Figure 1. Normal, Gestation Day 18. B6C3F1 mouse. The structure of the adrenal gland a gestation day 18 clearly shows the zona glomerulosa, zona fasciculata, and the medulla. Zona glomerulosa cells are arranged in small nests or packets and Zona fasciculata cells have begun to align in columns perpendicular to the capsular surface. Some intermingling of zona fasciculata and medulla cells can be seen as the corticomedullary junction is not sharply delineated.

Figure 2 (A81956) Normal cortex: Control B6C3F1 male in a 90-day study. The male adrenal cortex consists of the zona glomerulosa and zona fasiculata. The zona glomerulosa is a narrow poorly delimited band of nest of cells with round nuclei and scant cytoplasm located immediately below the capsule. The junction with the zona fasiculata is often difficult to clearly define as in this case. The zona fasiculata cells are polygonal to round with a centrally located nucleus and are arranged in single cell columns perpendicular to the capsular surface with fine capillaries between adjacent columns. The columnar arrangement is less obvious near the corticomedullary junction. Varying degrees of cytoplasmic vacuolation reflecting steroid synthesis and storage are present in the zona fasiculata. In this control male mouse from a 90-day toxicity study, the degree of cortical cytoplasmic vacuolation is within normal limits and would not typically be diagnosed and graded in a conventional toxicology study. Some degree of zona fasciculata cytoplasmic vacuolation is expected as a reflection of the circadian metabolic status of steroidogenesis.

Figure 3 (A81845). Normal adrenal: Control B6C3F1 male in a chronic study. In comparison to Figure 2 (A81956), the zona glomerulosa is more readily apparent and comprised of nests of cells with pale staining cytoplasm beneath the capsule. In this adrenal the columns of the zona fasiculata are less distinct in comparison with Figure 2 (A81956). A rich capillary network is present in the cortex and medulla. Also, the degree of cortical cell vacuolation is greater than in Figure 2 (A81956) and involves both zona glomerulosa and zona fasciculata.

Figure 4 (A81846). Normal adrenal: Control B6C3F1 male in a chronic study.
This is the contralateral adrenal in the same mouse as Figure 3 (A81845) and shows the extensive vascular system in the medulla. The subtle subcapsular cellular proliferation evident by closely packed basophilic nuclei is a normal occurrence in older mice. This degree of subcapsular hyperplasia is minimal and is an expected background change. It would typically not be diagnosed in evaluating a study unless there was a need to establish a grade for comparison to a potential treatment related response in mice exposed to a test agent or procedure. A small cluster of cells in the zona fasciculata consists of cells that are larger and more vacuolated than the surrounding zona fasciculata. These cells presumably have stored more cortical hormone but the reason for such a focal change is uncertain.

Figures 5a and 5b (A81973 & A81974). Normal adrenal. Adrenal from a normal male B6C3F1 mouse in a 90-day study. The cortex and medulla are normal and the low magnification of the adrenal (Figure 5a) shows a subtle variation in staining within the zona fasciculata. The paler staining areas of the zona fasciculata have slightly enlarged cells with finely vacuolated cytoplasm consistent with storage of cortical hormone. Present in the low magnification and more clearly in the higher magnification (Figure 5b) is the absence of an x-zone characteristic of the male mouse adrenal at this age. The normal medulla is characterized by small nests and packets of medullary cells.

Figures 6a and 6b (A81899 & A81900). Normal X-zone. Untreated female B6C3F1 mouse in a 90-day study. At the end of a 90-day study female B6C3F1 mice would be approximately 18 weeks old with a prominent adrenal x-zone as shown in right and left adrenal glands in this mouse. A small number of vacuolated cells are present within this x-zone but their presence or absence may vary among control female mice. Since this x-zone is within normal limits for this age mouse, a specific diagnosis is not necessary but its extent and appearance should be considered when evaluating possible accelerated x-zone involution in treated study cohorts. It is noted that the volume of the x-zone may vary depending a cross-section (Figure 6a) versus a more longitudinal section (Figure 6b).

Figure 7a and Figure 7b (A81923 & A81924). Normal x-zone: Control female B6C3F1 mouse in a 90-day study. The x-zone contains a mild scattering of vacuolated cells consistent with fatty change. X-zone fatty change in normal female B6C3F1 mice approximately 18 weeks of age is highly variable. This amount of x-zone fatty change is slightly greater than in Figures 6a and 6b. The degree of vacuolation of zona fasciculata cells is within normal physiological limits.

