Histology

THE UNIVERSITY OF MEDICINE AND PHARMACY TRGU MURE DISCIPLINE OF HISTOLOGY
Histology
PRACTICAL WORKS
Guide
Prof. Dr. Angela Borda Senior lecturer Dr. Flp Emke Senior lecturer Dr. Camelia Gliga Senior lecturer Dr. Andrada Loghin Senior lecturer Dr. Marcu Simona Tnde Senior lecturer Dr. Cosmin Moldovan
2013
Contents
INTRODUCTION ............................................................................................................... 1 Principles of histological tissue processing ..................................................................... 1 Histological diagnosis ...................................................................................................... 3 Execution of drawing ....................................................................................................... 5 EPITHELIA ......................................................................................................................... 7 Covering and lining epithelia ........................................................................................... 7 Glandular epithelia ......................................................................................................... 14 THE CONNECTIVE TISSUE........................................................................................... 18 Cells of the connective tissue..................................................................................... 18 Fibers of the connective tissue ................................................................................... 21 TYPES OF CONNECTIVE TISSUE ............................................................................ 23 SOFT CONNECTIVE TISSUES .............................................................................. 23 CARTILAGE ............................................................................................................. 26 BONE ......................................................................................................................... 28 MUSCLE TISSUES........................................................................................................... 31 SMOOTH MUSCLE ..................................................................................................... 31 SKELETAL MUSCLE .................................................................................................. 32 CARDIAC MUSCLE .................................................................................................... 33 HEMATOPOIETIC AND LYMPHOPOIETIC ORGANS ............................................... 35 RED BONE MARROW ................................................................................................ 35 THYMUS ....................................................................................................................... 36 LYMPH NODE ............................................................................................................. 38 SPLEEN ......................................................................................................................... 39 MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)......................................... 41 Palatine tonsil ............................................................................................................. 41 NERVOUS TISSUE AND NERVOUS SYSTEM ............................................................ 43 NEURON ....................................................................................................................... 43 GLIAL CELLS .............................................................................................................. 44 CENTRAL NERVOUS SYSTEM ................................................................................ 45 PERIPHERAL NERVOUS SYSTEM (PNS)................................................................ 48 THE CARDIOVASCULAR SYSTEM ............................................................................. 49 THE VASCULAR SYSTEM ........................................................................................ 49 DIGESTIVE SYSTEM ...................................................................................................... 55 THE ORAL CAVITY .................................................................................................... 55 Lips ............................................................................................................................ 55 The tongue ................................................................................................................. 57 DIGESTIVE TUBE ....................................................................................................... 61 Esophagus .................................................................................................................. 62 The stomach ............................................................................................................... 63 Small intestine............................................................................................................ 67 Large intestine ............................................................................................................ 71 DIGESTIVE GLANDS ................................................................................................. 72 MAJOR SALIVARY GLANDS................................................................................ 72 THE PANCREAS ...................................................................................................... 76 THE LIVER ............................................................................................................... 78 RESPIRATORY SISTEM ................................................................................................. 80 TRACHEA..................................................................................................................... 80
THE LUNG .................................................................................................................... 82 URINARY SYSTEM......................................................................................................... 85 THE KIDNEY ............................................................................................................... 85 MALE REPRODUCTIVE SYSTEM ................................................................................ 90 THE TESTES................................................................................................................. 90 ACCESSORY GENITAL GLANDS ............................................................................ 92 FEMALE REPRODUCTIVE TRACT .............................................................................. 94 OVARIES ...................................................................................................................... 94 THE UTERUS ............................................................................................................... 97 THE VAGINA ............................................................................................................. 101 PLACENTA ................................................................................................................. 102 MAMMARY GLAND................................................................................................. 105 SKIN ................................................................................................................................ 108 ENDOCRINE GLANDS ................................................................................................. 111 HYPOHYSIS ............................................................................................................... 111 THYROID GLAND..................................................................................................... 114 ADRENAL GLAND ................................................................................................... 115
INTRODUCTION
The aim of histology practical works is to familiarize students with the structural features of different tissues and organs. They consist in microscopic selfstudy of histological slides obtained from different tissues and organs, observation of microscopic details based on which these can be recognized and then drawing elements that actually will determine the histological diagnosis. For a tissue to end up on a slide in order to be studied on the microscope, it has to undergo some complex and laborious histological processing techniques.
Principles of histological tissue processing

Histological tissue processing involves performing rigurous successive steps, each having a decisive influence on the final result. By not respecting these steps artifacts (distortions of cell or tissue architecture) can be produced and difficulties may appear in establishing histological diagnosis. The most important steps of this technique are: tissue sampling, fixation, embedding, sectioning and mounting. 1. Tissue sampling. It consists of sampling small tissue or organ fragments. For histology practical works, sampling material originates mainly in humans (cadavers or surgically removed parts) but also in experimental animals. If sampling is done from cadavers, it should be taken into account that the structure of tissue alters rapidly after death (for example the medulla of the adrenal gland, the mucosa of the digestive tube). As a result, sampling will be made from fresh cadavers, and the fragments will be introduced immediately in a fixative solution. 2. Fixation. It represents the second stage and has the role to stop metabolic processes on one hand and to preserve the structure of the tissue on the other hand. In histological practice, the most common fixative is the formalin, an aqueous solution of formaldehyde. It is used in different concentrations, but the most common is 10%. Formalin can be used by itself or in combination with other chemical substances (for example, formalin together with a saturated solution of picric acid and acetic acid, forming the Bouin fixative solution). Fixation is approximately 24-48 hours long, depending on the size of the fragment of sampled tissue. After fixation the sample is washed with tap water to remove the fixative. 3. Embedding in paraffin. For a piece of tissue to be embedded in paraffin it must undergo the following operations: - dehydration is the removal of water from the tissues, since water is not miscible with paraffin. Dehydration is done with alcohols with increasing concentrations (70%, 90%, 95%, 100% or absolute alcohol)
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- clarification or impregnation with a paraffin solvent that has the role to remove the alcohol used for dehydrating the tissue, because it is not miscible with the paraffin. It is necessary to remove it with a reagent that makes the sample translucent. Xylene, toluene or benzene is used for clarification. - impregnation with paraffin is a phase that is performed in a thermostat (55-56 C) with the aim to penetrate the samples with paraffin down to the deepest gaps. - embedding in paraffin. Fluid paraffin will be poured over the paraffin impregnated sample, placed in a special container. Paraffin will solidify through cooling and thus we will obtain a block of paraffin containing the tissue fragment, which is impregnated and surrounded by a medium that allows cutting the tissue fragment into very fine slices, 5-7 thick. At the same time, paraffin acts as a physical support for the tissue, allowing sectioning without cell structure and architecture distortion. 4. Sectioning will be performed with a special device called microtome. Each section obtained will be fixed with an adhesive on the histological slide (for example egg white albumin). The adhesive prevents the sample from detaching from the slide and its loss during staining. 5. Section staining is mandatory in order to be able to examine the tissue under a microscope, because cells are colorless. The first step in staining the sample is the removal of paraffin with xylene or toluene. Then, since the colorants are aqueous solutions (and the tissue was dehydrated prior to embedding), the sample on the slide must be rehydrated. This is done gradually using alcohols with decreasing concentrations, from 100%, to 95% - 90% - 70%, and then water. Gradual rehydration has the role to prevent the structural alteration of the tissue. In this state, the tissue can be stained. The most commonly used staining is hematoxylin eosin. 6. Mounting consists of applying a cover slip over the sample. This step is required in order to maintain tissue integrity during successive manipulations, to preserve the chromatic unchanged and to ensure a transparent medium necessary for examination with transmitted light. This transparent environment can be Canada balsam or synthetic resin, which are also not miscible with water. Under these circumstances, the preparation should be again dehydrated with alcohols with increasing concentrations and then cleared in xylene or toluene, substance that will mix with the mounting material. After the mounting material dries, the preparation can be examined under the microscope. Microscopic examination of the preparation is aimed at establishing a histological diagnosis, and is performed in several stages.
Histological diagnosis
A correct histological diagnosis should include:
1. Diagnosis of the staining In histological and histopathological practice two types of staining are used: common/usual stains and special stains. Usual/common stains color cellular and tissue structures in different ways, allowing histological diagnosis. The most frequently used common/usual stain is that with hematoxylin-eosin (HE), with which most of the tissues and organs presented during the Histology practical works are stained. Special or histochemical stains are used to identify and locate some particular chemical or enzymatic components of the cells. For example, polysaccharides such as glycogen can be highlighted by the periodic acid Schiff (PAS) technique. Periodic acid oxidizes the glycogen into aldehydes, which in turn will be stained in purple-red with Schiffs reagent. Lipids can be identified with lipid-soluble dyes: Sudan, Red Oil O (sections on ice will be used for lipid staining). A subset of special staining are histo-enzymatic techniques. These techniques are used to identify and locate a large number of enzymes such as alkaline and acid phosphatase, dehydrogenase, and ATP-ase etc. Since most enzymes are very labile and might be destroyed by fixation, cryosections are used for these techniques. In recent years, histochemical techniques have lost their importance compared to the modern techniques of immunohistochemistry, which can be also regarded as a subset of special staining. Immunohistochemistry is one of the most important innovations in histology, which has rapidly developed after its discovery. The principle in immunohistochemistry is based on immunology: monoclonal antibodies are used against some cellular components that play the role of an antigen. The antigen-antibody reaction will be highlighted by a visible marker. The number of currently used antibodies in histology and pathology rises to several hundreds. Examples of antibodies used in the histology and histopathology practice: -anti-cytokeratin antibodies for epithelia -anti-desmin antibodies for muscle tissues -anti-chromogranin antibodies to highlight neuroendocrine cells -anti-thyroglobulin antibodies to highlight the follicular cells of thyroid -anti-smooth muscle actin antibodies for smooth muscle cells Further, we are presenting the chromatic results/features of some stains commonly used in histology:
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- hematoxylin-eosin (HE) stains the cytoplasm in different intensities of red (eosinophilic or acidophilic cytoplasm) or blue (basophilic cytoplasm), and nuclei in blue-violet. They may be pale (hypochromatic nuclei) or intensely stained (hyperchromatic nuclei) or may have an intermediate shade (euchromatic or normochromatic nuclei). -van Gieson staining stains collagen fibers in red, muscle fibers in yellow, nuclei in black. It is usually used to differentiate muscle fibers from collagen fibers. -trichrome stains are using a mixture of three dyes to color differently some cell structures. There are many trichrome stains. For example, in Masson staining collagen turns green (or blue, depending on the dye used), muscle, erythrocytes and keratin are red, and nuclei are black-brown. Mallory stain colors the collagen fibers in intense blue, the cartilage, ossein, mucins in different shades of blue, the red blood cells and myelin sheaths in yellow, and nuclei and cytoplasm in red. - P.A.S. reaction (Periodic-Acid-Schiff) reveals the structures rich in acidic muco-polisaccharids (basement membranes, glycocalyx, goblet cells mucin), staining them in purple-red; - alcian blue staining is used for mucins, which appear coloured in blue; - Gmri silver impregnation specifically stains the reticular fibres in black; - resorcin-fuchsin method (Weigert) specifically stains the elastic fibers in dark blue. 2. Diagnosis of the tissue Recognition of a tissue on a histological slide is based on the following criteria: - general architecture of the tissue. An epithelium will be recognized because it is composed almost exclusively of cells of the same type, closely linked to each other by intercellular junctions. Epithelia always cover a connective tissue, from which they are separated by a basement membrane (that does not stain in HE). A connective tissue will be recognized because it consists of cells, fibers and ground substance in different proportions. The cells have different origins, are in smaller number than those of the epithelia and are separated from each other by the ground substance and fibers (extracellular matrix). - cell type, which we determine by analyzing the shape, size and arrangement of cells. We will deduce the shape of the cell after the shape of the nucleus, because the cell limits are sometimes hardly visible. Usually, a cuboidal (or polygonal) cell has a rounded nucleus, while a columnar or spindle-shaped cell has an oval nucleus, with the long axis perpendicular to the basement membrane if it belongs to an epithelial cell. We will examine whether the cells are arranged in groups (epithelia) or are isolated by the extracellular matrix (connective tissue). We will examine the feature of the cytoplasm: for example, the presence of orderly arranged transversal striations will guide us towards a skeletal muscle fiber.
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Nuclear staining intensity will give us information about the state of activity of the cell: an intensely hyperchromatic nucleus characterizes a resting cell (for example the fibrocyte); a hypochromatic nucleus corresponds to a metabolically active cell (for example the fibroblast). - intercellular matrix (fibers and ground substance): we will analyze quantity and contents, the variety and arrangement of fibers (collagen, elastic and/or reticular), ordered or disordered.
3. Diagnosis of the organ Organ diagnosis is based on specific structural characteristics. For example, the small intestine is recognized after its wall composed of four tunics: mucosa (epithelium and connective tissue), submucosa (connective tissue), muscular layer (muscle tissue) and serosa/adventitia (loose connective tissue and adipose tissue). The presence of numerous finger-like processes (villi) at the level of the mucosa, covered by a simple columnar epithelium, proves the diagnosis of small intestine. Than, the different segments of the small intestine will be recognized by their particular features: for example the duodenum has glands in the submucosa. The liver will be recognized from histological point of view due to its lobular arrangement. In the centre of the lobule we will find the central (centrolobular) vein, from which radial cords of hepatocytes and sinusoid capillaries emerge. Lobules are delimited (in the pig liver) by a vascular - connective tissue layer, that at the confluence of 3-5 lobules forms a triangular space (interlobular Kiernan space) were an artery, a vein, a bile duct and a lymph vessel will be found.
4. Differential diagnosis Differential diagnosis will be made between tissues and organs that are similar in many ways. It consists in highlighting the differences that exist between two such similar structures. For example differential diagnosis can be done between different types of epithelia, connective tissues or between the different segments of the digestive tube (stomach with duodenum, duodenum with ileum, etc.)
Execution of drawing
A histological drawing must reveal schematically all the specific (characteristic) structures of a tissue or an organ, in order to establish a positive diagnosis. Thus we will draw a sketch of a tissue, giving attention to: - the characteristic shape and size of the nuclei with their, their location in the cell, the feature of the chromatin; the shape of the nucleus indicates the shape of
the cell, while the staining intensity gives us information about its functional state; cytoplasm with the specific staining features for each type of cell; cell limits, where visible; relation of the cell with other structures (basement membrane, intercellular substance, etc;) ground substance and fibers.
In drawing an organ we will outline the particular features of each type of tissue that forms the organ, respectively the relationship between these tissues. There are some structures that, although they are not emphasized with usual stains, should be still drawn. Thus, the basement membrane that hardly stains in HE, will be always drawn under the epithelia in the form of a straight or wavy line (in stratified epithelia) stained in red.
EPITHELIA
Epithelia are tissues that lack direct blood supply, represented by a tightly cohesive sheet of cells that covers body surfaces or line cavities (covering and lining epithelia), forms the functional unit of secreting glands (glandular epithelia), and the receptors of some sensory organs (sensory epithelia). Their cells show various, relatively geometrical shapes: polygonal, cuboidal, columnar etc. Epithelia show some special features that allow us to differentiate them from other tissues and that determine their way of representation by drawings: - all epithelia are resting on a basement membrane that separates them from the underlying connective tissue. On light-microscopy (LM) this structure can be visualized only in special stains. Although it is not always visible with common Hematoxylin-Eosin (HE) staining, the basement membrane will always be drawn as a thin red line underneath any epithelium. - epithelial cells are strongly held together by intercellular junctional complexes; there are no free spaces between the cells; - individual cells have distinct sides or domains (poles): apical, lateral, and basal (with exceptions: some of the endocrine glandular epithelia). The apical domain may present differentiations of the cell membrane: cilia, microvilli, stereocilia; the lateral domain represents the site of the intercellular junctions, while the basal domain is anchored to the basement membrane.
Covering and lining epithelia

