ANAT D502- Basic Histology
Immune and lymphatic system pre-lab
revised 9.25.09
Objectives:
1. Distinguish between lymphoid tissue associated with organs (non-encapsulated)
and specialized lymphoid organs of the immune system (encapsulated).
2. Understand the functional structure of organ-associated lymphoid tissue with respect to its local function.
3. Know the type of lymphocytes (B and T ) present in the different locations within lymphoid tissue and between different lymphoid organs.
4. Identify and understand the substructure of lymph nodes (i. e. cortex, medulla, nodules, reticular fiber stroma) and what processes occur in these different locations.
5. Identify and describe the processes that occur in germinal centers.
6. Diagram or describe the lymph flow through a lymph node.
7. Identify and name lymphoid tissue (organ-associated lymph tissue, lymph nodes, spleen, thymus).
8. Understand the functional organization of the spleen. Identify red and white pulp. Identify the germinal centers and their associated central artery. Describe or diagram the flow of blood through the spleen.
9. Identify thymic (Hassall's) corpuscles in the thymus.
Slides
s24
lymph node, human, Wilder's reticular (silver) stain
s24
lymph node, human, lesioned
s26 spleen, human
s27 palatine tonsils
s28 spleen, monkey
s29 thymus
s36 lymph node
s59 appendix
s81 cervix
Lymphoid tissue can be divided into central (primary) and peripheral (secondary) lymphoid tissue. Central lymphoid tissue is the genesis site of undeveloped/immature lymphocytes (where their stem cells reside). This includes bone marrow in the adult and liver in the fetus. The thymus is the site of partial maturation of T-lymphocytes that originated from bone marrow and is also considered a central lymphatic organ. Peripheral lymphoid tissue is the site of antigen presentation for B and T-lymphocytes that is involved in the maturation/proliferation of these lymphocytes. This tissue includes encapsulated lymphoid tissue and un-encapsulated, the capsule being of collagenous connective tissue. Peripheral lymphoid tissue includes lymph nodes and the spleen which are encapsulated, while un-encapsulated includes the tonsils and lymphatic nodules found in the digestive, respiratory, urinary and reproductive tracts. Encapsulated peripheral lymphoid tissue acts as an in-line filter for the lymph (lymph nodes) and blood (spleen). Un-encapsulated peripheral lymphoid tissue monitors the epithelial lining of the body, and, at this point, monitors the system for foreign substances. In both cases, the peripheral lymphoid tissue is involved in monitoring the body for infection by foreign substances. Both B and T-lymphocytes reside in these tissues and are responsible for mounting the defense mechanism against foreign substances.
Begin your study of the thymus, a central lymphoid tissue, by viewing s29 at low power. Observe the dense collagenous connective tissue which forms the capsule. In some slides, the capsule is poorly stained and not readily apparent. The cells within the organ are divided into lobules by connective tissue septa that penetrate the parenchyma. These lobules can be further divided into a cortex (outer region of lobule) and a medulla (inner region of lobule). The cortex presents as a more densely-staining region at low power because of the higher density of cells in this region. The cortex is enriched in T-lymphocytes. The medulla presents with lesser staining caused by the lower density of cells. The medullary cells include medium to large lymphocytes that have a greater volume of cytoplasm than the T-cells of the cortex. Also present in the medulla are epithelial/reticular cells whose cytoplasmic processes act as the support matrix for the parenchyma. The epithelial/reticular cells present as cells with very large, euchromatic nuclei. An important feature which allows for easy identification of the thymus is thymic (Hassall's) corpuscles. These are concentrically wrapped, degrading epithelial/reticular cells which appear as concentric, eosinophilic bodies. The thymus has no afferent lymphatic supply, but does have a vascular supply allowing for migration of T-lymphocytes in from the bone marrow (immature) and out of the organ to populate peripheral lymphoid tissue.
