Pathogenesis of experimental adrenal hemorrhagic necrosis ("apoplexy"): ultrastructural, biochemical, neuropharmacologic, and blood coagulation studies with acrylonitrile in the rat

The pathogenesis of experimental adrenal hemorrhagic necrosis produced by acrylonitrile in the rat was investigated by various morphologic, biochemical, and pharmacologic methods. One dose of this chemical injected intravenously caused 100 per cent incidence of adrenal hemorrhage and necrosis in 90 to 120 minutes. Electron microscopy, histochemistry, and light microscopy combined with colloidal carbon labeling suggested an early damage (30 minutes after administration of acrylonitrile) to the vascular endothelium in the adrenal cortex, prominent at 60 minutes, when lesion to the parenchymal cells was not visible. The use of extracellular diffusion tracer horseradish peroxidase further indicated that parenchymal cell injury was a late event. Damage to the vascular endothelium in the adrenal cortex was associated with retrograde embolization of medullary cells and cell fragments into the cortical capillaries. The ultrastructurally demonstrated platelet aggregation and fibrin precipitation at the sites of discontinuous vascular endothelium were accompanied by a decrease in circulating platelets and fibrinogen as well as prolongation of prothrombin, partial thromboplastin, and thrombin time. The concentration of dopamine, unlike that of noradrenaline, in the adrenals but not in the brain of rats injected with acrylonitrile showed a time-dependent elevation. Pretreatment with phenoxybenzamine (alpha-adrenergic antagonist) or labetalol (alpha- and beta-adrenergic blocker) or metyrapone (11-beta-hydroxylase inhibitor) and the depletion of catecholamines by reserpine or prior medullectomy prevented the chemically induced adrenal necrosis. These results indicate that the presence of a functional adrenal cortex is necessary for the development of cortical damage which is associated with early vascular lesion caused and/or modulated by vasoactive amines from the medulla and/or (metabolites of) acrylonitrile.     

Adrenal pathology in the acquired immune deficiency syndrome

Adrenal pathology was examined in 41 autopsied patients with the acquired immune deficiency syndrome. This represents the largest series and the first study with quantitation of adrenal cortical necrosis. In 32 cases clinical data were analyzed for features of adrenal insufficiency. Common clinical findings included vomiting, diarrhea, fever, hypotension, and hyponatremia. None of the 32 patients showed characteristic skin hyperpigmentation. Two patients were suspected premortem to have adrenal insufficiency. In one of these patients, adrenocorticotrophic hormone (ACTH) stimulation resulted in an adequate rise in plasma cortisol values. In the other patient, the baseline plasma cortisol value was elevated and failed to rise significantly after ACTH stimulation. Pathologic findings included widespread lipid depletion, infection by cryptococcus, and acid-fast organisms consistent with Mycobacterium avium-intracellulare, involvement by Kaposi's sarcoma, and necrotizing adrenalitis due to cytomegalovirus (CMV). A point-counting method was used to quantitate adrenal cortical and medullary necrosis. Necrosis due to CMV was greater in the medulla than the cortex. The maximum amount of adrenal cortical necrosis in any case was 70%. The degree of cortical necrosis was less than that usually associated with adrenal insufficiency.     

Weight and shape of the human adrenal medulla in various age groups

Summary The weight and shape of the adrenal medulla were studied in 118 adrenal glands obtained at autopsies of 62 patients (38 males and 24 females) between 0 and 52 years of age. In adolescents 15 years and older and adults cases of sudden death only were entered in this study. The weight was calculated using morphometric measurements done on serial sections of the glands. In the new-born, the medulla accounts for less than 1% of the total volume of the adrenal gland. Though there is a rapid growth of the adrenal medulla after birth, the percentage of adrenal medullary volume at all age levels during childhood and adolescence is lower than in adults where it constitutes 9% of the total adrenal volume on the average. This corresponds to an average medullary weight of 0.43 g. In the new-born, the medulla consists of a thin plate made up of immature medulloblasts. Within a few months these are transformed into mature medullary cells; the shape of the medulla soon approaches that of the adult gland: an increase in thickness around the central vein and flat processes into the alae. With increasing age the cortico-medullary border becomes irregular and ragged. Especially in the vicinity of the central vein an intermingling of medullary and cortical cell complexes is found. An unequivocal diagnosis of genuine adrenal medullary hyperplasia can be established only by weighing the dissected medulla or by applying morphometric methods.     