Figures 8a & 8b (A81920 & A81921). Normal x-zone with cytoplasmic vacuolation: Untreated female B6C3F1 mouse in a 90-day study. Prominent cytoplasmic vacuolation is present in this x-zone representing a more extreme example of vacuolar change sometimes present in adrenal glands in untreated 18-week old female mice (see Figures 6a, 6b, 7a, and 7b for comparison). Since this degree of cytoplasmic vacuolation is part of the variable spectrum of x-zone changes in 18-week old female mice, it need not be diagnosed but because of its extent it could be described in the study pathology narrative. While some pathologists have considered a diagnosis of fatty degeneration for this feature of the x-zone in a control of this age, this feature of the x-zone is a normal part of natural x-zone involution. It should be kept in mind that x-zone vacuolation (fatty change) is a non-exclusive form of normal x-zone involution. Involution can occur without any evidence of vacuolation (fatty change). A minimal to mild degree of subcapsular hyperplasia is present in this adrenal gland.

Figure 9 (A81958). Normal medulla: Control B6C3F1 male in a 90-day study. The adrenal medulla is comprised of nests of cells with closely spaced leptochromatice nuclei and finely granular slightly basophilic cytoplasm. The cellular nests are separated by intervening capillaries and larger vascular channels that provide a rich vascular network draining blood from the cortex. Although not apparent in this example, subtle cytoplasmic tinctorial variations may allow identification of epinephrine versus norepinephrine medullary cells. Some cortical cells at the corticomedullary junction have condensed hyperchromatic as well as pyknotic nuclei reflecting the final stages of normal cell death of cortical cells. Dying cell fragments are most likely carried away via the vascular system.

Figures 10 (A81904), 11 (A81867) & 12 (A81948). Accessory cortical nodule: Figures 10 and 11 are untreated controls and Figure 12 is a treated B6C3F1 mouse. All are males. Accessory cortical nodules are relative common in both male and female mice, have a strain-dependent variable incidence, and may be attached to or just beneath the adrenal capsule or separate in adjacent periadrenal adipose tissue. The consist of both zona glomerulosa and zona fasciculata cells and are generally diagnosed even though their incidence does not appear to be influenced by treatment.

Figure 13 (A81975). Diffuse cortical hypertrophy: Treated B6C3F1 female in a 90-day study. There are two notable changes present in this adrenal. The zona fasiculata is comprised of uniformly enlarge cells is finely vacuolated cytoplasm. This change is consistent with storage of steroid hormone or precursors and was originally diagnosed as increased cytoplasmic vacuolation. However, it could also represent a physiological change rather than a pathological process; thus, a diagnosis of increased cytoplasmic vacuolation should be based on comparison to age-matched concurrent controls. Conclusions regarding the importance of this type of change can be supported by significant increase in adrenal weight. The second notable change in this adrenal gland is a narrowing of the x-zone associated with increased nuclear density and minimal congestion. Since this is a 90-day study in a female mouse, the X-zone should normally be much wider (see Figure 6a for comparison). Consequently, this change represents premature x-zone involution and should be diagnosed. Premature involution of the x-zone may occur as a direct effect of treatment or as a hormonal andromimetic response.

Figures 14a (A81888), 14b (A81891) & 14c (A81890). Focal cortical hypertrophy: Vehicle control B6C3F1 male in a chronic study. In addition to presence of subcapsular hyperplasia (Figures 14a & 14b), there is an irregular patch of enlarged zona fasiculata round to polyhedral cells with uniformly stained, eosinophilic finely granular cytoplasm and centrally located nuclei (Figures 14b & 14c). Nuclei in this focus of hypertrophic cells vary in size suggesting some degree of polyploidy (Figure 14c). The function of this focus of hypertrophic cells is uncertain but may reflect a localized area of hormonal synthesis. A small irregular collection of enlarge cells with pale staining stipulated cytoplasm and somewhat condensed nuclei between the focus of hypertrophy and the medulla suggests early degeneration. In evaluating a toxicity study, the cytomorphological features of the hypertrophic cortical cells warrants description in the pathology narrative.