterochromaffin cells, argentaffin cells) are solitary elements and are few in number. They are usually identified by special staining. Your textbook mentions several types of them which secrete different substances. It is claimed that their highest number is found in the duodenum close to the pylorus. Trace the continuation of the mucosal epithelium into the crypt of Lieberkuhn. Notice that the epithelium in the upper half of a crypt is similar to that lining the villi. In the lower half of the gland, the goblet cells are usually absent and the majority of the cells have lost the characteristic appearance that you saw previously. You may see mitotic figures here. In addition, the bottom of the crypt contains Paneth cells. These are pyramidal cells with a round nucleus and large eosinophilic granules in the supranuclear cytoplasm. The basal cytoplasm is baso- philic. Paneth cells are characteristic of the crypts in the small intestine. You may have a better chance to identify the enteroendocrine cell here because it is more likely to be ovoid or pyramidal in shape compared to those in the general epithelium which is usually columnar. If cytoplasmic granules are seen, these should be mainly located in the basal cytoplasm. Examine the lamina propria. Note the lymphatic nature of the tissue. Compare its appearance in the three segments of the intestine. Confirm the fact that the lyn- phoid cells tend to increase caudad along the intestine. Solitary nodules increase in number and aggregated lymph follicles (Peyer's patches) are encountered in the ileum. Finally, examine the submucosa, muscularis externa, and serosa. The submucosa has a coarser and denser texture compared to the lamina propria. Its blood vessels are larger compared to those in the lawina propria (similar to what you saw in the stomach), Find out why this is so. In the duodenum, the submucosa is occupied by Brunner's glands, compoind tubular glands, apparently mucous secreting, which may disrupt oF break the continuity of the muscularis mucosae if they are big. Their ducts penetrate the mucosa to open at the bottom of the crypts. They are better developed in the upper duodenum, and may be found in the pylorus. They may be ab- sent in the lower duodenum. There are several nerve plexuses in the gastrointes- tinal tract, but with H&E, only one can be found easily - the myenteric plexus. Nore difficult but not impossible to find is the submucous plexus of Meissner. It ts also a plexus of nerve fibers with ganglion cells in the part of the submucosa close to the muscularis externa. You have found it if you can see the ganglion cells. Lastly, examine how the serosa is attached to the muscularis externa. LARGE INTESTINE Slides available: 32, Appendix, H&E, or Masson and H&E 33a, Rectum, H&E, or Masson and H&E 33b, Rectum, Masson and H&E The large intestine has the following segments: cecum, colon, and rectum. Devel- opmentally, the vermiform appendix is part of the large intestine but it has been reduced to a small appendage. Examine the slides and confirm that the large ‘intestine is also made up of the basic layers. Unlike the small intestine, it has no intestinal villi, but it has more goblet cells in both general epithelium and crypts. Paneth cells are usually absent. There are no plicae circulares. The crypts are longer in the colon, espec~69 XVI.9 ially the rectum. They are small in the appendix. The vermiform appendix is small and vestigeal. Therefore, a complete cross section of the organ is accommodated in the slide. Its lumen is often triangular in profile and relatively small. Its lumen may contain cellular debris, food Particles, parasitic eggs, or a section of a parasitic worm. Paneth cells may be seen occasionally in the crypts. Observe that both lamina propria and submucosa are intensely infiltrated with lymphoid cells. There are many lymphoid nodules whose closeness to each other is reminiscent of a Peyer's patch. The muscularis mucosae is often interrupted by lymphatic nodules. The tunica muscularis is thin and may be reduced to strands separated by connective tissue. A serosa covers the appendix. In the cecum and colon, the longitudinal layer of the muscularis externa is gathered into three thick bands called taeneae coli. There is usually a thin layer between these bands. You will not see taeneae in the rectum. Here, the longitudi- hal muscle forms a uniform coat although it might be somewhat thicker at the anter- jor and posterior aspects of the wall. There are many lymphoid nodules in the ce- cum and colon, but these decrease in number in the rectum. You may encounter part of a transverse semilunar fold (valve of Houston) of the rectum. It consists of a fold of mucosa and submucosa, with a core of circular miscle of the tunica muscu- laris. Look for the nerve plexuses here also. The serosa becomes an adventitia in the lower part of the rectum. LIVER Slides available: 34a, Liver, H&E 34b, Liver, Silver 34c, Liver, H&E Using the LP lens, examine the borders of the H&E preparation.If the capsule (Glisson's capsule) is included, note that it is a thin dense connective tissue. Except the bare area of the liver, the rest of the capsule is covered by meso- thelium (peritoneum) - a serosa. The connective tissue of Glisson's capsule con- times into the interior of the liver at the porta hepatis, accompanying and "en- veloping” the entering portal vein and hepatic artery, and the emerging bile ducts and lymphatics. Henceforth, it will be called periportal connective tissue and will always surround the branches of the portal vein and hepatic artery, the bile ducts and lymphatics. The areas occupied by the periportal connective tissue are referred to as portal areas. The liver is the largest gland of the body. Its parenchyma consists of only one type of cell ~ the hepatocyte. Hepatocytes arearranged in the form of plates that are usually one-cell thick. The plates anastomose with each other, and there are frequent openings in the plates so that the spaces between them are often continu ous with each other. These spaces are called hepatic lacunae. They are occupied by the hepatic sinusoids and supporting connective tissue. Scan your specimen with the LP lens. Note that it is a very vascular organ be- cause the hepatic plates are usually associated on both “free” surfaces with sinusoids. Look for the central veins. These are usually seen as cross or oblique sections of solitary vessels with a diameter larger than that of the sinusoids. The hepatic plates often appear to radiate from each central vein - producing aXVI.10 70 iepulat pattern. Each"lobule” is called the hepatic lobule or classicalliver jobule. Note that each lobule is not completely separated by connective tissue from those adjacent to it. There 1s contiguity of hepatic Plates of one lobule Pith those of the adjacent ones at the areas “lacking” in comertece tissue. It Ze usually at the junction of three lobules that you will see ea appreciable cent Loe ion Of connective tissue which may send thin extensions boeeecs adj; Jaonedrgee; LE this area contains branches of the hepatic portal vein, it is jdentified as periportal connective tissue. Establish the topography of the hepatic lobule. Its periphery 1s at the portal areas and an imaginary line con- cectsng addacent portal areas around that lobule: The center of che lobule is the Shera eins ,The lobule can then be divided into three zones. namely, the peri~ between the two other zones. Knowledge of these zones will help you later in identifying sites of pathology in the organ. Ty You look carefully, you may be able to appreciate that the hepatic plates fizected towards the periportal connective tissue form a "eels" around it consist are ofa circumferentially oriented plate. This is called rhe periportal limiting Plate or lamina limitans. Note also that hepatocytes in che lamina limitans are wing tobule. The lamina limitans has many perforations ico, Thus, you will see periporrerruptions in it. The “tube” formed by the lamina limiters contains the porter eal connective tissue, The theoretical lumen of thie tabs ve called the portal canal, Te tare the following structures, often collectively called tne Portal tr: 1z,lmterlobular vein - a branch of the portal vein, os 3 branch of the interlobular vein itself (terminal portal venule). These portal venous branches are usually bigger in diameter than the other structures in this enumeration. Occasionally, ing thrown on czy, Small venule arising from the terminal portel vesate or pass— p's through an aperture of the lamina limitans. ‘This te an inlet _venule. Topentetbebular artery - a branch of the hepatic artery, ore branch of the inter- zrginterlobular bile duct ~a radicle of the hepatic bile duct. It is lined by pimple cuboidal epithelium. Occasionally, you may see a enali tadicle of this duct snopes Section or passing through an aperture of the laniea imitans. This is a cholangiole. In addition, there are lymphatic vessels in the periportal connective tissue but they are often collapsed so that they are seen lene frequently. Small nerves here) °° DC Seen. There are also capillaries that are branches ot the arterioles here. Newt, examine tho hepatic sinusoids. Look for their endothelial cells. These are flat elements, usually seen only as flat nuclei, Identify the von Kupffer cells any ne Sinusoidal wall. These are macrophages. ‘They usually bulge into the lumen or Containe ohare ee git tour tdentification will be more definitive te the cell Simzerg Phagocytosed matter. There is usually an appreciable Bap between the Sinusoid and the hepatic plate beside it. Normally thie space should be easily Genonstrable only in electron micrographs. This 12 the erisinusoidal space of Pisse. Tissue processing usually causes its enlargement tee space of Disse 1s Gontimous through the apertures in the lamina limitans wrch another very narrow Thig the weed between the lamina Limitans and the periportal connective tissue. This the periportal space of Mall. It is believed thac tissue fluid in the spaceXVE.LL m1 of Disse reaches the space of Mall and percolates through the periportal connect- ive tissue to enter the lymphatic vessels. Examine the central veins. They are venules. Their wall is very thin. Note that there is no limiting plate around the vein and the lumen of the sinusoids converg- ing into it appear to be continuous with it. The central vein is the smallest tributary of the hepatic vein. Several central veins drain into a larger venule which is usually perpendicular to the longitudinal axis of the central vein. This is the intercalated vein (sublobular vein). This is also thin walled. but connect- ive tissue elements can be seen around it. It is surrounded by a limiting plate (hepatic limiting plate). Occasional sinusoids pass through perforations in the Limiting plate to drain into this vein. Examine the hepatocytes under the IP lens. Observe the heterogeneity of their cytoplasm and the presence of small, round, and relatively unstained or light ly stained areas representing lipid inclusions. Using both the H&E preparation and the silver stained tissue, examine the peri- sinusoidal spaces. There may be some cells there that are difficult to identify under the LN. If you have established the location of the sinusoidal wall, the cells in the perisinusoidal area could be the lipocytes mentioned in your textbook. Some investigators claim that fibroblasts may also be found here. But since the Lipocyte may be similar in appearance to the fibroblast, you cannot make a solid "guess" of its identity. Using the silver stained preparation, study the locations of the reticular fibers. Study similar slides of your classmates to have a more complete idea of their distribution. Note that generally the black reticular fibers are approx- imately at the perisinusoidal areas and around the central veins. GALL BLADDER, Slide 35, H&E The gall bladder is a sac-like organ. It is closely applied to the visceral surface of the liver. Its cystic duct is connected to the common bile duct. Identify the following layers of the gall bladder: 1, Mucosa, composed of a simple columnar lining epithelium supported by a lamina propria. There are a number of small blood vessels here. Lymphatic nodules may be seen at times. 