Immunocytochemical component of endocrine cells in pancreatic islets of horses

The endocrine cell components in the pancreatic islets of the following 4 pancreatic regions of the horse were investigated by immunohistochemical methods: lobus pancreatis sinister (left lobe); lobus pancreatis dexter (right lobe); and 2 regions of Corpus pancreatis (body), the duodenal lobe which lies along the cranial duodenal flexure and descending duodenum, and the intermediate lobe which is situated around the portal vein. The islets in the left and intermediate lobes contained a central mass of glucagon cells surrounded by insulin cells, a few somatostatin cells and sporadic pancreatic polypeptide (PP) cells. On the other hand, the islets in the duodenal lobe were small in size compared with the other 3 regions, and were predominant in insulin and pancreatic polypeptide (PP) cells, but almost lacked in glucagon cells. These findings suggested that the duodenal lobe was derived from the ventral pancreatic primordium, and the left and intermediate lobes were originated from the dorsal pancreatic primordium. In the right lobe, the composition and distribution of the islet cells were almost the same as those in the left and intermediate lobes, but there were several lobules containing numerous PP cells as seen in the duodenal lobe.     

Immunocytochemistry of normal pancreatic islets and spontaneous islet cell tumors in dogs

Immunocytochemical studies of the distribution of glucagon, gastrin, insulin, and somatostatin in normal canine pancreatic islets and 20 canine islet cell tumors were done using the peroxidase-anti-peroxidase (PAP) technique. In the normal adult canine pancreas, islets typically consisted of clusters of 20-30 cells, but smaller foci and even individual cells were identified. Alpha cells (glucagon) were often peripherally located, beta cells (insulin) were centrally located and most numerous, and delta cells (somatostatin) were the least numerous and randomly located. Both juvenile and adult canine pancreases did not stain for gastrin. Of the 20 tumors examined, 18 had positive immunoreactivity for insulin, nine for glucagon, 14 for somatostatin, and one for gastrin. Two tumors were uninterpretable due to autolysis. Three tumors were pure insulinomas, but no pure somatostatinomas, glucagonomas, or gastrinomas were identified. Most tumors and metastases had mixed positive immunoreactivity; one neoplastic cell type predominated with lesser numbers of other cell types. Metastatic sites (liver and lymph node) stained for insulin and somatostatin, only. Foci of non-neoplastic islet cell tissue (nesidioblastosis), often located at the pancreatic-mesenteric junction, stained strongly positive for insulin, glucagon, and somatostatin but not for gastrin. The tumor staining pattern did not consistently correlate with tumor function, as determined by blood glucose and serum insulin assays. The PAP technique works well on paraffin-embedded, formalin-fixed tissue using rabbit or guinea pig antisera as the primary antibody. Staining occurred on sections of paraffin blocks stored for up to 7 years.     

Immunoreactivity of cytotoxic T-lymphocyte antigen 2 alpha in mouse pancreatic islet cells

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.     

Leucine markedly regulates pancreatic exocrine secretion in goats

Four goats (30.1 ± 1.3 kg) with common bile duct re‐entrant catheter and duodenal catheter were used to evaluate the effects of duodenal leucine infusion on pancreatic exocrine secretion and plasma parameters with two 4 × 4 Latin square design experiments. In the long‐term infusion experiment, goats were fed twice daily [700 g/day, dry matter (DM) basis] at 8:00 and 18:00 hours and were duodenally infused with 0, 3, 6, 9 g/day leucine for 14 days. Pancreatic juice and jugular blood samples were collected over 1‐h intervals for 6 h daily from d 11 to 14 days to encompass a 24‐h day. In the short‐term experiment, goats were infused leucine for 10 h continuously at the same infusion rate with Experiment 1 after feed deprivation for 24 h repeated every 10 days. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The results showed that the long‐term leucine infusion did not affect pancreatic juice secretion, protein output, trypsin and lipase secretion and plasma insulin concentration, but linearly increased α‐amylase secretion. No changes in pancreatic protein and lipase secretion were observed in the short‐term infusion. Pancreatic juice and α‐amylase secretion responded quadratically, with the greatest values observed in the 3 and 6 g/day leucine respectively. Trypsin secretion linearly decreased, while plasma insulin concentration increased linearly with increased leucine infusion. The results demonstrated that duodenal leucine infusion dose and time dependently regulated pancreatic enzyme secretion not associated with the change in plasma insulin concentration.     

Immunohistochemical morphometry of pancreatic islets in the cat.