These epithelia are classified by combining two criteria: the number of cell layers and the shape of the cells in the outermost layer. Thus, we will study the following types of epithelia: Simple epithelia (one layer of cells): - squamous - cuboidal - columnar - pseudostratified Stratified epithelia (two or more cell layers): - nonkeratinized squamous - urothelium Simple squamous epithelium is formed by a single layer of flattened cells. This type of epithelium has specific names, depending on its localization: mesothelium in the serosa (mesentery, pleura, pericardium), and endothelium when it lines the blood and lymphatic vessels.
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Simple squamous epithelium, silver impregnation, mesentery. This tissue sample is studied after spreading the mesentery on the slide. Watching it from above, we can observe the shape and the specific arrangement of these cells. - on low magnification: we can observe the irregular, polygonal shape of the cells, tightly attached to one another, like pavement stones. - on high magnification: we will observe the crenellated, irregular outer limits of the epithelial cells highlighted by the precipitation of silver in the intercellular spaces. The other cellular structures (nucleus, nucleolus, and cytoplasm) do not stain under usual circumstances in case of silver impregnation.
Microscopic image
Schematic representation
Simple squamous epithelium: observed from above, only the cell limits are visible.
Simple cuboidal epithelium is formed by one layer of cuboidal cells. We will study this type of epithelium in the collecting tubules present in the kidneys medulla. Simple cuboidal epithelium, HE stain, kidney. - on low magnification: we will study the epithelium that lines the renal collecting tube. The cells are placed in one layer on the basement membrane (BM), delineating with their apical pole the lumen of the collecting tubule. - on high magnification: we can observe the fine intercellular limits. Cells are square shaped, with sides of approximately equal dimensions. The cytoplasm is pale eosinophilic. The nuclei are euchromatic, rounded and placed in the center of the cell.
3 Microscopic image Schematic representation
Simple cuboidal epithelium 1 Nucleus; 2 Intercellular limits; 3 Basement membrane (BM).
Simple columnar epithelium is formed of one layer of tall columnar cells. This type of epithelium can be found, for example, in the mucosa of the gallbladder. Simple columnar epithelium, HE stain, gallbladder. - on low magnification: the simple columnar epithelium consists of elongated cells placed in one layer on the basement membrane. The cytoplasm is pale eosinophilic. The nuclei follow the cells shape, as they are also elongated (oval), placed parallel to each other and perpendicular to the basement membrane, in the lower half of the cell. - on high magnification: the fine intercellular limits can be observed only towards the apical pole of the cells, while in the lower half, the limits are not visible, because of the crowded nuclei. Also with this magnification, the apical pole of these cells appears thickened and striated due to the presence of microvilli, which form the brushed border.
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1 4 Microscopic image Schematic representation
Simple columnar epithelium 1 Nucleus; 2 Intercellular limits; 3 Brush border; 4 BM.
Pseudostratified epithelium is a particular type of columnar epithelium, consisting of a single layer of cells. All cells are resting on the basement membrane (so it is a simple epithelium), but not all cells reach the surface of the epithelium. The cells have different heights; consequently, the nuclei are also placed at different heights, thus giving a false impression of stratification. This type of epithelium lines the tracheal lumen. Pseudostratified epithelium, HE stain, trachea. - on low magnification: we can observe a tall columnar epithelium, in which the nuclei occupy the lower 2/3rds of the cells. The nuclei may be rounded (near the basement membrane), oval or elongated, and they placed at different levels inside the cells. The upper third of the cell shows an eosinophilic cytoplasm. - on high magnification: we will observe differently shaped nuclei: round nuclei placed near the basement membrane, rounded-oval nuclei in the middle zone, rod-shaped nuclei placed in the upper half of the epithelium. Usually the nuclei occupy the lower two thirds of the cell, while in the rest of the cells upper one third we may observe the eosinophilic cytoplasm and very fine intercellular limits. The cells that reach the surface of the epithelium have cilia on their apical pole. Also in this epithelium, some tall cells with a vacuolar supranuclear cytoplasm, very palely stained and without cilia can be observed - the goblet cells.
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Pseudostratified epithelium 1 Basal cell; 2 Columnar cells with nuclei at different heights; 3 Cilia; 4 Goblet cell; 5 BM.
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Differential diagnosis of simple epithelia Simple squamous epithelium Localization endothelium, mesothelium lining, exchanges flattened sparse, mainly around the nucleus Simple cuboidal epithelium collector tubules in kidney, thyroid follicles etc. lining, absorption, secretion square equally distributed around the nucleus Simple columnar epithelium gallbladder, stomach, intestine, colon, etc lining, absorption, secretion rectangular abundant, especially in the upper half of the cell oval, in the lower 1/3rd of the cell, perpendicular to the BM brush border, cilia invisible or poorly visible at the upper pole HE Pseudostratified epithelium trachea, bronchi, epididymis lining, secretion, absorption multiform: cuboidal, columnar, goblet abundant, especially in the upper half of the cell multiform: multiform: rounded, oval cilia
Functions Shape of the cell
Cytoplasm
Nucleus
oval, parallel to round, in the the BM centre of the cell no differentiations visible by impregnations, invisible in HE Silver impregnation no differentiations visible
Apical pole Cellular limits Stain
invisible
HE
HE
Stratified squamous epithelia are composed of several cell layers placed in overlapped layers. The cells of this epithelium suffer morphological (and functional) changes from the basal layer towards the surface. Stratified squamous non-keratinized epithelium, HE stain, esophagus - on low magnification: we will recognize an epithelium consisting of several overlapped cell layers, resting on a wavy basement membrane that separates the epithelium from the connective tissue placed underneath; - on high magnification: we will observe three zones or cell layers: the basal or germinal layer (stratum basale) formed of one single row of cuboidalcolumnar cells with a basophilic cytoplasm and hyperchromatic, rounded-oval nuclei; the middle or intermediary layer (stratum spinosum) consisting of several rows of polygonal cells, strongly attached to one another, with eosinophilic
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cytoplasm, with clearly visible cell boundaries and an euchromatic, rounded nucleus placed in the center of the cell; the superficial layer formed of several rows of flattened polygonal cells, with their long axis parallel to the basement membrane. The cells in the superficial layer become more and more flattened, and as we head towards the surface, their cytoplasm becomes more and more clear, while the nucleus becomes smaller and smaller, hyperchromatic (pyknotic) and eccentrically placed in the cell.
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Stratified squamous non-keratinized epithelium 1 Basal layer; 2 Intermediary layer; 3 Superficial layer; 4 BM.
Transitional epithelium or urothelium is a stratified epithelium with a particular morphological appearance that lines only the urinary passages. Transitional epithelium (urothelium), HE stain, urinary bladder. - on low magnification: we will observe the stratified appearance of this epithelium, resting on an invisible (in this staining) basement membrane; - on high magnification: we will observe the cells appearance in the three layers of the epithelium: basal layer, intermediary and superficial layer. The basal or germinal layer consists of one row of cuboidal-columnar cells, with basophilic cytoplasm and a rounded-oval nucleus. The middle layer or intermediary layer consists of several rows of cells with a pear shaped cell body. These cells have a wide apical part, containing a rounded, euchromatic nucleus placed centrally, and a thin cytoplasm expansion that inserts itself between the cells of the underlying layer, thus resembling to a tennis racket. The superficial layer or layer of umbrella-like cells consists of one single row of large cells that cover 2-3 cells of the intermediary layer and send short projections between them. The umbrella-like cells have their long axis oriented parallel to the basement membrane, an intensely eosinophilic cytoplasm and one, seldom two, rounded or elongated hyperchromatic nuclei.
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Urothelium 1 Basal layer; 2 Intermediary layer of tennis racket-like cells; 3 Superficial layer of umbrella-like cells; 4 BM.
Differential diagnosis of stratified epithelia Non-keratinized Urothelium stratified squamous epithelium oral cavity, pharynx, Ureter, urinary bladder, prostatic esophagus, anus, urethra vagina barrier, protection barrier, distension cuboidal-columnar cuboidal-columnar cells, basophilic cells, basophilic cytoplasm, oval nucleus, perpendicular cytoplasm, oval to the basement membrane nucleus, perpendicular to the basement membrane polygonal cells, tennis racket-like cells, with a rounded eosinophilic apical pole, thin elongated basal pole cytoplasm, rounded nucleus, located centrally flattened cells, clear umbrella-like cells parallel with the cytoplasm, palely BM, intense eosinophilic cytoplasm, 1eosinophilic, 2 nuclei pyknotic eccentrically placed nucleus HE HE
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Localization
Functions Basal layer (identical)
Middle layer (several rows of cells)
Superficial layer
Stain
Glandular epithelia
These are epithelia specialized in synthesizing and secreting certain products. Morphologically they are organized into glands that may be exocrine or endocrine. Exocrine epithelial glands form glands that remain connected to the surface of the epithelium (from which they originate) by an excretory duct that transports the secreted product to the exterior. Endocrine glands are glands that have lost their connection with the epithelium they originate from, thus lacking excretory ducts. They show a typically rich capillary network in that the excretory products, the hormones, are released. The following exocrine epithelial glands will be studied: - without excretory duct the goblet cell (that will be studied together with the simple tubular gland) - with excretory duct: -simple tubular gland; -simple alveolar gland; -compound tubuloacinary gland Exocrine epithelial glands with excretory duct develop as epithelial invaginations into the underlying connective tissue (lamina propria of the mucosa). The terminal part of this invagination consists of secreting cells (secreting portion) being called adenomere, while its connecting structure with the surface forms the excretory duct. Simple tubular gland (Lieberkhn gland), HE stain, colon. - on low magnification: we will observe glands that are formed by invaginations of the surface epithelium into the underlying connective tissue in the mucosa of the colon. Tubular glands are finger-like structures or tubes consisting of three segments: fundus, corpus/body and the neck of the gland that communicates directly with the surface of the mucosa. Tubular glands are delimited by a basement membrane (glandilemma) on which a simple columnar epithelium is resting. The epithelium is composed of goblet cells, with a vacuolar appearance of their apical pole (which remains unstained) and tall columnar cells, the absorptive cells. - on high magnification: we will study the morphological features of both types of cells that form the glandular epithelium. The goblet cells, with an appearance resembling that of a champagne glass (gobletlike), have two domains: a narrow basal domain containing a hyperchromatic, oval or flattened nucleus, surrounded by a small amount of slightly basophilic cytoplasm, and a prominent cup or goblet shaped apical domain with a colorless, sometimes vacuolar cytoplasm, due to the large mucus granules present in the cytoplasm that do not stain in HE.
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Microscopic image
Simple tubular gland (Lieberkhn gland) 1 Lumen; 2 Absorptive cells; 3 Goblet cell; 4 BM.
The absorptive cells are tall columnar cells with an eosinophilic cytoplasm and hyperchromatic oval nuclei placed within the lower third of the cell. These cells are delimiting with their apical pole the very thin lumen of the tubular gland, in which they release the secreting product of the goblet cells. Simple alveolar gland (sebaceous gland), HE stain, lip. - on low magnification: we will observe the sac-like appearance of the sebaceous gland, that continues into a short excretory duct, and opens into the neck of the hair follicle; - on high magnification: we will observe the structure of the cells in relationship with their position inside the gland. On the basement membrane we can see one layer of cuboidal, basal cells, with basophilic cytoplasm and hardly visible cell boundaries. Their role is to intervene in the glands regeneration. Towards the center of the gland there are large polygonal cells with visible cell boundaries and a vacuolar cytoplasm, due to their secreting product (sebum), which dissolves during slide processing and does not stain in HE. As they approach the excretory duct, cells show a more and more vacuolar and paler cytoplasm, due to the accumulation of the sebum. The nucleus of these cells, at first rounded and located centrally, becomes smaller and smaller (pyknotic), intensely colored (hyperchromatic), and eccentric. The cells near the excretory duct gradually decay by losing their cell membrane and transforming into cellular
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fragments without nuclei. Together they will form the product elaborated by the gland, the sebum (holocrine type of secretion, by cellular decay).
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3 Microscopic image
Simple alveolar gland (sebaceous gland) 1- Adenomere; 2 Excretory duct; 3 Basal cells; 3 Cells filled with sebum; 4 Decaying cells; 5 BM.
Compound tubuloacinar gland, HE stain, submandibular gland. - on low magnification: we will notice the glandular acini, of different shapes, sizes and cytological appearances. - on high magnification: we will recognize the three main types of secreting acini: serous, mucous and mixed. The serous acinus is the smallest one and consists of 6-8 tall columnar cells placed on a basement membrane. Below the nucleus the cells have an intensely colored, basophilic cytoplasm while above the nucleus the cytoplasm is eosinophilic (containing zymogen granules). The nucleus is rounded, euchromatic and located centrally. With their apical pole the cells delineate a very narrow lumen, sometimes difficult to be seen. The mucous acinus is medium sized, with a large, more apparent lumen delimited by 4-6 large columnar cells placed on the basement membrane. Cells show an abundant cytoplasm, which is pale basophilic because of their mucus content. The nuclei are hyperchromatic, flattened, pushed towards the basal pole of the cell membrane and parallel to the basement membrane. The mixed acinus (serous and mucous) is the largest one. It is made up by a mucous acinus and a small cap of 3-5 serous cells on one side, with a crescent moon shape, called the Gianuzzis demilune. The mixed acinus has a wide lumen, delineated exclusively by mucous cells.
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In all three types of acini, thin contractile myoepithelial cells are located between the basement membrane and the basal pole of secreting cells. Myoepithelial cells have oval, hyperchromatic nuclei, with their long axis parallel to the basement membrane.
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5 Microscopic image
Secretory acini of the compound tubuloacinar gland 1 Serous acinus; 2 Mucous acinus, 3 Mixed acinus; 4 Gianuzzis demilune; 5 Myoepithelial cells; 6 BM.
The differential diagnosis of the acini Serous acinus small 6-8 serous cells Cytoplasm basophilic under the nucleus, eosinophilic at the apical pole round, centrally located narrow, point-like Mucous acinus medium 4-6 mucous cells pale, basophilic tint Mixed acinus the largest 4-6 mucous cells and 3-4 attached serous cells according to the cell type
Size Cells
Nucleus Lumen
flattened, basally located large
according to the cell type large
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THE CONNECTIVE TISSUE

The connective tissue is one of the best represented tissues in the body. It is formed of three main elements, which are present in different proportions: cells, fibers and ground substance; fibers, together with the ground substance, form the extracellular matrix ECM. Connective tissues have a very good blood supply.
Cells of the connective tissue

During the differentiation of the connective tissue, the first elements that develop are the cells that will elaborate the other two components, the fibers and the ground substance. The two types of cells in the connective tissue that we will study are: - proper (fixed) cells of the connective tissue: fibroblast/fibrocyte, chondroblast/chondrocyte, osteoblast/osteocyte , adipocyte, cells that are always present in the different types of connective tissue - cells migrated from the blood: macrophage, plasma cell, lymphocyte, granulocyte, cells that are not constantly present in the connective tissues. Cells of the connective tissue, HE stain, granulation tissue. During the proliferating phase of a wound healing process, a granulation tissue is formed. This is a repair tissue, which contains newly developed collagen fibers and all types of cells of the connective tissue, both proper cells and cells migrated from the blood. - on low magnification: numerous cells can be observed, along with collagen fibers (isolated or arranged in relatively disordered bundles), adipose tissue and numerous blood vessels, as the abundant vascularization is characteristic for this type of tissue. - on high magnification: the following cell types can be recognized: The fibroblast: medium sized, ribbon-shaped, elongated cell, with pale basophilic cytoplasm, which can be recognized by the large, oval, hypochromatic and centrally located nucleus, with visible nucleoli.
Microscopic image
Fibroblast 18
The fibrocyte: characterized by the elongated, intensely hyperchromatic and centrally placed nucleus. The cell is also elongated, spindle-shaped, with a low quantity of hardly visible eosinophilic cytoplasm.
Microscopic image
Fibrocyte
The adipocyte (adipose cell): a large, round or polygonal cell, with a single lipid-storage droplet that pushes the cytoplasm to the periphery and a flattened, hyperchromatic nucleus.
Microscopic image
Adipocyte
Plasma cell: round or oval cells with a dark, basophilic cytoplasm. The appearance of the nucleus is characteristic for this cell: rounded, eccentric and a particular distribution of the chromatin. The intensely stained granules of heterochromatin and the pale granules of euchromatin have an alternative arrangement, in radial fields, showing a cartwheel configuration.
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Microscopic image
Plasma cell
Macrophages: cells with variable size according to the quantity of the phagocytosed material. They have an irregular shape and a pale eosinophilic cytoplasm that contains the phagocytosed material. Sometimes macrophages are able to merge, forming multinucleated giant cells: large cells, with abundant cytoplasm and several nuclei.
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Microscopic image
Macrophage 1 Macrophage (histiocyte); 2 Multinucleated giant cells.
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Lymphocytes: small cells, with a round, hyperchromatic nucleus, and a small amount of basophilic cytoplasm appearing as a thin crescent-shaped rim at the periphery.
Microscopic image
Lymphocyte
Fibers of the connective tissue

The connective tissue has three types of fibers: collagen, reticular (they are a variety of collagen fibers, formed by type III collagen) and elastic ones. Excepting the collagen fibers, which stain in light pink, the other ones are not visible in HE. To view the fibers, special stains are used. Collagen fibers, van Giesons stain (acid fuchsin), mammary gland. Van Giesons stain is used for visualizing and differentiating collagen fibers from other structures. With this stain collagen fibers are colored in bright red and the epithelial tissue and the nuclei in brown-black. - on low magnification: we can see the collagen fibers stained in red, with a regular or an irregular arrangement. - on high magnification: we can observe the collagen fibers as more or less wavy, sinuous lines. They can be either isolated or grouped in fine or rough bundles. Although the collagen fibers of a bundle can detach and join the ones in the neighboring bundle, they never branch or merge (anastomose) with other fibers. Among collagen fibers, we can see the brown-black nuclei of the fibrocytes: oval, flattened, and intensely colored (hyperchromatic). The fibrocytes are often tightly bonded to the collagen fibers that they synthesize. Thus the cytoplasm, which is in a very small quantity anyway, and the cellular boundaries cannot be observed. Fibrocytes will be recognized and represented only by their nuclei.
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Collagen fibers 1 Isolated and bundle-grouped fibers; 2 Fibrocyte nuclei.
Reticulin fibers, silver nitrate impregnation, kidney. The reticulin fibers are argyrophilic and they can be visualized by silver impregnation, which colors them in black. - on low magnification: a network of reticular fibers stained in black can be seen; - on high magnification: we can observe the reticular fibers as irregular lines. They are thinner than collagen fibers. They branch and anastomose with other fibers forming networks with gaps of different shapes and sizes. They do not group in bundles.
Microscopic image
Reticular fibers
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TYPES OF CONNECTIVE TISSUE

Connective tissues can be divided into two large categories: embryonic connective tissue and adult connective tissue. Adult connective tissue can be also classified according to the consistency of the ground substance in: soft connective tissue, semi-hard connective tissue (cartilage) and hard connective tissue (bone). Soft connective tissues also include more categories, according to the proportion of the 3 components: cells, fibers and ground substance. The following types of connective tissue will be studied: I. Soft connective tissue 1. in which the 3 components (cells, fibers, ground substance) are present in the same proportion - Loose connective tissue 2. in which cells predominate (specialized tissues) - Reticular connective tissue - Adipose connective tissue 3. in which fibers predominate (fibrous tissue) - Tendinous connective tissue (tendon) II. Cartilage - Hyaline cartilage III. Bone - Compact bone - Spongy or cancellous bone
SOFT CONNECTIVE TISSUES