2. Muscularis, composed of smooth muscle fibers arranged in small bundles separ- ated by relatively wide areas of connective tissue that are directly continuous with the lamina propria. 3, Perimuscular connective tissue and serosa ~ a fibrous connective tissue with collagenous and elastic fibers. The portion adjacent to the muscularis often ap- pears denser compared to the more superficial part. This denser part is referred to as the perimuscular layer. The superficial and looser connective tissue, if covered by peritoneum, is a serosa. The aspect of the organ that is attached to and is continuous with Glisson's capsule is a fibrosa or adventitia. This looser connective tissue contains the larger blood vessels that provide smaller ones to the rest of the organ. It also contains nerves and lymphatics. ‘The mucosa has many’anastomosing folds. These folds are not fixed but shorten and flatten out when the organ is distended. In perpendicular sections of the wall, a portion of a fold may be included. This could appear like a cross section of a Jarge tubular structure in the lamina propria.XVI.12 72 Rokitansky-Aschoff sinuses are sections of epithelial invaginations into the underlying lamina propria, or even tunica muscularis. They may be mistaken for sections of tubular glands, but if you study their epithelium, you will find that this is identical to the lining epithelium of the organ. True ducts of Luschka - Sections of small ducts lined by simple cuboidal epi- thelium may be encountered in the perimuscular layer in some specimens, particu- larly in sections of the part that is attached to the liver. They would look similar to cholangioles. They are believe to be aberrant bile ducts. They are not connected to the epithelium of the géll bladder. ‘Small mucous secreting glands may be encountered in the mucosa at the "neck" of the gall bladder, or near its continuity with the cystic duct. PANCREAS, Slide 36, H&E The pancreas is the second largest gland of the body. It is covered by a thin loose connective tissue which sends thin strands into the interlobular areas. It is no easily seen in many places, being increased in amount only around the inter- lobular ducts. However, if you scan your specimen with the LP lens, the lobular organization of the gland will become very evident. Using the LP lens, examine the parenchyma. Identify its exocrine and endocrine areas. The exocrine part constitutes most of the pancreatic parenchyma. Its secretory portion is tubuloalveolar but acinar profiles predominate in the microscopic field. The round to oval acinus is lined by pyramidal shaped cells. The lumen of the acinus is very small. Under the HP lens, the acinar cell shows basophilia in its basal portion. The apical cytoplasm often has acidophilic "granules" which are zymogenic droplets.A supranuclear negative Golgi image may sometimes be seen. The endocrine portion is represented by the pancreatic islets (islets of Langerhans). There are more islets in the tail than in the body and head of the organ. They are easier to spot under the LP lens. In routine H&E preparations, the islets are less intensely colored than the acini. The islet cells are smaller than acinar cells so that their nuclei often appear to be closer to each other. Observe also that the islet cells vary in shape and size. The islets vary in size. However, , you will usually see that the diameter of the islet is appreciably larger than that of the acinus. You will not be able to identify the various cellular compon- ents of the islets in H&E sections. Examine the islets under the HP lens. Observe that there are many capillaries among the cells compared to the exocirne portion. The main duct of the pancreas (Wirsung) runs the entire length of the organ. The accessory duct (Santorini) is cranial to the main duct and is smaller in diameter. Throughout their course,both ducts give off interlobular branches. Both large and interlobular ducts are lined by simple columnar epithelium. There may be occasional goblet cells in the epithelium. Mucous glands may arise from the jarger ducts. A fairly thick and dense connective tissue surrounds these ducts. Nerves and the larger blood vessels enrouteto and from the area that they supply or drain accompany these ducts in the same connective tissue. Each interlobular duct gives offintralobular ducts. The latter is a thin tub~ ule lined by low columnar epithelium at its entrance into the lobule. The epithelium becomes low cuboidal as the duct nears the acinus that it will drain.XVI. 23 13 It is not surrounded by abundant connective tissue so that it would be harder to find. [t gives off branches (intercalated ducts) that are lined by a pale low cuboidal to squamous epithelium. This duct is the one that is directly connected to the acinus. Its terminal segment is often telescoped into the lumen of the acimis, in which case, the lining cells will appear as small pale cells in the center of the acinus and be identified as centroacinar cells. You may sometimes see a very tiny duct connected to a pancreatic islet. The Lining epithelium is composed of small squamous or low cuboidal cells. An obvious lumen is not always seen. This duct has been traced directly to the main ducts. It is not connected to an acinus or any intralobular duct.7h XVII.1 XVII. THE URINARY SYSTEM ‘The urinary system is composed of the kidneys and extrarenal parts, namely, the calyces, renal pelvis, ureters, urinary bladder, and urethra. In the male, the urethra is also part of the reproductive system. The kidney is functional- ly associated with the blood vascular system. You will observe that the orgaa is very vascular. The main functions of the kidney are the removal of nitrogenous waste pro- ducts of metabolism and the maintenance of a fluid and electrolyte balance. ‘The kidney also eliminates certain substances that are toxic or are in excess. In addition, it influences the blood pressure, and certain of its cells sec- Fete substances into the blood that influence the activity of cells or organs elsewhere in the body. It also conserves certain substances that could have been lost otherwise during the process of urine formation. ‘The Kidney Slide 41a, or b ‘The section of the kidney is usually made perpendicular to its outer surface. Scan the tissue using the LP lens. Look for the border of the tissue that is covered by its "true" capsule. ob- serve that the capsule is thin. The portion of the tissue immediately beneath the capsule and to which it is attached is the renal cortex. Look for two characteristic areas in the cor- tex: 1. Granular Cortex - This is more extensive than the other part. It consists chiefly of cross and oblique sections of uriniferous tubules, mainly parts of the nephron. It also contains sections of renal corpuscles which possess the widest diameter among the rounded profiles in this area. 2. Striate Cortex (Medullary Rays of Ferrein) - This consists of sections of straight tubular structures which are parallel to each other and are more or Jess perpendicular to the capsule. Observe that generally the diameter of the tubules in the striate cortex is less than that of the tubules in the granular cortex. Each medulJary ray is considered as the center of a renal lobule. An inter- lobular area is approximately between adjacent medullary rays. This 4s in the granular cortex and blood vessels coursing through this area are usually inter- Jobular arteries and veins. Descend through the cortex until you reach the part of the tissue where all the tubules are generally parallel to each other. This is the renal medulla. Notice that the change in texture or orientation of the tubules (profiles of cut tubules) is abrupt if you began your descent from the granular cortex. If you chose to descend from the striate cortex, the tubules in the medulla will appear to be directly Continuous with those of the former; thus, no marked change of texture or tubular orientation will be observed at this area. The junction of the renal cortex and medulla (cortico-medullary area) is best observed at the parts where the granular cortex is adjacent to the medulla. ‘The border of the medulla here is the so-called base of the renal pyramid.75 XVII.2 ‘The juxtamedullary area of the cortex is the cortical portion of the corti- comedullary area. Large arterioles and venules, generally paralle) to the base of the pyramid, may be seen here, These vessels are the arcuate arteries and veins. In some areas, these vessels will show their connection with the inter- lobular vessels in the cortex, or the interlobar vessels in the medulla. Using your HP lens, observe that there is an abundance of capillaries, usual- ly cut in cross or oblique sections, closely associated with the uriniferous tubules in both the cortex and medulla. These are the peritubular (intertubular) capillaries. In addition, there are vessels that are somewhat larger than the typical or ‘ordinary* capillaries that run parallel to the tubules in the medulla. These are the vasa rec! Using the LP lens, examine the areas surrounding the uriniferous tubules and blood vessels in the cortex and medulla.. These areas contain connective tissue fibers and small cells. Collectively, these areas are referred to as the renal interstitium. Note that there is a progressive increase in the amount of interstitial tissue as you examine adjacent parts beginning at the subcapsular area of the cortex, proceeding towards the medulla and ending at the tip of the renal pyramid. At the renal papilla, notice that the tubular elements are widely separated by interstitial elements in contrast to what you find in the cortex where the tubular elements are close to each other. The consecutive segments of the uriniferous tubules are listed below with their location and a short description to help you find and identify them. It is up to you to look for the complete description of these parts in the text- book. Not all parts described in the textbook may be seen in the specimens because of autolysis, damage to the tissue during preparation, or because they may be too small for the light microscope. Renal Corpuscle - This is found in the granular cortex. It is seen as a round body and has the largest diameter among the rounded profiles in the granular cortex. Since the part is randomly sliced, as are the other components of the organ, examine several renal corpuscles to ensure that you would see all parts that