The application of immunohistochemical technique with antisera for glucagon (Glu), insulin (Ins), somatostatin (Som) and pancreatic polypeptide (PP) to serial sections of the cat pancreas permitted the quantitative evaluation of the population of 4 endocrine cell types and that of the area, larger diameter and density of islets. The pancreas was divided macroscopically into the 4 portions, duodenal, gastric, anastomotic and splenic. The duodenal portion was characterized by the localization of PP-immunoreactive (IR) cell-rich islets, the dissemination of PP-IR cells in the exocrine parenchyma and the absence of Glu-IR cells. In the duodenal portion, the area, the larger diameter and the density of islets were significantly smaller than those in the other 3 portions. On the contrary, the other 3 portions were marked with the deficiency of PP-IR cells and the existence of Glu-IR cell-rich islets. Ins-IR cells, identified as compact cell masses without any other types of cells, occupied a major part of every islet, composing much the same population throughout the 4 portions. The Som-IR cell population appeared to be closely in parallel with the Glu-IR cell population in all of the portions. It is concluded that all islets are similar in the Ins-IR cell population, but different in the complementary arrangement of Glu- and PP-IR cells. Based on this difference, 2 types of islets can be classified.     

Regulation of pancreatic exocrine secretion in goats: differential effects of short‐ and long‐term duodenal phenylalanine treatment

Four yearling goats (31.2 ± 2.5 kg), surgically fitted with common bile duct reentrant and duodenal catheter, were used in two 4 × 4 Latin square design experiments to investigate the effects of duodenal infusion of phenylalanine for different times on pancreatic exocrine secretion (PES). In experiment 1 (the long‐term experiment), goats were duodenally infused with 0, 2, 4 or 8 g/day phenylalanine for 14 day. Pancreatic juice and jugular blood samples were collected over 1‐h intervals for 6 h daily from day 11 to day 14 to encompass a 24‐h day. In experiment 2 (the short‐term experiment), goats were infused with phenylalanine for 10 h continuously at the same infusion rate as experiment 1 after feed deprivation for 24 h repeated every 10 day. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The volume and pH of pancreatic juice were measured, and a 5% subsample was composited and frozen until analysis of enzyme activities. Plasma was frozen until analysis of insulin and cholecystokinin (CCK). In experiment 1, pancreatic juice, α‐amylase secretion and plasma CCK concentration responded quadratically (p < 0.05), with the top value observed at the 2 g/day phenylalanine. Trypsin secretion had a quadratic response (p < 0.05), with secretion increasing up to 4 g/day phenylalanine and decreasing thereafter. Phenylalanine linearly decreased pancreatic protein and lipase secretion (p < 0.05). The results of correlation analysis showed significant correlations (p < 0.05) between plasma CCK concentration and secretion of α‐amylase and trypsin. However, the short‐term phenylalanine infusion did not influence (p > 0.05) pancreatic juice, protein, α‐amylase, lipase, trypsin secretion and plasma CCK concentration. These results indicate PES of ruminants is stimulated by phenylalanine and is potentially mediated by CCK in the long‐term duodenal infusion treatment, but is not influenced by phenylalanine in the short‐term duodenal infusion treatment.     

CXCL14-like immunoreactivity in somatostatin-producing cells of the Japanese quail (Coturnix japonica) pancreas

This study examines chemokine CXCL14-like peptide distribution in the Japanese quail (Coturnix japonica) pancreas using a specific anti-human CXCL14 antibody. CXCL14-immunoreactive cells were observed in the pancreatic islet peripheral region. The staining was abolished after pre-absorbing the antibody with recombinant human CXCL14. CXCL14-immunoreactive cells were immuno-positive for somatostatin, but not glucagon and insulin. CXCL14 secreted from somatostatin-producing cells might participate in insulin secretion modulation together with somatostatin. In addition, CXCL14 might participate in glucose homeostasis in co-operation with somatostatin and growth hormone.     

Neoplasia and hyperplasia of pancreatic endocrine tissue in the rat: an immunocytochemical study

Spontaneously occurring neoplasms and non-neoplastic proliferative changes of the pancreatic cells in aging Sprague-Dawley and Long-Evans rats were examined for the presence and distribution of pancreatic hormones using immunocytochemical techniques. Islet cell tumors were indistinguishable in the two rat strains. They were composed principally of insulin-containing beta cells, but had additional and variable small proportions of cells that stained for somatostatin, glucagon, or rarely, pancreatic polypeptide. The heterogeneity in these spontaneous islet cell neoplasms was similar to that reported in humans as well as those induced in rats by streptozotocin. Hyperplasia of the islet cells also mainly affected the beta cells, but the overall pattern of immunocytochemical staining usually remained similar to that of normal islets, a point of distinction from islet cell neoplasms. In addition, rats with exocrine atrophy and fibrosis were found to have considerable disruption and focal proliferation of the islets.     

