Identification of immunohistochemical localization of irisin in the dwarf hamster (Phodopus roborovskii) tissues

Irisin is mainly secreted by heart and skeletal muscle cells. It is an exercise‐induced protein that converts white adipose tissue to brown. Increased irisin expression was lead to weight loss and improved glucose tolerance. We investigated irisin immunoreactivity in various tissues of the dwarf hamsters (Phodopus roborovskii). Tissues were processed, embedded in paraffin, sectioned at 5 μm and stained immunohistochemically for irisin. In the retina, irisin was found almost all layers, except outer nuclear layer. Also, irisin immunoreactivity was observed in the skin, cornea, striated muscle, parotid gland, tongue, oesophagus, stomach and small intestine. The findings from this study support the notion that skeletal muscle is not the primary source of irisin.     

Colocalization of gamma-aminobutyric acid immunoreactivity and acetylcholinesterase activity in nerve fibers of the mouse adrenal gland.

The present immunohistochemical and enzyme histochemical study showed gamma-aminobutyric acid (GABA) immunoreactivity and acetylcholinesterase (AChE) activity in the mouse adrenal gland. Weak GABA immunoreactivity was seen in clusters of chromaffin cells showing noradrenaline fluorescence. This finding suggests that both GABA and noradrenaline may be released from the granules of noradrenaline cells by adequate stimuli. GABA-immunoreactive varicose nerve fibers densely contacted adrenaline cells and large ganglion cells, but they were sparse in the periphery of clusters of noradrenaline cells. AChE activity was strong in a few large ganglion cells and weak in chromaffin cells showing noradrenaline fluorescence, and was found in numerous nerve bundles and fibers of the medulla. AChE-active nerve fibers more densely contacted noradrenaline cells than adrenaline cells. By using double labeling technique, numerous GABA-immunoreactive nerve fibers in the medulla were reactive for AChE in the same sections. These results suggest that both GABA and acetylcholine may be colocalized in the intra-adrenal nerve fibers and may have some secretory effects on the chromaffin cells.     

Cytochrome b5 expression in gonadectomy-induced adrenocortical neoplasms of the domestic ferret (Mustela putorius furo)

Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b(5) (cyt b(5)), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b(5) immunoreactivity was evident in 24 of 25 (96%) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b(5), which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were coexpressed with cyt b(5) in some of the tumors. We concluded that cyt b(5) is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors.     

Fecal cortisol metabolite analysis for noninvasive monitoring of adrenocortical function in the cheetah (Acinonyx jubatus).

A radioimmunoassay was validated for quantifying excreted cortisol metabolites in cheetah (Acinonyx jubatus) feces. High-performance liquid chromatography analysis indicated that immunoreactivity was associated with a water-soluble metabolite in fecal extracts from males and females. None of the immunoreactivity corresponded with free cortisol or corticosterone but rather was associated with a more polar, unidentified metabolite. To determine the biologic relevance of excreted immunoreactive cortisol metabolites, cheetahs were exposed to a variety of situations anticipated to increase cortisol secretion. First, to assess acute changes in adrenal activity, adrenocorticotropic hormone (ACTH; 400 IU i.m.) was administered to two adult males and two adult females. Pre-ACTH baseline serum cortisol and fecal cortisol metabolite concentrations varied among individuals. Serum cortisol concentrations were elevated above baseline within 10 min of ACTH injection, followed by corresponding increases in fecal cortisol metabolite concentrations (690-4,194% above baseline) 48 hr later in three of four cheetahs. In the fourth cheetah, a smaller increase (334% above baseline) in fecal cortisol metabolite excretion was observed 96 hr after ACTH injection. Seven cheetah females also were subjected to a variety of potentially stressful manipulations, including immobilization, translocation, and introduction to a male to assess the ability of this technique to detect physiologic changes in adrenal activity. Increased fecal corticoid metabolite excretion was observed 24-72 hr after exposure to these exogenous stressors. Results indicate that adrenocortical activity can be monitored noninvasively in the cheetah through analysis of these metabolites. This technique could be valuable for evaluating, and thus optimizing, environmental and management conditions and for investigating the role of stress in disease pathogenesis and the usually poor reproductive performance of this species in captivity.     

Acetylcholinesterase activity, and neurofilament protein, and catecholamine synthesizing enzymes immunoreactivities in the mouse adrenal gland during postnatal development.