BCL-2 expression and inhibin-A in adrenal neoplasms: a comparative study

Differential diagnosis between adrenal cortical and adrenal medullary lesions may be difficult in many cases. Different immunohistochemical, histochemical tools as well as ultrastructural diagnostic techniques have been employed to aid in differentiating between these lesions. Recently, both inhibin-A and BCL-2 have been shown to stain selectively adrenal cortical tissue and its derived neoplasms but not adrenal medulla or pheochromocytomas. In this study we compared the staining reactions of inhibin-A and BCL-2 in cases of adrenal cortical adenomas and carcinomas as well as pheochromocytomas. We found that both inhibin-A and BCL-2 stained cortical derived tissues, but not medullary derived tissues. Staining intensity for inhibin-A was significantly weaker than for BCL-2. We found that fixation techniques may influence the staining reactivity, as some cases did not immunoreact with any of the antibodies. We conclude that both inhibin-A, and, preferentially, BCL-2 are useful additions to a staining protocol to help in the differential diagnosis of cortical and medullary neoplasms.     

Immunofluorescence microscopic evaluation of the intermediate filament expression of the adrenal cortex and medulla and their tumors.

Normal adrenal glands (10 specimens) and adrenal gland tumors (58 cases) were immunohistochemically evaluated for different types of intermediate filament (IF) proteins. Some of the normal cortical cells showed cytokeratin positivity, and no positivity was seen for epidermal keratin or other types of IF. In the adrenal medulla, neurofilament positivity was seen in nerve axons, some ganglion cells, and chromaffin cells; and cytokeratin-positive cells could not be detected. Only the vascular and connective tissue elements showed vimentin positivity in both cortical and medullary areas. In half of the cortical carcinomas (13/25), cytokeratin-positive tumor cells were found. Furthermore, vimentin-positive tumor cells were present in 10 of 25 cases, in some of them together with cytokeratin-positive cells. Thus, the results show heterogeneity among the adrenal cortical carcinomas. Interestingly, many benign adrenal cortical tissues and some carcinomas lacked immunoreactivity for all types of IF, suggesting a poorly developed IF system in these tissues. In contrast to adrenal cortical tumors, pheochromocytomas contained neurofilamentlike immunoreactivity. These results reflect the different cellular nature of adrenal cortical and medullary tumors, which apparently can be distinguished from each other with antibodies to intermediate filament proteins.     

Blood vascular beds of rat adrenal and accessory adrenal glands, with special reference to the corticomedullary portal system: a further scanning electron microscopic study of corrosion casts and tissue specimens

Blood vascular casts of the rat adrenal glands were observed with a scanning electron microscope. The cortical capillary plexus drains, through the corticomedullary venous radicles, into the subcortical veins continuous with the medullary collecting veins. The medullary capillary plexus drains into the corticomedullary venous radicles, subcortical veins and medullary collecting veins. No portal vessel was noted between the cortical and medullary capillaries. These findings indicate that the cortical blood rich in glucocorticoids preferentially and continuously flows into the corticomedullary venous radicles, subcortical veins and medullary collecting veins all three of which are fenestrated in type, and also suggest that the vascular route from the cortical capillaries to the medullary collecting veins functions as a substitute for the portal system, controlling the biosynthesis of catecholamines in the adrenal medulla. The vascular bed of the accessory adrenal gland (extra-adrenal cortical or chromaffin body) is sometimes annexed to that of the adrenal gland. On rare occasions, the vascular beds of the extra-adrenal cortical and chromaffin bodies fuse with each other. Additional scanning of tissue samples confirmed the direct drainage of cortical capillaries into the medullary veins and also the endothelial fenestrations of these capillaries and veins.     

