Spleen and thymus
Objectives
You should be able to identify the following in the
class slide sets:
| Active thymus | Spleen | ||||
| lobule | capsule | ||||
| capsule | trabeculae | ||||
| connective tissue septa | trabecular arteries | ||||
| cortex | trabecular veins | ||||
| lymphocytes | white pulp | ||||
| epithelial reticular cells | central artery | ||||
| medulla | PALS | ||||
| lymphocytes | lymphatic nodules | ||||
| epithelial reticular cells | marginal zone | ||||
| Hassall’s corpuscles | red pulp | ||||
| sinusoids | |||||
| Involuting thymus | endothelium | ||||
| lymphatic tissue | splenic cords | ||||
| adipose tissue | reticular cells | ||||
| Hassall's corpuscles | reticular fibers | ||||
| macrophages | |||||
| plasma cells | |||||
| various blood cells | |||||
Blue Histology: The spleen and thymus are in different sections in the program. Ignore (or review) the other aspects of the immune system that are presented in these two sections.
 | Thymus | |
|
Spleen |
Slides
| | D-36 | Thymus, involuted (H&E) | |
| D-37 | Thymus, active (H&E) | |
| D-38 | Spleen, dog perfused (H&E) | |
| D-39 | Spleen, human, at autopsy (H&E) |
Slide descriptions
D-37 Thymus, active (H&E)
The thymus has extremely sophisticated functions but they are not reflected in structure visible on routine histological slides. You will be able to distinguish only two types of tissue, cortical and medullary, and two classes of cells, T cells and epithelial reticular cells. Examine this slide grossly and note that it is divided into lobules, each with a darker-staining cortex and lighter-staining medulla (illustration).
Look at a cortical region of a lobule under your microscope. The cells are predominantly T-lymphocytes and their immediate precursors. T-lymphoblasts actively divide here. Most of the newly formed T-cells die at the hands of macrophages. Others with appropriately rearranged T-cell receptor genes escape to the capillaries at the boundary with the medulla and contribute to the circulating pool of T-cells. The only blood vessels in the cortex are capillaries. This turns out to be significant. There is a blood-thymus barrier in the cortex.
Observe the sparse
fixed supporting cells with larger pale nuclei (illustration).
From their general appearance they have been called "reticular cells," but this
is a mistake. They are actually epithelial in form. Their embryonic origins trace
back to ectodermal epithelium of the embryonic foregut and they retain their fundamental
epithelial properties. Immunologists now subdivide these epithelial reticular
cells into five or six different types but these are indistinguishable from each
other on slides. Can you remember their various functions? It might be worthwhile
to make a list of them now.
| 1.____________________________________________________________ | |
| 2.____________________________________________________________ | |
| 3.____________________________________________________________ | |
| 4.____________________________________________________________ | |
| 5.____________________________________________________________ |
There are also a substantial number of macrophages in the cortex but you will not be able to distinguish them from epithelial reticular cells in this H&E preparation. A really good histologist probably could recognize them, but I cannot. If a large pale nucleus has a substantial amount of cytoplasm around it it probably belongs to a macrophage instead of an epithelial reticular cell.
The medullary regions are paler because they have far fewer thymocytes. This makes the epithelial reticular cells easier to observe. In this part of the thymus they have come from endoderm. Some of them aggregate into easily seen pink structures called Hassall's corpuscles. The function and significance of these structures are unknown (likely they have none). They are composed of swirls of epithelial reticular cells around a keratinized or calcified core. The blood-thymus barrier is interrupted in the medulla, and here one can find blood vessels larger than capillaries. This allows T-lymphocytes to escape from the medulla into the circulation.
Finally, look at the seams of connective tissue that separate the lobules of the thymus and invest them with tenuous capsules (illustration). What specialized cell types do you see in them (illustration)? There is a poorly understood but obvious association between the thymus and mast cells.
D-36 Thymus, involuted (H&E)
The thymus is largest during childhood and progressively involutes after puberty. This slide is from an old individual. Note that most of the parenchyma has been replaced with adipose tissue, but some lymphopoiesis continues even into old age, illustration. Many large Hassall's corpuscles are present. These structures can continue to grow until some are big enough to see with the unaided eye.
D-38 Spleen, dog perfused (H&E)
The spleen has a complex organization. It is organized into lobules, but these can only be imagined in a slide because the connective tissue is too scant to outline them. It is important for you to read about this lobular structure in your text so that this organ will not seem to be just a chaotic jumble of pulp and trabeculae.
Hold your slide up to the light and note the blue dots of white pulp in a red background (illustration). The irregular empty spaces are veins running in the dark red trabeculae. At low power distinguish red from white pulp. The white pulp looks like small islands of dense lymphatic tissue and is always accompanied by an arteriole, the central artery (illustration). Observe several islands of white pulp at 100 X. They are purple because the predominant cell types are small lymphocytes with dark nuclei and scant cytoplasm. In three dimensions the white pulp has the geometry of a branching tree surrounding branching central arteries. Actually the white pulp can be thought of as the tunica adventitia of these arteries, but composed of lymphocyte-laden reticular tissue instead of the usual ordinary dense connective tissue. No wonder that a central artery invariably is present in areas of white pulp. These tiny arteries are called "central arteries" because they run in the interior of the elusive splenic lobule, not because they run down the middle of the white pulp. Typically central arteries are off to one side in their area of white pulp. (Don't believe a textbook which tells you differently).
