Enhanced histopathology of the thymus

Abstract

The thymus is a primary or central lymphoid organ in which T lymphocytes undergo differentiation and maturation autonomously within the cortex, without the need for antigenic stimulation, and it is essential for the normal development and function of the immune system. The thymus has been shown to be a sensitive target organ following exposure to immunotoxicants and endogenous corticosteroids, and a decrease in size or weight is often one of the first noted measures of compound-induced effects with cortical lymphocytes (thymocytes) being especially susceptible. Therefore, changes in thymus histopathology and architecture are considered to be of particular relevance for immunotoxicity screening. The separate compartments in each lymphoid organ should be evaluated separately and descriptive rather than interpretive terminology should be used to characterize changes within those compartments (Haley et al., 2005). Therefore, enhanced histopathological evaluation of the thymus involves the determination of the size and cellularity of the cortex and medulla, which should be noted separately. Other changes to evaluate include, but are not limited to, increased lymphocyte apoptosis, lymphocyte necrosis, cortex:medulla ratio and an increase or decrease in the epithelial component of the thymus.

Figure 1

Figures 1a and 1b are low and high magnifications of normal thymus cortex from a control 30-day-old male Sprague–Dawley rat. These can be compared with the low and high magnifications of thymus cortex in Figures 1c and 1d from an age- and sex-matched rat treated 3 hours previously with 1 mg/kg bodyweight of dexamethasone. In the control thymus there are only rare apoptotic bodies (arrow, Figure 1b). In the images from the treated rat, there are increased numbers of apoptotic lymphocytes and tingible body macrophages (macrophages containing stainable bodies or cellular debris) giving this tissue a classic “starry sky” appearance at low magnification (Figure 1c). The arrows in Figure 1d point to tingible body macrophages with intracytoplasmic apoptotic bodies. Note the absence of inflammatory cells. Figure 1e is an image of the thymus from the medullary region of the treated rat in figures 1c and 1d. There are fewer apoptotic cells than in the cortical region due to fewer steroid receptors on the medullary lymphocytes. The arrowhead indicates a single apoptotic lymphocyte whereas the arrow indicates a tingible body macrophage.

Figure 2

Figure 2a is a transmission electron photomicrograph of normal lymphocytes from the thymus cortex of a control 30-day-old male Sprague–Dawley rat. Figure 2b shows apoptotic lymphocytes from the same region in a rat treated 3 hours previously with 1 mg/kg bodyweight dexamethasone. The lymphocytes are shrunken with irregular shapes and have condensed and peripheralized chromatin. The arrow shows fragmented nuclear material and the arrowhead indicates a portion of cytoplasm that has been extruded. Figure 2c illustrates more advanced lymphocyte apoptosis within the thymus cortex. The apoptotic lymphocytes are smaller with more electron dense condensed nuclear material. The small arrow indicates one of the apoptotic lymphocytes with nuclear fragmentation. The large arrow indicates a tingible body macrophage with intracytoplasmic apoptotic bodies.

Figure 3

Figure 3a is an image of cortical necrosis in a 30-day-old male Sprague–Dawley rat treated 48 hours previously with 1 mg/kg dexamethasone. The large arrow indicates an enlarged medullary region that is being repopulated with small, mature lymphocytes. The small arrow indicates a region of cortical necrosis and the arrowhead points to the capsule that is expanded by edematous fluid with a few interspersed inflammatory cells. Figures 3b and 3c are higher magnifications of the image in 3a. Figure 3b shows the area of necrosis with a few interspersed apoptotic lymphocytes (arrows). Figure 3c shows the inflammatory cell component which is predominately neutrophils.