Soft connective tissues are formed of ground substance with a low consistency, respectively cells and fibers in a various proportion. Loose connective tissue, HE stain, mammary gland. It is a type of connective tissue in which cells, fibers and the ground substance are found in almost the same proportion. - on low magnification: two types of connective tissues are observed: a dense connective tissue and a loose connective tissue in which glandular structures are found, forming the mammary gland lobules; on this slide we will study only the appearance of the loose connective tissue (in the lobules). - on high magnification: pale pink stained collagen fibers can be observed. They appear as isolated fibers or arranged in bundles, with a variable orientation. The connective tissue cells and ground substance are found between them. The fibroblasts and fibrocytes can be recognized, due to their hypo- respectively hyperchromatic nuclei, placed in the vicinity of the collagen fibers. Also some other cells can be seen: the plasma cells, the lymphocytes, adipocytes.
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Since it is a well vascularized tissue, capillaries can be also observed. On longitudinal sections, capillaries appear as structures with a thin lumen, in which the elongated, protruding, hyperchromatic nuclei of the endothelial cells can be recognized. These are placed alternatively on each side of the blood vessels wall, like saw teeth; sometimes red blood cells can be distinguished lining up within the tube. On cross sections the capillaries appear like small, rounded spaces delineated by one, maximum two nuclei bulging into the lumen. The loose connective tissue contains also other types of fibers (elastic and reticulin fibers) as well as cells, migrated out of the blood, other than the above mentioned, but that are not visible in HE staining. In case of other localization (adventitial layer, the organs capsule) this type of tissue can contain a variable amount of adipose tissue.
4 3
8 7
6 1 2 Microscopic image Schematic representation 5
Loose connective tissue 1 Collagen fibers; 2 Nucleus of a fibrocyte; 3 Nucleus of a fibroblast; 4 Plasma cell; 5 Adipocyte; 6 Lymphocyte; 7 Capillary in longitudinal section; 8 Capillary in cross section.
Reticular connective tissue, HE stain, lymph node. It is a type of connective tissue consisting of cells (reticular cells) and reticulin fibers. This tissue forms the stroma of the lymphoid organs (lymph node, spleen, bone marrow etc). - on low magnification: we will search the profound, medullar zone of the lymph node, where the lymphocytes are rare and where we can observe the reticular connective tissue, with a lace, network-like appearance; - on high magnification: we will study the appearance of the reticular cells: star shaped cells, with eosinophilic cytoplasm and a large, rounded and hypochromatic nucleus. They have long and less numerous processes that link with processes of neighboring cells and form a network-like appearance that doubles the network of fibers. The reticulin fibers cannot be seen in this staining. In the gaps of the network that forms the stroma of the lymph node, lymphoid components can be recognized small cells with a round and hyperchromatic nucleus and scanty, crescent shaped cytoplasm (lymphocytes).
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Reticular connective tissue 1 Reticular cell; 2 Lymphocyte.
Adipose connective tissue, HE stain, mammary gland. It is a tissue in which the cells, closely placed to each other, predominate, while the ground substance and the fibers are sparse. - on low magnification: a connective tissue can be seen, consisting almost exclusively of large adipose cells, with well defined, visible cell limits and a colorless cytoplasm; - on high magnification: adipose cells can be observed, isolated or closely placed next to each other, in nests, forming the so called paniculus adiposum. They are large cells, oval or polygonal (when placed in groups), with well defined cell membranes. Most of the cell is occupied by a single, large vacuole, colorless in HE staining (the cell seems empty inside), that pushes the cytoplasm and the nucleus to the periphery. The nucleus appears flattened, hyperchromatic and eccentrically located, while the cytoplasm is reduced to a narrow ring that surrounds the vacuole. So adipocytes take the aspect of a signet-ring. The very narrow intercellular spaces contain just rare, fine reticular fibers and a lot of capillaries.
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2 1 Schematic representation
Microscopic image
Adipose tissue 1 Adipose cell; 2 Capillary. 25
Tendinous connective tissue, HE stain, tendon. It is a connective tissue in which regularly oriented collagen fibers predominate, with just a few cells and little ground substance. - on low magnification: we can see a connective tissue consisting mainly of collagen fibers, arranged in regular bundles; between the fibers connective tissue cells, called tenocytes (tendon cells), can be found; - on high magnification: thick, pale eosinophilic collagen fibers, with a regular arrangement may be observed. On longitudinal section they show a slightly wavy trajectory and are organized in dense bundles, parallel to each other. Between the bundles respectively between the fibers, a small number of fibrocytes (tenocytes) are present. They are elongated, rectangular shaped cells, with slightly visible cell limits and eosinophilic cytoplasm, closely attached to the connective tissue fibers. The nucleus is oval, hyperchromatic. Often the nuclei are placed head to head, yielding an aspect of twin nuclei, which is typical for the tendinous tissue.
Tendinous connective tissue 1 Tenocytes (twin nuclei); 2 Bundles of collagen fibers.
Differential diagnosis of different types of soft connective tissue can be done according to the proportion of the components and to their organization.
CARTILAGE
Cartilage is a special form of connective tissue consisting of a denser yet flexible ground substance, collagen and/or elastic fibers and specialized cells: chondroblasts, active cells that synthesize the abundant extracellular matrix of the cartilage, and chondrocytes, inactive cells.
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Based on the characteristics of the matrix (nature and architecture of fibers, amount of ground substance) we may distinguish three types of cartilage: hyaline, elastic and fibro-cartilage. Hyaline cartilage, HE stain, trachea. It is a tissue with abundant ground substance, cells and a few collagen fibers, masked by the ground substance. - on low magnification: we will see the hyaline cartilage located in the middle layer of the tracheal wall. We observe the homogenous (pale blue) ground substance in which chondrocytes are placed, individually or in clusters called isogenous groups. At the periphery the cartilage is surrounded by the perichondrium covering the cartilage and separating it from the connective tissue around it. - on high magnification: we can observe the perichondrium and the structure of the cartilage. The perichondrium has two layers: an outer fibro-vascular layer and an inner cellular layer. The outer layer consists mainly of collagen fibers, few cell components represented by fibrocytes, and blood vessels. The inner cellular layer contains chondroblasts, differentiating cells that synthesis the extracellular matrix. They appear as flattened, lens-like cells, with their long axis parallel to the surface of the cartilage. Chondroblasts have a basophilic cytoplasm, an elongated or oval euchromatic nucleus, with an obvious nucleolus. Between the perichondrium and the cartilage no precise boundaries may be observed, as the transition from one another is gradual. In the structure of the hyaline cartilage we observe an abundant, nonhomogeneous, pale basophilic ground substance. Collagen fibers are very thin, fine and thus they cannot be distinguished from the ground substance. Chondrocytes are placed inside spaces (lacunae) in the ground substance called chondroplasts. They are rounded or oval cells, respectively polygonal when they are organized in clusters. Chondrocytes have a basophilic cytoplasm and a round or oval, euchromatic nucleus. Chondrocytes appear as isolated cells or they are arranged in so called isogenous groups, formed of 3-8 chondrocytes. We distinguish axial isogenous groups, within which cells are arranged in rows, one above another like coins in a stack, and crown-like isogenous groups, in which cells are arranged in a ring (radial) or crown like pattern. Around the isolated cells and also around the isogenous groups, ground substance is more condensed and stains intensely basophilic (dark blue), surrounding the cells or the groups like a capsule called territorial matrix. The paler stained matrix between chondrocytes respectively groups of cells, is called interterritorial matrix.
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2 Microscopic image
5 Schematic representation
Hyaline cartilage 1 Fibro-vascular perichondrium; 2 Cellular perichondrium (chondroblasts); 3 Chondrocytes (crown-like isogenous groups) with territorial matrix; 4 Chondrocytes (axial isogenous groups) with territorial matrix; 5 Interterritorial matrix.
BONE
Bone is a variety of connective tissue, rigid and inflexible because of the hard, mineralized, ground substance. Together with the connective tissue fibers (collagen fibers), the ground substance forms the bone matrix in which cells are placed: the osteoblasts, osteocytes and osteoclasts. Two types of bone will be studied: compact bone and spongy bone. Compact bone, dried and polished The sample is obtained after drying and polishing a bone cross-section until it reaches a suitable thickness for placing under a cover slip. On the dried and polished bone section we will study the mineral component of the bone. Compact bone consists of Havers or haversian systems (osteons) and interhaversian systems. An osteon is formed by a central, osteonal (Havers) canal surrounded by a few concentrically arranged lamellae. - on low magnification: we will study the structure of an osteon. On cross section the osteon appears as a structure formed of a rounded central space, the haversian canal colored in black, surrounded by 8-15 concentrically arranged bone lamellae. At the margins of the lamellae we can see lens-like or oval cavities, which are lacunae, dug in the ground substance, that stain in black and are called osteoplasts. From osteoplasts long, thin, branched canaliculi emerge, oriented in all directions, yielding the appearance of a spider. Through these canaliculi osteoplasts communicate with the neighboring ones.
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1 Microscopic image
Compact bone, dried and polished 1 Havers system; 2 Interhaversian system; 3 Haversian or central canal; 4 Volkmanns canal; 5 Interstitial bone lamellae; 6 Osteoplasts.
Also large, oblique or transverse canals not surrounded by lamellae can be observed - the perforating canals or Volkmanns canals (stained in black) connecting the haversian canals. Between the osteons, remnants of previous concentric lamellae called interstitial lamellae can be seen. They are arched interstitial lamellae with osteoplasts, binding osteons to each other. - on high magnification: we will study the spider-like appearance of the osteoplasts. Spongy bone (trabecular or cancellous spongy bone), HE stain, spongy bone. It is a haversian bone tissue in which the marrow spaces represent the main component, while the osseous component is poorly represented. - on low magnification: we will recognize the morphology of the spongy bone. It has a spongy appearance, due to the existence of areolas (cavities) separated by bone trabeculae. The bone trabeculae of variable length are usually short and oriented in different directions. They intersect and anastomose, delimiting the areolar cavities. These cavities have different sizes and shapes, contain bone marrow and sometimes they communicate. - on high magnification: we can recognize the cells present in the bone tissue, namely the osteocytes, osteoblasts and osteoclasts. We can observe the trabeculae formed by a few bone lamellae oriented parallel to each other, and osteocytes in parallel rows, placed within osteoplasts between the lamellae. Osteocytes appear as oval, flattened, lens-like cells with reduced, eosinophilic cytoplasm. The nucleus is large, oval or elongated, hyperchromatic and centrally located. Osteoblasts are placed like strands of beads on the surface of the trabeculae. They are metabolically active cells with a cuboidal, oval or often triangular shape. Their nucleus is large, oval, euchromatic and the abundant cytoplasm is basophilic.
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3 4
Spongy bone 1 Bone lamellae with osteocytes; 2 Osteoblasts; 3 Osteoclast; 4 Areola; 5 Howships lacuna.
Osteoclasts are present in a small number, only in areas of bone reshuffling. They can be found at the periphery of areolar cavities, in contact with the trabecula. This paler contact area (bone resorption area) is called Howships lacuna. Osteoclasts are very large cells, with an abundant eosinophilic cytoplasm and numerous hyperchromatic nuclei (multinucleated cell), irregularly arranged in the cell. Differential diagnosis of the two types of bone can be done according to their architectural organization.
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MUSCLE TISSUES
Muscle tissues are formed of cells specialized for contraction, muscle cells, which due to their elongated shape are also called muscle fibers. Around the muscle fibers a more or less abundant connective tissue with blood vessels and nerves can be seen. According to the morphological and functional features of the muscle fibers, there are three types of muscle tissues: - smooth muscle tissue (formed of fibers with homogenous cytoplasm, with no striations) - skeletal muscle tissue (formed of fibers with longitudinal and transverse striations) - cardiac muscle tissue (a type of striated muscle different from the skeletal muscle, that forms the muscle of the heart).
SMOOTH MUSCLE
The smooth muscle is formed of elongated muscle cells (fibers), with sharp endings, and a prominent central part (spindle-shape cells). They are organized in bundles. Between the bundles a variable amount of connective tissue can be found. Smooth muscle, HE stain, urinary bladder. This tissue sample was obtained by spreading the wall of a frog urinary bladder on the slide (it is not a histological section). By brushing it, the mucosa of the urinary bladder was removed and muscle fibers partially dissociated, so that isolated fibers could also be observed. - on low magnification: we will identify the smooth muscle fibers, isolated or placed in bundles with different orientation, forming a network-like aspect, and also some cell components of the connective tissue, like fibrocytes. - on high magnification: we will examine the appearance of an isolated smooth muscle fiber, and also its arrangement in bundles. The smooth muscle fiber appears as a spindle-shaped cell with thin endings and a prominent central area. The cytoplasm of the cell is homogeneous and eosinophilic. It contains only one nucleus, which is centrally located, hypochromatic, elongated and with the long axis oriented parallel to the long axis of the cell. Most of the fibers are arranged in bundles, in which the extremity of a fiber is in the vicinity of the central area of the neighboring fiber.
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Smooth muscle tissue 1 Isolated muscle fiber; 2 Smooth muscle bundle; 3 Nucleus of a fibrocyte.
SKELETAL MUSCLE
This type of tissue is formed of elongated cells, with a ribbon-like shape and rounded endings. They are arranged parallel to each other, in bundles and are surrounded by a small amount of connective tissue, blood vessels and nerves (10% of the muscular mass). Skeletal muscle, HE stain, tongue. - on low magnification: we can observe the striated skeletal muscle fibers with ribbon-like shape. They have numerous elongated, hyperchromatic nuclei placed with their long axis parallel to the long axis of the muscle cell. These nuclei are placed at the periphery of the cell, under the cell membrane (sarcolemma). - on high magnification: on longitudinal section we will see the muscle fibers with an eosinophilic and heterogeneous cytoplasm (sarcoplasm), with orderly transverse and longitudinal striations. The longitudinal striations or Leydigs columns (observed on longitudinal sections) are given by the arrangement of the myofibrils organized in longitudinal bundles, stretching from one end to the other inside the muscle fiber. Transverse striations are given by the regular alternation of clear and dark bands present in the structure of the myofibrils. On histological slides transverse striations appear as intensely eosinophilic (dark red) bands corresponding to the dark bands; they alternate with pale eosinophilic bands which correspond to the clear bands. These bands are perpendicular to the long axis of the muscle fiber, and placed at equal distance. On cross section, myofibrils appear as fine point-like areas, placed in groups that are delimited by thin septa of cytoplasm (Cohnheim fields).
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Microscopic image 5 7 4 8 3
6
Striated skeletal muscle 1 Longitudinal section; 2 Cross section; 3 Longitudinal striations; 4 Transverse striations; 5 Peripheral nuclei; 6 Sarcolemma; 7 Cohnheim fields; 8 Connective tissue.
CARDIAC MUSCLE
It is formed of muscle fibers (cells) much shorter than the skeletal fibers. Cardiac muscle fibers are rectangular and have branched endings that merge (anastomose) with the neighboring fibers. This junction between the endings of muscle fibers is called stria scalariformis (or intercalated discs). Between the fibers we can observe an abundant connective tissue and numerous blood vessels. Cardiac muscle, ferric hematoxylin stain, myocardium. Ferric hematoxylin is a special stain, which reveals the striations of the muscle fiber: clear bands remain colorless while the dark ones appear like black bands. The intercalated disk (stria scalariformis), the nucleus and the components of the connective tissue between the fibers stain also in black. - on low magnification: on longitudinal section, we will observe the rectangular shape of the myocardial muscle fibers and their arrangement in bundles, with a network appearance.
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- on high magnification: we will study in detail the structure of the cardiac muscle cells. They are rectangular cells, with branched endings (bi- or trifurcated), organized in parallel bundles and linked at their distal endings through the intercalated disks (stria scalariformis). Intercalated disks appear like densely stained cross-bands, oriented perpendicularly to the muscle fibers long axis, with a linear fashion or in the way that resembles with the risers of a stairway. Also we may notice the single nucleus, centrally located, with an elongated, oval shape, stained in black. The cytoplasm (sarcoplasm) shows transverse striations on the longitudinal section, with a register-like arrangement, given by the alternation of the clear and dark bands present in the structure of myofibrils, and longitudinal striations (Leydigs columns) due to the presence of myofibrils. Between cells a larger amount of connective tissue than that of the skeletal muscle is present, which is rich in capillaries.
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3 Microscopic image
Cardiac muscle tissue 1 Stria scalariformis; 2 Single, centrally located nucleus; 3 Connective tissue.
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HEMATOPOIETIC AND LYMPHOPOIETIC ORGANS

Hematopoietic and lymphopoietic organs are the central compartment of the blood tissue. They contain young, immature cellular components representing the precursors of the blood cells. In these organs processes like hematopoiesis and lymphopoiesis are taking place. All these organs have a common plan of histological organization, all of them being built up by a capsule, stroma and parenchyma. The capsule is formed by a fibrous connective tissue showing some variations between different organs. Septa consisting of connective tissue and blood vessels detach from the internal surface of the capsule and divide the organ. The stroma. Excepting the thymus, whose stroma is of epithelial origin, all the other lymphoid organs have a stroma formed by reticular connective tissue. This tissue (see the reticular connective tissue) is built up by a tridimensional meshwork of reticular fibers, with two types of meshes: some of them are small and contain the components of the parenchyma, and some of them are large, irregular, lined from place to place by endothelial cells, forming sinusoid capillaries, through which lymph or blood will circulate. Besides reticular fibers, the connective tissue of the stroma is also formed of star-shaped reticular cells with numerous processes linked with the processes of the neighboring cells. The structure of the parenchyma will be studied separately for each hemato- and lymphopoietic organ.
RED BONE MARROW

It is located inside the areoles, cavities of the spongy bone and is formed of stroma and parenchyma. Red bone marrow, HE stain, spongy bone. - on low magnification: we will observe a tissue rich in cells, located within the areoles of the spongy bone, and formed by numerous islands of myeloid cells that are morphologically unidentifiable in this staining and on this magnification. Also a variable number of adipose cells and several giant cells with an eosinophilic cytoplasm (megakaryocytes) can be seen. - on high magnification: we will study first the megakaryocyte, which is a huge, giant cell, with abundant eosinophilic cytoplasm and a large, centrally located, sprouted, lobulated or ring-shaped nucleus. The presence of the megakaryocyte is indispensable for recognizing and diagnosing the red bone marrow. Adipose cells may be recognized by their signet-ring cell appearance. The rest of the cells are represented by young components of the myeloid cell population. In order to identify them special staining is required.
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2 Microscopic image
Red bone marrow 1 Megakaryocyte; 2 Adipose cell; 3 Young components of the myeloid cell population.
THYMUS
The thymus is a lymphoepithelial and endocrine organ with a lobulated structure. It is covered by a connective tissue capsule from which septa of connective tissue detach and divide the organ in lobes and lobules. The stroma differs from that of the peripheral lymphoid organs, because of its epithelial origin. The parenchyma has two distinct areas: a darker, peripheral area called cortex and a paler, centrally located area, called medulla. The peripheral area is clearly divided by the capsules connective tissue extensions (septa), giving the organ a lobulated aspect. The central area forms a common core for all the lobules. In the medulla we can see eosinophilic lamellar structures, the so-called Hassalls corpuscles. Thymus, HE stain. - on low magnification: we will study the morphological organization of the thymus, surrounded by a capsule of connective tissue rich in adipose cells. From the capsule septa of connective tissue detach and delimit well individualized thymic lobules. The thymic lobules with polygonal shape have a central, paler area (medulla) and a peripheral, darker area (cortex). - on high magnification: we will study the cytological structure of the two areas in the thymic lobules. The cortex is rich in cells and is composed especially by small lymphocytes (thymocytes). The medulla, besides the thymocytes that are less numerous and much more loosely arranged, contains stromal epithelio-reticular cells (thymic epithelial cells). These are larger cells, with an abundant pale, eosinophilic cytoplasm and a
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large, centrally located, hypochromatic nucleus. Also in the medulla, we will find some structures which are characteristic for the thymus: the Hassalls corpuscles. They appear as rounded or oval eosinophilic structures, composed of concentrically placed lamellae, giving corpuscles the appearance of an onion bulb. The lamellae in the Hassalls corpuscles correspond to isolated masses of closely packed, concentrically arranged epithelio-reticular cells, partially or completely keratinized that exhibit flattened nuclei. The number of corpuscles increases with age.
Microscopic image 1 2 3 6
Thymus 1 Capsule; 2 Incomplete septa; 3 Cortex; 4 Medulla; 5 Epithelio-reticular cells; 6 Hassalls corpuscle.
Differential diagnosis: with other hemato- and lymphopoietic organs, according to the organization of the thymus parenchyma in lobules delimited by septa of connective tissue, with its two areas, cortex and medulla and the presence of the Hassalls corpuscles in the medulla.
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LYMPH NODE
The lymph node is a lymphoid organ, placed along lymphatic vessels and surrounded by a connective tissue capsule from which short trabeculae extend to divide only the periphery of the organ. The lymph node has a stroma, composed of a reticular connective tissue (anastomosed processes of the reticular cells forming a network, doubled by the network of reticular fibers) and a parenchyma divided into three regions: the outer cortex, the inner cortex (or paracortex) and the medulla, which are joined by a system of lymphatic sinuses (subcapsular sinus, paratrabecular sinus and medullar sinus). Lymph node, HE stain. - on low magnification: at the periphery it is surrounded by a connective tissue capsule, containing adipose cells. Narrow and short trabeculae extend from the capsule and divide only the periphery of the organ. Under the capsule we will notice the three regions of the parenchyma: the outer cortex, composed of lymphoid follicles with a crown-like arrangement, the inner cortex (paracortex), as a dense and diffuse lymphoid tissue and the medulla formed by cords of lymphocytes separated by narrow and irregular spaces (the medullar sinuses). - on high magnification: we will study the cytological structure of each region of the node and that of the lymphoid sinuses. The outer cortex is formed of secondary lymphoid follicles. Secondary lymphoid follicles have a pale central zone (germinal center) and a dark peripheral zone (mantle zone or corona). The germinal center consists of larger cells, with hypochromatic nuclei and an abundant cytoplasm, these explaining its light appearance comparative to that of the corona. These cells, called centro-follicular cells (cells of the center of the follicle), are activated lymphocytes from the mantle zone. Among these cells macrophages are also found, but immunohistochemical staining is necessary in order to highlight them. The mantle zone consists of small lymphocytes with hyperchromatic nucleus and thin cytoplasm. The paracortex consists of a diffuse lymphoid infiltrate, in which lymphocytes are densely arranged. In this region are also found the so called Schultzes postcapillary venules, lined by a tall endothelium of cuboidal or columnar endothelial cells. The medulla, near the hilum, is built up by cords of lymphocytes, the medullar cords separated by the medullar sinuses. In the medulla, near the medullar sinuses, the number of lymphocytes is lower, so that stromal components are easier to be seen. The sinuses of the lymph node. At the periphery of the lymph node, between the capsule and the cortex, a paler area can be seen, the subcapsular (cortical) sinus. It is clearly visible as an optical empty space that covers the cortex and that contains rare components of the stroma. The paratrabecular (perifollicular) sinuses are placed along the trabeculae, and are not always visible on histological slides, due to the high density of lymphocytes in the cortex and paracortex. They are originating from subcapsular sinuses, and drain into medullar sinuses in the medullar region, located between the medullar cords.
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1 8 2 5 6 7 3
9 Microscopic image
Lymph node 1 Capsule; 2 Outer cortex; 3 Paracortex (inner cortex); 4 Medulla; 5 Secondary lymphoid follicles; 6 Mantle zone; 7 Germinal centre; 8 - Subcapsular sinus; 9 Trabecular sinus; 10 Medullary sinus; 11 Schultzes postcapillary venules.
Differential diagnosis can be made with other hemato- and lymphopoietic organs, based on the presence of the three regions and of the lymphoid sinuses.
SPLEEN
The spleen is a lymphoid organ, situated on the trajectory of the blood vessels, representing the place where the circulating blood meets the lymphoid tissue. The spleen is surrounded by a connective tissue capsule, containing also smooth muscle cells. From the capsule, trabeculae composed of connective and muscular tissue that divide incompletely the organ, are detaching. The stroma is formed of reticular connective tissue (a network of reticular fibers and large, star shaped reticular cells). The parenchyma consists of two zones: the white pulp and the red pulp. Spleen, HE stain. - on low magnification: we will observe the histological organization of the spleen. The parenchyma consists of two interspersed zones: the white pulp and the red pulp. The white pulp, that represents about 20% of the parenchyma, has two components: Malpighian corpuscles or splenic nodules (secondary lymphoid follicles and 1-2 central or peripheral arteries) and periarterial lymphoid sheath (PALS) (a sheath of lymphocytes surrounding a central artery). The red pulp, located between the lymphoid structures of the white pulp, consists of a vascular network (splenic sinusoids) and cells cords (splenic cords), formed especially by red blood cells.
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- on high magnification: we will observe the cytological structure of both zones of the splenic parenchyma: white pulp (formed of Malpighian corpuscles and periarterial lymphoid sheath) and red pulp. The Malpighian corpuscle is a spherical or oval lymphoid structure, consisting of a secondary lymphoid follicle, with a germinal center, a mantle zone (a microscopic structure similar to that of lymphoid follicles in the lymph node) and a marginal zone at the periphery, where the white pulp comes in contact with the red pulp. At the periphery of the Malpighian corpuscle 1-2 arterioles with characteristic microscopic features can be found. The periarterial lymphoid sheath appears like an aggregate of small lymphocytes, surrounding a central artery. The red pulp consists of cell cords placed between the splenic sinusoids. The cells of the red pulp are mainly red blood cells, macrophages and lymphocytes.
1 2 4
6 3
Microscopic image
Spleen 1 Capsule; 2 Trabecula; 3 Malpighian corpuscle; 4 Periarteriolar lymphoid sheath; 5 Red pulp; 6 Arteriole. 40
Differential diagnosis: with the other hemato- and lymphopoietic organs, based on the presence of the capsule and the thick trabeculae of connective and muscular tissue and on the characteristic structure of the parenchyma formed of the two zones: white pulp and red pulp.
MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)

The lymphoid organs, that are part of the MALT system, are localized in the lamina propria of the mucosa of different organs like the digestive tube, respiratory system, etc. Unlike other lymphoid organs, these are not delimited by a capsule. Palatine tonsil The palatine tonsil is a peripheral lymphoid organ located in the oral cavity. The surface of the tonsil is covered by a nonkeratinized stratified squamous epithelium. From place to place, the epithelium penetrates the subjacent connective tissue and forms the tonsil crypts deep invaginations, sometimes with ramifications. Under the surface epithelium and around the crypts, in the lamina propria of the mucosa, strings of lymphoid follicles with germinal centers and interfollicular diffuse lymphatic infiltrations can be seen. Palatine tonsil, HE stain. - on low magnification: we will study the histological structure of the palatine tonsil, covered by the mucosa of the oral cavity, represented by a stratified squamous nonkeratinized surface epithelium and a lamina propria consisting of connective tissue. We may observe the deep tonsil crypts, lined by the surface epithelium of the palatine tonsil. The lamina propria is smooth, without papillae, rich in secondary lymphoid follicles consisting of a germinal center and mantle zone, which are placed around the crypts. Between and around the lymphoid follicles, an abundant diffuse lymphoid infiltrate interfollicular tissue - can be seen. - on high magnification: we will study the structure of the surface epithelium, with numerous lymphoid infiltrations around the crypts that sometimes mask the epithelium. The lumen of the crypts may be empty or may contain cell debris (desquamated epithelial cells), lymphocytes, etc. The lymphoid follicles have the characteristic cytological structure of the lymphoid follicles in the lymph node.
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2 1
Microscopic image
Palatine tonsil 1 Epithelium of the oral cavity; 2 Crypt; 3 Secondary lympoid follicles; 4 Dense interfollicular lymphoid tissue.
Differential diagnosis: with other hemato- and lymphopoietic organs, based on the presence of crypts that are not lined by a capsule but by a stratified squamous nonkeratinized epithelium of the oral cavity and through the presence of secondary lymphoid follicles, located on both sides of the tonsil crypts.
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NERVOUS TISSUE AND NERVOUS SYSTEM