The lymph node is our first example of peripheral lymphoid tissue which is encapsulated. Begin your study of the lymph node using s24 and we will investigate the lymph flow through the node. View the slide with the naked eye and you will observe an ovoid structure. Study the section at low power and scan around the surface of the organ. You will note a dense collagenous connective tissue capsule. Scan the surface until you find a region with an indentation of the connective tissue capsule. In this region, you should see blood vessels sectioned longitudinally, a lymphatic vessel filled with lymph and leukocytes (but no red blood cells), and possibly some fat cells. This region is the hilum of the node and is the site of entry of arteries and nerves, and the exit of veins and lymphatic vessels. This lymphatic vessel is called the efferent lymphatic vessel. Continue scanning the capsule at low power and you may observe some lymphatic vessels penetrating the capsule. The vessel lumens will be filled with leukocytes and colloid (protein) but no red blood cells. These lymphatic vessels are the site of entry of lymph into the node and are called the afferent lymphatic vessels. In some of these vessels, you may observe smooth muscle cells which concentrically wrap around the endothelial cells. The afferent lymphatic vessels empty into the subcapsular space of the node. This space is not readily apparent in s24 but shows to more advantage in s36. Study s36 at low to medium power and scan around the capsule. You should find a region beneath the capsule where the cells are somewhat sparsely distributed. Continue with s36 and follow the connective tissue septa which penetrates the parenchyma. These are called trabeculae and the space on each side of a trabecula contains loosely packed cells. This is the trabecular (cortical) sinus. The lymph flows from the afferent lymphatic vessels, into the subcapsular sinuses, then through the cortex via the trabecular (cortical) sinuses, then through the medulla via the medullary sinuses and then back into the lymphatic circulatory system via the efferent lymphatic vessels.
Return to s24 and we will study the features of the parenchyma of the node. You will observe that the parenchyma is arranged roughly into a cortex and a medulla. The cortex includes lymphatic nodules and tightly packed lymphocytes. The medulla contains cords of lymphocytes interspersed between more loosely packed lymphocytes which reside in the medullary sinuses. The cortex is further divided into an outer cortex containing the lymphatic nodules and an inner cortex (paracortical zone or thymic-dependent zone) which contains no nodules or cords of cells but presents as packed lymphocytes. The inner cortex (paracortical zone) may not be readily apparent in your slide. Many of the lymphatic nodules contain a germinal center which appears as a more lightly stained region since the cells are packed less tightly; the germinal center is surrounded by the more basophilic follicular mantle. The germinal centers are enriched in B cells. You will also observe mitotic figures (40x, 100x) in the germinal centers resulting from the mitotic activity of the B-cells. Lymphatic nodules without germinal centers are called primary nodules and those with are called secondary nodules. The inner cortex and the inter-nodular region in the outer cortex are enriched in T-cells. The lymphocytes can migrate out of the node and into the vasculature via unique venules found in the non-germinal center region of the parenchyma. These structures are called high endothelial venules (postcapillary venules) (cross-section, longitudinal section) and present as structures looking like capillaries but with cuboidal endothelial cells instead of flattened cells. This route allows lymphocytes to directly exit the blood stream without having to travel along the efferent lymphatic vessel. The other route for lymphocyte to exit is via the medullary sinuses which empty into the efferent lymphatic vessel found at the hilum. Reticular cells are present throughout the parenchyma, but are most readily observed in the medullary sinuses. The cells present with very large, euchromatic nuclei and a poorly to non-staining cytoplasm. These cells synthesize and secrete the reticular fibers forming the stroma to which the lymphocytes attach forming the medullary cords. To visualize the reticular fibers specifically, re-examine the lymph node slide (s22 ) from the connective tissue lab which has been stained with Wilder's silver method. This slide also shows the nature of the medullary cords and sinuses to good advantage.