Figure 4

Figures 4a—l are examples of thymuses from a dexamethasone study in 30-day-old male Sprague–Dawley rats. Figure 4a is the normal saline treated control rat thymus. Figures 4b and 4c are high magnifications of the cortical and medullary regions, respectively. Figure 4d is from a rat 24 hours posttreatment. Figures 4e and 4f are high magnifications of the cortical and medullary regions, respectively. Figures 4g and 4j are thymuses from rats 48 hours after treatment. Figures 4h and 4k are high magnifications of the corresponding cortical regions while Figures 4i and 4l are high magnifications of the corresponding medullary regions. When Figures 4a and 4d are compared, a striking tinctorial change is obvious in the treated thymus, especially in the cortical regions due to marked apoptosis of the lymphocytes in this region of the treated rat (Figure 4e). The arrows in Figure 4d indicate regions of tissue tearing, most likely due to lack of cell-to-cell adhesion among the apoptotic cells. The medullary region in figure 4d has increased in area and is less cellular (Figure 4f). This decreased cellularity in the medulla is due to mild lymphocyte apoptosis at 12 hours posttreatment (data not shown). The cortical:medullary ratio has changed significantly in Figure 4d due to a minimal decrease in the cortical area and a mild increase in the medullary region. A qualitative assessment would indicate that the cortical:medullary ratio in Figure 4d is overall approximately 1:2 compared to the normal 2:1 ratio in Figure 4a. Due to tangential cuts through these regions and the generation of different values depending on where the measurement is taken, it is difficult to make a quantitative assessment of this ratio. Although the animals in Figures 4g and 4j were in the same treatment group, there was variability in the tissue response to treatment. Compared to control (Figure 4a), there is a dramatic decrease in the size of the organ. The thymus in Figure 4g shows recovery of the cortical (Figure 4h) and medullary (Figure 4i) thymic tissue with repopulation of the thymocytes. At low magnification, the overall cortical:medullary ratio is approximately 1:10. The arrow in Figure 4g indicates a parathymic lymph node. The thymus in Figure 4j was more severely affected with necrosis and inflammation of the cortical tissue (Figure 4k). However, there is a robust recovery of the medullary thymocytes evidenced by the darker medullary regions in Figure 4j and the presence of increased numbers of small dark lymphocytes in Figure 4l. The cortical:medullary ratio in Figure 4j is difficult to determine due to the lack of normal cortical tissue. The arrow indicates a parathymic lymph node.

Figure 4

Figures 4a—l are examples of thymuses from a dexamethasone study in 30-day-old male Sprague–Dawley rats. Figure 4a is the normal saline treated control rat thymus. Figures 4b and 4c are high magnifications of the cortical and medullary regions, respectively. Figure 4d is from a rat 24 hours posttreatment. Figures 4e and 4f are high magnifications of the cortical and medullary regions, respectively. Figures 4g and 4j are thymuses from rats 48 hours after treatment. Figures 4h and 4k are high magnifications of the corresponding cortical regions while Figures 4i and 4l are high magnifications of the corresponding medullary regions. When Figures 4a and 4d are compared, a striking tinctorial change is obvious in the treated thymus, especially in the cortical regions due to marked apoptosis of the lymphocytes in this region of the treated rat (Figure 4e). The arrows in Figure 4d indicate regions of tissue tearing, most likely due to lack of cell-to-cell adhesion among the apoptotic cells. The medullary region in figure 4d has increased in area and is less cellular (Figure 4f). This decreased cellularity in the medulla is due to mild lymphocyte apoptosis at 12 hours posttreatment (data not shown). The cortical:medullary ratio has changed significantly in Figure 4d due to a minimal decrease in the cortical area and a mild increase in the medullary region. A qualitative assessment would indicate that the cortical:medullary ratio in Figure 4d is overall approximately 1:2 compared to the normal 2:1 ratio in Figure 4a. Due to tangential cuts through these regions and the generation of different values depending on where the measurement is taken, it is difficult to make a quantitative assessment of this ratio. Although the animals in Figures 4g and 4j were in the same treatment group, there was variability in the tissue response to treatment. Compared to control (Figure 4a), there is a dramatic decrease in the size of the organ. The thymus in Figure 4g shows recovery of the cortical (Figure 4h) and medullary (Figure 4i) thymic tissue with repopulation of the thymocytes. At low magnification, the overall cortical:medullary ratio is approximately 1:10. The arrow in Figure 4g indicates a parathymic lymph node. The thymus in Figure 4j was more severely affected with necrosis and inflammation of the cortical tissue (Figure 4k). However, there is a robust recovery of the medullary thymocytes evidenced by the darker medullary regions in Figure 4j and the presence of increased numbers of small dark lymphocytes in Figure 4l. The cortical:medullary ratio in Figure 4j is difficult to determine due to the lack of normal cortical tissue. The arrow indicates a parathymic lymph node.