Nervous tissue is integrated in both the central nervous system, CNS (spinal cord, cerebellum, cerebral hemispheres) and the peripheral nervous system, PNS (spinal and vegetative ganglia, peripheral nerves). It is composed of highly differentiated cells called neurons and supporting cells. The latter are the glial cells (neuroglias) in CNS, respectively the satellite cells and Schwann cells in the peripheral nervous system.
NEURON
The neuron is the functional and morphological unit of the nervous tissue and system consisting of a cell body (perikaryon) and a variable number of cytoplasm processes (dendrites and axon). It is a cell with: - multiform appearance (stellate, pyramidal etc.) with 2 or more processes that may be highlighted by silver stain - a cell body (perikaryon) containing a large round, vesicular nucleus, with a prominent nucleolus - in the cytoplasm, using appropriate special stains, specific organelles can be visualized: neurofilaments (silver stains) and Nissl bodies (toluidine blue, cresyl violet staining). The shape of neurons is variable and depends on the size of the perikaryon and the number of processes that arise from the cell body. Thus, neurons may be star shaped, oval, pyramidal, pear-shaped or round. Neurons can be visualized by the usual staining with HE or using silver staining (Golgi method, Cajal method, etc), which stains the cell and its processes in black. Pyramidal neuron, Golgi silver stain, cerebral cortex. - on low magnification: we will identify the pyramidal cells in the cerebral cortex; - on high magnification: the perikaryon has a triangular shape. From its corners arise one or more richly branched dendrites (dendritic tree), while from the middle base emerges an axon. The cell is placed so that the tip of the triangle is oriented towards the cortex surface and the base to the depth of the cortex.
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3 1
4 4 2
Microscopic image
Pyramidal cell 1 Cell body; 2 Axon; 3 Main dendrite; 4 Secondary/auxiliary dendrites.
GLIAL CELLS
Glial cells (neuroglia) are supporting cells of CNS. In light microscopy, specific staining techniques with nitrate salts of silver and gold (Golgi, Cajal, Del Rio Hortega) are used to reveal glial cells. In these stainings the glial cells appear as cells with a very small cell body and numerous processes (starfish shape). Some glial cells are found in the gray matter (protoplasmic astrocyte, microglia, oligodendroglia) while others reside within the white matter (fibrous astrocyte, oligodendroglia, microglia).
Protoplasmic astrocyte, Golgi silver stain, cerebral cortex. - on low magnification: we will locate in the microscopic field those areas of the cortex around pyramidal neurons in which we will recognize the protoplasmic astrocyte, stained in black with silver nitrate; - on high magnification: the protoplasmic astrocyte appears as a cell with a very small cell body, with very numerous short, thick and richly branched processes; sometimes the processes show asperities on their surface.
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Microscopic image
Protoplasmic astrocytes
CENTRAL NERVOUS SYSTEM

Spinal cord, HE stain. - on low magnification: we will observe on cross section the organization of the spinal cord: the central canal surrounded by the H-shaped gray matter (anterior horn, posterior horn), and at the periphery the white matter, covered by the meninges. We will study the microscopic appearance of the gray matter in which we will recognize the multipolar neurons that are larger in the anterior horns and smaller in the posterior horns, and also the numerous, small, hyperchromatic nuclei of the neuroglia and small association neurons. Between these cells we may see an eosinophilic mass with fibrillar aspect, formed by the processes of the neurons. - on high magnification: multipolar neurons in the anterior horns are large cells, with an abundant, eosinophilic cytoplasm and a round, centrally located, vesicular nucleus with a large nucleolus. Due to their large number of processes, these neurons have a star-like shape. In the gray commissure we can see the central canal lined by a layer of columnar cells called ependymal cells. They are tall, columnar cells, with a pseudoepithelial appearance (closely joined together without being placed on a basement membrane). They have an oval nucleus, with coarse chromatin, placed towards the gray matter. At the apical pole of these cells we can see microvilli. In the white matter we will find myelinated nerve fibers composed of: an axial filament (neuronal process, usually an axon, as a centrally located eosinophilic dot), surrounded by the myelin sheath (ring-like, colorless space) and glial cells (a thin circular rim of cytoplasm and a hyperchromatic nucleus).
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3 6
C Microscopic images
Spinal cord general view 1 White matter; 2 Anterior horn; 3 Posterior horn; 4 Gray comissure; 5 Central canal; 6 Ependymal cells; A Anterior horn; B Central canal; C Posterior horn; D White matter.
Differential diagnosis: with the cerebellum, based on the localization of the gray and white matter, respectively the individual histological features. Cerebellum, HE stain. - on low magnification: we will observe the general appearance of the whole cerebellum, with the gray matter at the surface (cerebellar cortex) and the white matter in the interior (arbor vitae). In the cerebellar cortex we can recognize three characteristic layers: molecular layer, at the surface; Purkinje cells layer, in the middle; granular layer, adjacent to the white matter; - on high magnification: we will study the cytological organization of each layer of the cerebellar cortex:
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- the molecular layer, placed right under the meninges, has a fibrillar appearance, consisting mainly of neuronal processes from the underlying layers. It has few star-shaped cells, some of them being small and placed superficially while the others, larger, are profoundly located; - the Pukinje cells layer (intermediary layer) is built up by only one row of pear-shaped cells, with their basal part oriented towards the granular layer. From the top of each cell 1-3 thick dendritic processes emerge, that will richly branch in the molecular layer, forming a tree-like structure; from the bottom of the cells a single axon emerges, descending towards the white matter. Purkinje cells have an eosinophilic cytoplasm and a large, hypochromatic and centrally located nucleus, in the most prominent part of the cell. -the granular layer is close to the white matter, being the thickest layer, very rich in cells. In HE staining only the nuclei of the neurons forming this layer, and those of the glial cells are visible. These numerous, small, rounded, hyperchromatic nuclei yield the granular aspect of the layer. Under the cerebellar cortex we can observe the white matter, as a central shaft with fibrous aspect, consisting of myelinated and unmyelinated nerve fibers.
2 3 4
Microscopic image
Cerebellar cortex 1 Molecular layer; 2 Layer of Purkinje cells; 3 Granular layer; 4 White matter.
Differential diagnosis: with the spinal cord, based on the localization of the gray and white matter, respectively the individual histological features.
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PERIPHERAL NERVOUS SYSTEM (PNS)

PNS consists of ganglia (dorsal root or spinal and autonomic or vegetative ganglia) and of peripheral nerves (expansions of the motor and sensory neurons). Autonomic (vegetative) ganglia, HE stain. - on low magnification: we will study the general organization of the ganglia. It is surrounded by a thin capsule of connective tissue, and contains randomly arranged neurons, each of them surrounded by a thin capsule. Between the neurons unmyelinated nerve fiber can be seen. - on high magnification: the features of the neurons can be visualized. These are small, star-shaped neurons due to their numerous processes (multipolar neurons) with 1-2 nuclei placed at the periphery of the cell (eccentrically located). Each neuron is surrounded by an incomplete row of satellite cells and a very thin, fibrous capsule. Between the neurons unmyelinated nerve fibers can be observed, appearing as fine, randomly arranged eosinophilic filaments.
Vegetative ganglion 1 Neuron; 2 Satellite cell; 3 Nerve fibers.
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THE CARDIOVASCULAR SYSTEM

The circulatory system consists of the heart, a muscular cavitary organ (a pumping organ) and the vascular system, system of tubules (vessels) that transports the blood throughout the whole body: capillaries, arteries and veins.
THE VASCULAR SYSTEM

Capillaries are small caliber vessels (with a diameter of 7-14 microns), which allow the red blood cells to pass only in a single file. Structurally, the capillary wall is very thin, as it is formed only by an endothelium that rests on a basement membrane. In between the layers of the basement membrane, sometimes we can see an undifferentiated cell, named pericyte. Capillary, Masson-Goldner trichrome stain. - on low magnification: we will study the general structure of a cross section through the tongue, and observe the dorsal surface of the mucosa, that covers the papillae of the tongue. This mucosa consists of a stratified squamous epithelium, stained in brown, with a keratin layer here and there, stained in orange, and a lamina propria, in which the collagen fibers are stained in green. In the lamina propria we will see the capillaries under different sections with a narrow lumen, delimited by a very thin wall.
Cross section Microscopic image
Longitudinal section 1
Capillary 1 Nucleus of endothelial cell; 2 Nucleus of the pericyte; 3 Red blood cells. 49
- on high magnification: in longitudinal section, we will observe the capillary wall formed by a single layer of flattened squamous cells (endothelial cells) placed on a basement membrane. The endothelial cells have an oval, hyperchromatic nucleus, oriented parallel to the basement membrane, stained in brown-black and that protrude into the vessels lumen in an alternating manner, like saw teeth. Adjacent to the capillaries wall we can see the nucleus of the pericyte, with a different appearance than the nucleus of the endothelial cell: it is larger and hypochromatic. In cross section, the capillary has a small, rounded lumen, in which at most one nucleus protrudes (the nucleus of the endothelial cell). The arteries. Arteries have a wall composed of three tunics or layers: - the tunica intima lines the lumen of the vessel and consists of: endothelium and subendothelial layer. - the tunica media is the most developed layer, formed of smooth muscle fibers and elastic lamellae in different proportions in different types of arteries, concentrically arranged around the lumen. - the tunica adventitia formed especially of collagen fibers and only few elastic fibers and smooth muscle fibers, placed longitudinally (along the vessels). Here we will also see the vasa vasorum and nervi vasorum. The media is delimited from the intima by the internal elastic lamina, respectively from the adventitia by the external elastic lamina. We will study two types of arteries: the large elastic arteries and the medium-sized muscular arteries. Large elastic artery, HE stain, aorta. - on low magnification: on cross section we will see the large, round or oval, regular lumen of the aorta, with a thick wall, consisting of three tunics: the tunica intima, which represents about 10%, the tunica media, 80%, and the tunica adventitia, 10% of the thickness of the wall. In HE staining, the elastic structures in the arterial wall are colorless, and appear as empty or paler stained (bright) spaces. - on high magnification: we will study the components of the three tunics: - the intima, with an obvious endothelium recognized due to the hyperchromatic nuclei that protrude into the lumen; the subendothelial layer formed of collagen fibers and fibroblasts concentrically arranged; - the internal elastic lamina consisting of condensed elastic lamellae that do not stain in HE, appears as a narrow, wavy, colorless space, which separates the tunica intima from the tunica media, . - the tunica media consists mainly of elastic lamellae, which are colorless in HE staining and that appear as thick, undulated, interrupted spaces because of their slightly spiraling arrangement. Among them we will observe smooth muscle fibers (longitudinally sectioned) with their specific morphology, rare collagen fibers and ground substance. The fibroblasts are missing, their function being performed by smooth muscle fibers.
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- the external elastic lamina is placed between the media and the adventitia, poorly represented and hardly visible in this staining. - the tunica adventitia is formed of collagen fibers, elastic fibers which are not arranged in lamellae and fibroblasts placed along the vessel. At the periphery of the adventitia we observe vessels: vasa vasorum (arterioles and venules) and nervi vasorum (nerves).
2 A 3
5 C
Microscopic image
Large elastic artery, HE overview A Intima; B Media; C Adventitia; 1 Endothelial cell; 2 Internal elastic lamina; 3 Elastic lamellae; 4 Smooth muscle cells; 5 External elastic lamina; 6 Vasa vasorum.
Large elastic artery, Weigert resorcin-fuchsin stain (RF), aorta. Resorcin-fuchsin is a special stain for elastic fibers, which appear blueviolet. Only elastic elements stain on this slide. - on low magnification: the high amount of elastic elements (fibers and lamellae) colored in blue-violet is highlighted; - on high magnification: we will study the presence and arrangement of elastic elements in each tunic. Intima presents rare and fine elastic fibers in the subendothelial layer. The internal elastic lamina consists of thin, sinuous, overlapping elastic lamellae. The media contains numerous, wavy elastic lamellae, concentrically oriented around the lumen and linked together by fine, oblique elastic fibers. The external elastic lamina appears as a condensation of elastic
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lamellae. In the adventitia we can see longitudinally placed elastic fibers. Also the elastic fibers of vasa vasorum appear as stained.
3 4
Microscopic image
Large elastic artery, RF staining 1 Internal elastic lamina; 2 Elastic lamellae; 3 External elastic lamina; 4 Adventitia.
Medium-sized muscular artery and vein, HE stain, vascular-nervous bundle. - on low magnification: we will see on cross section the artery with a circular, regular lumen and a thick wall of connective and muscular tissue, consisting of intima (5-10%), media (50%) and adventitia (40-45%); near the artery we will see the vein with a large, irregular and collapsed lumen and a thinner and less organized wall composed also of the three layers: intima (5%), media (15%) and adventitia (80%); - on high magnification: in the wall of the artery, the intima is recognized by the endothelium with oval, hyperchromatic nuclei bulging into the lumen, the very thin subendothelial layer and a very well represented internal elastic lamina, that appears as a colorless, narrow and very sinuous linear space. The media is made up of circular oriented smooth muscle fibers arranged in several layers with rare collagen fibers and elastic lamellae among them. At the limit between the media and the adventitia, the external elastic lamina is highlighted as a condensation of elastic fibers, usually hardly visible in HE staining. The adventitia has a similar structure with the adventitia of the elastic artery.
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Nearby the artery we will observe the structure of the vein, with an endothelium, a very fine subendothelial layer and very thin internal elastic lamina, less sinuous and colorless. In the relative thin media, we will see collagen fibers, some fibroblasts and a few smooth muscle fibers. The veins adventitia is the best represented tunic, formed of smooth muscle cells bundles, collagen fibers and a network of elastic fibers (invisible on this slide) distributed longitudinally. The adventitia also contains vasa vasorum and nervi vasorum.
1
Microscopic image
Muscular artery, HE 1 Intima; 2 Media; 3 Adventitia.
2 3
Microscopic image
Vein, HE 1 Intima; 2 Media, 3 Adventitia. 53
Differential diagnosis: between the muscular artery and elastic artery (HE and RF stain), respectively between the vein and the muscular artery, based on the proportion of the three tunics and their structure.
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DIGESTIVE SYSTEM
The digestive system is formed by the (gastrointestinal) tract and associated digestive glands. oral cavity, digestive
THE ORAL CAVITY