Macrophages are phagocytic and antigen presenting cells found in the stroma of the lymph node. Although lymph nodes are filled with macrophages, they are difficult to observe in most histological sections due to the tight packing of cells in both the cortex and medulla. However, in the subcapsular sinus isolated macrophages can sometimes be observed. Examine slide s24 and look for large, eosinophilic cells with irregular nuclei in this more open region. The animal in slide 36 was injected with india ink which was subsequently taken up by the macrophages. This method of labeling macrophages tends to obscure their morphology but nicely demonstrates their abundance.The spleen is also a peripheral lymphoid organ that is also involved in removal of aged red blood cells. As such, it is highly vascularized and contains a considerable amount of red blood cells. The organ is encapsulated and has a hilum as observed in lymph nodes. Examine s28 (monkey spleen) at low power and you will observe the collagenous capsule and a hilum which should be apparent on one aspect of the section. Study the parenchyma at low power and you should observe nodular structures surrounded by strong basophilia. This region is called the white pulp of the organ. In fresh, unstained specimens, these regions have a white appearance (mostly white blood cells). Between the nodules is eosinophilic material, which upon closer examination, is seen to be predominantly red blood cells. This region is called the red pulp. Carefully study the nodules at higher power and you will observe larger cells in the interior of the germinal center. This region is occupied primarily by B-lymphocytes. Around the germinal center is the follicular mantle, a zone of smaller cells which gives rise to the basophilia seen at low power. This region is also occupied primarily by B-lymphocytes. The next structures that we want to study are shown to better advantage in s26 (human spleen), so switch to that slide and locate a germinal center. In many cases, you will observe an arteriole in cross section, eccentrically located relative to the germinal center. This is called the central (or sheathed) artery, and T-cells are enriched around this artery (technically, an arteriole but called and artery) forming the peri-arteriolar lymphatic sheath (PALS). You may observe these structures in isolation (not next to a germinal center) throughout the organ both in cross and longitudinal section. Views of longitudinal sections gives you a better understanding of the concept of the PALS. The red pulp is conceptually further divided into splenic cords and splenic sinuses. These structures are not easily discernable in your slides because the blood was not removed prior to fixation. A demonstration slide of a partially exsanguinated spleen (i.e., one in which the blood was removed) shows these structures to advantage. The splenic cords are made of blood cells (RBCs and leukocytes), reticular cells, and lastly macrophages which are involved in removal of aged RBCs. The sinusoids are the site of re-entry of blood cells from the splenic cords. Examples of ink-filled macrophages can be readily observed in the red pulp of s28 (monkey spleen). Another way to label macrophages in the spleen and simultaneously demonstrate their erythroclastic function is to stain for iron using Perls’ histochemical method. A demonstration slide of mouse spleen stained with Perls’ iron method is available in the lab. In contrast to india ink, the iron deposits within the macrophages appear bright blue (Prussian blue) but again this method makes discernment of cell morphology difficult.
Next we will study examples of un-encapsulated, peripheral lymphoid tissue. This form of lymphoid tissue is typically adjacent to epithelia (just beneath). The first example we will study is the palatine tonsil which is partially encapsulated by connective tissue and is involved in monitoring the oral mucosa. Study s27 (palatine tonsil) at low power and scan around the surface of the tissue. One region of the tissue will be covered by stratified squamous epithelia which lines the oral cavity/upper digestive tract. Other regions of the lymphoid tissue will be covered with connective tissue. Observation of the epithelial-covered surface shows that there are crypts or invaginations of the surface epithelia into the lymphoid tissue. These crypts allow for an increased surface area for monitoring the mucosa. Within the lymphoid tissue, you will observe germinal centers that may contain mitotic figures and high endothelial venules between the germinal centers.
Further along the digestive mucosa you will also observe lymph nodules in the sub-epithelial space. Examples of this can be found in the esophagus (s52), jejunum (s95), colon (s99) and appendix (s59). They are most readily found in s59 which contains both an inflamed and non-inflamed appendix. You will observe aggregates (nodules) of lymphoid cells. Some os these aggregates will demonstrate germinal centers, others may not.
The last example of non-encapsulated peripheral lymphoid tissue that we will observe is that found in the cervix of the reproductive tract of the female. Study s81 at low power and scan around the surface of the tissue for a region covered with stratified squamous, non-keratinized epithelia. Along this surface, look for a region shaped in the form of a V. This is the junction between the vaginal and cervical canals. Just beneath the epithelia in this region, you will observe a preponderance of lymphocytes. The epithelia is usually very thin over these regions. There will likely not be any germinal centers present in the section, thus, these nodules are considered central lymphoid nodules (although the nodular structure is not readily apparent).