Neurogenic signals regulate chromaffin cell proliferation and mediate the mitogenic effect of reserpine in the adult rat adrenal medulla

The adrenal medulla is innervated by nerve fibers from several sources, which synapse on chromaffin cells and stimulate the secretion of catecholamines. The antihypertensive agent reserpine is known to reflexively increase this neurogenic stimulation by depleting catecholamine stores, and long-term administration of reserpine is associated with adrenal medullary hyperplasia and neoplasia. To determine the role of neurogenic signals in regulating normal and reserpine-stimulated proliferation of chromaffin cells, the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating nuclei was assessed in the adrenal medulla of adult rats. Unilateral adrenal denervation caused a 4-5 fold decrease in chromaffin cell labeling by 5-bromo-2'-deoxyuridine during a 2-week labeling period. Denervation also prevented stimulation of labeling in animals receiving reserpine in their diet. These findings suggest that neurogenic control of cell proliferation may play an important role in the pathogenesis of adrenal medullary hyperplasia and neoplasia, and in the normal development of the peripheral and central nervous systems.     

The role of calretinin, inhibin, melan-A, BCL-2, and C-kit in differentiating adrenal cortical and medullary tumors: an immunohistochemical study.

Morphologic distinction between adrenal cortical and medullary tumors can be difficult. Previous studies have shown inhibin, melan-A, and BCL-2 to be useful markers for adrenal cortical tumors. We have recently observed a high level of calretinin expression in normal adrenal cortex but not the medulla and therefore evaluated its diagnostic application for adrenal tumors in comparison with inhibin, melan-A, and BCL-2. C-kit is a transmembrane tyrosine kinase receptor. Immunodetection of c-kit expression has been recently used for tumor diagnosis, and c-kit-positive tumors can potentially benefit from kit kinase inhibitor treatment. Although c-kit expression was reported in adrenal medulla and pheochromocytoma, it has not been evaluated in adrenal cortical tumors. In this study, 28 adrenal cortical tumors (12 carcinomas, 16 adenomas), 20 pheochromocytomas, and 20 extraadrenal paragangliomas were evaluated for calretinin, inhibin, melan-A, BCL-2, and c-kit expression by standard immunohistochemical assays on paraffin sections. The percentage of immunoreactivity in adrenal cortical tumors was as follows: calretinin, 96%; melan-A, 89%; inhibin, 92%; BCL-2, 20%; and c-kit, 5%. Normal adrenal medulla did not stain for c-kit but was positive for BCL-2. Eighty-six percent of pheochromocytomas stained for BCL-2 and none for calretinin, with the exception of the ganglioneuromatous areas in composite pheochromocytomas (n = 5). Extraadrenal paragangliomas showed reactivity with calretinin in 25%, melan-A in 5%, inhibin in 16%, BCL-2 in 38%, and c-kit in 8% of the cases. Our results indicate that calretinin is the most sensitive among all the adrenal markers tested. Like melan-A and inhibin, calretinin is also a very specific marker in differentiating cortical from medullary adrenal tumors. In addition, calretinin can be used to confirm a composite pheochromocytoma. BCL-2 does not appear to be useful in differentiating adrenal cortical from medullary tumors. C-kit is not useful in the diagnosis of adrenal tumors, and kit kinase inhibitor might have a limited role in the treatment of adrenal tumors and paraganglioma because of the low frequency of c-kit expression in these tumors.     