The present study showed the acetylcholinesterase (AChE) activity, and neurofilament protein (NFP), catecholamine-synthesizing enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) immunoreactivities in the mouse adrenal gland during postnatal development. From birth to postnatal-1-day, AChE activity was weakly and diffusely found in some medullary cells and in very few nerve fibers whereas strong NFP immunoreactivity was seen in a few ganglion cells and in remarkably numerous nerve fibers in the medulla. Almost all meduallary cells were reactive for both DBH and PNMT during this period. From postnatal-2- or -3-day to postnatal-1-week, strong AChE activity was observed in a few large ganglion cells, but the reaction was weak in clusters of chromaffin cells, and the number of strong AChE-active nerve fibers in the medulla was rapidly increased. From postnatal-2-day onwards, the number of NFP-immunoreactive nerve fibers in the medulla were remarkably numerous. Numerous chromaffin cells were reactive for both DBH and PNMT whereas some chromaffin cells were reactive for only DBH from postnatal-2-day onwards. These results suggest that drastic changes such as an increase of acetylcholine in the nerve fibers, differentiation of noradrenaline and adrenaline cells of the medulla may occur during this period. From postnatal-2-week to postnatal-3-week, weak AChE activity was seen in the clusters of several chromaffin cells and a few ganglion cells, and the number of AChE-active nerve fibers in the medulla was gradually increased. From postnatal-4-week to postnatal-8-week (adult), the distribution and frequency of AChE activity in the adrenal gland were similar to those at postnatal-3-week. In the adult, AChE activity was weakly seen in the clusters of several chromaffin cells showing noradrenaline fluorescence in the adrenal medulla. The noradrenaline cells were contacted by denser AChE-reactive nerve fibers than adrenaline cells. These results suggest that the development of cholinergic nervous system in the mouse adrenal medulla may be completed by postnatal-3-week.     

CHANGES IN ULTRASONOGRAPHIC APPEARANCE OF ADRENAL GLANDS IN DOGS WITH PITUITARY-DEPENDENT HYPERADRENOCORTICISM TREATED WITH TRILOSTANE

Trilostane, a 3beta-hydroxysteroid dehydrogenase inhibitor, has been used successfully over the last few years for the treatment of canine pituitary-dependent hyperadrenocorticism. In a prospective study of 19 dogs with pituitary-dependent hyperadrenocorticism, the adrenal glands were measured before and at least 6 months after initiation of trilostane therapy. Right adrenal gland length and caudal pole thickness and left adrenal gland caudal pole thickness increased significantly (p < or = 0.05); there was no significant change in left adrenal gland length. Enlargement of adrenal glands during trilostane therapy may occur as a result of suppression of the negative feedback mechanism affecting cortisol production.     

Immunohistochemical localization of chromogranin A in normal tissues from laboratory animals

To analyze the distribution of Chromogranin A in endocrine cells of various species of laboratory animals (dog, gerbil, guinea pig, hamster, monkey, mouse, and fetal, neonatal, and adult rats), normal tissues were stained immunohistochemically with polyclonal anti-bovine Chromogranin A antiserum (SP-1). Selected tissues (pituitary, adrenal, thyroid, parathyroid, pancreas, brain, peripheral nerve, stomach, small and large intestine, bone marrow, spleen, thymus, lymph node, and liver) from these species and from the rabbit were stained with two monoclonal anti-human Chromogranin A antibodies (LK2H10 and PHE5) to compare the immunoreactivities of the monoclonal antibodies and polyclonal antiserum. Staining with the polyclonal antiserum (SP-1) resulted in a broader spectrum of immunoreactivity but had more nonspecific background staining than either monoclonal antibody. Immunoreactivity and staining intensity with SP-1 varied between species, but most endocrine tissues (pituitary cells in the anterior and intermediate lobes, thyroid "C" cells, adrenal medulla, parathyroid, pancreatic islets, and enterochromaffin cells) from most species stained positively. In some species, pancreatic alpha cells stained more intensely, and two populations of adrenal medullary cells with different staining intensities were observed. Sciatic nerve (axonal area) was immunoreactive with monoclonal antibodies and/or the polyclonal antiserum in several species. The spectrum of immunoreactive tissues from fetal and neonatal rats increased with age. There was good cross-reactivity between species with SP-1, but not with either LK2H10 or PHE5. These results indicate that many endocrine cells with secretory granules in laboratory animals express Chromogranin A and that a polyclonal antiserum, such as SP-1, is more sensitive in detecting this protein in various species than monoclonal antibodies such as LK2H10 or PHE5.     