Occasional nodules develop in the white pulp. They are especially abundant in children and in people with blood-borne disease. If the section goes through a nodule, the area of white pulp looks much larger and denser (illustration). You may even find germinal centers in some sections. It is important to realize, however, that white pulp is more than just nodules. Some atlases which show all white pulp as nodules floating in a sea of red pulp are wrong and confusing. Read your text about the distribution of B and T cells within the white pulp because this is important and you cannot distinguish B from T cells by eye. Note that two stated objectives of this lab section are to know the locations of the PALS and the marginal zone.
The red pulp is composed of a lattice of reticular cells infiltrated by enormous quantities of blood cells. Many venous sinusoids traverse it. They are expanded and empty on this slide because this tissue was deliberately perfused with saline before it was fixed. The areas of tissue between sinusoids are called "cords" (a poor term), so that red pulp is a mixture of cords and sinusoids (higher mag.).
The sinusoids are large, irregular, elongated structures. They are lined with specialized endothelial cells that are cigar shaped instead of flat. In cross section the endothelial cell nuclei look round and bulge into the lumen of the sinusoid (illustration). Where the section misses the nucleus the cell looks like a tiny bleb at the edge of the sinusoid. The nuclei of the endothelial cells look elongate when sectioned longitudinally (illustration). The cells are held in place by reticular fibers wrapped circumferentially around the outside of the sinusoid. The endothelial cells do not form a continuous sheet. Slits between them allow blood cells to percolate in and out. It may be worthwhile to get out your immersion oil to look at these specialized lining cells of the sinusoids.
Oil immersion also will allow you to examine the reticular cells of the cords. Seek out an area of red pulp that has been well flushed of red blood cells. Do this before putting oil on your slide. Some areas along the connective tissue trabeculae are especially free of blood cells. The strings of cytoplasm that you see are arms of reticular cells stretched out along reticular fibers to form a meshwork (illustration). Find the nuclei of these cells. Reticular cells in the right orientation will show a thin coat of pale, bluish cytoplasm surrounding their nuclei and extending out into long arms (example 1, example 2, example 3).
Next look for macrophages that are ingesting red blood cells. They are easy to spot because the engulfed erythrocytes are bright orange/yellow. You will find many near the trabeculae (especially at the lower left hand side of your slide as you look through the microscope at it. You may want to scan for these large cells first at 100 X). Observe that their nuclei are large and pale staining.
Now turn to the free cells wandering through the reticulum. Again, look in places in the cords where the cells are not too crowded. Small lymphocytes and plasma cells are the most abundant type. Plasma cells have a round nucleus about the size of that of a lymphocyte, but surrounded by a substantial amount of basophilic cytoplasm. The grains of chromatin in their nuclei are more distinct and contrasty than is the case with lymphocytes. Find and examine at least half a dozen plasma cells. Monocytes are recognized by their small bean-shaped nuclei but are not easy to find. You would have to scan at 430 X power to pick them out. During this scanning neutrophils are a pleasure to come across. Their complex nuclei make them unmistakable. Even more pleasurable is to come across a megakaryocyte! That's right, these are to be found in the spleen of the dog, although not the human.
You can now wipe the oil from your slide and turn to the trabeculae that crisscross the red pulp. These finger-like projections of connective tissue have two functions. One is to physically support the mass of splenic pulp which otherwise would have the consistency of very soft jelly. The other is to provide passage ways for the larger arteries and almost all of the veins. In the dog, which provided this tissue, the trabeculae happen to be composed of smooth muscle instead of ordinary dense connective tissue. This will be apparent to you. The trabecular arteries and veins run right down the middle of the trabeculae (illustration). Remember that the blood has been flushed from them. The veins have no wall of their own other than a sheet of endothelium (illustration). Thus, they look like large, irregular cavities in the trabeculae. 430 X magnification will show the flattened nuclei of the lining endothelium which identifies them as blood vessels instead of just artifactual spaces.
The trabecular arteries have a more definitive structure, with a well developed tunica media. The smooth muscle of that layer is circularly arranged while that of the surrounding trabeculae runs longitudinally. Thus it is easy to distinguish between the two. The trabecular arteries also have an adventitia layer of dense connective tissue. In some cases this is hard to see but in places where the tissue has shrunk the adventitia has pulled away from the wall of the trabeculum. The artery then appears to be floating in a hole in the trabeculum. One warning is that arteries run for a shorter distance in the trabeculae than veins. Therefore you see more sections of trabecular veins. The reason for the difference is that the arteries run for a considerable distance in the pulp as central veins. Pulp veins are almost too short to notice. If you look around you can find places where the sinusoids seem to drain directly into trabecular veins (illustration) because the pulp veins are so short.
D-39 Human spleen at autopsy (H&E)
Repeat your identification of the gross features as in D-38. This human spleen is more difficult to study because the tissue was allowed to collapse when it was fixed (illustration). Also, the blood was not flushed from the sinusoids. Both slides of the spleen, of course, have the same cell types. One small difference is that the macrophages here are laden with small granules of dense pigment instead of the whole red blood cells as in D-38 (high mag). I suspect that when the dog spleen was flushed with saline the altered environment stimulated the macrophages to gobble up even young erythrocytes but then the tissue was fixed before any digestion could occur. In contrast, the human spleen acquired at autopsy probably sat around for a long time before fixing allowing the macrophages to rather completely digest all of the hemoglobin molecules. A second difference between the two tissues is that is that the capsule and trabeculae are made up of dense ordinary connective tissue in the human instead of smooth muscle as in the case of the dog, (illustration).
You can still easily tell the trabecular arteries (transverse) from trabecular veins by looking at their wall. The numerous sinusoids are collapsed, (illustration) but are lined with modified endothelial cells as you saw in the animal slide.
Perhaps fewer of the sections of white pulp cut through nodules than was the case with the dog spleen in D-38. What do you think?