are visible under the LM. Look for the following: 1. Vascular Pole, together wwith the afferent and efferent glomerular arterioles, 2. Urinary Pole, together with the neck of the proximal tubule, 3. Parietal Layer of Bowman's Capsule, or Capsular Epithelium, 4. Capsular Space ox Bowman's Space, or Glomerular Space, 5. Renal Glomerulus, and components of the visceral layer of Bowman's capsule. Proximal Tubule - This is the longest part of the nephron. It consists of a convoluted and a straight segment. After a short neck that continues from the Bowman's capsule at the urinary pole, the proximal tubule becomes coiled and is thus called the proximal convoluted tubule. It is located in the granular cortex. It has the larger diameter, longer length, and tighter coiling com- pared to the distal convoluted tubule. Its lining cells are usually more acid- ophilic than those of the other tubular elements. In well prepared specimens, the apical cytoplasm shows a brush border. As the proximal tubule enters the striate cortex, it loses its convolutions and descends here as a straight tube, now referred to as the thick descending segment of Henle's loop. Compare the appearance of the convoluted with the straight segment. You will notice that the latter has a smaller diameter. Under the LM, the cytological appearance of both segments appear similar. The straight segment may be found in both the striate cortex and the "outer" medulla. Thin Segment of Henle's Loop - At the end of the proximal tubule, there is an abrupt decrease in the diameter of the next segment. This is the thin segment76 XVII.3 ef Henle’s loop. Tt is located in both the ‘outer* and ‘inner* medulla. It is the part of Henle's loop that 1s most variable in length and position withis see yacge It may be absent in short nephrons with corresponding short loope Segienle; Under the LM this tubule may be mistaken for a capillary because of {ES gmaii diameter and simple squamous lining epithelium. If you look cerefoi- ty, however, you will see that this tubule has a larger diameter than the capillaries and that its squamous epithelium is thicker than that of the Uhtter: The longest thin segment is found in the longest nephron and octupies three sections of Henle's loop, namely, the thin descending segment, the gna the thin ascending segment of Henle's loop. In this casa, the thin dese Ang seament may be found in both the “outer* and “inner” medulla, the cesst thin pau Ascending segment are in the "inner" medulla. In the short loop, the thin segment may occupy only the terminal portion of thedescending link of Henle's loop. In the loops that are intermediate in length, the thin ascending segment may Also be found in the "outer" medulla. te Distal Tubule - This 1s much shorter than the proximal tubule. It consists of sie adhe and convoluted sogment. The straight portion is dizectiy contiauous vith the thin segment of Henle's loop. In the casa of a short os abeest thin tnaments the straight portion of the distal tubule also occupies the creat of the loop. The straight portion is also ing soutent of Menlo's loop. It is found in both the "outer" medulla and the striate Sortex. Upon leaving the striate cortex, the straight portion goes to the serene bole Of {ts own renal corpuscle and a portion of its wall becom seegchet te the afferent glomerular arteriole, and participates in the form Qiton of the so-called juxtaglomerular complex. Beyond this poiat, the cascre becomes coiled and is now called the distal convoluted tubule, Although the Ghameter of the distal convoluted tubule is less than that of the provimel G2avoluted tubule, its lumen may be slightly wider. The lining celle of ene Ape Ae find it easier to locate a section of a distal convoluted tubers at the area close to the vascular pole of the renal corpuscles. Gollecting Tubules (Ducts) - The distal convoluted tubule ends in the granular sorte The Desinaing of the system of collecting tubules is, therefore, found ab the granular’ cortex. This 1e the arch collecting tubule which 1s ccarectad tp, 'he, fexminal portion of the distal convoluted tubule, It is the emallecc ot She collecting tubules. Its diameter is slightly less than that of the aietay sonnouuted tubule. It may be distinguished from the tubular elements of the Bephron by its low cuboidal epithelium, clear cellular boundaries, and hove seep ut pater cytoplasm. It enters the striate cortex and unites with a straight collecting tubule. This straight tubule usually has the sane cyto- dogical appearance as the arch collecting tubule in routine Hee preparations. A ceraight collecting tubule, while descending through the striate sostex, ageives several arch collecting tubules befora entering the medulla, te fuses with another of the same category as it descends in the sinases medulla, proaveing @ bigger straight collecting tubule. The lining epithelium at thie {aver As cuboidal. It ie claimed that this kind of fusion occurs seven thas ib the inner medulla. The biggest straight collecting tubule 4s formed ac ene jast fusion. It has a low columnar epithelium. This is the papillary duct which ends at the tip of the renal papilla by opening into a minor calyre At the region of the renal papilla where there are only papillary ducts,XVII.4 77 thin segments of Henle's loops, and elements of the vasa recta, you can easily identify the papillary duct. Sometimes, the staining of the collecting tubules in the medulla is more intense and acidophilic than those in the cortex. Renal Vasculature ‘The following blood vessels may be seen in routine H&E preparations: 1. Interlobar Artery and Vein - You may see these only if your specimen includes a side of the renal pyramid and/or its adjacent base. They are the Jargest blood vessels of their category that may be seen in routine sections. 2. Arcuate Artery and Vein - These are located in the corticomedullary area, usually in the juxtamedullary cortex. If they have been cut longitudinally,. you will seo that their long axis is more or less parallel to the base of the renal pyramid. In some instances the part may show the vessel's connection with its interlobular vessel. In rare instances, you may also see the con- nection with the interlobar vessel. 3. Interlobular Artery and Vein - These are located in the interlobular area of the cortex. They are radially directed towards the renal capsule, more or Jess parallel with the medullary rays. In many instances, you will see an intralobular branch arising from the artery. Often, this intralobular branch is the beginning of the afferent glomerular arteriol 4. Afferent Glomerular Arteriole - This is easy to locate at the vascular pole of the renal corpuscle. At the part most proximal to the glomerulus, many of the cells of its tunica media are the juxtaglomerular cells (JG celis). In routine H&E sections, these cells appear paler than ordinary smooth muscle fibers and are epithelioid in arrangement. The afferent arteriole has a larger diameter than the efferent glomerular arteriole in the same renal corpuscle. 5.Renal Glomerulus - This is composed of loops of capillaries which are inti- mately associated with the visceral layer of Bowman's capsule whose component cells are called podocytes. In a fortuitous slice of this area, you may be able to distinguish between the endothelial cell nucleus and the podocyte because of their position. The endothelial cell nucleus bulges into the lumen of the glomerular capillary. The podocyte with its nucleus projects towards Bowman's space. The internal messangeal cell is only rarely identifiable in routine HE sections. The important basement membrane between the capillary and the podocytes is demonstrated by special staining. 6. Efferent Glomerular Arteriole - In addition to being smaller in diameter than the afferent vessel, this structure has a relatively thinner wall. It gives rise to the peritubular (intertubular) capillaries of the cortex and medulla. 7. Peritubular (intertubular) Capillaries - These are small capillaries that are closely associated with the uriniferous tubules. They forma rich network around the tubules, especially in the cortex. 8. Vasa Recta - Thesé are branches of the efferent arterioles of glomeruli jocated in the juxtamedullary cortex. They are capillary loops that accompa- ny the loops of Henle and collecting tubules in the medulla. Their diameter is greater than that of ordinary capillaries. The ascending limb of these vessels is larger and often irregular in cross section compared to the78 XVIL.S descending limb. The crest of the loops are found at various levels in the medulla, so that less of these vessels will be seen at the inner medulla. Peritubular capillaries in the medulla and juxtamedullary cortex also arise from these vessels. 9. Cortical Veins - These are radially oriented vessels in the cortex. There are two groups of cortical veins: (a) The superficial cortical veins are small in diameter and number. They are found in the superficial cortex and drain the peritubular capillaries in the same area. They bring the blood towards the cortical surface and empty it into the stellate veins.(b) The deep cortical veins are found in the deeper cortex, especially in the juxta- medullary area. They are mich more numerous and wider in diameter than the superifical group. They drain the peritubular capillaries in the deep cortex and in the medulla, tacluding the vasa ractn. Those vassels empty dato the interlobular veins or arcuate veins. 10, Stellate Veins - These ara cortical vessels located close to the renal capsule. They run parallel to the renal surface. They converge on interlobular veins. Juxtaglomerular complex (JG complex) - This is located in the vascular pole of the renal corpuscle. The following components can be identified in routine preparations: 1. duxtaglomerular cells or JG cells 2. Macula densa - This is the area of the distal tubule that is "attached" to the region of the JG cells. The wall of the distal tubule at the area of attachment shows crowding of nuclei and the epithelial celle at the site may also be taller. Lacis cells (Polkissen cells, extraglomerular cells, polar cushion, or external mesangeal cells) - The cells are chiefly found at the indentation or hollow produced by the diverging afferent and efferent arterioles in the vascular pole. They are small and pale staining. The macula densa may extend over them, ‘The Extrarenal Parte ‘The Renal Calyces, Pelvis, Ureter, and Urinary Bladder Slides Available: Ureter, HéE, Slide 42 Urinary bladder, HGE or HEC, Slide 43a Urinary bladder, H6E, Slide 43> The basic structure of the wall of the calyc! bladder is similar and consists of the following: pelvis, ureter, and composed of (a) a transitional type of lining epithelium, and (b) lamina propria 2. Supporting connective tissue - This is not a distinct submucosa. It blends with the lamina propria. It has many elastic fibers and its basal or periphor- al part is looser in character. This loose connective tissue is well developed in the urinary bladder.79 XVIT.6 3. Muscularis - This consists of an inner longitudinal and an outer circular smooth muscle layer. Boundaries between layers are obscured by intermingling of the muscle fibers of these layers. At the lower third of the ureter, and in the bladder, the muscle layers are not only thicker but there is a third outer longitudinal layer. If your specimen includes the intramural part of the ureter, the circular muscle layer is absent here. 4, Adventitia - This is loose connective tissue. Compare the ureter with the urinary bladder. Notice that the main difference between the two is the thickness of the component layers. ‘The superior portion of the urinary bladder is covered by peritoneum. This part may be said to have a serosa instead of an adventitia. Only the urinary bladder has mucous secreting glands. These are close to the internal urethral orifice. They are similar to the glands of Littr: Female Urethra, H6E, Slide 44 Unlike the male urethra, the female urethra is purely a pact of the urinary system. It is about an inch Jong. Its mucosa consists of a stratified squamous epithelium (sometimes, psoudostratified columnar epithelium) supported by a lamina propria which has many elastic fibers, venous plexuses, and smooth muscles. Beneath the lamina propria is a miscularis consisting of inner longitudinal and outer circular smooth muscle layers. At the terminal portion of the urethra, there is an added outer circular layer of skeletal muscle fibers.8 XVIII. 