Pancreatic Polypeptide and Insulin-Secreting Tumor in a Dog With Duodenal Ulcers and Hypertrophic Gastritis

A 7-year-old spayed female Cocker Spaniel was hospitalized with a history of chronic vomiting, anorexia, and weight loss. Laboratory abnormalities included leukocytosis, metabolic alkalosis, hypoglycemia, hypoproteinemia, and hyperinsulinemia. Gastroscopy and ultrasonography revealed multiple gastric masses and a possible pancreatic mass, respectively. Examination of tissues obtained at necropsy showed a pancreatic adenocarcinoma with hepatic metastasis, gastric hypertrophy, and multiple duodenal ulcers. Immunocytochemical staining of the neoplasia was positive for pancreatic polypeptide (PP) and insulin and negative for gastrin, calcitonin, adrenocorticotropic hormone (ACTH), serotonin, L-enkephalin, chromagranin, glucagon, and somatostatin. Subsequent serum gastrin and PP assays showed a fasting hypergastrinemia with a normal response of gastrin to provocative testing and extremely increased PP values. The high PP values may have resulted in the vomiting and gastrointestinal ulceration. A PP-secreting tumor has not previously been reported in the dog.     

Cell-specific localization of the cholecystokininA receptor in the porcine pancreas

Cholecystokinin (CCK) produced in the mucosa of the upper small intestine exerts several biological functions. Its secretion in physiological amounts is modulated by the interaction of extracellular regulators and by binding to intracellular receptors of the target cells. The relative affinity of CCK to its receptor has been characterized in various biological and pharmacological studies and it is now well established that CCK has a higher affinity to the CCKA than to the CCKB receptor. Furthermore CCK influences the secretion of pancreatic enzymes in several species but very little is known about the relationship between CCK and the islet hormone-producing cells in the pig pancreas. The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in pigs. The aim of the present study was to characterize the precise cellular location of the CCKA receptor in the porcine pancreas. Polyclonal antiserum was raised against the N-terminal epitope of the CCKA receptor molecule and used for localization studies. Using immunohistochemistry on methanol/acetic acid-fixed, paraffin-embedded pancreas, the CCKA receptor could successfully be localized in islet cells. Parallel staining of serial sections with antibodies directed against insulin and glucagon revealed colocalization with glucagon in alpha cells. No immunoreaction was found in the exocrine pancreas. Our results support the concept that in the porcine species the stimulation of the exocrine pancreas is mediated by the CCKB rather than the CCKA receptor, as it is known for the rat species.     

Hypoglycemia in Four Dogs With Smooth Muscle Tumors

Tumor-associated hypoglycemia has been reported in dogs with pancreatic β-cell tumors, hepatic tumors, and, rarely, with other neoplasms. This article describes 4 dogs with marked hypoglycemia associated with smooth muscle tumors (jejunal leiomyoma, gastric leiomyoma and leiomyosarcoma, and splenic leiomyosarcoma). Presenting clinical signs included grand mal seizures, lethargy, weakness, ataxia, and, in 1 dog, polyuria/polydipsia. The serum insulin concentration was low in 1 dog and normal in the other dog evaluated. Immunohistochemical staining for insulin was negative in the 4 tumors; the 3 tumors arising from the stomach and jejunum stained diffusely positive for glucagon. Blood glucose concentrations rapidly returned to normal after complete surgical resection of the tumors, and clinical signs associated with hypoglycemia resolved. Long-term follow-up available in 3 of the 4 dogs found no recurrence of clinical signs related to hypoglycemia at 15, 31, and 38 months after surgery, respectively.     