Figure 4

Figures 4a—l are examples of thymuses from a dexamethasone study in 30-day-old male Sprague–Dawley rats. Figure 4a is the normal saline treated control rat thymus. Figures 4b and 4c are high magnifications of the cortical and medullary regions, respectively. Figure 4d is from a rat 24 hours posttreatment. Figures 4e and 4f are high magnifications of the cortical and medullary regions, respectively. Figures 4g and 4j are thymuses from rats 48 hours after treatment. Figures 4h and 4k are high magnifications of the corresponding cortical regions while Figures 4i and 4l are high magnifications of the corresponding medullary regions. When Figures 4a and 4d are compared, a striking tinctorial change is obvious in the treated thymus, especially in the cortical regions due to marked apoptosis of the lymphocytes in this region of the treated rat (Figure 4e). The arrows in Figure 4d indicate regions of tissue tearing, most likely due to lack of cell-to-cell adhesion among the apoptotic cells. The medullary region in figure 4d has increased in area and is less cellular (Figure 4f). This decreased cellularity in the medulla is due to mild lymphocyte apoptosis at 12 hours posttreatment (data not shown). The cortical:medullary ratio has changed significantly in Figure 4d due to a minimal decrease in the cortical area and a mild increase in the medullary region. A qualitative assessment would indicate that the cortical:medullary ratio in Figure 4d is overall approximately 1:2 compared to the normal 2:1 ratio in Figure 4a. Due to tangential cuts through these regions and the generation of different values depending on where the measurement is taken, it is difficult to make a quantitative assessment of this ratio. Although the animals in Figures 4g and 4j were in the same treatment group, there was variability in the tissue response to treatment. Compared to control (Figure 4a), there is a dramatic decrease in the size of the organ. The thymus in Figure 4g shows recovery of the cortical (Figure 4h) and medullary (Figure 4i) thymic tissue with repopulation of the thymocytes. At low magnification, the overall cortical:medullary ratio is approximately 1:10. The arrow in Figure 4g indicates a parathymic lymph node. The thymus in Figure 4j was more severely affected with necrosis and inflammation of the cortical tissue (Figure 4k). However, there is a robust recovery of the medullary thymocytes evidenced by the darker medullary regions in Figure 4j and the presence of increased numbers of small dark lymphocytes in Figure 4l. The cortical:medullary ratio in Figure 4j is difficult to determine due to the lack of normal cortical tissue. The arrow indicates a parathymic lymph node.

Figure 4

Figures 4a—l are examples of thymuses from a dexamethasone study in 30-day-old male Sprague–Dawley rats. Figure 4a is the normal saline treated control rat thymus. Figures 4b and 4c are high magnifications of the cortical and medullary regions, respectively. Figure 4d is from a rat 24 hours posttreatment. Figures 4e and 4f are high magnifications of the cortical and medullary regions, respectively. Figures 4g and 4j are thymuses from rats 48 hours after treatment. Figures 4h and 4k are high magnifications of the corresponding cortical regions while Figures 4i and 4l are high magnifications of the corresponding medullary regions. When Figures 4a and 4d are compared, a striking tinctorial change is obvious in the treated thymus, especially in the cortical regions due to marked apoptosis of the lymphocytes in this region of the treated rat (Figure 4e). The arrows in Figure 4d indicate regions of tissue tearing, most likely due to lack of cell-to-cell adhesion among the apoptotic cells. The medullary region in figure 4d has increased in area and is less cellular (Figure 4f). This decreased cellularity in the medulla is due to mild lymphocyte apoptosis at 12 hours posttreatment (data not shown). The cortical:medullary ratio has changed significantly in Figure 4d due to a minimal decrease in the cortical area and a mild increase in the medullary region. A qualitative assessment would indicate that the cortical:medullary ratio in Figure 4d is overall approximately 1:2 compared to the normal 2:1 ratio in Figure 4a. Due to tangential cuts through these regions and the generation of different values depending on where the measurement is taken, it is difficult to make a quantitative assessment of this ratio. Although the animals in Figures 4g and 4j were in the same treatment group, there was variability in the tissue response to treatment. Compared to control (Figure 4a), there is a dramatic decrease in the size of the organ. The thymus in Figure 4g shows recovery of the cortical (Figure 4h) and medullary (Figure 4i) thymic tissue with repopulation of the thymocytes. At low magnification, the overall cortical:medullary ratio is approximately 1:10. The arrow in Figure 4g indicates a parathymic lymph node. The thymus in Figure 4j was more severely affected with necrosis and inflammation of the cortical tissue (Figure 4k). However, there is a robust recovery of the medullary thymocytes evidenced by the darker medullary regions in Figure 4j and the presence of increased numbers of small dark lymphocytes in Figure 4l. The cortical:medullary ratio in Figure 4j is difficult to determine due to the lack of normal cortical tissue. The arrow indicates a parathymic lymph node.