1 0 XVIII, MALE REPRODUCTIVE sysrEn Slides available: 52a, Testis, H&E 52b, ‘Testis, Iron Hematoxylin 5c, Testis and Epididyais, &E or Iron Hematoxylin sing the LP lens, examine the periphery of the organ. It is enveloped in a At che po uense fibro~elastic connective tissue calles tumica albuginea testis. aretne Posterior aspect of the organ the connection tissue is thickened to form the mediastinum testis. There are many blood aad lymph vessels here. You may also cmeenter Profiles of several segments of the duct system. Mesothelium covers the lesan de ttface of the capsule except at the region cr the mediastinum. Connective quagte SePtarcalled septula testig radiate tron tne mediastinum to the capsule and divide the parenchyma into pyranidal compartments containing seminiferous tubules foiled Jobult testis. The septa are frequent ly tite or deficient near the capsule the Tat the Parenchmaltissue appears to be contincoo, in this area. Internal to interstitial connective tissue or stroma of the testis. Blood vessels in the stroma ang ggntula testis. A number of lymphatic capiliacion are also found in the stroma trontntea vasculosa. and to a small extent, in ene tunica albuginea. Lymph draine through vessels in the mediastinum. SEMINIFEROUS TUBULES Stt11 using the LP lens examine the seminiferous tubules. Note that the peri- Ferent ee sack tubule is well demarcated by a thick basement membrane and circun- ferentially disposed collagenous fibers and flee cells. Next, scan the lining epi- cree, Comedtm that it is a stratified epitheling The component cells of the epithelium are of two types, namely the epermatogenic cells, seen in various stages se, tevelopment and constituting the majoriey of the epithelial cells, and the Sertoli cells (supporting cells or sustentaculer cells). Generally, spermatogenic Seite are roundish in shape with a round nucloas (except the spermatozoa and the gate stages of the spermatid). The Sertoli ceil 44 large and columnar, with an SeiT Ructeus that is oriented perpendicular to the basement membrane. The Sertoli Sort is adjacent to the basement membrane but ine apical portion is usually found steste Same level as that of the spermatids and spermatozoa. Its boundaries are not sacar; [ts lateral surfaces’ have processesthar connect with those of adjacent sSertoli cells, but these are not usually appreciated under the LM. Spermatogenic certs, tie within recesses of the Sertoli ceil se two cells are not fused, but thts fact is not clarified by the LM so thae Spermatogenic cells appear to lie within the Sertoli cytoplasm.XVIIL.2 81 Using the LP and HP lenses alternatively, examine the spermatogenic cells and identify their stage of development. Spermatogonia occupy the base of the epithel- ium, Some possess an ovoid nucleus which may be light or dark staining. Others have a round nucleus with some heterochromatin arranged near the nuclear envelope. Primary spermatocytes are found at a higher level than the spermatogonia. They are the biggest elements among the developing cells. Their chromatin are often filamentous or ribbon like. Secondary spermatocytes often occupy a higher level in the epithelium than the primary sppermatocytes. They may be smaller than the spermatogonium. Their nuclear chromatin are often granular. Spermatids are very small and occupy the apical parts of the Sertoli cell. They are at their largest size at their early stages of development. Their nucleus is dark staining, compact and round. Their cytoplasm may still be seen. In later development, their nucleus is wedge shaped and the remaining cytoplasm may be hard to see under the HP lens. Spermatozoa are usually the most superficial cells in the epithelium. They are also the smallest. They appear to be free in the lumen or may seem to "occupy" the apical cytoplasm of the Sertoli cell. You should see its flagellum to identify a Spermatozoon definitively. In a given section of the seminiferous tubule, it is uncommon that all the stages of development of the spermatogenic cells will be represented. At the end of the loops of seminiferous tubules, at the apex of the lobule, there are fewer spermatogenic cells and greater number of Sertoli cells. Interstitial Tissue In addition to a few connective tissue cells (fibroblasts, macrophages, mast cells, and undifferentiated mesenchymal cells) the interstitial cells of Leydig are found in the stroma and tunica vasculosa, and occasionally in the tunica albuginea. The Leydig cell is large (14-20 micrometers) and are variable in shape. Its acidophilic cytoplasm may have small vacuoles representing areas formerly oc- cupied by lipid droplets which have been removed during tissue processing. They may also contain yellowish lipochrome pigment. The cytoplasmic crystal of Reinke is not seen in H& & preparations. However, its presence may be inferred when its negative image is seen. The vesicular nucleus is large and often eccentric in position, There are one or two prominent nucleoli. Leydig cells tend to be poly- hedral when they are in a cluster. When they are solitary, they may be elongated or fusiform. The cell clusters are scattered seemingly at random in the stroma. However, the cells may be arranged in short rows along capillaries or venules. Binucleated Leydig cells are not uncommon. TUBULI RECTI (Sometimes seen in Slide 52c, Testis & Epididymis) These are short and straight tubules that usually begin in the region of the interstitial connective tissue and run the major part of their course in the mediastinum testis. A tubule is somewhat dilated at its beginning. Its lining of simple cuboidal to columnar epithelium begins abruptly at its junction with a seminiferous tubule and continues without much alteration into the next segment. The basement membrane is still well developed but is thinner than that of the seminiferous tubule.ea XVIIT.3 RNTE TESTIS (Sometimes seen in Slide 52c, Testis & Epididyais) a consists of anastomosing wide channels in the mediastinum testis. The ghithelium is usually lower compared to that of the tubuli rectl. It may be simple Junction cup eome areas. The basement membrane is no longer seen under ene iH Its junction with the succeeding segment is located in the upper posterior edge of the mediastinum, PUCTUL EFFERENTES (Sometimes seen in Slide 52c, Testis & Epididymis) yea asin in the mediastinum testis as relatively straight tubules but soon jeave it and becone highly convoluted ducts forming the vascatne cones that cons~ Edtute the bulk of the head of the epididymis. These ducts are Lincs by a pseudo~ sieathfied epithelium that presents a wavy luminal profile because of irregularly alternating groups of tall and low cells. The basal profile of the epithelium is sate peatta may be present in both cell types but are more conseage sn the tall. celts: The tall cells are darker staining than the low cells ber may show cyto~ Plasmicvacuoles. The low cells have a brush border and a cestrat flagellum (not usually seen in the class slides) on their luminal surface, Secretory granules Tay be Seen in the light staining cytoplasm. Although both ceil types are claimed sett neeeeretory in nature, the low cells are evidently so and may soon form intra epithelial glands. A third cell type, called basal cell, 1g somevincs Seen in the gpitheldun, It 4s extremely light staining and varies fron rou dion to a somewhat qugular shape. The connective tissue surrounding the convoluted Portion of the ducts 4s Loose in character although it is dense and lanelleted immediately exter- see eoanhe, basement membrane, Sone circumferentially arranged emonre muscle fibers Secec There 48 also an increase in the number of smooth euccie fibers around the ducts. DUCTUS EPLDIDYMIS Slide 52c, Testis & Epididymis, Hae, jhe epididdyais is an organ that is oriented along the longitudinal axis of the festis and is related posteriorly to the same. The ductus epididymis, together with its supporting connective tissue , forms a amall part of tne head, the whole body aid tail of the epididymis, The ends of the ductuli efferentes tase to form this panste duct. Le is very tortuous at the region of the head and body but becomes ieee 30 in the tail. Its thick pseudostratified epitheliua hee. smooth basal and puminal profile. The epithelium consists mostly of tall celta with sterocilia. Basal cells are irregularly distributed on the basement membrane; they are much fomag’s the tail where three layers of larger sized snooth mecie fibers may be found, namely, inner longitudinal, intermediate circumferential, and outer longi- Gnd ne, 1a¥eFs~ The height of the epithelium progressively decreases towards the sien’ the duct. In certain instances, small longitudinal folde nt ene epithelium deen Gore of lamina propria may be seen - preeaging the noxe segment, the ductus deferens. DUCTUS DEFERENS Slide 53a, Spermatic cord, H&E 53by Spermatic cord, Masson, H&E/HaC his 4s a much bigger duct than the previous seguent. Its Junction with the ductus epididymis is marked by an abrupt enlargement of the lumen and thickening83 XVIIL.4 of the muscular coat. While the ductus deferens is more frequently thought of as a felativel straight duct in the spermatic cord, its beginning may form a part of the tail of the epididymis and may be slightly coiled. The pseudo stratified epi~ thelium is lower in height but is similar in composition and function to that of the ductus epididymis. The luminal profile is irregular in cross section because of longitudinal mucosal folds. The smooth muscle coat is very thick and consists of 'an inner and outer longitudinal layer with a middle circumferential layer. An adventitia is recognizable as a coat in the straight portion of the duct, but in its initial coiled portion the convolutions are embedded in a common connective tissue contiguous with that embedding the coils of the ductus epididymis. The ductus deferens is the important component of the gross structure called the spermatic cord. It is possible, therefore, that you will encounter in your slide the other components of the cord, such as arterioles, venules or larger vessels, lymphatics, nerves, and strands of skeletal muscle. AMPULLA OF THE DUCTUS DEFERENS (No slide available) This