Canine pancreatic endocrine tumors: immunohistochemical analysis of hormone content and amyloid

Thirty-one primary canine pancreatic endocrine tumors and their metastases were studied histologically and immunohistochemically for the presence of insulin, glucagon, somatostatin, pancreatic polypeptide (PP), gastrin, and adrenocorticotrophic hormone (ACTH). Tumors were also evaluated for the presence of amyloid. The cytoarchitectural pattern of 25 of 31 primary tumors was predominantly solid, whereas three tumors were mostly glandular, two were unclassified, and one had a gyriform pattern. Cells with insulin immunoreactivity were found in 30 of 31 tumors and were found in all cases in which there was clinical evidence of inappropriate insulin secretion. Insulin was the only hormone demonstrable in three of the 30 tumors, but cells immunoreactive for other hormones were also present in various combinations in most tumors [i.e., glucagon (13 of 30), somatostatin (17 of 30), PP (25 of 30), and gastrin (2 of 30)]. One tumor contained only cells with glucagon and PP immunoreactivity. Amyloid was found in ten of 31 primary tumors but was not detected in metastases. Cells with insulin immunoreactivity were the only cell type consistently present in tumors containing amyloid. Amyloid deposits did not immunoreact with any of the antisera. Seventeen of 31 dogs had metastasis of the pancreatic endocrine tumor to regional lymph nodes, liver, or both. All metastases available for study (15 of 17) contained cells with insulin immunoreactivity and some contained cells with PP or somatostatin immunoreactivity. No statistically significant (P greater than 0.05) differences in tendency to metastasize were found when pancreatic endocrine tumors were compared by region of origin, cytoarchitectural pattern, presence of amyloid, or by number of hormones contained within the tumor.     

Localization of amylin‐like immunoreactivity in the striped velvet gecko pancreas

Immunohistochemical techniques were employed to investigate the distribution of amylin‐like immunoreactive cells in the pancreas of gecko Homopholis fasciata. Four types of endocrine cells were distinguished: insulin immunoreactive (B cells), pancreatic polypeptide immunoreactive (PP cells), glucagon and pancreatic polypeptide immunoreactive (A/PP cells) and somatostatin immunoreactive cells (D cells). Pancreatic islets contained B, A/PP and D cells, whereas extrainsular regions contained B, D and PP cells. In the pancreatic islets, amylin‐like immunoreactive cells corresponded to B cells, but not to A/PP or D cells. In the extrainsular regions, amylin‐like immunoreactive cells corresponded to either B or PP cells. Amylin secreted from intrainsular B cells may regulate pancreatic hormone secretion in an autocrine and/or a paracrine fashion. On the other hand, amylin secreted from extrainsular PP and B cells, and/or intrainsular B cells may participate in the modulation of calcium homoeostasis in an endocrine fashion.     

Ghrelin,insulin and pancreatic activity in the peri‐weaning period of goat kids

Two groups of 3‐day‐old Saanen goat kids (MILK and WMIX) were studied in order to investigate the effect of weaning on plasma ghrelin and insulin and on pancreatic activity. MILK kids received goat milk to age 50 days; WMIX kids were initially fed milk but started weaning at 30 days and were completely weaned by 48 days. Dry matter intake and body weights were recorded, and plasma samples were analysed for metabolites, ghrelin, insulin, leptin and α‐amylase. At 50 days, all the animals were slaughtered, pancreas samples were analysed for α‐amylase, zymogen, DNA and ribosomal capacity (RNA/zymogen). Seven days after the beginning of the weaning program, dry matter intake in the WMIX group began to decrease in relation to the MILK group. Nonetheless, body weight did not differ throughout the study period. Weaning significantly decreased plasma levels of glucose, amino acids, urea and insulin, but increased creatinine and ghrelin. In weaning kids ghrelin secretion may help minimize the negative consequences of the new diet on dry matter intake. Pancreatic zymogen and ribosomal capacity did not differ between the groups, whereas pancreatic amylase activity was over three times higher in MILK than WMIX kids even though the former had no dietary starch. This finding could be a consequence of lower pancreatic secretion of amylase in the MILK group due to the lack of dietary starch, resulting in pancreatic accumulation, but could also be due to higher plasma insulin in the MILK group, suggesting a role for insulin in the functional development of the pancreas in weaning goats.     

鸭胰腺内高血糖素—,胰岛素—和生长抑素—免疫反应细胞的形态及分布

用ABC免疫组织化学方法,对1 周龄绍鸭胰腺内的高血糖素(A)、胰岛素(B)和生长抑素(D)免疫反应细胞的形态及分布进行了观察。结果表明,上述3 种细胞在全胰的分布及形态有差异。A 细胞主要成群散在于A 胰岛,少数位于混合型胰岛的边缘。D 细胞主要散在于A 胰岛中,少数位于B胰岛和混合型胰岛的边缘。B细胞主要呈团块状分布于B胰岛,少数位于混合型胰岛的中央。在胰外分泌部有散在的A 和D 细胞,位于腺泡及导管上皮细胞之间或结缔组织中。A 细胞形态各异,以多边形为主,多数细胞伸出形态多样的胞质突起,伸达胰岛或其他细胞间。D细胞的形态与A 细胞相似。B细胞形态均一,呈圆形或卵圆形,未见胞质突起,在外分泌部未见到B细胞。     

Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47–65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.     