It represents the anterior compartment of the digestive tract through which it communicates with the exterior. It is composed of: - oral mucosa - lips - tongue - dental organ, formed by the teeth and the supporting system of them in the dental arch, the periodontium - salivary glands, associated glands of the digestive system The lips Lips are two muscular - cutaneous folds that delimit the anterior opening of the mouth. On a sagittal section through the lip we may observe that it is composed of a core mass of muscle and connective tissue, covered by: - skin on the external side - vestibular mucosa on the internal side (oriented towards the oral cavity) - free margin or vermilion, between the two sides. Lip, HE stain. - on low magnification: we will observe the general microscopic aspect of the section, with bundles of striated muscle fibers sectioned in different incidents and connective tissue rich in blood vessels in the center, covered by the three types of coatings. - on high magnification: we will study the structure of each side and that of the muscular and connective tissue central skeleton: - the external side, covered by a fine skin composed of epidermis and dermis, with hair follicles, sebaceous glands and sweat glands - the internal side, covered by the vestibular mucosa (part of the oral mucosa), consisting of a nonkeratinized stratified squamous epithelium with an underlining lamina propria and submucosa containing minor salivary glands. - the vermilion, representing a transition zone between the skin and the lips mucosa. The surface epithelium is a stratified keratinized squamous epithelium with some particularities: it is thin and the superficial keratinized layer is very fine. The lamina propria of this zone presents numerous papillae which protrude deeply into the thickness of the epithelium, reaching almost its surface. In the papillae we observe numerous capillaries. Due to the arrangement of the papillae and the reduced thickness of the epithelium, the red color of the blood in the capillaries
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can be visualized. This morphological aspect explains the reddish color of the free margin, specific for the lips. The vermilion does lack hair follicles, sweat and sebaceous glands. The central part of the lip is formed of bundles of striated muscle fibers belonging to the orbicular muscle of the lip, surrounded by connective tissue that contains small salivary glands.
II 9 2
5 I 8 4 6 1
III
Internal side
Vermillion Microscopic images
External side
Lip - sagittal section I Internal side; II Vermilion III External side. 1 Stratified nonkeratinized squamous epithelium; 2 Deep papilla in the vermilion; 3 Epidermis; 4 Sweat gland; 5 Hair follicle; 6 Sebaceous gland; 7 Striated muscle fibers; 8 Small salivary glands; 9 Nerves; 10 Blood vessel.
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The tongue The tongue is a muscular - epithelial organ that occupies the greatest part of the oral cavity. It consists of a core mass of connective and muscle tissue covered by a mucosa that is part of the oral mucosa, and has a different aspect on both sides of the tongue: smooth on the ventral (inferior) side and with small projections on the dorsal side. Tongue, HE stain. - on low magnification: we will observe the general morphological appearance of the section, recognizing: - the core mass consisting of bundles of striated muscle fibers with different orientations. Between the muscle bundles we may observe a connective tissue with blood vessels, nerves, adipose tissue and small salivary glands (serous and mucous) with excretory ducts extending through the lamina propria and opening on the surface of the epithelium - the mucosa with a typical appearance on both surfaces: Mucosa of the inferior surface of the tongue is thin and smooth and consists of: - a nonkeratinized stratified squamous epithelium and - a lamina propria (chorion) formed by a loose connective tissue, with numerous short papillae. Mucosa of the dorsal surface of the tongue is thick and irregular and it is formed by: - a thick keratinized stratified squamous epithelium and a lamina propria. This epithelium presents numerous projections, called lingual papillae. Each lingual papilla is formed by a core of connective tissue with blood vessels, covered by a nonkeratinized stratified squamous epithelium. After the shape, structure and localization four types of lingual papillae can be distinguished: filiform, fungiform, foliate and circumvallate papillae. The filiform papillae may be recognized on low magnification due to their flame-like shape, with a narrow tip. The tip of the papilla is covered by a hood of keratin. Filiform papillae have no taste buds. The fungiform papillae are mushroom-shaped projections, with a narrower basal part and smooth, slightly flattened, rounded upper part. They are covered by a nonkeratinized stratified squamous epithelium that contains a few taste buds. In the centre of the papilla, the connective tissue forms secondary papillae in contact with the epithelium. The foliate papillae are rudimentary in humans; they can be observed on the lateral surface of the tongue. They consist of low ridges parallel between them separated by deep mucosal clefts. They are covered by a nonkeratinized stratified squamous epithelium that contains few taste buds on the lateral sides.
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I 2 1
II
5 4 6
III Schematic representation
Microscopic image
Tongue I dorsal (upper) surface; II lateral surface; III ventral (inferior) surface. 1 - Filiform papilla; 2 - Fungiform papilla; 3 - Foliate papilla; 4 Small salivary glands; 5 Skeletal muscle fibers; 6 Epithelium of the ventral surface.
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The circumvallate papillae can be found only in the posterior part of the tongue, before the sulcus terminalis. They have some special characters, distinguishing them from the rest of the buds: - they are large, dome-shaped structures and thus they do not fit into the microscopic field, even examined on low magnification - they do not protrude from the surface of the tongue, residing in the thickness of the lingual mucosa - they are surrounded by a moat-like invagination, called vallum, which separates them from the rest of the lingual epithelium - the nonkeratinized stratified squamous epithelium that covers them, contains on the lateral sides of the papilla numerous taste buds - the connective tissue core of the papilla forms a lot of secondary papillae in contact with the epithelium - the lamina propria at the base of the papillae contains small, serous salivary glands (Ebners glands) with their excretory ducts opening into the base of the moats.
1 2 2
Microscopic image
Circumvallate papilla 1 Epithelium of the tongue; 2 Vallum; 3 Taste bud; 4 Ebners serous glands.
The taste buds represent the receptor organ of the gustatory system. - on low magnification: we will observe the taste buds that appear like small, barrel-shaped or oval pale-staining structures, located in the thickness of the epithelium of some lingual papillae. They start at the basement membrane, extend through the entire thickness of the epithelium and open onto the surface of the epithelium through a small opening called taste pore.
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- on high magnification: we will study in detail the histological structure of the taste buds. A taste bud is made up by three types of cells: taste receptor cells, supporting cells and basal cells. The taste receptor cells and the supporting cells are spindle-shaped, bent, arranged like the staves of a barrel; they extend from the basement membrane onto the surface of the epithelium. The basal cells can be found only near the basement membrane. On histological sample we may recognize them after the following features: - the supporting cells are thin cells located towards the periphery of the taste bud, but also in its interior part, surrounding and in the same time separating the other cells from each other. They have a darker eosinophilic cytoplasm and a centrally located, elongated and hyperchromatic nucleus. - the taste receptor cells are tall cells with a paler eosinophilic cytoplasm and a centrally located, oval, hypochromatic nucleus. At their apical pole, next to the taste pore, they have a long process with the role of a surface receptor for taste. - the basal cells are placed next to the basement membrane, are smaller, cuboidal with a rounded nucleus and do not reach the surface of the epithelium.
1 2
3 Microscopic image
Taste bud 1 Taste pore; 2 Supporting cell; 3 Basal cell; 4 Taste receptor cell.
Differential diagnosis of the tongue can be made with the lip, from which it differs by the absence of the skin and of the transition zone typical for the lip (vermilion), by the presence of the lingual papillae and by the typical structure of the core mass of the tongue with connective, muscular and glandular (small salivary glands) tissue.
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DIGESTIVE TUBE
The digestive tube, composed of different segments, with some structural particularities, has a common plan of organization. The wall of the digestive tube consists of four layers (tunics): 1. The mucosa, the innermost layer lining the cavity of the tube, is composed of: - surface epithelium, of different types according to the localization and function of each segment (stratified squamous or simple columnar, monomorphic or polymorph) - lamina propria (chorion) consisting of loose connective tissue with blood vessels, nerves, lymphocytes diffusely arranged or in form of lymphoid follicles and, in some segments of the digestive tube, glands (stomach, duodenum) and - muscularis mucosae which is a fine, thin muscular layer, composed of smooth muscle fibers arranged in two layers, an internal circular layer and an external, longitudinal layer. 2. The submucosa, composed of a dense irregular connective tissue, with large blood vessels, lymph vessels and nerves. Also here we may find small autonomic, vegetative ganglia that form the plexus of the submucosa, Meissners plexus, and glands, in some parts of the digestive tube (esophagus and duodenum). 3. The muscular layer (muscularis externa, muscularis propria) consisting mostly of two thick layers of smooth muscle fibers, an internal layer with circular muscle fibers and an external layer with longitudinal muscle fibers. Exceptions from this arrangement are represented by: the stomach that presents a third layer of oblique muscle fibers placed above the circular layer, and the colon where the bundles of the outer muscular longitudinal layer fuse to form three equidistant longitudinal strips, called taeniae coli. Between the muscular layers a small amount of connective tissue with blood vessels, nerves and the myenteric nerve plexus or Auerbachs plexus (small vegetative ganglia and nerve fibers) can be found. On histological sections the muscle fibers in the circular layer appear longitudinally sectioned while those in the longitudinal layer appear transversally sectioned. 4. The external layer can be the serosa or the adventitia. The serosa, which covers at the exterior only those parts of the digestive tube located inside the peritoneal cavity, is composed of: a mesothelium, a simple squamous epithelium and a subserosal layer, situated between the mesothelium and the muscular layer, formed by a small amount of loose connective tissue rich in blood vessels, lymph vessels and nerves. The adventitia is present in the segments of the digestive tube located outside the peritoneal cavity and it is composed only of loose connective tissue, adipose tissue, blood vessels and nerves.
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Esophagus The esophagus is a long, hollow, tubular organ that links the oropharynx to the stomach. Esophagus, HE stain. - on low magnification: we will distinguish the four typical layers in the wall of the esophagus: mucosa, submucosa, muscular layer and the external layer. At this magnification we will observe that the mucosa exhibits numerous longitudinal folds, giving the lumen, on cross section, an irregular, star-shaped outline. - on high magnification: we will study the features of the four layers. The mucosa has three layers: - surface epithelium is a nonkeratinized stratified squamous epithelium, separated from the underlying lamina propria by a basement membrane which is strongly curled. - lamina propria (chorion) with a structure that has been already described in the general part and that lacks glands; it invaginates into the thickness of the epithelium generating papillae - muscularis mucosae with the characteristic structure described in the general part (an internal layer of circular oriented smooth muscle fibers and an external layer of longitudinal oriented smooth muscle fibers). The submucosa is formed by a loose connective tissue rich in blood vessels, lymph vessels, Meissners nerve plexus, lymphocytes diffusely arranged or as small lymphoid follicles and mucous tubulo-acinar (esophageal) glands. The mucous acini release their secretion through an excretory duct with a dilated lumen, lined by a thin stratified squamous epithelium that crosses the mucosal thickness and opens at the surface of the epithelium. The muscularis propria consists of two layers: an internal circular and an external longitudinal one, but with a different structure in different parts of the esophagus. Thus, in the upper third the muscular layer is formed almost entirely by striated muscle fibers, in the middle third by striated and smooth muscle fibers, while in the lower third the smooth muscle fibers predominate. The external layer is represented mostly by adventitia and only in a small portion, before the gastroesophageal junction, by the serosa (with the structure described in the general part). Differential diagnosis can be made with other segments of the digestive tube, the most important elements of diagnosis typical for the esophagus being the surface epithelium, of nonkeratinized stratified squamous type, and the presence of glands in the submucosa.
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1 2 3 4 5 6
7 8 9 10
Microscopic image
Esophagus 1 Nonkeratinized stratified squamous epithelium; 2- Lamina propria; 3 Muscularis mucosae; 4 Submucosa; 5 mucous acini; 6 Meissners plexus; 7 - Internal muscular layer (circular); 8 Auerbachs plexus; 9 - External muscular layer (longitudinal); 10 Adventitia;
The stomach It is a muscular and hollow organ that extends from the cardia to the pyloric sphincter, with a wall formed by the characteristic four layers. From histological and anatomical point of view the stomach has three distinct regions: the cardia situated under the gastro-esophageal junction, the fundus located between the cardia and pyloric sphincter, and the pyloric region, located proximally to the pyloric sphincter. All these regions differ one from each other only upon the aspect of the glands in the mucosa, the histological structure of the other layers being identical. Stomach, fundus region, HE stain. - on low magnification: we will observe the arrangement and the structure of the four layers of the gastric wall. - on high magnification: we will study in detail the structure of each layer.
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The mucosa: The surface epithelium is a simple columnar and monomorphic (composed of only one type of cells). It invaginates from place to place into the lamina propria forming the gastric pits or foveolae. The cells of the epithelium are tall, columnar, eosinophilic, with a paler stained and vacuolar apical pole due to its mucin content, the secretory product of these cells. They have an oval, euchromatic nucleus, located in the lower third of the cell, perpendicular to the basement membrane. The surface epithelium of the stomach in its entirety forms a gland-like structure (membraniform gland), an exocrine gland that secrets mucin. Lamina propria contains the gastric glands, called fundic glands, and a small amount of loose connective tissue. The fundic glands are simple or branched, long tubular glands, consisting of neck, a body and a basal, fundus portion. They open into the gastric pits and occupy the whole thickness of the lamina propria. The fundic glands are lined by a simple columnar epithelium composed of different types of cells: - mucous neck cells just beneath the opening of the glands into the gastric pits; they have a similar aspect with the mucous cells of the surface epithelium, but they are smaller and often difficult to be observed in HE stain. - chief cells (peptic cells) or basophile cells are the most numerous cells present in the lining epithelium of the fundic glands. They are columnar and through their apical pole they are delineating the lumen of the gland. Chief cells have a basophilic cytoplasm, while the nuclei are oval, euchromatic and located in the lower third of the cell. The role of these cells is to secrete the pepsinogen. - parietal cells (oxyntic cells) or oxyphilic (acidophilic) cells are more numerous in the upper part of the glands. They are large cells, polygonal (triangular on section) resting on the basement membrane which they slightly deflect outwardly. Their apical pole becomes narrower being compressed by the neighbouring chief cells. The nuclei of the parietal cells are rounded, centrally located and their cytoplasm is intensely eosinophilic. They secrete hydrochloric acid (HCl) and the intrinsic factor. - besides these cells, in the glandular epithelium enteroendocrine cells and stem cells, which are not visible in HE stain, appear. They can be highlighted using immunohistochemical reactions with monoclonal antibodies: for instance with anti-chromogranin antibodies. By this method the cytoplasm of enteroendocrine cells appears stained in brown. The muscularis mucosae is composed of the two layers: internal circular and external longitudinal. The submucosa with the structure already described in the general part, has no glands. The muscularis externa of the stomach is a thick layer and it differs from the standard structure of the digestive tube by presenting a third layer of smooth muscle fibers oblique oriented. Thus the muscular layer of the stomach is formed of three muscular layers: internal oblique, intermediary circular and external longitudinal. The serosa of the stomach has the characteristic structure described above at the general outline of the digestive tube.
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2 1 3 4 I 5 6
7 8 9 II
10
III 11 12 IV 13 14 15
Microscopic image
Stomach, fundic region I Mucosa; II- Submucosa; III Muscularis externa; IV Serosa. 1 Monomorphic simple columnar epithelium; 2 Gastric pits; 3 Mucous neck cells; 4 Chief cells; 5 Parietal cells; 6 Lamina propria; 7 Fundus glands in cross section; 8 Muscularis mucosae; 9 Meissners plexus; 10 Internal layer of muscularis externa (oblique); 11 Middle layer of muscularis externa (circular); 12 Auerbachs plexus; 13 External layer of muscularis externa (longitudinal); 14 Subserosal layer; 15 Mesothelium.
Differential diagnosis will be made with the pyloric region of the stomach based on the different structure of the glands in the lamina propria.
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Stomach, pyloric region, HE stain. - on low magnification: we will study the structure and arrangement of the four layers of the gastric wall.
1 2
4 5 II
6 III 7 8 9 10 11
IV
Microscopic image
Stomach, pyloric region 1 Monomorphic simple columnar epithelium; 2 Deep gastric pits; 3 Pyloric glands; 4 Muscularis mucosae; 5 Meissners plexus; 6 Internal muscular layer (oblique); 7 Middle muscular layer (circular); 8 Auerbachs plexus; 9 External muscular layer (longitudinal); 10 Subserosal layer; 11 Mesothelium.
- on high magnification: we will study the particular features of each layer. The wall of the pyloric region of the stomach has an identical structure with that of the fundic region. Differential diagnosis is based on the presence of shorter pyloric glands opening in deeper and larger gastric pits than the fundic glands. They are branched, coiled, tubular glands, delineated almost entirely by mucous cells, similar to the surface mucous cells. Glands have a relatively wide lumen due to the high viscosity of the secretion product (mucus).
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Differential diagnosis can be made with the fundic region of the stomach based on the different structure of glands in the lamina propria. Small intestine The small intestine is the longest portion of the digestive tube. It consists of three segments: duodenum, jejunum and ileum. The lumen of the intestine is not smooth due to the presence at this level of differentiations of the intestinal wall in order to enlarge the contact surface with the intestinal content. These differentiations, visible on low magnification are the circular folds (plicae circulares) also known as valves of Kerckring and the intestinal villi. The valves are permanent transverse folds that contain a core of submucosa, each valve extending on half or on three quarters of the way around the circumference of the lumens. The intestinal villi are finger-like projections of the mucosa extending into the lumen. Duodenum The duodenum is the first segment of the small intestine after the pyloric region of the stomach. Duodenum, HE stain. - on low magnification: we will study the structure and arrangement of the four layers of the intestinal wall and that of the villi. In the duodenum the intestinal villi are leaf-shaped. - on high magnification: we will study the structure of the intestinal villi and also the structure of each layer. The intestinal villi are covered by the surface epithelium of the mucosa and are centered by a core of connective tissue, which is an extension of the lamina propria. In the centre of the villus the so called lacteal can be seen (a lymphatic capillary with a narrow lumen, oriented longitudinal, lined by a row of flattened, squamous cells from which only the elongated, hyperchromatic nuclei protruding into the lumen are visible). Around the lacteal smooth muscle fibers from the muscularis mucosae can be found. The mucosa The surface epithelium covers the intestinal villi and afterwards it protrudes into the lamina propria forming the crypts of Lieberkhn or Lieberkhn (intestinal) glands: simple tubular glands that occupy most of the lamina propria and that are separated one from each other by thin septa of connective tissue. The Lieberkhn glands are opening on the surface of the mucosa at the base of intestinal villi. The surface epithelium is a simple columnar, polymorph epithelium composed of various cell types: - enterocytes that are tall columnar cells with oval nuclei, located in the lower third of the cell, perpendicular to the basement membrane. At the apical pole
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these cells present the striated border (brush border) as a slightly striated, eosinophilic band. - goblet cells that are placed between the other cells of the surface epithelium. They are barrel or champagne glass shaped cells with a more prominent cell body and apical pole, colorless in HE due to its content in mucus. At the basal pole a flattened, hyperchromatic nucleus can be seen. - Paneth cells which are cells present only in the small intestine. They are located in the epithelium lining the base of the Lieberkhn glands, in groups of 4-8 cells. They are tall, columnar cells, with a round or oval nucleus, hypochromatic and basally located. The cytoplasm above the nucleus is intensely eosinophilic and granular. - the rest of the cells in the lining epithelium and in the Lieberkhn glands (enteroendocrine cells, stem cells and M cells) cannot be visualized in usual stains with HE. The lamina propria is built up by a connective tissue rich in blood vessels, lymph vessels, lymphocytes, lymphoid follicles and nerves surrounding the Lieberkhn glands. The muscularis mucosae is composed of the two layers of smooth muscle fibers. From the muscularis mucosae smooth muscle fibers are starting, that will pass through the lamina propria and will reach the core of the villi. The submucosa has the structure already described at the general outline of the digestive tube. It contains branched tubulo-acinar mucous glands, called Brunners glands, made up by mucous acini and excretory ducts that are opening into Lieberkhn glands. The muscularis propria also has the structure described at the general outline of the digestive tube (with an internal circular layer and an external longitudinal layer). The external layer at the level of the duodenum is represented by the adventitia or serosa, depending on the extra- or intraperitoneal localization of the organ. Differential diagnosis will be made with the other segments of the small intestine (better developed villi, leaf-like shaped, a smaller number of goblet cells and presence of Brunners glands in the submucosa) and with the esophagus containing also glands in the submucosa, but without intestinal villi.
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3 4 II
5 6 III 7 8 IV
Microscopic image
Duodenum I Mucosa; II Submucosa; III Muscularis propria; IV Adventitia. 1 Villus; 2 Lieberkhn gland; 3 Muscularis mucosae; 4 Brunners glands; 5 Meissners plexus; 6 Inner muscular layer (circular); 7 Auerbachs plexus; 8 External muscular layer (longitudinal).
Ileum It is the last segment of the small intestine that continues with the large intestine at the level of the ileocecal junction. Ileum, HE stain. - on low magnification: we will study the structure and arrangement of the four layers of the intestinal wall. The circular folds (Kerckrings valves) and intestinal villi are also present, the last ones being shorter and thinner (finger-like).
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- on high magnification: we will study the structure of each layer. The general outline of the ileum is similar to that of the duodenum, the difference being represented by: - the structure and the shape of the intestinal villi - the increasing number of goblet cells in the ileum - the presence in the lamina propria and submucosa of some large aggregates of lymphoid tissue (MALT type) consisting of a few lymphoid follicles and an interfollicular, diffuse lymphoid tissue. This structure is called the Peyers patch. The Peyers patches are located in that part of the ileum placed at the opposite of the mesenterys insertion region. In the region where Peyers patches are located, intestinal villi are infiltrated with lymphocytes, are atrophic and sometimes they even lack.
II
3 4 5
III IV
6 7 8 9 10
Microscopic image
Ileum I Mucosa; II Submucosa; III Muscularis externa; IV Serosa. 1 - Vilus; 2 Lieberkhn gland; 3 Muscularis mucosae; 4 Peyers patch; 5 Meissners plexus; 6 Internal muscular layer (circular); 7 Auerbachs plexus; 8 External muscular layer (longitudinal); 9 Subserosal layer; 10 Mesothelium.
Differential diagnosis will be made with the other segments of the small intestine, based on the shape of the villi, lack of glands in the submucosa and the presence of the Peyers patches.
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Large intestine The large intestine is the end portion of the digestive tube formed by the following segments: cecum, appendix, colon (ascendant, transverse, descendent and sigmoid), rectum and the anus. Colon, HE stain. - on low magnification: we will study the structure and arrangement of the four layers of the intestinal wall. We will observe that in the large intestine the mucosa has a smooth surface, because the intestinal villi will disappear at this level. - on high magnification: we will study the structure of each layer. The mucosa is smooth, without circular folds and villi. The surface epithelium of the mucosa is a polymorph simple columnar epithelium. The invaginations of the epithelium will form the crypts of Lieberkhn or intestinal glands. These are simple tubular glands, which extend in the whole thickness of the lamina propria and that open at the mucosal surface. They have a neck, a body and a basal, fundus part placed near the muscularis mucosae. The glands wall is lined by the same type of epithelium as that on the surface and consists of following cell types: absorptive cells, goblet cells, whose number increases as we approach the terminal part of the large intestine, stem cells, enteroendocrine cells and M cells, the last three types being visible only by complementary examinations (immunohistochemistry, electron microscopy). Lamina propria has in general the same structure with that of the whole digestive tube, but it shows an increasing development of the mucosa-associated lymphoid tissue (of MALT type) isolated lymphoid follicles and an interfollicular tissue. The muscularis mucosa is formed of the two characteristic layers of smooth muscle fibers. The submucosa has the same structure with that described in the general outline of the digestive tube. In the submucosa of the large intestine we will find no glands, while the lymphoid tissue is more abundant. The muscularis propria has a characteristic structure in the colon. It is formed also by two layers of smooth muscle fibers (inner circular and outer longitudinal) but the outer longitudinal layer is divided into three longitudinally oriented ribbon-like bands, called taeniae coli. The external layer is represented by the serosa with its typical structure described above. Differential diagnosis will be made with the small intestine, based on the absence of the intestinal villi, the aspect of the surface epithelium, presence of the taeniae coli.
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2 II 3
4 III 5 IV Microscopic image Schematic representation 6
Colon I Mucosa; II Submucosa; III Muscularis propria; IV Serosa. 1 - Lieberkhn glands; 2 - Muscularis mucosae; 3 Meissners plexus; 4 Internal muscular layer (circular); 5 Auerbachs plexus; 6 External muscular layer (longitudinal).
DIGESTIVE GLANDS