is the segment of the ductus deferens that stretches from its ureteral Grossing to the surface of the prostate. It is dilated and fusiform in shape. Its fetucture is different enough from the preceding segment of the ductus deferens co be listed separately. The mucosa forms numerous complicated branching folds that fuse in many places to form net-like partitions seen in tissue sections. The epi- thelium is pseudostratified; its cells appear to have secretory function. Between the bases of the folds, there may be gland-like invaginations lined by simple columnar epithelium. The smooth muscle of the ampulla is much less regularly arranged. SEMINAL VESICLE Slide 55, Seminal Vesicle, H&E ais As an evagination of the distal end of the ampulla and consists of @ long highly coiled tubule that has essentially the same structure as the latter. However, its mucosal folds are longer and more intricate, and the smooth muscle layer is Fhinner. Connective tissue that is rich in elastic fibers holds the coils of the tubule together, EJACULATORY DUCT (No slide available) {his segment passes through the prostate and opens into the urethra beside the colliculus seminalis. The epithelium is simple or pseudostratified columnar hich nay Possibly be secretory. The epithelium may become transitional in type near its opening in the urethra. Thin mucosal folds project into the lumens PROSTATE GLANDS Slide 54a, Prostate, H&E, or Masson & H&E 54b, Old Prostate, H&E This 1s composed of 30 to 50 tubulo-alveolar glands held together by a thick ftbromuscular connective tissue. There are about 16 to 32 excretory ducts that qhen Andependently into the prostatic urethra. There is a tendency towards saccular formation of the alveoli. The glandular epithelium is usually simple o peesdoccnan [ified columnar although some areas may be simple cuboidal or even squamous, Prostatic concretions called corpora amylacea, are rounded bodies found in cheBh XVIIL.S fambe, of the alveoli. They are absent or rare in the very young but increase in umber with age. They may become calcified. The prostace surrounds the prostatic tn vardouc gs umerous smooth muscle fibers in the connective tiecve are scattered sphincter. MALE URETHRA (No available clide, but some slides of the Prostate may show a €ross section of the prostatic urethra.) ‘This ts the final conmon pathway for spermatozoa and urine. The male urethra Tay be divided into three segments, based on location. Each segment has a charac Feristic cross-sectional profile of its lumen. FROSTALIC URETHRA ~ This 4s the initial segent. It 4s surrounded by the pee aes ete luminal cross section 1s sometimes likened to on inverted " Pecause of the colliculus seminalis on ite posterior weil’ Its liming epi- thelium is transitional in type. NENBRANOUS URETHRA - This is the middle segment. It passes through the uro- Senital diaphragn and, therefore, is surrounded by skeletal muscle. It is the Shortest segment. The lumen 1s stellate in cross cection because of the pre~ CAVERNOUS URETHRA (or penile urethra) - This is the longest segment. It passes through the corpus spongiosum (urethrae). In cross Section, its lumen is a tngueverse slit in the region of the penile shaft, but is vertical slit at the Tesion of the fossa navicularis. The Lining epithelium is usually strati- fied or pseudost ratified columnar throughout tes length, except in the fossa navicularis which is lined by stratified squamous epithelium. The epithelium of the urethra, especially the last two Segments, is supported Pyreose connective tissue. Smooth muscle fibers in thie tissue are predominantly jongitudinally oriented near the epithelium. Some circularly arranged strands may Pe encountered more peripherally. Goblet cells may be found in the epithelium. Intra-epithelial cysts occur throughout the length of the urethra. Hucosal recesses called lacunae of Horgagni, are found Particularly in the plands or uetitd Segments of the urethra. At the bottom of vice recesses are the Slands of Littre which are invaginations lined by essentially the same epithelium as that of its urethral segment of origin. Intraepithelial cysts are common in the Slands of Littre. BULBOURETHRAL GLANDS (of COWPER) (No slide available) ‘hese consist of a pair of compound tubulo-alveolar glands that are apparently the secret mature. While the excretory ducts enpty through the cavernous urethra, the secretory portions are associated with the membranous urethra and are embedded in the sphincter urethrae.8s XIX.1 XIX. FEMALE REPRODUCTIVE SYSTEM The female reproductive system consists of the ovaries, oviducts, uterus, vagina, external genitalia, and accessory glands. During pregnancy and lactation, the pla- centa and mammary glands are also important organs. During the reproductive years of a woman, histologic changes occur in the uterus, oviducts and ovaries. The uterus undergoes marked changes in its endometrium. Less marked changes occur also in the oviducts. Changes in the ovaries involve maturation of the ova and the accompanying development of associated granulosa cells and con- nective tissue elements resulting in the formation of mature ovarian follicles and their transformation after ovulation. Tissue changes in theseorgans are coordinated with each other. These are greatly influenced by hormones secreted by the hypophysis, hypothalamus and ovarian follicular cells acting on their respective target organs. During pregnancy and lactation the placenta secretes hormones that result in the prolongation of certain phases of the ovarian cycle. After the reproductive years, regressive changes occur in the uterus which are apparently due partly to the absence of ovarian hormones. Since the chief activity of the ovaries is the development of the ova, when the store of ova has been exhaust- ed, ovarian regression becomes final and complete. THE OVARY Slides available: 45a, Ovary, Graafian follicle, H&E/i&¢ 45c, Ovary, Corpus luteum, H&E 45c, Ovary, Corpus albicans, H&E Using the LP lens, scan the sections of ovaries in slides 45a, b, and c. Identify the following: germinal epithelium, cortex, and medulla. The germinal epithelium is either simple squamous or cuboidal. It covers the surface of the ovary except the hilus. Beneath the epithelium is a thin layer of dense connective tissue called tumica albuginea. It is not sharply demarcated from the cortex but its elements are parallel to the surface of the ovary while those of the cortex form whorls. The cortex isa thick layer beneath the tunica albuginea. It encloses the centrally place medulla except at the region of the hilus. The cortex consists of a stroma of fine collagenous and reticular fibers, and many spindle-shaped or fusiform cells arranged in many curving directions, imparting upon this layer its characteristic “swirly” texture. Blood vessels in the cortex are smaller than those in the medulla. It is in this layer where you will find the ovarian follicles in various stages of develpment, and their derivatives and remnants. The earliest stage of follicular developmentis seen in the superficial part of the cortex, close to the tunica al- buginea. The later stages are found in the deeper part of the cortex. However, since the follicle increases in size as it matures, you will find that it progressively occupies a greater portion of the thickness of the cortex. Although they differ in texture, the cortex is not sharply demarcated from the medulla. ‘The medulla consists of looser connective tissue with coarser collagenous fibers than in the cortex. It has many elastic fibers and fibroblasts. Smooth muscle fibers and adipocytes may also be seen especially near the hilus. (The hilus is not always included in the section.) The presence of many coiled arteries is characteristic in the medulla. In a tissue section these would present as many cross sections of Jarge arterioles and small muscular arteries. Still using your LP lens, look for the three stages of follicular development chronologically summarized as follows: 1, Primordial Follicle (unilaminar follicle). Use Slide 45a, This is the earliestXIX.2 86 stage of a follicle. It consists of an ovum (primary oocyte) surrounded by a single layer (unilaminar) of squamous follicular cells. The follicle is in the superficial part of the cortex, close to the tunica albuginea. 2. Primary Follicle (multilaminar follicle). Use Slide 45a. This is the second stage of development. The follicle is situated deeper in the cortex. It is char~ acterized by the following: - The ovam is bigger than the one in the preceding stage. = The zona pellucida develops around the ovum, and becomes progressively thicker. - Follicular cells grow in height and proliferate to become stratified (multi~ laminar). As the follicle develops further, the layers of follicular cells (now more appropriately called granulosa cells) continue to increase. Observe that unlike other stratified epithelia, the granulosa cells are not compactly arrang- ed but instead show spaces around each coll. = A basement membrane becomes very evident between the granulosa cells and the cortical stroma. = The cortical stroma immediately surrounding the follicle has specialized to form the theca folliculi.Note that the cells of the theca show a predominantly circumferential orientation around the follicle. In the more advanced stage of development of the primary follicle the theca folliculi shows differentiation into two layers, namely, the theca interna, adjacent to the granulosa cells, and the theca externa. The theca interna is darker staining and more cellular. The theca externa is lighter staining, is less cellular, and its fibers are looser and less circumferentially oriented. 