Ultrasonographic measurement of the adrenal gland-to-abdominal aortic ratio as a valuable method of estimating normal adrenal size in rabbits

A wide range has been reported for the ultrasonographic measurements of the normal adrenal gland in rabbits. Therefore, having sufficient information about the normal measurements of the adrenal gland and their relationship with indicators such as weight, sex and the diameter of the internal abdominal aorta will be of great help in diagnosing diseases of the adrenal gland. In the present study, 21 healthy adult intact rabbits were selected. The abdominal cavity of the animals was examined using ultrasound; adrenal gland parameters such as length, width, height, circumference and area on the right and left sides were measured in both sagittal and transverse planes. Additionally, the diameter of the abdominal aorta in the sagittal plane was imaged. After statistical analysis, the parameters of the adrenal glands on the right and left showed a positive significant correlation with the weight and the diameter of the abdominal aorta, although no significant correlation was found between these parameters and sex. The ratio of adrenal gland parameters to abdominal aortic diameter was calculated and the statistical analysis of the values showed that, except for the ratio of left adrenal area to aortic diameter, the other ratio of adrenal gland parameters to abdominal aortic diameter was not significantly related to weight. Therefore, these ratios can be used as suitable indicators for assessing the change in size of the adrenal gland of rabbits of different sizes. Knowing the relationship between normal adrenal measurements and the indices such as weight, sex and diameter of the abdominal aorta can help clinicians and researchers evaluate the changes in the size of the adrenal gland.     

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.     

Distribution of neutral amino acid transporter ASCT 1 in the non-neuronal tissues of mice

Distribution of ASCT1, a neutral amino acid transporter, in non-neuronal peripheral tissues of adult and developing mice was examined by immunohistochemistry and immunoelectron microscopy. Immunoreactivity for ASCT1 in the digestive system was localized in basal cells of stratified squamous epithelia from oral parietes to nonglandular region of the stomach, chief cells of the glandular stomach, acinar cells of the salivary gland and exocrine pancreas, and Paneth's cells of the small intestine, in all of which the basolateral membrane was selectively immuno-labeled. In the liver of adult mice, ASCT1 immunoreactivity was detected on the plasma membrane of hepatocytes surrounding central veins, and a temporal expansion of immunoreactive hepatocytes was observed in the embryonic and CCl4-treated adult livers. ASCT1 was also localized on the plasma membranes of proximal uriniferous tubule epithelial cells in the kidney of adult mice, and those of supporting cells in the medulla of adrenal gland. These results suggest that ASCT1 is expressed in various non-neuronal peripheral tissues in mice, and it contributes to the amino acid transport throughout non-neuronal tissues.     

Comparative study on calretinin immunoreactivity in gerbil and rat retina

Expression of calretinin in retina has been ascribed to multiple biological and functional aspects in the visual system. In this study, we examined the distribution patterns of calretinin immunoreactivity in gerbil and rat retina. In the gerbil, calretinin immunoreactivity was present in bipolar and amacrine cells of the inner nuclear layer and in neurones of the ganglion cell layer. In the rat, amacrine and ganglion cells showed calretinin immunoreactivity, but bipolar cells did not contain calretinin immunoreactivity. In both species, calretinin immunoreactivity was absent in cones, cone bipolars, and horizontal cells. In conclusion, gerbil as well as rat has a rod-dominant retina. The differences in calretinin expression between rat and gerbil require further investigations under various functional and developmental conditions.     

Adrenal necrosis in a dog receiving trilostane for the treatment of hyperadrenocorticism

Clinical and biochemical changes suggestive of hypoadrenocorticism were observed in a 10-year-old male neutered Staffordshire bull terrier shortly after beginning therapy with trilostane for the treatment of hyperadrenocorticism. The dog's condition was stabilised with intravenous fluids, fludrocortisone and prednisolone. An exploratory laparotomy and excisional biopsy of the left adrenal gland were performed. Histopathological analysis showed adrenal cortical necrosis with reactive inflammation and fibrosis. Trilostane is a reversible inhibitor of steroid synthesis and this complication has not been reported previously. Clinicians should be aware that trilostane therapy may result in adrenal necrosis but that prompt treatment might correct a life-threatening situation.     