The digestive glands that we will study are: major salivary glands (parotid, submandibular and sublingual glands), pancreas with its double, exocrine and endocrine component, and the liver. MAJOR SALIVARY GLANDS The main or major salivary glands are: the parotid, submandibular and sublingual glands. Their role is to secrete saliva. All major salivary glands have the same general plan of organization: - at the exterior they are surrounded by a connective tissue capsule, consisting of connective tissue fibers, blood vessels, nerves, adipose tissue. The capsule sends septa, with the same structure, into the parenchyma dividing the gland into lobules. - the parenchyma consists of secretory acini (serous, mucous and mixed) and an intralobular system of excretory canaliculi or ducts (intercalated ducts or Boll passage, and striated ducts). - the stroma of the salivary glands consists of all the connective tissue septa that divide the gland into lobules, and also of the intralobular stroma (surrounding
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the acini) which includes: connective tissue, blood vessels, nerves, a variable number of adipose cells that increase with age and lymphoid infiltrates. In the connective tissue septa extralobular excretory ducts can be found. The parotid gland Its structure follows the above described general plan of organization of the salivary glands. The parotid gland can be easily recognized because the parenchyma is formed exclusively by serous acini. Parotid gland, HE stain. - on low magnification: we will observe the general structure of the gland. At the periphery we will see a connective tissue capsule, rich in adipocytes, and which contains blood vessels. The capsule sends septa into the gland dividing it into lobules. In these septa we can recognize blood vessels and the large, interlobular excretory ducts. Intralobular parenchyma consists only of serous acini and of intralobular excretory ducts, represented by the intercalated duct or the Boll passage and the striated ducts. - on high magnification: we will study the appearance of the serous acini and that of the intra- and extralobular excretory ducts of the parotid gland. The serous acini are small and each of them consists of 6-8 columnar cells resting on a basement membrane. The cytoplasm above the nucleus is eosinophilic (it contains zymogen granules) while under the nucleus it is intensely basophilic. The nucleus is round, euchromatic and centrally located. With their apical pole cells delineate a very narrow, point-like lumen, sometimes hardly visible on histological sections. Between the basement membrane and the basal pole of the serous cells we can find some elongated cells with an oval, hyperchromatic nucleus, placed parallel to the basement membrane, called myoepithelial cells. These cells have the role of helping the excretion of secretory products into the lumen. The intercalated duct or the Boll passage continues the lumen of the serous acini and consequently it has a very small diameter. It is lined by a simple cuboidal epithelium and a discontinuous row of myoepithelial cells. The striated duct continues the intercalated duct, and it can be found in the glandular parenchyma (surrounded by serous acini). It consists of a single layer of tall, columnar cells, with a round, centrally placed nucleus and an eosinophilic cytoplasm with striated subnuclear appearance. The lumen of this duct is well delineated and it is much larger than that of the intercalated duct and of the acini. The interlobular excretory ducts can be found in the interlobular septa, being surrounded by connective tissue and having a large lumen. Their wall consists in the initial part of a tall columnar epithelium and as the lumen becomes larger and larger, the epithelium becomes bistratified.
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3 6 7 4
Parotid gland 1 Capsule; 2 Interlobular septum; 3 Serous acinus; 4 Intercalated duct (Boll); 5 Striated duct; 6 Interlobular duct; 7 Adipose cells; 8 Capillary.
Differential diagnosis between the excretory ducts
Intercalated duct Size Localization Epithelium Lumen very small intralobular, surrounded by acini cuboidal very small
Striated duct Same as an acinus intralobular, surrounded by acini tall columnar regular, larger than that of the acini
Extralobulary ducts large extralobular, surrounded by connective tissue bistratified large, irregular
The submandibular (submaxillary) gland It follows the general organization of the major salivary glands. The gland has a very thin capsule. The structure of the parenchyma is characteristic consisting of serous, mucous and mixed acini, the submandibular gland being a mixed, serous-mucous exocrine gland. Submandibular (submaxillary) gland, HE stain. - on low magnification: we will observe the capsule and the septa that divide the gland into lobules and the parenchyma of the submaxillary gland formed by three types of acini (serous, mucous and mixed) with different shape, size and staining.
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1 3
10 4 8 7
6 2 5 Schematic representation
Microscopic image
Submandibular gland 1 Capsule; 2 Interlobular septum; 3 Serous acinus; 4 Mucous acinus; 5 Mixed acinus; 6 Intercalated duct (Boll); 7 Striated duct; 8 Interlobular duct; 9 Adipose cells; 10 Capillary.
- on high magnification: we will study the histological features of the three types of acini. The serous acini are the smallest ones and more intensely stained. The mucous acini are larger and consist of columnar mucous cells with a paler stained cytoplasm (it contains mucus that does not stain in HE) and a flattened nucleus pushed to the periphery. The mixed acini are built up by a mucous acinus capped at one of the poles by a group of serous cells, forming the demilune of Gianuzzi. In all types of acini, between the basement membrane and the secretory cells, myoepithelial cells can be seen. The striated duct is long and so it can be easily recognized on the histological section. The intra- and extralobular excretory ducts have the same localization and structure like that in the parotid gland. The sublingual gland It follows the general organization of the major salivary glands. Characteristic is the fact that the parenchyma is formed especially of mucous acini and a few mixed acini (serous-mucous gland) and that the intralobular ducts are shorter and thus apparently less numerous. Sublingual gland, HE stain. - on low magnification: we will notice the lighter staining of the section through the sublingual gland, due to the predominance of the mucous acini, that stain paler in HE. We will also see a smaller number of intercalated (Boll passage) and striated ducts in the parenchyma. The Boll passage is very short and thus difficult to be seen.
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- on high magnification: we will study the same components of the gland as in the parotid and submandibular gland.
1
6 8
4 7
3 5 9 Microscopic image 10 Schematic representation
Sublingual gland 1 Capsule; 2 Interlobular septum; 3 Mucous acinus; 4 Mixed acinus; 5 Serous acinus; 6 Intercalated duct (Boll); 7 Striated duct; 8 Interlobular duct; 9 Adipose cells; 10 Capillary.
Differential diagnosis will be made between the three types of salivary glands, based on their microscopic structure. THE PANCREAS It is a parenchymatous organ forming a large gland, exocrine and endocrine in the same time. The exocrine component of the pancreas represents the largest part of the glandular parenchyma and consists of serous acini and excretory ducts. It secrets an alkaline fluid rich in enzymes and with an important role in digestion, that will be released into the duodenum through the pancreatic duct. The endocrine component represents only a small part of the parenchyma and consists of all the islets of Langerhans. It is responsible for the secretion of some hormones with major role in regulation of glucose metabolism. Pancreas, HE stain. - on low magnification: we will see a very thin connective tissue capsule that sends into the interior fine septa, dividing the gland incompletely into lobules. The exocrine pancreas resembles from a histological point of view with the parotid, consisting of serous acini and excretory ducts. The islets of Langerhans appear as rounded or oval islets, paler stained, interspersed between the pancreatic acini.
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- on high magnification: we will study the characteristic appearance of the pancreatic acini. They consist of 6-8 tall columnar cells placed on a basement membrane. Their cytoplasm presents a double staining: basophilic under the nucleus and eosinophilic above the nucleus. Above the nucleus the cytoplasm displays a granular aspect due to the presence of the zymogen granules secreted by the cell. In the centre of the acini we can see 1-2 pale, round or oval nuclei surrounded by a thin layer of cytoplasm (almost invisible in HE staining) that belong to the centroacinar cells. These cells represent the lining cells of the smallest intercalated ducts that extend on small distances into the lumen of the acinus; they are absent in all other acinar glands. The intercalated ducts converge to form intralobular ducts placed in the parenchyma of the pancreas, and these are continuous with interlobular ducts, lined by a bistratified epithelium and surrounded by the connective tissue of the septa. Striated ducts and myoepithelial cell are lacking in the exocrine pancreas. The islets of Langerhans are delineated at the periphery by a fine connective tissue capsule. Their cells are arranged in nests, with a fine capillary network between them. These secretory cells are small (comparative to the cells of the serous acini), have a granular and pale cytoplasm. In the islets of Langerhans four major types of cells can be described: alpha, beta, delta and F or PP. Nowadays, their identification can be made by using monoclonal antibodies. Differential diagnosis: can be made with the parotid gland.
3
1 4
Pancreas 1 Serous acini; 2 Intralobular duct; 3 Interlobular duct; 4 Islet of Langerhans.
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THE LIVER It is a lobulated, parenchymatous organ. The hexagonal-shaped lobules consist of cell plates radially oriented starting from a blood vessel placed in the core of the lobule, the central vein (centrolobulary vein). At the convergence of 3-4 hepatic lobules, some triangular spaces called the Kiernans portal spaces consisting of connective tissue, vascular elements and bile ducts can be observed. For illustration we will present sections from a pigs liver, were the liver lobules are much better seen than in humans due to the fact that they are better delimited by an abundant perilobular stroma. Liver, pig liver, HE stain. - on low magnification: we will notice the organization of the pig liver in lobules. These lobules have a polygonal shape and consist of hepatocyte plates convergent towards a vascular structure, placed in the centre of the lobule, central (centrolobulary) vein (CV). Between the hepatic lobules the Kiernan portal spaces can be observed. - on high magnification: we will study the appearance of the hepatocytes: polygonal-shaped cells, with a granular cytoplasm and a round, centrally located nucleus. Between the hepatocyte plates narrow, sinuous spaces called hepatic sinusoids can be observed. Their wall consists of endothelial cells and Kupffer cells, visible by their nuclei. In the Kiernan space, embedded in a relative abundant connective tissue we will see 4 components: an artery, a vein, 1-2 bile ducts and lymphatic vessels. - the vein can be recognized after its collapsed lumen, the thin and poorly structured wall - the artery has a regular, circular lumen, and a wall with a visible structure, corresponding to that of a small muscular artery (arteriole) - the lymphatic vessels appear as clefts lined by an endothelium, with no red blood cells in the lumen - the bile duct has a circular lumen, delineated by a simple cuboidal epithelium, with rounded nuclei, suggesting the aspect of a string of beads.
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1 7 5 4
2 6 3 Microscopic image Schematic representation
Liver 1 Central vein; 2 Plates of hepatocytes; 3 Hepatic sinusoids; 4 Arteriole; 5 Venule; 6 Bile duct; 7 Lymphatic capillary
Liver injected with China ink and stained with HE This staining is used for visualizing the Kupffer cells in the wall of hepatic sinusoids. In their cytoplasm different quantities of black granules of different sizes can be seen. These granules represent in fact the China ink with which the experimental animal was injected before slaughter and that was ingested by the Kupffer cells.
1 2
Microscopic image
Liver parenchyma 1 Kupffer cells; 2 Hepatic sinusoids; 3 Hepatocytes.
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RESPIRATORY SISTEM
The respiratory system consists of two portions: an air-conducting portion, represented by all the structures that conduct the air to the alveoli, and a respiratory portion, consisting of all the structures that take part in gas exchange. The air-conducting portion has two components: extrapulmonary and intrapulmonary. The extrapulmonary component consists of: - nasal cavities - nasopharynx and oropharynx - larynx - trachea - major bronchi (primary) The intrapulmonary component consists of: - lobar bronchi (secondary) - segmental bronchi (tertiary) - bronchioles - terminal bronchioles. The respiratory portion is the segment where gas exchanges take place and includes: - respiratory bronchioles - alveolar ducts - alveolar sacs - alveoli The intra- and extrapulmonary respiratory ducts are tubular structures morphologically adapted to the respiration. Therefore they will have a general plan of organization, with structural changes at the level of the different segments, linked strictly to the functional aspect of the specific segment.
TRACHEA
It is a tubular structure that will be studied on a cross section. Trachea, HE stain. - on low magnification: we will identify the 4 layers of the tracheal wall: mucosa, submucosa, fibro-cartilaginous layer, and adventitia. The cartilaginous layer is C-shaped, thus incomplete at the posterior side, the endings being joined by the trachealis muscle. Because the section is made through the cartilage, the fibrous layer located between the cartilage rings cannot be seen. - on high magnification: we will study the structure of the 4 layers of the tracheal wall in detail.
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The mucosa consists, like every other mucosa, of a surface epithelium and a lamina propria. The epithelium is of respiratory type, that is pseudostratified columnar ciliated, consisting of 5 types of different cells. In HE stain only 3 types can be identified: basal cells, with a round nucleus placed near the basement membrane, columnar ciliated cells and goblet cells, with their known structure. The lamina propria consists of a loose connective tissue that may present a diffuse lymphoid infiltrate belonging to the MALT system (Mucosa-Associated Lymphoid Tissue). The submucosa is a layer of loose connective tissue, with tracheal glands consisting of mixed acini which are releasing their secretory product at the surface of the epithelium, through a duct lined by a simple cuboidal epithelium. The tracheal cartilage is of hyaline type, having the above described incomplete structure. On the posterior side of the trachea, the free endings of the cartilage are united through bundles of smooth muscle fibers that form the trachealis muscle. They are longitudinally sectioned and have the characteristic structure of the smooth muscle fibers. At this level, the epithelium is folded. The adventitia consists of a loose connective tissue, adipose cells, blood vessels and nerves.
4 3 6
Microscopic image
Trachea 1 Respiratory type epithelium; 2 Lamina propria; 3 Submucosa with tracheal glands; 4 Hyaline cartilage; 5 Trachealis muscle; 6 Adventitia.
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THE LUNG
The lung has a structure determined by the distribution at this level of the bronchial tree and of the intrapulmonary vascular system. It is covered on its outer side by the visceral layer of the pleura that insinuates at the level of the scissurae, separating the pulmonary lobes. The connective tissue septa divide the parenchyma of the lobes in segments, and the segments in lobules. The pulmonary lobules are separated through very fine septa that are hardly visible on histological sections. Every pulmonary lobule consists of pulmonary acini, the smallest structural units of the lung. Lung, HE stain. On a cross section through the lung we may study the structure of different segments of the intrapulmonary bronchial tree (intrapulmonary bronchus, bronchiole, respiratory bronchiole, alveolar duct, alveolar sac and pulmonary alveolus) and that of the pulmonary stroma. - on low magnification: we will notice the lace-like appearance of the lung, given by the pulmonary alveoli and we will observe the different, intrapulmonary segments of the bronchial tree seen on the section, such as that of the intrapulmonary bronchus, bronchiole, respiratory bronchiole, alveolar ducts, alveolar sacs and pulmonary alveoli. - on high magnification: we will first identify each segment of the bronchial tree according to their specific histological structure. Afterwards we will make a differential diagnosis between these different components. The intrapulmonary bronchi have a large lumen, with a wall consisting of the following five layers: - the mucosa is folded, and consists of a respiratory type of epithelium (pseudostratified columnar ciliated) and a lamina propria that reduces gradually in thickness with the bronchi diameter. In the epithelium we can recognize the same cells as in that of the tracheal epithelium: columnar ciliated cells, goblet cells, basal cells; - the muscularis formed by bundles of smooth muscle fibers, circular arranged; - the submucosa consisting of loose connective tissue and glandular acini with a mixed secretion; - the cartilage layer is represented by islets of hyaline cartilage that are becoming smaller and smaller, as we approach the bronchiolus; - adventitia, which continues with the adventitia of the adjacent structures (arterial ramifications, interstitial tissue of the pulmonary parenchyma) is represented by a fine layer of loose connective tissue. The bronchioles are airways with a diameter less than 1 mm. Their initial part is lined by a pseudostratified ciliated columnar epithelium that turns gradually into a simple columnar ciliated epithelium, as its diameter reduces. The goblet cells, still present in larger bronchioles, are missing in the smaller ones. Also the glands under the epithelium and the islets of hyaline cartilage are missing. The muscular layer is very well developed, with circular and spiral muscle fibers.
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4 5
6 7 A
Bronchus and bronchiole A Bronchus; 1 Respiratory epithelium; 2 Lamina propria; 3 Muscularis; 4 Islets of cartilage; 5 Glandular acini; 6 Adventitia; 7 Pulmonary parenchyma. B Bronchiole; 1 Ciliated simple columnar epithelium; 2 Lamina propria; 3 Reisseissen muscle; 4 Adventitia.
The respiratory bronchioles have a narrow lumen lined by a cuboidal epithelium that still has in its initial part some ciliated cells. The wall of the respiratory bronchiole is discontinuous, a characteristic feature of this segment; from place to place alveoli are opening in the lumen, directly or through alveolar ducts. In the wall of the respiratory bronchioles the muscle fibers are still present. The alveolar ducts are elongated tubular structures, with a discontinuous wall, due to the numerous openings of the alveolar sacs or of the alveoli. The only visible segment of the alveolar ducts is the very short, continuous part of the wall. In this part, the ducts are lined by a simple squamous epithelium, the alveolar epithelium, under which some smooth muscle fibers can be found, forming a ring around the openings of the alveolar sacs or the alveoli.
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Microscopic image
A AS AD RB
RB
TB AD AS A Schematic representation
Lung
TB Terminal bronchiole; RB Respiratory bronchiole; AD Alveolar ducts; AS Alveolar sacs; A Alveoli.
The alveolar sacs are hollow structures with an irregular lumen, surrounded by groups of pulmonary alveoli that are opening in a common space before their open into the alveolar ducts. The continuous portions are lined by an alveolar epithelium, with no muscle fibers underneath. The pulmonary alveoli appear like small, polygonal spaces. Every alveolus is delineated by its own, very thin wall, consisting of an alveolar epithelium, a simple squamous epithelium. The pulmonary alveoli are separated one from each other by alveolar septa that consist of a very thin layer of loose connective tissue very rich in blood vessels and pulmonary macrophages (dust cells).
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URINARY SYSTEM
The urinary system is composed of the kidneys (paired organs), ureters, urinary bladder and the urethra. The urine produced in the kidney will be conducted through the ureter into the urinary bladder, where it will be deposited until its excretion through the urethra. Their histological structure is very different, reflecting their different function.
THE KIDNEY
The kidney is an organ formed of parenchyma and stroma, covered by a thin connective tissue capsule. It has a medial concave and a lateral convex surface. The concave surface presents the renal hilum, through which the renal artery enters the organ, while the renal vein and ureter leave. The parenchyma of the kidney has two distinct zones, visible to the naked eye on a sagittal section through the kidney: - cortex, with a granular aspect - medulla, with longitudinally striated aspect Structurally, the parenchyma of the kidney consists of nephrons and collecting tubules. The nephron represents the structural and functional unit of the kidney with the role to filter blood and to ensure control of the urine concentration and composition. The nephron has two components, the renal Malpighi corpuscle and renal tubules. The stroma of the kidney called also renal interstitium, surrounds the renal corpuscles, renal tubules and blood vessels. It is formed of connective tissue with fibroblasts, collagen and reticular fibers. In order to be able to recognize and to describe microscopically the components of the kidney, a short description of the semi-microscopic structure of the kidney is necessary, structure that can be seen also by the naked eye on cross section through the kidney. The semi-microscopic structure of the kidney The medulla consists of the medullary pyramids of Malpighi and the Ferrein pyramids. The medullary pyramids of Malpighi are triangular structures, with their basal part oriented towards the renal capsule and their tip towards the renal hilum, forming the renal papilla. The Ferrein pyramids appear like parallel fascicles of the medulla that start from the basal part of the medullary pyramids towards the renal capsule, without reaching the surface of the kidney.
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The cortex consists of three zones: cortex cortices, cortical labyrinth and the renal columns of Bertin. The main zone of the cortex, also called cortex corticis, is situated immediately under the renal capsule and contains numerous red granular elements, the renal or malpighian corpuscles (corpuscles of Malpighi). The cortical labyrinth represents that zone of the cortex situated between the Ferrein pyramids; it also contains renal corpuscles. The renal columns of Bertin are formed by cortical substance that descends into the medulla, separating from each other the medullary pyramids of Malpighi. A Malpighi pyramid together with the Ferrein pyramids starting from its basal part and the related cortex (cortical labyrinth, columns of Bertin) are forming a renal lobe. A Ferrein pyramid together with the surrounding cortex (cortical labyrinth) forms a renal lobule. Kidney, HE stain. - on low magnification: we will recognize the cortex with the renal or malpighian corpuscles and the medulla represented by the large renal pyramids of Malpighi and by the small Ferrein pyramids, with a longitudinally striated aspect. - on high magnification: we will study the microscopic structure and the arrangement of the components of the nephron and that of the collecting tubules. The renal corpuscles of Malpighi are located in the cortex, in the cortex cortices and the cortical labyrinth. They are spherical and consist of the capsule of Bowman and the glomerulus. Each renal corpuscle has two poles: - a vascular pole, through which the afferent glomerular arteriole enters the corpuscle while the efferent arteriole goes out - a urinary pole, the site where the urinary space is continuous with the proximal convoluted tubule. The capsule of Bowman covers at the exterior the glomerulus and consists of: - a parietal layer formed of a simple squamous epithelium resting on a basement membrane, - a visceral layer, attached to the capillaries of the glomerulus, formed by flattened epithelial cells, called podocytes with a structure difficult to be observed in LM. Between the two layers of the capsule of Bowman, the urinary space can be observed, that appears colorless on the histological slides. The renal glomerulus consists of a converging capillary network and mesangium. Capillaries will be recognized after the nuclei of the endothelial cells and the content in red blood cells. The mesangium is formed by mesangial cells and mesangial matrix. Identification of the mesangial cells in LM and HE staining might be difficult. In fact in the renal glomerulus there are three different types of cells: podocytes, endothelial cells of capillaries and mesangial cells. Identification of
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these cells might be done by the appearance of their nuclei (3 types of nuclei), but this is difficult and uncertain in the normal kidney and in LM.
8 1 3 7 4 7 2 8 4 2 1 3
9 5 9 6 5
10 6 10 Schematic representations 11
Microscopic images
Kidney 1 Renal glomerulus; 2 Parietal layer of the Bowman capsule; 3 Visceral layer of the Bowman capsule; 4 Convoluted part of proximal tubule; 5 Straight part of proximal tubule; 6 Loop of Henle; 7 Straight part of distal tubule; 8 Convoluted part of distal tubule; 9 Collecting tubule; 10 Ducts of Bellini; 11 Capillary.
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The renal tubule continues the capsule of Bowman until the junction with the collecting tubules and has four distinct histological regions: The proximal tubule (PT) has a convoluted portion located in the cortex, around the renal glomerulus and a straight portion, pars recta, that descends into the medulla and is continuous with the loop of Henle. On a section it can be recognized after some particular features: - it is lined by a simple tall cuboidal/columnar epithelium, with cells having an intensely eosinophilic cytoplasm (rich in mitochondria) and a brushed border at their apical pole. The brushed border is formed by microvilli occupying almost the whole lumen of the tubule. Because of these structures the lumen of the PT is imprecisely defined. The loop of Henle, situated in the medulla, continues the proximal tubule. It is a straight tubule with a thin wall and two portions, a thin descending limb and an ascending limb. The wall of the loop of Henle is lined by a simple squamous epithelium, similar to that of blood vessels. It differs from the blood vessels in that the lumen contains no red blood cells. The distal tubule (DT) has a straight segment located in the medulla that continues the ascending limb of the loop of Henle, and a convoluted segment, located in the cortex, around the renal glomerulus, together with the similar segment of the PT. We may recognize it after some features that distinguish it from the PT: - it has a large lumen that is better delineated due to the absence of the brushed border - it is lined by a simple cuboidal epithelium, with more nuclei on the section, because the cells are smaller than those of the proximal tubule - cells are paler stained, because of the smaller number of the organelles present in the cytoplasm. The connecting tubules link the DT with the collecting tubules. These are very short ducts and cannot be identified on histological sections. The collecting tubules descend through the Ferrein pyramids into the renal pyramids of Malpighi. These tubules are lined by a simple cuboidal epithelium, with clear cell limits, pale cytoplasm and without the brushed border at their apical pole. The collecting tubules merge to form the papillary ducts or ducts of Bellini. The ducts of Bellini are located in the profound medulla, having the same histological structure as the collecting tubules. Between the ducts of Bellini a more abundant stroma of connective tissue can be found, than in the rest of the kidney. Urinary bladder The urinary bladder is a hollow organ with a wall consisting of 3 layers: mucosa, covered by the urothelium, the muscular layer, a thick layer formed by three overlapping layers and at the exterior the serosa or the adventitia. Urinary bladder, HE stain. - on low magnification: we will observe the three layers of the urinary bladder, the mucosa, composed of urothelium and lamina propria, the muscular
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layer and the serosa or the adventitia. In the empty bladder the mucosa is intensely folded while the urothelium shows several layers of cells. The relaxed bladder has a smooth mucosa and an urothelium formed of 2-3 cell layers. - on high magnification: we will study the structure of the three layers of the bladder wall. The mucosa is covered by the urothelium, while the lamina propria consists of a loose connective tissue with numerous blood vessels and nerves. In the lamina propria fine bundles of smooth muscle fibers may appear. The muscular layer is composed of three layers of smooth muscle fibers, inner longitudinal, intermediary circular and outer longitudinal, that are more often difficulat to be identified due the complex spatial arrangement. At the exterior the bladder is covered by the serosa or the adventitia both with the already known structure.
I 1
2 3
II 4
5 III
Microscopic image
Urinary bladder I Mucosa; II Muscularis; III Adventitia. 1 Urothelium; 2 Lamina propria with bundles of smooth muscle fibers; 3, 5 Longitudinal smooth muscle fibers; 4 Circular smooth muscle fibers.
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MALE REPRODUCTIVE SYSTEM