Chronic renal lesions in the uninephrectomized Gunn rat after analgesic mixtures

Chronic cortical and medullary damage have been produced in uninephrectomized homozygous Gunn rats by single doses of the analgesics aspirin, paracetamol and phenazone, and by analgesic mixtures. The lesions are more severe than those of other experimental models of analgesic nephropathy, and the appearances of the cortical lesions suggest that they are ultimately due to the effects of papillary necrosis rather than to acute tubular necrosis observed in acute experiments with this model. The presence of an acute inflammatory reaction in both cortex and medulla in a number of animals one month after administration of analgesics indicates the possibility that the observed chronic renal damage may result from the intervention of additional complicating factors rather than from a single direct effect of analgesics.     

Epithelial-lined (true) cyst of the adrenal gland: a case report

A case of an epithelial-lined (true) adrenal cyst is reported. Although over 300 adrenal cysts have been reported in the literature, true cysts are rare. In this case, a 4.0 cm cyst lined by cuboidal to flattened cells with bland cytologic features was incidentally found at autopsy. Immunologic studies performed on formalin-fixed, paraffin-embedded sections demonstrated that the cells expressed keratins (AE1/AE3+, CAM 5.2+, and MAK-6+) and were negative for epithelial membrane antigen, vimentin, factor VIII, and desmin. Normal adrenal cortical and medullary cells did not express keratins, suggesting that the cyst lining was not derived from either adrenal cortex or medulla. A mesothelial origin, with a pathogenesis analogous to the formation of primary cysts of the spleen, is proposed.     

Adrenal gland: chemically induced structural and functional changes in the cortex

The adrenal cortex is the target of a surprisingly large number of exogenous chemicals. Until recently, the toxic action of these chemicals was discovered serendipitously. Following our observations that acrylonitrile, cysteamine or pyrazole induces hemorrhagic adrenocortical necrosis in the rat, we recently recognized a structure-activity correlation which predicts the adrenocorticolytic property of alkyl chemicals, i.e., 2-3 carbons with double or triple bonds and with nucleophilic terminal radicals (e.g., -CN, -SH, -NH2). On the basis of our results obtained with electron microscopic, histochemical and biochemical studies as well as those of others, we propose the following sequence of events in the pathogenesis of chemically induced adrenocortical necrosis: 1) Depletion of glutathione and increased dopamine concentration in the adrenals; 2) Endothelial damage and rupture of capillary walls in the adrenal cortex due to either direct attack by the chemicals (metabolites) and/or released monoamines; 3) Retrograde embolization of medullary tissue fragments into the cortical capillaries; 4) Enhanced destruction of cortical vascular walls with subsequent platelet aggregation, fibrin deposition which is often associated with a systemic drop in platelet counts, and changes in blood coagulation; 5) Escape of plasma and cellular elements of blood into extravascular spaces and damage of adrenocortical parenchymal cells; and 6) Hemorrhage and necrosis in the adrenal cortex. This pathogenetic sequence was investigated in detail with acrylonitrile, and studied in various aspects with thioguanine, cysteamine and pyrazole.     

Up-regulation of ret by reserpine in the adult rat adrenal medulla

The receptor tyrosine kinase, ret, is activated by glial cell line-derived neurotrophic factor, neurturin and related ligands that bind to glycosylphosphatidylinositol-tailed receptors GFRalpha1-4. Ret expression is developmentally regulated and detectable only at very low levels in adult adrenal medulla. However, mutations of ret that cause constitutive activation or alter signal transduction give rise to adrenal medullary hyperplasia and pheochromocytomas in humans with hereditary multiple endocrine neoplasia (MEN) syndromes 2A and 2B and in animal models. These discordant observations pose the conundrum of how a molecule barely detectable in the adult adrenal can contribute to development of adrenal medullary pathology that typically occurs in adults. We recently reported that depolarization and phorbol esters that activate protein kinase C act synergistically with neurturin to up-regulate ret protein and mRNA expression in adult rat chromaffin cell cultures. Those findings suggested that ret expression in vivo is not static and might be regulated in part by neurally derived signals. We show here that the anti-hypertensive agent reserpine, which is known to cause a reflex increase in trans-synaptic stimulation of chromaffin cells, increases expression of ret mRNA and protein in adult rat adrenal medullary tissue in vivo. Elevated ret protein levels are detectable both by immunoblots and immunohistochemistry, which shows immunoreactive ret in chromaffin cells and neurons after reserpine administration. The finding that ret expression is subject to up-regulation by environmental signals in vivo suggests that epigenetic factors might influence the development of adrenal medullary disease by affecting the expression of ret. It is known that long-term administration of reserpine leads to the development of adrenal medullary hyperplasia and pheochromocytomas in rats. Our findings suggest potential utility of the rat model for studying the roles of ret in the adrenal medulla and the mechanisms of its involvement in MEN 2 and other pheochromocytoma syndromes.     