Figure 5

The arrow in Figure 5a indicates a focal area of increased lymphocytes that are large and pale-staining, with a higher magnification depicted in Figure 5b. An increase in the number of lymphocytes may affect either the cortex or the medulla. These lymphocytes can also be organized into lymphocytic nodules that can resemble germinal centers. Lymphocyte hyperplasia in the thymus with lymphoid follicle formation has not been observed in F344 rats. The formation of actual germinal centers in the thymus of humans and dogs may be an indication of autoimmune disease. Either focal or diffuse lymphoid hyperplasia can also occur after involution of the thymus. Generally, the cortex will show focal accumulations of lymphocytes as well as patchy atrophic changes. Figure 5 photomicrographs are courtesy of Drs. C. Frith and J. Ward.

Figure 6

These images are from a 2-year-old male B6C3F1 mouse that was treated with phenolphthalein. The arrow in Figure 6a indicates a region of the thymus with a diffuse increase in lymphocyte numbers. These pale lymphocytes appear to diffusely expand the medullary region with no visible cortical tissue remaining. The higher magnifications in Figures 6b and 6c indicate an increase in the number of large, pale lymphocytes, prominent vessels and occasional scattered macrophages with pale golden aggregates of pigmented material (arrows). Figure 6 photomicrographs are courtesy of Drs. Frith and Ward.

Figure 7

A focal or diffuse increase in thymic lymphocytes may occur in older mice and may involve either the cortex or medulla. This type of lesion usually occurs in thymuses that have undergone physiological involution. In the mouse, it generally occurs in mice older than 6 months of age and is more common in female B6C3F1 mice. In the mouse, lymphocyte hyperplasia is usually characterized by infiltration of the medulla with small, uniform lymphocytes. Figure 7 is an example of diffuse increased lymphocyte numbers in the thymus cortex. The low magnification in Figure 7a illustrates that there is no invasion of the medullary region and the higher magnification in Figure 7b illustrates the small, uniform lymphocytes without a prominent epithelial component. These features help to distinguish this lesion from thymic neoplasias. Figure 7 photomicrographs are courtesy of Drs. Frith and Ward.

Figure 8

Figure 8a is an example of focal prominent epithelium (arrows) associated with thymic involution in a male 2-year-old F334 rat. This lesion is characterized by prominent tubular structures lined by cuboidal epithelium seen in the high magnification image (Figure 8b). This lesion should be distinguished from thymic neoplasias, such as thymomas, with a prominent epithelial component. Figure 8c and the higher magnification in 8d are images from a 2-year-old male F344 control rat with a focal area of increased epithelial cells at the intersection of two lobules (arrow). Focal areas of increased epithelial cells can be a common finding in the F344 rat.

Figure 9

The epithelium free areas (EFAs) in the thymus are lymphocyte-rich areas that run from the subcapsular region to deep in the cortex, occasionally bordering the medullary regions. These structures are found in the rat and are considered to be strain-dependent. Special stains are helpful to visualize these regions. Figure 9 is a section of thymus from a control Wistar rat with prominent subcapsular EFAs (arrows). These regions consist of tightly packed CD4/CD8 positive lymphocytes and appear to be lightly encapsulated. These regions would not stain with laminin or keratin, allowing differentiation from the rest of the thymus. Figure 9 photomicrograph is courtesy of Dr. F. Kuper.