3, Secondary Follicle (antral follicle). Use Slide 45a. Slide 45b may also show this. This is the last stage of development of the follicle. It is characterized by the following: = The ovum will attain its maximum size at this stage, and will complete its first maturation division to form the. secondary oocyte and first polar body just before ovulation. The second maturation division will immediately start, but since it will go to completion only if it is fertilized after having been ovul- ated, the mature ovum and the second polar body is not usually seen in the ovary. - The follicular antrum appears at this stage so that the ovum becomes eccentric in position and is attached to the membrana granulosa only through the cumulus oophorus. = The theca interna and externa are fully developed. The theca interna is highly vascular but you have to look closely to appreciate this because the vessels are capillaries. Many cells contain small lipid droplets. In the theca externa, the cells are smaller and look like fibroblasts. Arterioles, that supply the capill- aries of the theca interna, are found in this layer. Because the blood vessels are larger here, you may form the impression that the theca externa is more vascular. - The Graafian Follicle is the fully developed or mature follicle. It has attain~ ed maximum size and usually occupies the full thickness of the cortex. The big follicular antrum containing the liquor folliculi is mainly responsible for the large size of the follicle. Atretic Follicle - Atresia or degeneration of a follicle can occur at any time during its devel opment. Initial changes may appear either in the ovum or in the follicular cells, depending on the age and/or size of the follicle. The type where Anitial changes occur in the ovam are seen in primordial and primary follicles. Atresia starting in the follicular wall (including theca interna) occurs in the bigger follicles, especially secondary follicles; this type is usually seen in the sexually mature woman. Atresia of a small follicle usually leaves very little trace of its4IX.3 87 disappear, co resi# of @ large follicle entails a longer time for tissue changes to qiseppear, ‘so that there is a better chance of sceing it in the speciacn: tee fol- lowing structural characteristics are indicative of the latter type of atresia: Tree profile of the follicle is not smoothly round or oval, but is irregular. ‘The follicle may appear to be collapsing. The ovum may still be preseac. be seen. anrebenerated follicular cells and connective tissue invasion of the follicular antrum may be seen. Corpus Luteum. Use Slide 45b, Ovary, Corpus luteum, H&E. After ovulation, changes gecur in the follicle that transform it into a corpus luteum. The following are important structural features of the corpus luteum: crete et may show evidence of hemorrhage in specimens obtained soon after Rurgation: Those taken long after the event show a hyaline type of locge cow nective tissue in the center. celine) rantiosa cells have transformed into granulosa lutein cells (true lutein Show oyind form thick folds of stratified cells around the center. The celine show evidence of lipid content in the form of small vacuoles, cyene theca interna cells have transformed into theca lutein cells. ‘They are cytologically and functionally similar to the true lutein cells although they seiner: darker staining, and have a more compact nucleus. Thece loved the gate Scattered in groups around the periphery, esp. between the folds of the true lutein cells. ~ There are many small blood vessels, esp. capillaries, among the lutein cells. Trang Corpus luteum attains various degrees of development. The atretic follicle tpansforms into a corpus luteum atreticum before becoming hyaline connective eonaue and finally disappearing. If ow lation without fertilization occurs, the poriicle transforms into a corpus luteum of menstruation and isco Le days fefore regressing into a hyaline tissue called corpus elbicane, Te there is fertilization, a corpus luteum of pregnancy is formed which laste ance six Tonths before gradually regressing until term. The corpus luteum of pregnancy is the biggest corpus luteum. Gorpus Albicans. Use Slide 45¢, Ovary, Corpus albicans, H&E. This is composed of Bary fwaline elements anong collagenous fibers. Cello are few. The size nt the how Long aneuends on the size of the corpus luteum from which it originated od how long after its formation the specimen was obtained. The largest and most per- Sistent corpus albicans is derived from the corpus luteum of pregnancy. ‘THE ovipucTs Slide Available: 46, Fallopian tube, H&E. ee hes paized organ. The oviduct is a tubular structure. Its medial end Cavity veg an the wall of the uterus. Its lateral end opens into the pecicveal GRIALY Wery close to the ovary. It has four segments, nanely, the intramural part which passes through the uterine wall, followed by the tethves which is the slender fee Eine ence cxPanded ampulla, and finally the funnel-like infundiberen with ite finger-like fimbriae very close to the ovary. re phas three layers: mucosa, muscularis, and serosa. The mucosa is composed of qisauble columnar lining epithelium supported by a lamina propsias There are no Blands here. The epithelial cells are of two types: ciliated velig which are more Minerous in the ampulla and infundibulum, and secretory cells. The tntcor predom-88 XIX.4 inate in the whole epithelium. The mucosa is thrown into longitudinal folds. The folds are tall and richly branching in the ampulla and infundibulum. These folds decrease in number, height and complexity towards the medial end of the tube. The mucosal folds are reduced to mere longitudinal ridges in the intramural segment. The muscularis is composed of inner circular and outer longitudinal layers of smooth muscle. The investing peritoneum is the serosa. ‘THE UTERUS Slides Available: 47a, Uterus, early estrogenic, H&E, proliferative phase 47b, Uterus, late estrogenic, H&E, proliferative phase 47c, Uterus, secretory, H&E The uterus has three layers: 1, Endometrium - This corresponds to the mucosa and consists of a. a simple columnar epithelium similar to that of the oviduct. Mucous secretory endometrial glands derived from the epithelium is invaginated into the underly- ing stroma. The glands are simple tubular with slight branchings at the bottom and, during active sexual life, vary considerably in height, diameter, coiling, and secretory activity, depending on the functional state of the organ. b. endometrial stroma - This corresponds to a lamina propria. Its amount and character varies with the stage of the endometrial cycle or pregnancy. ‘THE ENDOMETRIAL CYCLE: roliferative Phase - The endometrium is thinnest immediately after menstruation. After this, it will progressively increase in height in response to estrogenic hormones being secreted by the ripening ovarian follicles. This is called the Proliferative phase because there is proliferation of the endometrial components. The glands are lengthening and the endometrial cells are increasing in number, with many mitoses. The perpendicular endometrial blood vessels are also length- ening but do not reach the more superficial part of the endometrium. This stage is also called the follicular phasereferring to the fact that the changes going on in the endometrium are due to the activity of the ripening follicles which are secreting estrogen. Secretory Phase - Here, the endometrium may still increase in height but this is mainly due to a form of edema in the endometrial stroma, and accumulation of secretory products in the lumen of the endometrial glands. This phase is char- acterized by the widening and coiling of the glands with evidence of much secre- tory activity. The arterioles have lengthened further, reaching the superficial Parts of the endometrium, and are very coiled. The venules have wide lumina (sinuses). Just before the onset of the menstrual period, exten- sive coiling and widening of the glands have reached such an extent that the endometrium appears to have plenty of holes under the LP lens. This is some- times referred to as "Swiss cheese endometrium". This period is called secretory phase because the glands are actively sec- retory. It is also called the luteal phase because the changes occuring at this time are chiefly the result of stimulation by progesterone which is being sec- reted by lutein cells in the ovary. This is the period when the endometrium becomes ready to receive the fert- ilized ovum. The endometrial cells are the ones that will transform into decidual cells should implantation of the owm occur.XIX.5 89 Menstrual Phase - Onset of this phase is characterized by a decrease in height of the endometrium due to loss of interstitial fluid and cessation of secretory activity of the glands. The interstitial elements of the endometrium are brought closer together because of shrinkage. The endometrium looks darker because the nuclei of endometrial cells are closer to each other. Blood vessels are less evident. The glands appear to be collapsing. Later in this period, blood vessels break up so that you may see interstitial hemorrhage in the endometrium. The superficial part of the endometrium (functionalis) sloughs off leaving behind its basal part (basalis). The raw surface of the endometrium is relined by epithelium through proli- feration of cells in the remaining part of the glands in the basalis. 2. Myometrium - This is composed of about four smooth muscle layers. The muscle fibers increase in size and number during pregnancy. The supporting connective tissue consists of collagenous and reticular fibers that pass between individual muscle fibers and fiber bundles. 3. Perimetrium - This is actually covering peritoneum. This is present only in the fundus, the posterior surface of the corpus down to the supravaginal portion of the cervix, and approximately the upper two third of the anterior surface of the corpus. Over the rest of the uterus and supravaginal part of the cervix, only loose connective tissue make up the third layer; sometimes this is referred to as the parametriua. In the vaginal portion of the cervix the third layer is a continuation of the epithelium of the vagina. CERVIX UTERI Slide Available: 48a, Vagina and Cervix, H&B Using the LP lens, scan the specimen. Identify the part of the cervix that projects into the lumen of the vagina. Observe that the vaginal epithelium, which is stratified squamous, is reflected unto the vaginal surface of the cervix. Notice that this epithelium is abruptly replaced by a simple columnar type somewhere at the beginning of the cervical canal, near the external os. Examine the mucosa of the cervical canal. Although the specimen is a longitudinal section, you will see parts of its branching folds. There are depressions in the mucosa some of which lead into large simple branched tubular glands that are characteristically oriented obliquely or almost parallel to the surface of the mucosa. Epithelial cells of the mucosa and cervical glands are apparently mucus secreting. The lamina propria is somewhat dense and cellular. The middle layer of the cervix is mostly dense fibrous connective tissue com- posed of collagenous and elastic fibers, and fibroblasts. There are few smooth muscle fibers, especially in the vaginal part of the cervix which some investi- gators claim do not contain any. VAGINA Slides Available: 48a, Vagina and Gervix, N&E 48b, Vagina, H&E Using the LP lens, identify the three laryers of the vagina, as follows: 1, Mucosa - This is lined by stratified squamous non-keratinizing epithelium sup- Ported by a lamina propria that is somewhat dense immediately beneath the epithel- ium, becoming looser towards the muscularis. There are transverse folds in the mucosa. There are plenty of elastic fibers just beneath the epithelium. There are many lymphocytes in the connective tissue. Lymph nodules are not uncommon.90 XIX.6 2. Muscularis - There are interlacing bundles of longitudinally and circularly arranged smooth muscle, The longitudinal fibers are more abundant, and superior~ ly are continuous with the myometrium of the uterus. The circalar fibers are more superficial. Skeletal muscle from the bulbocavernosus is arranged around the vagina at the region of the introitus and constitute its sphincter. 