Relationship between chromaffin cells and blood vessels in the rat adrenal medulla: A transmission electron microscopic study combined with blood vessel reconstructions

The rat adrenal medulla architecture was examined using a combination of medullary blood vessel reconstructions and transmission electron microscopy. The peripheral radicles of the central vein and the medullary capillaries of the medullary arteries were thus precisely identified in the electron microscopic observations. The observations confirmed that the peripheral segments of the central vein were sinusoidal vessels with an attenuated and fenestrated endothelial wall. No ultrastructural differences were observed between segments lined by epinephrine-storing cells and those lined by norepinephrine-storing cells. The findings suggest that these peripheral segments of the adrenal central vein were sites of cortical hormonal effects on the adrenal medulla. The vessel structure does not support the hypothesis that medullary chromaffin-cell development is controlled by selective distribution of adrenal blood vessels.     

Adrenal medullary hyperplasia: A clinicopathologic study of four cases

This study presents the clinicopathologic findings in four new cases of adrenal medullary hyperplasia. The patients presented with episodic hypertension frequently associated with palpitations headache, and diaphoresis. All four had elevated urinary catecholamine levels during attacks, and were thought clinically to have a pheochromocytoma. In each case laparotomy revealed a diffusely enlarged adrenal gland without a discrete tumor. Histologic examination of the adrenals demonstrated a diffuse or diffuse and nodular expansion of the medulla confirmed by morphometric study. Of the four patients, three underwent unilateral and one bilateral adrenalectomy. Two patients who underwent unilateral adrenalectomy have been free of symptoms for three years. Thus, it would appear that adrenal medullary hyperplasia may occur unilaterally or asynchronously in the two glands. The bilaterally adrenalectomized patient has had persistent attacks, suggesting that the stimulus to adrenal medullary hyperplasia may possibly affect other chromaffin tissues. On the basis of our cases and a review of the literature, we propose the following criteria for the diagnosis of adrenal medullary hyperplasia: a clinical history of episodic attacks suggesting pheochromocytoma (generally with associated increased urinary catecholamine levels), an adrenal gland showing diffuse expansion of the medulla into the alae or tail of the gland with or without nodule formation, a medulla composed of enlarged cells with or without pleomorphism, and, most important, an increased medulla/cortex ratio, together with an increased calculated medullary weight asddetermined by morphometric analysis.     

Autoantibodies to adrenal medullary and thyroid calcitonin cells in type I diabetes mellitus--a prospective study

Autoantibodies to adrenal medulla that gave a diffuse immunofluorescent staining pattern were detected in 17 out of 107 (16%) patients with newly diagnosed Type I diabetes mellitus, in 13 out of 178 (7%) patients with long lasting disease but in none of 80 mixed control sera tested. The antibodies were of IgG class and 31 of the 33 positive sera also fixed complement. In 32 out of 34 cases, detection of the antibodies was correlated with the presence in the serum of pancreatic islet cell antibodies (ICA). The specificity of adrenal medullary antibodies is distinct from ICA and from C-cell antibodies since their reactivity was not abolished by preabsorption with extracts from human insulinoma or thyroid C-cell carcinoma. The presence in the serum of antibodies to adrenal medulla is not related to a functional defect of the adrenal medulla, but this new specificity indicates a further autoimmune reaction related to the natural history of Type I diabetes.     