Histological evaluation of the adrenal glands of seven dogs with hyperadrenocorticism treated with trilostane

The lesions in the adrenal glands of seven dogs with hyperadrenocorticism that had been treated with trilostane were studied histologically. The glands of the six dogs with pituitary-dependent hyperadrenocorticism had moderate to severe cortical hyperplasia that was either diffuse or nodular. The lesions were more pronounced in the zona fasciculata than in the zona reticularis, and the zona glomerulosa was normal. In the dog with a functional adrenal tumour the non-tumour bearing adrenal gland showed mild nodular hyperplasia. Five of the seven dogs had variable degrees of adrenal necrosis, which was severe in two of them. The terminal deoxynucleotidyl transferase-mediated DUTP nick-end labelling (TUNEL) reaction specified areas of cell death as apoptosis in three of the dogs, and was positive in one of the dogs without visible areas of cell death. There were variable degrees of cortical haemorrhage in three of the dogs. In some of the dogs the lesions were severe enough to lead to hypoadrenocorticism.     

Diagnostic efficacy of plasma ACTH-measurement by a chemiluminometric assay in canine hyperadrenocorticism

This retrospective study was performed to investigate the diagnostic efficacy of the chemiluminometric ACTH-measurement to differentiate between pituitary and adrenal dependent hyperadrenocorticism (HAC) in dogs. 49 dogs with pituitary HAC, 10 dogs with adrenal HAC and 1 dog with a combination of both pathologies were included. Dogs with HAC like symptoms, where HAC had been ruled out, served as controls (n = 18). All dogs with adrenal HAC, as well as 9 dogs with pituitary HAC had an ACTH concentration below the detection limit of 2.2 pmol/l (10 pg/dl) plasma. Using 2.2 pmol/l as a cut-off the sensitivity and specificity to diagnose pituitary HAC was 0.82 (95 % CI 0.686 - 0.914) and 1 (95 % CI 0.692 - 1), respectively. With the help of the chemiluminometric assay, a correct classification was possible in 85 % of patients with HAC. As an ACTH-concentration below the detection limit was found in dogs with adrenal as well as pituitary HAC, additional discriminatory tests are necessary in these cases.     

Evaluation of feline oral squamous cell carcinomas for p16 protein immunoreactivity and the presence of papillomaviral DNA

Oral squamous cell carcinomas (OSCCs) develop commonly in cats. While the cause of the feline neoplasms is unknown, a quarter of human OSCCs are caused by papillomavirus (PV) infection. As PV DNA has been previously detected in a feline OSCC, it was hypothesised that PV infection could be a significant cause of feline OSCCs. Human OSCCs that are caused by PVs contain increased p16^CDKN2A protein (p16), which can be detected using immunohistochemistry. In cats, increased p16 immunoreactivity has been reported within PV-associated skin lesions. This study evaluated p16 immunoreactivity within 30 feline OSCCs. Additionally, PCR was used to amplify PV DNA from the OSCCs. Increased p16 immunoreactivity was present within 2 OSCCs. However, as PV DNA was not amplified from any OSCC in this study, it cannot be confirmed that the increased p16 was caused by PV infection. Therefore, these results do not support the hypothesis that PVs are a significant cause of OSCCs in cats. Loss of p16 expression is considered an important process in the development of human non-PV-induced OSCCs. In contrast, loss of p16 immunoreactivity was only present in 2 feline OSCCs. This suggests that human and feline OSCCs develop due to different molecular mechanisms.     

鸢尾素-前体基因:分子结构、表达调控和生物学功能及其与骨骼肌纤维类型转化

鸢尾素(irisin)是新近发现的可分泌型肌源细胞因子,来源于Ⅲ型纤连蛋白组件包含蛋白5水解形成的多肽片段。目前证明鸢尾素及其前体基因在糖脂代谢疾病发生与运动治疗中发挥作用,可能与骨骼肌纤维类型转化存在关联。本文通过总结鸢尾素及其前体基因分子结构特点、表达调控和生物学功能最新研究进展,结合骨骼肌纤维类型转化机制,探讨分析鸢尾素-前体基因途径与骨骼肌纤维类型转化潜在分子关联,以期为畜禽肉质改良提供新思路。     