3. Adventitia - This is dense connective tissue with many coarse elastic fibers. This layer blends with the connective tissue of the surrounding structures. There are many venous plexuses here. There are also many nerves and some ganglion cells. THE PLACENTA Slides Available: 49a, Placenta, H&E 49b, Placenta, early, N&e 49c, Placenta, late, Had The placenta is a temporary organ which develops during gestation. Lt is made up of components from the embryo (chorion) and the endometrial stroma (decidua basalis) of the uterus. You will have to read your textbook before trying to inter- pret the histology of the placenta in the laboratory. The following might help you The placenta develops adjacent to the amnion of an embryo during the Lith to 16th gestational day. Syncitiotrophoblasts in this area surround the blood vessels of the endometrial stroma. The wall of these blood vessels become eroded (opened up by the invasive trophoblastic cells). When the intima of the blood vessels disappear, the vessels are called lacunae. Lacuaae intercommunicate with each other and with the venous sinuses of the endometrium. The first villi arising from the chorionic plate are solid cords of trophoblastic cells that grow outward through the labyrinth of syncitial trophoblasts and lacunae. These are called primary chorionic villi. The base of the primary villi are then invaded by mesenchyme from the chorion. These villi are now called secondary villi. They consist of an outer layer of syncitiotrophoblastic cells beneath which is a layer of cytotrophoblastic cells. The core of the villus is mesenchymal connective tissue. The intercommuni- cating and expanded lacunae are now collectively called intervillous space. When the connective tissue core of the villi become vascularized, they become tertiary villi or definitive placental villi. The name of these three villi, therefore, refer to their degree of development. The primary villus will not be seen in older placenta. Transient secondary villi will continue to arise as long as the placenta is still growing in size. ‘The next set of terms refer to the position or function of the villi. From the tips of the secondary villi, solid cords of cytotrophoblastic cells grow toward the opposite wall (basal plate). These are the cytotophoblastic cell columns, The cells from these columns, upon reaching the opposite wall, spread out laterally and coalesce with similar ones from neighboring cell columns, resulting in the formation of the so-called trophoblastic shell. These cell columns are called anchoring villi. The villi from which they arose are called stem villi. The stem villi branch extensively, establishing several generations of branches. The finest branches are very slender and their tips remain free in the intervillous space. They are called terminal villi.-In the well established placenta, most of the cytotrophoblasts are gone, and the syncitiotrophoblastic cells form a very thin lining in which large areas may be devoid of nuclei. Capillaries in the core of these villi are situated very close to these thin areas. The maternal component of the placenta is derived from decidua basalis. This is not always seen in the class specimens. Decidual cells are large and light stain-91 XIX.7 ing, and may appear finely vacuolated. In this vicinity, relatively large arteri- oles and venous sinuses, not enclosed by trophoblastic cells belong to the maternal part. THE PEMALE MAMMARY GLAND Slides Available:5la, Resting Mammary Gland, H&E 51b, Gestational Mammary Gland, H&E Slc, Lactating Mammary Gland, H&E ‘The mammary gland is compound tubulo-alveolar in type. Since each lobe has a duct (lactiferous duct) that empties independently to the outside through the nipple, the mammary gland is actually a group of glands. Each lobe is separated by dense connective tissue and adipose tissue. The lobe is divided into lobules through a system of branching ducts. The secretory portions consist of alveoli that arise from alveolar ducts. The interlobular connective tissue is dense and contains plenty of adipocytes during the resting state of the gland. The intralobular connective tissue is looser in consistency and is cellular in character. There are usually no adipocytes here. The lining epithelium of alveoli and alveolar ducts is simple cuboidal to columnar. In the hierarchy of ducts, there is an increase in height of the epithelium as bigger ducts are examined, becoming stratified squamous in the main or lactiferous duct. Changes in the mammary gland during active sex life are chiefly seen in two areas of the tissue, namely, the connective tissue and the tubular systen. There seems to be a suggestive reciprocal relationship between these two components in which the tubular elements are extensive while the connective tissues are very much reduced in the active gland, especially during pregnancy and lactation. The reverse is found in the resting state. It is from this point of view that you may want to guide your study of the mammary gland. The LP lens is most useful here. The HP lens perhaps will be useful only if you want to look for myoepithelial cells in the alveoli. Resting Mammary Gland, Slide 5la. Observe that there is abundance of adipose tissue in the specimen, You will see “islands” of relatively dense connective tissue containing a few cross sections of alveolar ducts. Gestational Mammary Gland, Slide 51b. Lobulation is easily perceived here. The lobules are in the form of large compact groups of alveoli which predominate in the parenchymal areas. The lumen of the alveoli vary in size. They are empty and small in some, and somewhat larger in others, and may contain some acidophilic material, presumably, colostrum. ‘The lobules are surrounded by relatively thinner dense connective tissue. Ducts are easier to find in the connective tissue septa. Observe that the number of adipocytes is greatly reduced. Lactating Mammary Gland, Slide 5lc. Lobulation is also very evident here. The connective tissue septa are much thinner compared to the gestational gland. Dilataion of the alveoli and ducts is very common. The degree of dilatation of the alveoli in the various lobules is not uaiform. Zhe ducts in the septa are also frequently dilated. In many alveoli and ducts, the lumen contains acidophilic granular material. This should be milk.92 XX.1 XX. ENDOCRINE GLANDS Endocrine glands are ductless glands whose secretions (hormones) reach the cells or tissue they will influence through blood vessels (and lymphatics). Small amounts of the hormones are sufficient to affect the target cells and only small amounts are normally secreted by these glands. There are many cells or organs that are endocrine in nature. Only the big ones that are permanent and fixed in loca- tion are included in this chapter. These are the hypophysis, thyroid, parathyroids, adrenals, and epiphysis cerebri. HYPOPHYSIS Slide recommended for study: 19b, Hypophysis, H&E The hypophysis develops from two sources, the dorsal outpocketing from the the roof of the embryonic pharynx (Rathke's pouch), and the ventral growth from the floor of the diencephalon. The hypophysis, therefore, has two general compo- nents with contrasting textures. The adenohypophysis is cellular in texture. It is derived from the pharyngeal growth. The neurohypophysis is fibrous in texture. It is derived from the diencephalon. Scan the specimen to locate the subdivisions and various regions of the organ. Identify the body and hypophyseal stalk. The stalk marks the superior part of the organ. Examine the fibrous connective tissue capsule. It is somewhat loose in character. There may be some adipocytes present. Note that there are some in~ ward extensions of the capsule, in the form of trabeculae. ext locate the adeno- hypophysis and neurohypophysis and their respective components. ADENOIYPOPHLYSIS This is anterior to the neurohypopiysis. It has three regions: 1, PARS DISTALIS (anterior lobe) - This constitutes the bulk of the adenohypophysis (about two thirds of the gland). 2. PARS TUBERALIS This is the thin upward extension of the pars distalis. It forms an incomplete cuff around the infundibular stem, and occupies the anterior and la~ teral boundaries of the hypophyseal stalk. 3. PARS INTERMEDIA - This is interposed between the pars distalis anteriorly and the infundibular process posteriorly. Associated with this region are colloid- filled cystic follicles of various sizes, and lined by cuboidal to columnar epi- thelium Note the cellularity of the adenohypophysis. The cells are generally arrang- ed in round groups (cross section of cords) in most parts of the region. The cords are separated from each other by sinusoids, and a scanty amount of connective tissue fibers and cells. The sinusoids in most ‘class slides are empty or partially collapsed 80 that they may not be prominent in the tissue,XX.2. 93 PAKS DISTALIS Look for the cells that can be’identified in H&B preparations in the center of the adenohypophysis. There are three of ther 1. Acidophils - These possess eosinophilic (acidophilic) cytoplasmic granules. They are rouaded or polygonal in shape. Within a group of cells (section of a cord) they tend to occur in clusters next to a sinusoid , or singly in the interior of the cord. 2, Basophils - These have basophilic cytoplasmic granules. They occur as round, polygonal, or elongated cells. The elongated cells are more often found in the Anterior of the cord, while the round ones tend to be seen at the periphery, ad- jacent to a sinusoid. If you scan the entire adenohypophysis, you will notice that. in many instances, there are more basophils in the periphery than in the center of the lobe. Some of them may encroach into the anterior part of the neural lobe. 3, Chronophobes ~ These are pale cells. Sometimes the cytoplasm seems unstained. they are often clustered in the interior of the cord. While they are smaller than the acidophils and basophils, some of them may be as large as the other two. They tend to have a narrow cytoplasm around the nucleus. Some invetigators believe that chromophobes are a heterogenous group of cells composed of undifferentiated cells, and degranulated acidophils and basophils. You may be able to agree with this idea because these cells may sometimes be faintly acidophilic or basophilic, and some- times appear to have a few cytoplasmic granules. PARS TUBERALIS Go the periphery of the adenohypophysis, at the part above the pars intermedia. The pars tuberalis is included in your specimen if there is a marked formation of elongated cell cords that are parallel to one another. Correspondingly, the sinu- soids are also elongated and parallel to the cords. The area should be more vascu~ lar than the rest of the adenohypophysis. (Read up on the hypophyseal portal sys- tem.) The acidophils and basophils are smaller than those in the pars distalis. There seems to be more basophils here. PARS INTERMEDIA The cells here are generally smaller than those in the pars distalis. There is a greater number of basophilic staining cells here compared to the pars distalis. NEUROHYPOUYSIS The neurohypophysis is mainly composed of fibers of the hypothalamo-hypophy- seal tract. They are unmyelinated axons. This is why this part has a suggestive fibrous texture. This region 1s composed of three parts: (1) median eminence of the tuber cinereum, (2) infundibular stem (with the median eminence it is called neural stalk or infundibulum), and (3) infundibular process (also called pars nervosa; with the pars intermedia it is sometimes called the posterior lobe of the hypophysis . ‘ie slides usually contain the infundibular process only. Note that the fibrous components of the pars nervosa are distributed in curves and whorls, Note also the presence of small capillaries and small cells with dark staining nuclei, These cells are usually pituicytes. Some of them may have yellow94 XX.3. (1ipochrome) pigment granules in their cytoplasm and processes. The pituicytes are believed to be a form of neuroglia. Some investigators believe that other types of glial cells