Murine cytomegalovirus adrenalitis in athymic nude mice

Summary During studies of the pathogenesis of murine cytomegalovirus (MCMV) infection in athymic nude mice, we noted striking virus involvement of the adrenal glands. Because patients with the acquired immunodeficiency syndrome (AIDS) have recently been reported to have adrenal necrosis and evidence of infection of the adrenal gland with human cytomegalovirus (HCMV), we have further evaluated adrenal involvement during MCMV infection. Following virus inoculation, MCMV replicated to high titer in the adrenal glands of T-cell deficient, homozygous nude mice, but not heterozygous littermates with intact T-cell function. Concomitant with the high titers of virus, there appeared overt histological evidence of herpesvirus infection accompanied by patchy necrosis of adrenal cortical and medullary tissues. Acyclovir, which inhibits growth of MCMV, reduced virus replication in the adrenal gland. Similarly, virus replication was diminished in homozygous nude mice immunologically reconstituted by infusion of normal spleen cells three weeks prior to infection. Thus, in the absence of functioning T lymphocytes, MCMV can infect and replicate in adrenal tissues causing a progressive destructive adrenalitis.With 1 Figure     

Microcirculation of the rat adrenal gland: A scanning electron microscope study of vascular casts

Blood vascular beds of the rat adrenal gland were filled with methacrylate resin and observed with a scanning electron microscope. The classical findings on mammalian adrenal glands of Flint (1900), Bennett and Kilham (1940), and Gersh and Grollman (1941) were confirmed. The cortical capillaries arise from the cortical arteries and converge at the corticomedullary junction into the peripheral venous radicles which flow into the tributaries of the central vein. The medullary capillaries originate from the medullary arteries and drain through the deep venous radicles into the tributaries of the central vein. No direct connection between the cortical and medullary capillaries was noted except for rare communications via the peripheral venous radicles. These findings show that most of the cortical blood, rich in glucocorticoids, flows in the medulla, not through the medullary capillary plexus but exclusively through the radicles of the central vein. Evidence for adrenal portal vessels could not be found.     

Histochemically demonstrable catecholamines in the sympathetic nervous system of trisomic 16 and normal fetal mice.

The formaldehyde-induced fluorescence (FIF) and micro-spectrofluorometric techniques were used to study catecholamines in the sympathetic nervous system of normal and trisomy 16 fetal mice with a gestation age of 15 days, an animal model for human trisomy 21 (Down's syndrome). FIF intensity in the stellate sympathetic ganglion of trisomic embryos did not differ from that of controls, whereas in the adrenal medulla the FIF intensity was 38% less in trisomic than in control embryos. When adrenal medullary cells from embryos with a gestational age of 15 days were maintained in culture for 7-10 days, a difference in FIF intensity between groups was still evident. The rate of noradrenaline uptake in cultured adrenal medullary cells also was significantly less in trisomic than in control fetal mice. The results suggest that the development of adrenal medulla is slowed in trisomic 16 mice and that uptake of noradrenaline by trisomic adrenal medullary cells is impaired.     

Papillary necrosis in experimental renal transplantation in the rat

Renal papillary necrosis is a frequent complication of unsuccessful renal transplantation in rats, occurring in both isografts and allografts. Papillary necrosis does not occur alone, but only and inevitably in association with severe cortical damage. The pattern of the lesion is different from other forms of papillary necrosis in that the least severe lesions occur in the outer medulla and the more severe lesions involve both medulla and papilla. The incidence of papillary necrosis is increased in isografts, but not in allografts, by longer preservation times. It is suggested that the principal underlying cause may be damage to medullary capillaries, occurring either during preservation or as a consequence of rejection and leading to medullary ischemia.