The male reproductive system consists of: - generative glands (testes) - excretory spermatic ducts (intratesticular and extratesticular) - accessory genital glands (seminal vesicles, prostate gland, bulbourethral glands of Cowper) - copulatory organ (penis)
THE TESTES
The testes are paired organs and have a double function: - reproductive by producing the male gametes in the seminiferous tubules - endocrine by secreting the male sexual hormones (testosterone) by the Leydig cells in the testicular interstitial tissue (intertubular space) In the structure of the testis we can distinguish the following components: - an inextensible capsule covering the organ, thickened at the posterosuperior margin to form the testicular mediastinum - stroma built up by connective tissue septa, that originate from the mediastinum and have a radial orientation, dividing partially the tissue into testicular lobes (poorly individualized in humans). Inside the lobes, between the seminiferous tubules an interstitial connective tissue exists where the endocrine Leydig cells are widespread. - parenchyma, a component represented by all the seminiferous tubules, containing he seminal epithelium. Testis, HE stain. - on low magnification: we will observe the general morphologic outline typical for the testicle. The parenchyma is represented by numerous seminiferous tubules, sectioned in different incidences. Between the tubules a small amount of interstitial tissue can be seen. - on high magnification: we will study mainly the elements of the seminiferous epithelium and the components of the interstitial tissue. In the structure of a seminiferous tubule following components can be noticed: The tunica propria also called peritubular tissue consists of 2-4 concentrical layers of collagen fibers, with fibrocytes and smooth muscle fibers between them. Under the tunica propria a basement membrane can be found (not visible in this staining) on which the seminiferous epithelium is resting. The seminiferous epithelium structurally appears as a stratified polymorph epithelium, built up of various overlapping cells. We can distinguish two cell populations one represented by the Sertoli cells (supportive and nutritional role), and the other one represented by the germ cells (that are undergoing spermatogenesis). The Sertoli cells (supportive or sustentacular cells) can be recognized after their large nucleus, oval or triangular, vesicular (pale) with 1-2 conspicuous
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nucleoli, usually located centrally. These large nuclei are located from place to place close to the basement membrane; the presence of their nucleoli makes them easy to be mistaken for a cell. The cell membrane and the cytoplasm of these cells are poorly visible due to their close contact with the germ cells.
1 9 2
5 8 Microscopic image 7 Schematic representation 6
Seminiferous tubule 1 Tunica propria; 2 Spermatogonia; 3 Primary spermatocytes; 4 Secondary spermatocytes; 5 Spermatids; 6 Mature sperm cell; 7 Nucleus of Sertoli cell; 8 Interstitial Leydig cells; 9 Blood vessel.
The germ cells are represented by the spermatogonia, primary and secondary spermatocytes, spermatides and sperm cells. Each cellular element will be identified by the shape, structure and its position in the seminal epithelium: - spermatogonia are placed in one row on the basement membrane, slightly distanced one from each other, with a smaller diameter than that of the cells placed above them (spermatocytes). The cell limits being hardly visible they will be recognized after their nuclei: small, rounded and with a powdery chromatin (spermatogonia with powdery nuclei) or with coarse, dark chromatin (spermatogonia with coarse nuclei); - primary spermatocytes are the largest cells, located at some distance from the tubules wall (right above the spermatogonia), in one row. Their nuclei are the largest ones; they are rounded and have a network like chromatin, corresponding to the different phases of the first meiotic division. - secondary spermatocytes are rarely observed on histological slides, because they remain in this phase only a limited period of time. They resemble to the primary spematocytes, but they are smaller and situated closer to the lumen of the tubule. - spermatides are located nearby the lumen of the tubule, in several layers, having a rounded or elongated shape and medium sized diameter. Their cytoplasm is slightly eosinophilic while cell boundaries are distinct. They too can be recognized by their nucleus: spherical in the first stages, and then more and more
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elongated. The nuclei have a homogenous appearance due to a condensed chromatin. - sperm cells are the mature cells easy to be recognized after their location and the appearance of the nucleus. Representing the last maturation stage of the germ cell, the sperm cell is situated close to the lumen of the seminiferous tubule, with its head embedded in the seminiferous epithelium and its tail (flagellum) oriented towards the lumen. The head of the sperm cell can be recognized after the shape of the nucleus: much reduced in volume relative to the nucleus of the spermatid, oval, intensely hyperchromatic due to the condensed and homogenous chromatin. The interstitial tissue is composed of a loose connective tissue, blood vessels, lymph vessels and nests of interstitial cells of Leydig, which secrete testosterone. These are large cells, polygonal, with rounded or oval, euchromatic nuclei, with 1-2 nucleoli. The eosinophilic and fine granular cytoplasm may contain a yellowish-brown pigment (lipofuscin) and/or some crystalloid elements, intensely eosinophilic: crystals of Reinke. Between these cells capillaries can be found, because the cells have an endocrine function. All Leydig cells together form the interstitial gland.
ACCESSORY GENITAL GLANDS