Distribution of CD68‐, CD8‐, MHCI‐ and MHCII‐positive cells in the bull and ram testis and epididymis

The mammalian testis possesses a special immunological environment because of its properties of remarkable immune privilege and effective local innate immunity. The testicular immune privilege protects immunogenic germ cells from systemic immune attack, and local innate immunity is important in preventing testicular microbial infections. Thus, this study aimed to immunohistochemically demonstrate the distribution and localization of CD68‐, CD8‐, MHCI‐ and MHCII‐positive immune cells in the testes and epididymes. Negative immunoreactivity was detected in the seminiferous tubule epithelium and peritubular myoid cells of the testes upon staining in CD68, CD8 and MHC Class I. Positive CD68 immunoreaction was determined in the Sertoli cells and some Leydig cells. The detection of positive cells for CD8 clearly indicated the presence of lymphocytes. Furthermore, the staining with MHCI intensity was ascertained to vary from weak to moderate in the Sertoli and Leydig cells and connective tissue cells. MHCII‐positive immunoreactivity was determined in myoid cells and Leydig cells in the interstitial area. The epithelium of the epididymis showed positive staining for CD68 and CD8, but the stroma displayed a rather weak staining. In the ram epididymis, neither intraepithelial nor interstitial positive reaction was observed for MHCI. In the epididymis, the basal cells displayed a stronger staining for MHCII. In conclusion, these cells not only contribute to local immunity through their direct effects on the quality of fertility in males, but also contribute either directly or indirectly to immune privilege by minimizing the development of both autoimmune reactions and potentially harmful risks.     

Immunohistochemical differentiation of reactive from malignant mesothelium as a diagnostic aid in canine pericardial disease

Objectives

To develop a provisional immunohistochemistry panel for distinguishing reactive pericardium, atypical mesothelial proliferation and mesothelioma in dogs.

Materials and Methods

Archived pericardial biopsies were subject to haematoxylin and eosin staining, immunohistochemistry for cytokeratin, vimentin, insulin‐like growth factor II mRNA‐binding protein 3, glucose transporter 1 and desmin. Samples were scored for intensity and number of cells stained.

Results

Ten biopsies of reactive mesothelium, 17 of atypical mesothelial proliferation, 26 of mesothelioma and five of normal pericardium were identified on the basis of haematoxylin and eosin staining. Cytokeratin and vimentin were expressed in all biopsies, confirming mesothelial origin. Normal pericardial samples had the lowest scores for insulin‐like growth factor II mRNA‐binding protein 3, glucose transporter 1 and desmin. Mesothelioma and atypical proliferative samples were similar to each other, with higher scores for insulin‐like growth factor II mRNA‐binding protein 3 and glucose transporter 1 than the reactive samples. Desmin staining was variable. Insulin‐like growth factor II mRNA‐binding protein 3 was the best to distinguish between disease groups.

Clinical Significance

An immunohistochemistry panel of cytokeratin, vimentin, insulin‐like growth factor II mRNA‐binding protein 3 and glucose transporter 1 could provide superior information compared with haematoxylin and eosin staining alone in the diagnosis of cases of mesothelial proliferation in canine pericardium, but further validation is warranted.     

Dexamethasone‐induced alterations in glucose tolerance and,insulin, glucagon and adrenaline responses during the first month in white leghorn chicks

1. Alterations in glucose tolerance and, insulin, glucagon and adrenaline responses in dexamethasone (DXM)‐treated (induced hypocorticalism) chicks have been evaluated chronologically during the first month of development of White Leghorn chicks.

2. Alterations in glucose tolerance and, insulin resistance and glucagon and adrenalin responses in dexamethasonised chicks were recorded. Control chicks too displayed differential glucose tolerance and, insulin, glucagon and adrenaline responses on a chronological basis.

3. The observations suggest increased insulin and adrenaline sensitivity in the first fortnight and gradually increasing glucagon response in the second fortnight in neonatal chicks.

4. Furthermore, increased insulin sensitivity and attenuated glucagon responsiveness was also associated with DXM‐induced hypocorticalism.