are fouad here. Be alert to the presence of Herring bodies. These are basophilic granular structures found among nerve fibers. They are accumulations of neurosecretions along an axon or axon terminal. Although it is claimed that it is not satisfactorily stained by hematoxylin in routine ll&E preparations, nevertheless, you may see them in your slide. ‘THYROLD GLAND Slide Availabl 20, Thyroid, W&E This has two dense connective tissue capsules. The outer one is part of the deep cervical fascia. The inner one is the true capsule. In the class slides, only the true capsule is included with the gland. It is adherent to the gland and sends septa into the interior to divide the parenchyma into lobules. Each lobule is con- posed of follicles. Using the LP lens, look for the follicles. These are somewhat globular in shape. They vary in size but in humans the smaller ones predominate. The wall of the follicle is usually lined by simple cuboidal epithelium. Its cavity contains colloid, the storage form of the thyroid secretion. It is gel-like and has varied staining, but is more often acidophilic. The follicles are separated by scanty interstitial connective tissue composed of reticular fibers embedded in much ground substance. The fibers are directly con- tinuous with those of the capsule and septa. This stroma is similar to the lamina propria of the intestines in the sense that, not uncommonly, some areas look like diffuse lymphatic tissue and, occasionally, you may encounter a solitary lymphatic nodule. There is a rich capillary network, lymphatics, and nerves in this connect- ive tissue. The epithelium of the thyroid follicle is compo sed of two types of cells: the principal or follicular cell, constituting the major population, and the parafolli- cular cell. The parafollicular cell is very difficult to identify in N&B prepara- tions and uader the LM. It is usually larger than the follicular cell and is paler staining. Characteristically, it occupies a lower level than the follicular cells in the epithelium. [ts apical part does not reach the general level of the epithel- ial surface because it is often "bridged over" by the apical portions of adjacent follicular cells. The parafollicular cell has also been seen in the interstitial connective tissue of the gland. Variations in the structure of the follicle include differences in size and shape, type of epithelium, and, amount and staining charac~ teristics of the colloid. In the hypofunctioning gland, the follicles are usually bigger and contain more colloid which is acidophilic staining. The epithelium is low, even squamous in type. In the hyperfunctioning gland, the follicles are usually smaller and irregular in shape. [ts colloid may be basophilic and less in in amount. Its epithelium is often columnar and may be folded into the cavity of the follicle.95 XX.b PARATIYROIDS. Slides Available: 21a, Parathyroid, H&E 21b, Parathyroid, H&c There are usually four parathyroids at the back of the thyroid gland. Their capsule is usually shared with the true capsule of the thyroid. Reticular fibers in scanty ground substance support the parenchyma. Capillary networks and nerves between the masses of parenchymal cells are also supported by these fibers. Trab- eculae extend into the interior and divide the gland into incomplete lobules. Most blood vessels enter the gland through these trabeculae. Parenchymal cells form irregular cords or groups. Infrequently, these cells may form follicles containing colloid. Adipocytes are present in the interstitial tissue and increase with age. * The parenchyma is composed of two cell types: the principal cell or chief cell, and the oxyphil cell. Chief cells are more numerous than oxyphils. In gener- al, they are polygonal in shape and are smaller than oxyphils. Their vesicular nucleus is centrally located. Some investigators claim that there are two types of Principal cells. one shows a faintly acidophilic cytoplasm and contains large gly~ cogen deposits (undemonstrated in class slides). The other has a more intense cyto- Plasnic basophilia and contains less glycogen deposits. Perhaps these two "subtypes" are mere expressions of different physiologic phases of the same cell. However, you will notice that there are indeed some chief cells that are more acidophilic than others. The oxyphil cell is larger than the principal cell. It may be scattered as solitary elements among the chief cells, or several may form small clusters. It is often easier to find oxyphils in the periphery of the gland. Tae nucleus is smaller and darker staining than that of the chief cell. The cytoplasm is strongly acido- philic due to plenty of mitochondria. It is usually granular in appearance. Since the ‘smaller nucleus is darker staining and the acidophilic cytoplasm has greater volume than the chief cell, a cluster of oxyphils, will stand out among the chief cells due to the contrast of a group of wider spaced dark nuclei amid closer spaced paler ones. The oxyphils are usually absent at birth. They start to appear between 4% to seven years and increase in number with age, especially after puberty. In older persons, there is an increase of connective tissue, adipocytes, and oxyphils, There may also be an increased tendency to form swall follicles with colloid. ‘ADRENALS. Slide Available: 22, Adrenal gland, H&E, il&C The adrenals are relatively thin organs. At about the middle of each gland, and facing anteronedially, is a slight indentation called the hilus. The adrenal vein exits through the hilus. An artery may enter it, but most of the arterial supply comes from branches ramifying over the capsule and sending branches through96 XX.5, it to a subcapsular plexus. The fibrous capsule sends trabeculae into the interior and is increased in amount at the hilus. Reticular fibers continue from the capsule and trabeculae into the parenchyma to provide the main support for its cells and blood vessels. There are trabecular arteries from the capsular arteries. These are destined for the medulla. They are called medullary arterioles. Scan the specimen with your LP lens. Look for the thicker cortex which enclos- es the much thinner medulla. Cortical cells are usually acidophilic while medullary cells tend to be basophilic. Note that there is an easily recognizable pattern of cellular grouping in the cortex (cords) compared to the medulla. Also, large veins in the interior of the organ are usually found in the medulla. The cortex and medulla are actually two different glands that became incorporated into one organ during development. ADRENAL CORTEX - This has three zones that are not sharply demarcated from each other: 1. Zona glomerulosa - This is a narrow region, about two to three cells thick, loc~ ated immediately below the capsule. The short columnar cells are grouped in. irreg- ular masses and cords. The scanty cytoplasm is acidophilic but there might be some basophilic staining masses in it. There may be a few fine lipid droplets. The nucleus is often dark staining. 2. Zona fasciculata - This 1s the thickest zone. It is between the zona glomerulosa and zona reticularis. The cells are polyhedral and bigger than those of the zona glomerulosa. They are arranged in straight cords that are perpendicular to the cap- sule. The cytoplasm is acidophilic but contains many large lipid droplets that often impart a foamy appearance upon the cytoplasm, and lightens the color of the cells. Mitotic figures may sometimes be seen at the transition between this zone and the zona glomerulosa. 3. Zona reticularis - This is the innermost zone. It abuts into the medulla. The cells are arranged in irregular branching and anastomosing cords. The cells are generally acidophilic. They are smaller than those of the zona fasciculata, and contain less lipid in their cytoplasm. Two other cell "types" may be seen in this layer, the light cell, which has a pale staining cytoplasm and nucleus, and the dark cell, which has a dark staining almost pyknotic nucleus and a dark staining cytoplasm. It may contain lipofuscin in its cytoplasm. Because of the branching and anastomosing nature of the cords, you will find many cross and oblique sections of the cords surrounded by medullary tissue. Examine the spaces between the cell cords in the cortex. Note that these are occupied by sinusoids whose walls closely conform to the shape of the spaces. ADRENAL MEDULLA This 1s very thin and is surrounded on all sides by the cortex. It is easily distinguished from the latter by its basophilia and the presence of a thick-walled vein. This is the central vein (or medullary vein at the hilus). Note that the smooth muscle of this vein is longitudinally oriented and unevenly distributed, being thicker at one side. Also notice that the smooth muscles are arranged in bundles. The parenchyma is mainly composed of chromaffin cells, which are irregu- larly polyhedral with basophilic cytoplasm. They are epithelioid and are arranged in irregular masses and cords. There are also a few sympathetic ganglion cells (sometimes surrounded by satellite cells) that may be scattered singly or in small groups, and stall round cells with scanty basophilic cytoplasm (probably lympho-XX.6 97 cytes). There are many capillaries here. These are branches of the medullary arter- joles. Look for venules. in the medulla. The smaller ones are found near the corti-~ co-medullary junction. These receive the cortical sinusoids. Medullary capillaries are also connected to them. (This arrangment suggests a venous portal system.) Bigger venules are usually more centrally located. They drain the venules in the cortico-medullary border. Sometimes, you may see one connected to the central vein. EPIPHYSIS CEREBRI (PINEAL GLAND) Slides Available: 23a, Epiphysis cerebri, H&e 23b, Epiphysis cerebri, Masson, HaC, Silver This is a small flat conical body that is attached through its stalk to the Posterior part of the roof of the third ventricle of the brain. The third ventri- cle sends a shallow extension into the ventricular surface of the stalk (pineal recess) which is lined by ependyma of the ventricle. Pia mater invests the surface of the organ as its capsule, From the capsule, septa containing the blood supply of the parenchyma penetrate the interior and divide it into small masses and cords of cells that are sometimes called lobules. The parenchyma has two general types of coll Parenchymal cells) and interstitial cells. + pinealocytes (chief cells or In H&E sections, pinealocytes have ill-defined borders and pale cytoplasm. Variably staining cytoplasmic granules may be seen. The vesicular nucleus has a well defined border. Silver stained preparations demonstrate long thin cytoplasmic Processes with endings that often terminate around blood vessels. Interstitial cells are perivascular in location, or are distributed between Sroups of pinealocytes. ‘They are composed of astrocytes, that provide support to the parenchyma, and phagocytic microglial cells. They have basophilic cytoplasm. Nerve Fibers mix with the processes of the chief cells and astrocytes and are more numerous near the epiphyseal stalk. Acervuli (corpora arenacea or brain sand) may be found in the parenchyma or in the stalk. These are intensely basophilic bodies, often showing a lamellar concentric structure. They are intercellular calcified organic matrix. O.M.T. --00000--