The prostate Prostate is composed of a system of compound tubulo-alveolar gland, with a very well represented connective - muscular stroma. Prostate, HE stain. - on low magnification: we will observe the morphologic organization of the prostate. The alveoli (glands) and excretory ducts have the same histological structure, so on histological slides they can be hardly distinguished from one another. In some alveoli we can see 1-2 prostatic concretions, which are oval and eosinophilic, with a lamellar appearance. They result from the precipitation and storage of the remaining prostate secretion product, arranged in concentric layers (corpora amylacea or sympexions of Robin). - on high magnification: we will study the stroma and the prostate glands. The prostatic stroma is composed of connective tissue (paler stained) and bundles of smooth muscle fibers with their typical appearance. The lining epithelium of the glands and that of the excretory ducts shows folds that usually have a core of connective tissue and blood vessels. This bistratified epithelium (resting on the basement membrane) is formed by a single row of flattened basal cells (with an oval, hyperchromic nucleus that is parallel to the basement membrane), overlapped by a row of tall, columnar secreting cells (with an oval nucleus, situated near the basal pole of the cell).
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Prostate gland 1 Bistratified glandular epithelium; 2 Muscular-connective tissue stroma.
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FEMALE REPRODUCTIVE TRACT

The female reproductive tract is represented by: - internal genital organs: ovaries, Fallopian tubes (oviducts), uterus, vagina - external genital organs (external genitalia): vulva (with the labia majora and labia minora) and the clitoris. We will study the structure of the ovary and uterus. Also in this chapter we will study the placenta, due to its close relationship with the pregnant uterus, and the mammary gland, structurally and functionally linked to the genital tract.
OVARIES
Ovaries are paired organs, with an oval flattened shape. The surface of each ovary is covered by a simple cuboidal or columnar epithelium, that flattens with age and under which we will find the tunica albuginea, with a fibrous avascular structure. The main tissue of the ovary can be divided into three compartments, from the surface to its depth: cortex, medulla and hilum. - the cortex is situated at the periphery, immediately under the tunica albuginea. It is a zone very rich in cells, with ovarian follicles in different stages of development and/or the corpus luteum, respectively corpus albicans. - the medulla is profound and consists of loose connective tissue with blood vessels and nerves. - the hilum is the place where blood vessels, lymph vessels and nerves enter and leave the ovary; here we may find the cells of the ovarian hilum, similar to the Leydig cells in the testicle. Ovary, HE stain. - on low magnification: we will observe the general morphologic outline of the ovary. On the surface of the ovary we can see a simple cuboidal or squamous epithelial lining. The cells in the epithelium have rounded or flattened dark nuclei, respectively, and rest on a thick basement membrane. Under the epithelium the tunica albuginea can be found, a layer of a dense connective tissue without blood vessels. Under the tunica albuginea lies the cortex easily to be distinguished after the numerous ovarian follicles, with or without a cavity, delineated by one or more cellular layers (follicular cells) and eventually a cellular capsule, the so called theca, with one or two layers. Also in the cortex we may see the menstrual corpus luteum, and scar structures, corpus albicans and atretic follicles. Between these structures lies the stroma of the ovary, formed by a connective tissue which is very rich in cells: spindle shaped cells, fine collagen fibers and a few smooth muscle fibers. The medulla consists of a loose connective tissue, large blood vessels with a sinuous trajectory, lymph vessels and nerves.
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Ovary histological image
- on high magnification: we will study especially the cortex, where we will distinguish the ovarian follicles in different phases of development, the corpus luteum and the corpus albicans: The primordial follicle is situated in the superficial part of the cortex and consists of an oocyte (a large rounded cell with a fine granular cytoplasm, a large eccentrically located nucleus, with a fine chromatin and large nucleolus) that is surrounded by an incomplete layer of flattened cells, called follicular cells. The primary follicle, also located in the superficial part of the cortex, represents the initial phase in the development of the follicles. The oocyte, which is enlarged, is completely surrounded by a layer of follicular cells that become cuboidal or columnar. Between the cellular membrane of the oocyte and the follicular cells an intensely eosinophilic, homogenous zone can be seen, called the zona pellucida. The activated or growing primary follicle is characterized by a large oocyte surrounded by the zona pellucida. The follicular cells, which are placed in several rows, are also called granulosa cells. The outermost layer of the granulosa cells is resting on a basement membrane that separates them from the connective tissue of the ovarian stroma. The secondary follicle (cavitary) deeper located in the cortex, is formed by a larger oocyte surrounded by the zona pellucida and a thicker layer, the membrana granulosa. Between the follicular cells small intercellular spaces filled with follicular fluid are appearing. Next, two sheaths surrounding the follicle will develop, the theca interna and theca externa. The profound theca interna is situated around the membrana granulosa, from which it is separated by a basement membrane. It is formed by enlarged stromal cells (secreting cells) and a rich capillary network. The thin theca externa consists of connective tissue and smooth muscle fibers.
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4 1
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10 11 2 3 12 13 14 15
16 17 7 8
Ovarian follicles 1 Surface epithelium; 2 Tunica albuginea; 3 - Ovarian stroma; 4 Primordial follicle; 5 Primary follicle; 6 Primary activated follicle; 7 Secondary follicle; 8 Tertiary (de Graaf) follicle; 9 Theca externa; 10 Theca interna; 11 Follicular basement membrane; 12 Cells of the granulosa; 13 Antrum; 14 Corona radiata; 15 Zona pellucida; 16 Primary oocyte; 17 Cumulus oophorus.
The mature follicle (de Graaf follicle) represents the phase of complete development of an ovarian follicle. It occupies the whole cortex and it protrudes at the surface of the ovary. In the mature follicle, the numerous spaces between the granulosa cells fuse and form a single large cavity the antrum follicularum, containing follicular fluid. The antrum is delineated by the membrana granulosa (granulosa), pushed towards the periphery, consisting of 2-3 cell layers. It is resting on a basement membrane that separates the granulosa from the cells of the theca interna. It does not stain in HE but it can be highlighted using special stains (with PAS). The oocyte eccentrically placed inside the follicle (at one pole) is embedded in a thick zone of the granulosa, called cumulus oophorus (discus proligerus). Cells of cumulus oophorus close to the oocyte are elongated and radially arranged around the oocyte, forming the corona radiata. The oocyte is a large cell and has a coarse granular cytoplasm, with a large nucleus, which is eccentric and vesicular so-called germinal vesicle and a dense nucleolus the germinal spot. At the exterior the follicle is surrounded by the thecae of the follicle, which are well developed in the tertiary follicle:
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- theca interna formed by 3-4 layers of elongated polygonal cells, with an abundant, eosinophilic and vacuolar (because of the lipid droplets) cytoplasm. Between the cells numerous capillaries can be found. - theca externa, formed by collagen connective fibers placed concentrically around the follicle and by smooth muscle fibers. The corpus luteum, developed after discharging of the oocyte out of the follicle (ovulation), consists of rounded or polygonal cells, arranged in irregular anastomosing cords, with numerous capillaries between them. These are called lutein cells. We can distinguish two types of lutein cells: - follicular lutein cells, which occupy most of the corpus luteum, are large polygonal cells, with an abundant pale eosinophilic cytoplasm, that appears vacuolar because of the fine lipid droplets. These cells have a larger, rounded, hypochromatic nucleus, with a conspicuous nucleolus. - theca lutein cells, smaller cells situated at the periphery of the corpus luteum, scattered around the blood vessels. They have a more intensely stained cytoplasm, with larger lipid droplets, while their nucleus is smaller and darker than that of the follicular lutein cells.
Microscopic image
Corpus luteum 1 Follicular lutein cells; 2 Theca lutein cells; 3 Blood vessels.
THE UTERUS
The uterus, from anatomic point of view, presents: a dilated superior part, (body or corpus and fundus), a narrow and very short middle part (the isthmus uteri) and a lower cylindrical part (cervix uteri). The uterus has two cavities: the uterine cavity and the canal of the cervix with two orifices, the internal orifice oriented towards the uterine cavity and the external orifice towards the vagina. The
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uterine wall has three layers with uneven thickness, each of them having a specific name: - perimetrium is the outer layer, represented partly by the peritoneal serosa (the fundus and the posterior part of the body) and adventitia in the rest. - myometrium or the muscular layer - endometrium or the uterine mucosa. Uterus, HE stain. - on low magnification: we will observe the general outline of the uterine wall: a thick muscular layer (myometrium) lined to the interior by the endometrium, placed directly on the muscles (submucosa is missing), and covered at the exterior by the perimetrium represented by serosa or the adventitia. The endometrium consists of the surface epithelium and lamina propria. The surface epithelium invaginates into the lamina propria forming the endometrial glands. The lamina propria or endometrial stroma is represented by the connective tissue between these glands. In the myometrium we will notice numerous blood vessels. - on high magnification: we will study in detail the structure of the uterine wall. The endometrium is a hormone dependent mucosa and therefore it has different outlines in the four different phases of the menstrual cycle. At practical works we will study only two phases: the follicular or proliferative phase and the progestational or secretory phase. The surface epithelium is a columnar epithelium consisting of two types of cells that alternate: ciliated and secretory cells. The endometrial glands are simple tubular glands, seldom branched, lined by a simple columnar epithelium with few ciliated cells. Between the glands the endometrial stroma can be observed, consisting of a connective tissue very rich in cells. Under hormonal influences (estrogens, progesterone) these structures of the endometrium have different appearances in the two phases of the menstrual cycle. The follicular phase (proliferative) takes place in the first 14 days of the menstrual cycle; in this phase the endometrium becomes thicker, reaching 2-3 mm. The glands, the stroma and the arteries in the stroma proliferate. The glands, straight in the beginning and equally distributed in the stroma grow in length and will become wavy, sinuous. Their lumen is narrow and empty. The cells of the glandular epithelium have basally located nuclei, with prominent nucleoli. The endometrial stroma is dense, darker stained and very rich in cells. In the stroma next to the myometrium spiral arteries can be observed, arteries that in this phase of the endometrial cycle will not reach the surface of the endometrium. In the progestational phase (secretory) the endometrium is much more different from that in the previous phase. It reaches 5-6 mm in thickness. The endometrial glands, growing too much in length will become coiled, with an increasingly larger, more irregular lumen that may contain the secreting product of the glands, which stains eosinophilic. At the beginning of this phase cells of the glandular epithelium have their nuclei oriented towards the apical pole, due to the accumulation of vacuoles filled with secreting product under the nucleus. Later the
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vacuoles will move above the nucleus dilating the cells apical pole and pushing the nucleus towards the basal part of the cell. Swelling of the endometrial stroma will occur (edema, due to interstitial fluid accumulation); stromal cells will undergo a decidual reaction, by becoming larger with an abundant pale eosinophilic cytoplasm (containing glycogen). The spiral arteries will reach the surface of the epithelium and will become intensely coiled.
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Microscopic image
Uterus, proliferative phase I Endometrium; II Myometrium; III Perimetrium. 1 Surface epithelium; 2 Endometrial glands; 3 Endometrial stroma; 4 Spiral arteries; 5 Smooth muscle fibers; 6 Blood vessels; 7 Mesothelium.
The myometrium represents the biggest part of the uterus. It is composed of bundles of long smooth muscle fibers, oriented in all directions, in a complex
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architecture in which the layers cannot be distinguished. The myometrium has a rich arterial and venous blood supply. Blood vessels in the myometrium have no adventitia. The perimetrium is formed by the peritoneal serosa that covers mainly the fundus of the uterus, the rest being covered by the adventitia. The structure of the serosa and that of the adventitia are already known.
Microscopic image
Uterus, secretory phase 1 Surface epithelium; 2 Endometrial glands; 3 Endometrial stroma with edema and stromal cells with decidual reaction; 4 Spiral arteries.
The cervix, HE stain. The cervix is the lower extension of the uterus, with a cylindrical shape and two (inner and outer) orifices. The part of the cervix projecting into the vagina is called ectocervix, while the portion between the two orifices is the endocervix. Its structure presents 3 layers, of which only the muscular layer and the mucosa differ from that of the uterus. - on low magnification: we will observe the outline of the cervical mucosa. The epithelium lining the ectocervix is identical with that of the vagina: stratified squamous nonkeratinized epithelium. The epithelium of the endocervix is a simple columnar one with mucous secreting cells. It displays numerous projections into the lamina propria, showing a glandular appearance. Towards the vaginal margin of the cervix there is a transition zone between the endocervix and the ectocervix. - on high magnification: we will observe the simple columnar epithelium of the cervical mucosa formed mostly of mucous cells and rare ciliated cells.
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We will study the transition zone, respectively the zone where the stratified squamous nonkeratinized epithelium of the ectocervix transforms into a simple columnar epithelium of the endocervix. This squamo-columnar junction has a high clinical importance, representing the starting point of various pathologies. Glands of the cervix are lined by an identical epithelium with that of the endocervix, are less numerous than the endometrial glands, but more branched and contain mucus in the lumen. The muscular layer consists of smooth muscle cells embedded in collagen and elastic fibers. Unlike the body of the uterus, in the muscular layer the connective tissue predominates.
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Cervix 1 Endocervical epithelium; 2 Transitional squamo-columnar zone; 3 Epithelium of the ectocervix; 4 Glands of the cervix; 5 Lamina propria; 6 - Muscular layer.
THE VAGINA
It is a fibromuscular tube inserted with its upper part on the cervix, while the lower part communicates with the vulva. In the structure of the vaginal wall we can distinguish three layers: mucosa, muscular layer and the adventitia. Vagina, HE stain - on low magnification: we will recognize the three layers of the vaginal wall: the mucosa with a squamous stratified nonkeratinized epithelium and a thick lamina propria, with papillae and without glands, but with numerous blood vessels; muscular layer that has two layers of muscle fibers (internal circular, external longitudinal); the adventitia consisting of a loose connective tissue, rich in adipose cells.
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- on high magnification: we will study especially the outline of the vaginal mucosa, following the shape of the cells in the stratified squamous epithelium.
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4 II
III
Microscopic image
Vagina I Mucosa; II Muscular layer; III Adventitia; 1 Surface epithelium; 2 Lamina propria; 3 Blood vessels; 4 Circular muscular layer; 5 Longitudinal muscular layer.
PLACENTA
Placenta is a temporary organ that develops inside the uterus during pregnancy intervening in changes between mother and fetus. It has two components: a fetal placenta and maternal placenta, represented by a very thick and histological modified uterine mucosa (decidua basalis). Fetal placenta consists of the chorionic plate and chorionic villi. The chorionic plate is a membrane-like structure, composed of a connective tissue core, lined towards the amnion by a simple columnar or squamous epithelium (amniotic epithelium), and towards the intervillous space by the
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trophoblast. From the chorionic plate stem villi will emerge that will branch giving rise to chorionic villi. Villi are highly branched structures, bathed by maternal blood. On histological slides, chorionic villi appear sectioned in different incidents. Maternal placenta is represented by the decidua basalis. Placenta, HE stain. - on low magnification: we will recognize the chorionic villi sectioned in different incidents (oblique, transverse, longitudinal) and also portions of the maternal decidua. - on high magnification: we will study the histological structure of the sectioned villi and that of the fragments of maternal placenta. In cross section a chorionic villus appears as consisting of a core of connective tissue and blood vessels (a network of collagen and reticular fibers, with numerous smaller and larger blood vessels) lined by an epithelium (trophoblast). The appearance of chorionic villi permits us to establish the age of the pregnancy: early placenta (until the fifth month of pregnancy) or late placenta.
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A
4
B
Microscopic image
Early placenta A Chorionic villus; B Decidua; 1 Fetal placenta (chorionic villus); 2 Syncytiotrophoblast; 3 Cytotrophoblast; 4 Mesenchymal tissue; 5 Maternal placenta (decidua); 6 Decidual cells.
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a. Early fetal placenta presents chorionic villi lined by a bistratified trophoblastic epithelium: - the syncytiotrophoblast is the outer darker layer, represented by cytoplasm mass with numerous rounded, hyperchromatic nuclei. - the cytotrophoblast, placed under the syncytiotrophoblast, is composed of one layer of smaller, cuboidal cells (Langerhans cells) with a basophilic cytoplasm and a large, euchromatic nucleus with a visible nucleolus. The cytotrophoblast is separated from the connective tissue core of the villus by a relatively thick basement membrane. The connective tissue in the core of the villus is of mesenchymal origin and consists of star-shaped cells with numerous branched processes, a mucoid intercellular matrix and a capillary network in which primitive fetal (nucleated) erythrocytes can be recognized.
2 1
A 3
4 B Microscopic image Schematic representation
Late placenta A Chorionic villus; B Decidua; 1 Chorionic villus; 2 Syncytiotrophoblast; 3 Core of connective tissue and blood vessels; 4 Decidua.
b. Late fetal placenta is characterized by further branching of chorionic villi; thus, on the slide they will appear smaller. The internal layer of the trophoblast, the cytotrophoblast will undergo atrophy and it will disappear. Thus,
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villi will be delineated only by the syncytial epithelium, in which from place to place we will observe islets of nuclei. The connective tissue core will turn into a fibrous tissue containing a rich network of blood vessels, fibroblasts and an acidophilic hyaline substance, the fibrin. Maternal placenta or the decidua develops from the functional upper layer of the endometrium. It consists of decidual cells: large, polygonal cells with visible cell limits and with large, hypochromatic nuclei, with an obvious nucleolus. The cytoplasm of these cells is abundant, granular and/or vacuolar, eosinophilic or basophilic. Between the decidual cells a network of dilated blood vessels and scraps of heavily modified (from secretory point of view) endometrial glands can be observed. Also large, multinucleated cells can be present.
MAMMARY GLAND
The mammary glands are intensely modified apocrine sweat glands. Structurally they are tubulo-alveolar glands. Every mammary gland consists of 1520 mammary lobes, each of the lobes being individualized and separated from each other by a layer of connective tissue and drained by an excretory duct (lactiferous duct), that opens at the nipple. In the adult nulliparous women the mammary gland is represented only by excretory ducts and stroma. The secreting alveolar structures will develop only during gestation and lactation. Resting mammary gland, HE stain. - on low magnification: we will observe the structure of the mammary gland formed by stroma and parenchyma, that is divided into lobes and lobules. Mammary lobules are easily recognized after their lighter color. They contain a stroma formed by loose connective tissue (functional stroma) and glandular components, represented by branched intralobular ducts. Lobules are separated from each other by thick connective tissue septa, representing the nonfunctional stroma. In this stroma interlobular ducts, the large excretory ducts of the mammary gland, can be found. - on high magnification: we will study the structure of the stroma and that of the glandular parenchyma. In the mammary lobule, functional stroma consists of a loose connective tissue, blood vessels and nerves. It does not contain adipose tissue. The glandular component is represented by intralobular ducts. These ducts have a narrow, rounded lumen delimited by a bistratified epithelium: myoepithelial cells next to the basement membrane and cuboidal or small columnar secreting cells, delineating the lumen. Myoepithelial cells can be recognized after their oval, hyperchromatic nucleus, parallel to the basement membrane. The intralobular ducts are branched and are ending in a group of blind saccular structures called terminal duct lobular units (TDLUs). These are small spherical structures, without lumen, consisting of tall columnar cells, surrounded by myoepithelial cells and the basement membrane.
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Resting mammary gland 1 Terminal duct lobular units (TDLUs); 2 Intralobular ducts; 3 Intralobular stroma; 4 Interlobular stroma; 5 Interlobular duct; 6 Adipose tissue.
The nonfunctional stroma represented by interlobular septa, consists of a denser connective tissue and a variable amount of adipose tissue. The interlobular ducts present in this stroma, have a large, irregular lumen lined also by a bistratified epithelium, composed of a basal layer of myoepithelial cells and a layer of cuboidal or columnar cells. Around the basement membrane a connective tissue layer is present. These ducts converge into a lactiferous duct, while in depth they branch giving rise to the intralobular ducts. Mammary gland during pregnancy and lactation, HE stain. - on low magnification: unlike the resting mammary gland, mammary lobules enlarge gradually during pregnancy, becoming very large during lactation. In such a lobule the glandular component is well developed while stroma (functional stoma) is quantitatively reduced. By growing of the lobules nonfunctional stroma also decreases more and more. - on high magnification: we will study the features of the glandular component. Thus we will observe that secreting alveoli develop at the end of the intralobular ducts. During pregnancy these alveoli have a narrower lumen and towards the end of this phase they may contain an eosinophilic material, the colostrum. The secreting cells of the epithelium become tall columnar, with vacuolar apical cytoplasm (they contain a lipid secretion product). Myoepithelial cells are situated from place to place between the basal pole of secreting cells and the basement membrane. During lactation the secreting alveoli have the same structure, but they are much larger with a larger lumen containing a secreting product. The interlobular ducts are hard to distinguish from the secreting alveoli, because they have the same structure and are also dilated. In this phase both the functional and nonfunctional stroma are quantitatively reduced.
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Microscopic image
Mammary gland during pregnancy 1 Glandular alveoli and intralobular ducts; 2 Intralobular stroma; 3 Interlobular stroma; 7 Interlobular ducts.
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SKIN
The skin is an organ which due to its complex structure acts as a protective, receptor and excretory organ. It is built up by three overlapping layers: epidermis, dermis and hypodermis and also of: glands, sensory receptors and skin appendages (hair, fingernails).
Skin, HE stain, fingertip - on low and on high magnification we will recognize the components of the three layers of the skin. The epidermis consists of a stratified squamous keratinized epithelium in that five layers can be recognized: - basal layer (stratum basale or stratum germinativum) formed of one row of cuboidal or columnar cell, with reduced darker basophilic cytoplasm, and oval, elongated nuclei, oriented perpendicular to the basement membrane; - intermediary or spinous layer (stratum spinosum) consisting of 6-14 layers of large, irregular, polygonal cells, with visible cell boundaries; in the profound part of the intermediary layer cells have a dark basophilic cytoplasm while in the upper part of this layer, cytoplasm in cells becomes pale eosinophilic. Nuclei are rounded and euchromatic. - granular layer (stratum granulosum) is formed of 2-4 layers of flattened, rhomboidal cells, with their long axis oriented parallel to the surface of the epidermis. In the cytoplasm large, intense basophilic granules (keratohyaline granules) can be observed on high magnification. Nuclei of these cells are flattened, but hardly visible being masked by the granules. - clear, translucent layer (stratum lucidum) is a thin layer, consisting of 2-3 rows of flattened cells, with a bright, pale eosinophilic outline. Nuclei are absent in this layer. - cornified layer (stratum cornosum) appears like an intense eosinophilic structure, of variable thickness at the surface of the epidermis. In this layer flattened cellular shadows, which have an eosinophilic cytoplasm but no nuclei, can be observed. The eosinophilic staining is given by the presence of keratin. The superficial cells filled with keratin eventually are shed from the surface (desquamation). From place to place in the thickness of the epithelium the helical path of sweat glands can be observed, representing the continuation of the excretory ducts from the dermis. The dermis, according to its structure and localization, is formed of two layers: a superficial dermis or papillary layer and the profound dermis or reticular layer, in contact with the hypodermis. The papillary layer is formed of loose connective tissue with numerous capillaries and nerve endings. This tissue projects into the above placed epithelium forming the dermal papillae. In the tip of these papillae so-called Meissners sensitive corpuscles can be found. These corpuscles are oval and oriented
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perpendicular to the surface epithelium. They are surrounded by a connective tissue capsule, and in the interior they are formed of flattened Schwann cells arranged in overlapping lamellae. A single sensitive unmyelinated axon meanders between the lamellae.
1 2 3 I A 4 5
II
6 7
III
B 8 IV
C Microscopic images Schematic representation
Skin A Epidermis, general view; B Hypodermis; C Vater-Pacini corpuscle; I Epidermis; II Papillary layer of the dermis; III Reticular layer of the dermis; IV Hypodermis. 1 Stratum cornosum; 2 Stratum lucidum; 3 Stratum granulosum; 4 Stratum spinosum; 5 Stratum basale; 6 Meissners corpuscle; 7 Excretory duct of the sweat gland; 8 Sweat gland glomerulus; 9 Vater-Pacini corpuscle.
The reticular layer consists of a dense connective tissue, with thick bundles of collagen fibers, orientated tangential or perpendicular to the basement membrane. It contains blood vessels, hair follicles with sebaceous glands attached
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to them. It is crossed by the excretory ducts of the sweat glands. Due to their sinuous orientation, sweat glands appear sectioned in different incidences, with different shapes and narrow lumens, lined by 1-2 rows of cuboidal cells. The hypodermis is built up by a loose connective tissue and groups of adipose cells (paniculus adiposum). In the hypodermis we can observe oval structures that on cross section show an onion bulb appearance: Vater-Pacini corpuscles. These consist of concentrically placed cellular lamellae (flattened cells of the connective tissue and Schwann cells) and an unmyelinated nerve ending located in the centre of the corpuscle. In the profound hypodermis the glomerules of sweat glands can be found. Because sweat glands are coiled tubular glands on histological section they appear as acini-like structures, lined by tall, secretory cells doubled at the exterior by myoepithelial cells.
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ENDOCRINE GLANDS
Glands with inner secretion consist of endocrine cells surrounded by a stroma and a rich capillary network. The endocrine glands have no excretory ducts. The secretion product called hormone, will be released directly into the blood and will action at distance on some tissues or target organs, causing biochemical, functional and/or structural changes. Endocrine cells may group in three ways: a. endocrine cells that form individualized organs (glands): hypophysis, epiphysis (pineal gland), thyroid, parathyroid and adrenal gland. b. islets of endocrine cells, comprised in the stroma of other organs: interstitial gland in testis, corpus luteum and theca interna of the mature ovarian follicle in the ovary, islets of Langerhans in the pancreas and others. Their structure will be studied together with that of the organs in which they are contained. c. endocrine cells placed between cells of different epithelia, forming the diffuse neuro-endocrine system (DNES). In this chapter we will describe the structure of only those endocrine glands that form separate organs. They are different structurally, but have a common general organization plan: - the capsule, at the exterior, with a structure consisting of connective tissue and blood vessels; it sends septa that will divide glands into lobes and lobules; - the stroma formed of a loose connective tissue, numerous capillaries and lymph vessels, nerves. Present in all endocrine glands, stroma differs quantitatively from one gland to the other. - the parenchyma consists of anastomosing cords, nests or vesicles (follicles) of endocrine cells, in close contact with blood capillaries.
HYPOHYSIS
Hypophysis or the pituitary gland has an oval shape, is situated in the cranial cavity and is connected to the hypothalamus through the infundibular stem (pituitary stalk). In relation with its structure and origin, hypophysis consists of two distinct parts: a. Adenohypophysis, the largest part, is an epithelial structure. It consists of three lobes: - anterior lobe (pars distalis) - intermediate lobe (pars intermedia) - tuberal lobe (pars tuberalis) b. Neurohypophysis, the smallest part, with a fibrillar structure. It consists of two lobes:
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- posterior lobe (pars nervosa or neural lobe) - infundibulum consisting of the infundibular process and the median eminence. Hypophysis, HE stain. - on low magnification: we will observe the general outline of the gland, which is covered by a very thin capsule. We will identify the adenohypophysis, with an epithelial structure, that forms about 75-80% of the hypophyseal mass and the neurohypophysis with a fibrillar structure (connective tissue fibers, nerve fibers) with few cells only. - on high magnification: we will study the characteristic histological features of the two components of the hypophysis. Adenohypophysis: - anterior lobe: it contains various types of endocrine cells arranged in cords or nests. On HE stained histological slides two cell populations can be observed: chromophobic cells which cytoplasm does not stain or is very pale and chromophilic cells with variously colored cytoplasm. Chromophobic cells represent about 50% of the cell population and are located in the centre of cell cords. They appear as clusters of vesicular, hypochromatic nuclei. Their cytoplasm has no granules, thus being hardly visualized. Chromophilic cells represent the other 50% of the cell population of the anterior lobe. They are relative large cells, with distinct cell limits and a granular cytoplasm. Based on the color of these granules, we differentiate two types of chromophilic cells: acidophils and basophils. - acidophils are the most numerous chromophilic cells. They appear as relative large, rounded or oval cells, with distinct limits and with an eosinophilic cytoplasm due to the large granules, with high affinity for acidic dyes. Their nuclei are rounded, hypochromatic and centrally located. These cells have the role to secrete following hormones: STH (somatotrope hormone or growth hormone) and PRL (prolactin or lactotrope hormone). - basophils are less numerous chromophilic cells. They are larger than the acidophils, are rounded or oval, with visible cell limits, a basophilic cytoplasm, due to their granules with affinity for basic dyes. Nuclei are rounded, hypochromatic and centrally or eccentrically located. They have the role to secrete the following trop hormones: TSH (thyroid-stimulating hormone), ACTH (adrenocorticotropic hormone) and gonadotropins LH and FSH (luteinizing hormone and follicle-stimulating hormone). Between the cells, a quantitatively reduced stroma is present, formed by a network of fine reticular fibers (which cannot be visualized in HE staining) and numerous capillaries. - intermediate lobe is rudimentary in humans, being represented by a thin blade of cells located between the anterior lobe and the neurohypophysis. It consists of short cell cords and from place to place of vesicles with an eosinophilic homogeneous colloid. Cells present here are of two types, chromophobic and
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basophilic, the last ones with basophilic granules in the cytoplasm. Frequently cells and vesicles extend towards the posterior lobe of the hypophysis. Neurohypophysis: - posterior lobe or neural lobe: has especially a fibrillar structure, consisting of bundles of unmyelinated nerve fibers of the hypothalamohypophyseal tract, stromal connective tissue fibers, glial cells called pituicytes and few basophilic epithelial cells migrated from the intermediate lobe. Between the fibers we may see some rounded or oval structures, containing an eosinophilic material, called the Herring bodies, and a rich capillary network. These structures represent deposits of neurosecretory products, containing the two hormones: oxytocin and vasopressin, synthesized by neurons of the hypothalamus, transported along the axons and deposited in here. Cells of neurohypophysis are represented by: - pituicytes, small irregular glial cells, highly branched (around capillaries), with a round or oval, hypochromatic nucleus, a pale cytoplasm, and imprecise cell limits. - cells of the connective tissue with elongated, hyperchromatic nuclei - basophilic cells migrated from the epithelial lobes (anterior and intermediate lobe).
2 1 4 3 2 3
5 6 I II Schematic representation III
Microscopic images
Hypophysis I Anterior lobe; II Intermediary lobe; III Neurohypophysis; 1 Acidophilic chromophilic cell; 2 Basophilic chromophilic cell; 3 Chromophobic cell; 4 Unmyelinated nerve fibers; 5 - Basophilic chromophilic cell migrated from the intermediary lobe; 6 Capillary. 113
THYROID GLAND
Thyroid gland, the largest endocrine gland in the organism, is located in the neck region, in front of the larynx and of the trachea and consists of two lobes (left and right lobe) connected by the isthmus. The gland is delimited from the surrounding organs by a covering fine capsule, continuous with the profound fascia cervicalis. From the capsule arise septa of connective tissue and blood vessels, dividing the gland into irregular lobules. The parenchyma is composed of thyroid follicles while the stroma is fine and rich in blood vessels. Thyroid gland, HE stain. - on low magnification: we will observe the general histological structure of the gland. At the exterior there is a highly vascularized connective tissue capsule from which septa arise, dividing the gland. Inside the gland we will observe numerous thyroid follicles, in different functional phases and of different sizes.
2 4 Microscopic images Schematic representation
Thyroid gland 1 Follicular epithelium; 2 C cells; 3 Colloid; 4 Capillary
- on high magnification: we will study the structure of thyroid follicles. These are rounded or oval, delineated at the periphery by a simple epithelium, called follicular epithelium, and contain a cavity. The cavity is delineated by the apical pole of the follicular epithelial cells and contains an eosinophilic gelatinous mass, called colloid. Around the thyroid follicles there is a rich capillary network (fenestrated capillaries). The follicular epithelium consists of two types of cells, placed on a basement membrane: - follicular cells have different features depending on their functional state: flattened cells (endothelial like cells) are resting cells; cuboidal cells are cells that secrete the colloid and are the most numerous; tall columnar cells are cells that
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reabsorb the colloid and release active hormones into the blood. The nucleus has different shapes, in correlation with the shape of the cell. The role of follicular cells is to secrete the thyroid hormones: thyroxine (T4) and triiodothyronine (T3). - parafollicular cells or C cells appear isolated or in small groups located between the follicular cells, adjacent to the basement membrane. They do not reach the lumen of the follicle so they do not come into contact with the colloid. They are large cells, with a pale cytoplasm, and a large, rounded, pale nucleus. On histological slides stained with HE they cannot be differentiated from the follicular cells; to identify them immunohistochemical reactions with anti-calcitonin and anti-chromogranin are used. C cells secrete calcitonin (hence the designation C cells).
ADRENAL GLAND
Adrenal glands are retroperitoneal located glands placed on the top of the kidneys, included in the adipose tissue surrounding the kidney. On histological slides, the adrenal gland follows the general organization plan of the endocrine glands: - a thin connective tissue capsule that surrounds the gland. From the inner surface of the capsule septa arise that extend into the parenchyma, bringing blood vessels and nerves. - stroma consists of a network of reticular fibers and numerous capillaries located around the endocrine cells of the parenchyma; - parenchyma of the gland is formed by cords and nests of cells. We distinguish two main zones: - cortex (adrenal cortex) situated at the periphery (under the capsule, and around the medulla) and that represents the most part of the gland; - medulla (adrenal medulla) centrally located and representing about 20% of the gland. Adrenal gland, HE stain. - on low magnification we will study the architecture of the two zones of the adrenal gland; while on high magnification we will study the cytological aspects. Thus at the exterior we will notice the fine capsule of connective tissue, with adipose cells, blood vessels, nerves and sometimes vegetative ganglia. Under the capsule we will identify the two zones of the parenchyma: The cortex located immediately under the capsule is formed by cellular cords and capillaries. The trajectory of capillaries determines the arrangement of cell cords. Based on this arrangement we distinguish three zones: - zona glomerulosa is a thin layer of tissue, consisting of cells organized in nests or short cords, glomerulus-like arranged. Around them a small amount of stroma and a rich network of fenestrated capillaries are present. Cells are small, cuboidal or columnar, with a basophilic cytoplasm and a small, hyperchromatic rounded nucleus, located centrally or eccentrically, closer to the apical pole.
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- zona fasciculata is the thickest zone of the cortex; it is formed by long cell cords, like vertical columns, oriented perpendicular to the adrenal medulla, separated by fenestrated capillaries. The cells are large (the largest cells of the cortex), rounded or polygonal, with a pale vacuolar cytoplasm, due to their high lipid content, which is dissolved during tissue processing. They have one, rarely two large, hypochromatic nuclei, with 1-2 conspicuous nucleoli. Between the cell cords the elongated, hyperchromatic nuclei of endothelial cells lining capillaries can be observed. - zona reticularis is the deepest zone of the cortex. It consists of very short anastomosed cell cords (1 3 cells), in close contact with dilated capillaries. Their cells are much smaller than those of the zona fasciculata, intensely eosinophilic, and contain fewer lipid droplets. Some of the cells have a yellow pigment (lipofuscin pigment) in the cytoplasm.
1 2
Microscopic images
Adrenal gland 1 Capsule; 2 Zona glomerulosa; 3 Zona fasciculata; 4 Zona reticularis; 5 Adrenal medulla.
The medulla is the central zone, completely surrounded by the cortex; it consists of two types of cells: chromaffin cells (because of their reaction with chrome salts) of neuroendocrine origin, and supporting cells (sustentacular cells). Chromaffin cells (medullar cells) are organized in nests (islets) or short anastomosing cords. They have characteristic features: they are large, polygonal
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cells, with an abundant, granular, basophilic cytoplasm. Their nuclei present important variations in size, shape and localization inside the cell. Sustentacular cells are found at the periphery of the chromaffin cell nests and are recognized after their elongated, hyperchromatic nuclei, larger than that of the endothelial cells. These cells are difficult to be recognized in HE staining while with IHC they are easily highlighted (protein S-100 and vimentin). Around the cell nests a fine stroma and a delicate capillary network can also be found. In the medulla large, dilated blood vessels with a thin wall, can be observed (medullary veins).
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Recommended bibliography
Eroschenko V: diFiore's Atlas of Histology with Functional Correlations. Lippincott Williams & Wilkins, 2008 Gartner LP, Hiatt JL: Color Textbook of Histology. Saunders, 2006 Junqueira LC, Carneiro J: Basic Histology, Text and Atlas. Mcgraw Hill, 2007 Ross MH, Pawlina W: Histology. A Text and Atlas. Williams & Wilkins, 2010 Young B, Lowe JS, Stevens A, Heath J: Wheater's Functional Histology. A Text and Colour Atlas. Churchill Livingstone Elsevier, 2007

Histology

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THE UNIVERSITY OF MEDICINE AND PHARMACY TRGU MURE DISCIPLINE OF HISTOLOGY

Principles of histological tissue processing

Covering and lining epithelia

3 Microscopic image Schematic representation

Simple cuboidal epithelium 1 Nucleus; 2 Intercellular limits; 3 Basement membrane (BM).

1 4 Microscopic image Schematic representation

Simple columnar epithelium 1 Nucleus; 2 Intercellular limits; 3 Brush border; 4 BM.

5 1 Microscopic image Schematic representation

Functions Shape of the cell

Apical pole Cellular limits Stain

4 Microscopic image Schematic representation

1 4 Microscopic image Schematic representation

Functions Basal layer (identical)

Middle layer (several rows of cells)

flattened, basally located large

according to the cell type large

THE CONNECTIVE TISSUE

Cells of the connective tissue

Macrophage 1 Macrophage (histiocyte); 2 Multinucleated giant cells.

Fibers of the connective tissue

1 Microscopic image Schematic representation

Collagen fibers 1 Isolated and bundle-grouped fibers; 2 Fibrocyte nuclei.

TYPES OF CONNECTIVE TISSUE

SOFT CONNECTIVE TISSUES

6 1 2 Microscopic image Schematic representation 5

1 Microscopic image Schematic representation

Reticular connective tissue 1 Reticular cell; 2 Lymphocyte.

Adipose tissue 1 Adipose cell; 2 Capillary. 25

1 Microscopic image Schematic representation

Tendinous connective tissue 1 Tenocytes (twin nuclei); 2 Bundles of collagen fibers.

3 Microscopic image Schematic representation

3 Microscopic image Schematic representation

HEMATOPOIETIC AND LYMPHOPOIETIC ORGANS

RED BONE MARROW

MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)

NERVOUS TISSUE AND NERVOUS SYSTEM

Pyramidal cell 1 Cell body; 2 Axon; 3 Main dendrite; 4 Secondary/auxiliary dendrites.

CENTRAL NERVOUS SYSTEM

PERIPHERAL NERVOUS SYSTEM (PNS)

3 2 Microscopic image Schematic representation

Vegetative ganglion 1 Neuron; 2 Satellite cell; 3 Nerve fibers.

THE CARDIOVASCULAR SYSTEM

THE VASCULAR SYSTEM

Cross section Microscopic image

Muscular artery, HE 1 Intima; 2 Media; 3 Adventitia.

Vein, HE 1 Intima; 2 Media, 3 Adventitia. 53

THE ORAL CAVITY

Vermillion Microscopic images

III Schematic representation

4 III 5 IV Microscopic image Schematic representation 6

2 8 Microscopic image Schematic representation

Differential diagnosis between the excretory ducts

3 5 9 Microscopic image 10 Schematic representation

2 Microscopic image Schematic representation

Pancreas 1 Serous acini; 2 Intralobular duct; 3 Interlobular duct; 4 Islet of Langerhans.

2 6 3 Microscopic image Schematic representation

Liver parenchyma 1 Kupffer cells; 2 Hepatic sinusoids; 3 Hepatocytes.

4 Microscopic image Schematic representation

MALE REPRODUCTIVE SYSTEM

5 8 Microscopic image 7 Schematic representation 6

ACCESSORY GENITAL GLANDS

2 Microscopic image Schematic representation

Prostate gland 1 Bistratified glandular epithelium; 2 Muscular-connective tissue stroma.

FEMALE REPRODUCTIVE TRACT