Effects of nitrofurans on adrenal cortical tissue in chickens

In chicken adrenal glands, aspartate transaminase (AST) activity is concentrated in the adrenal cortical cells. Administration of a bolus dose of furazolidone (200 mg/kg) to the birds produced a decrease in AST activity in the adrenal glands and a general disorganization in the ultrastructure of the adrenal cortical cells. Many adrenal cortical mitochondria appeared to be moribund, being swollen, without cristae or apparently empty. These effects preceded atrophy of the adrenal cortical tissue, which was most marked in the central part of the adrenal glands. The atrophy was still present after 12 weeks, and it is therefore suggested that nitrofurans may be useful in the treatment of Cushing's disease in other animal species. When furazolidone or furaltadone was given in the diet at therapeutic levels, blebs of an electron-dense substance developed in adrenal cortical mitochondria and AST activity in the adrenal glands was reduced. These effects were found to be reversible, and the treatments did not produce atrophy of the adrenal cortical tissue.     

Effects of mitotane therapy in dogs with pituitary dependent Cushing syndrome on the adrenal gland size--an ultrasonographic study]

The effect of mitotane therapy on adrenal gland size was evaluated in 13 dogs with pituitary dependent hyperadrenocorticism. Ultrasonographic measurements were obtained before and during mitotane therapy. During therapy both adrenal glands were shorter and thinner (median during therapy: left adrenal gland 19.4 mm long, 5.4 mm thick, right adrenal gland 18.1 mm long, 6.1 mm thick) than before mitotane therapy (median before therapy: left adrenal gland 23.6 mm long, 8.3 mm thick, right adrenal gland 21.6 mm long, 8.1 mm thick). Statistical evaluation showed a significant reduction in size. But ultrasonographic measurement of adrenal gland size is not useful in the evaluation of adrenal reserve during mitotane therapy. Inadequate adrenal reserve was not identified and adrenal size measurement by ultrasonography was not helpful to differentiate adequate and inadequate control of adrenal cortisol secretion during mitotane therapy.     

Evaluating the correlation between adrenal gland dimensions and aortic diameter in healthy dogs

In the present study, the relationship of normal adrenal dimensions with weight, aortic diameter, age and sex was evaluated. Moreover, a formula for estimating the normal dimensions of adrenal gland was provided. Thirty‐two intact adult dogs that were considered healthy based on history, physical examination, routine blood works and specific adrenal tests as well as absence of any abnormal ultrasonographic findings were included. Adrenal glands and abdominal aorta were imaged in the sagittal plane by ultrasound. Length, maximal height of the cranial and caudal poles, area and circumference of the left and right adrenal glands and also the internal diameter of the abdominal aorta were measured. Analysis showed that there is a positive correlation between the adrenal gland dimensions and weight and aortic diameter. The ratio of adrenal gland dimensions and the aortic diameter was calculated, but this ratio showed a great amount of variability in tandem with a significant correlation to the weight; Meaning that the ratio of the adrenal gland dimensions to the aortic diameter was not a reliable index for evaluating the size of the normal adrenal gland in animals with different weights. Therefore, we used the weight along with adrenal measurements in a linear regression model, and then, we were able to estimate the adrenal gland dimensions in different weights. Knowing the relationship of normal adrenal dimensions with the indices such as weight, age, sex and diameter of abdominal aorta can help the clinicians to diagnose the adrenal gland diseases.     

Steroid hormone receptors ERα and PR characterised by immunohistochemistry in the mare adrenal gland

Background

Sex steroid hormone receptors have been identified in the adrenal gland of rat, sheep and rhesus monkey, indicating a direct effect of sex steroids on adrenal gland function.

Methods

In the present study, immunohistochemistry using two different mouse monoclonal antibodies was employed to determine the presence of oestrogen receptor alpha (ERalpha) and progesterone receptor (PR) in the mare adrenal gland. Adrenal glands from intact (n = 5) and ovariectomised (OVX) (n = 5) mares, as well as uterine tissue (n = 9), were collected after euthanasia. Three of the OVX mares were treated with a single intramuscular injection of oestradiol benzoate (2.5 mg) 18 – 22 hours prior to euthanasia and tissue collection (OVX+Oe). Uterine tissue was used as a positive control and showed positive staining for both ERalpha and PR.

Results

ERalpha staining was detected in the adrenal zona glomerulosa, fasciculata and reticularis of all mare groups. Ovariectomy increased cortical ERalpha staining intensity. In OVX mares and one intact mare, positive ERalpha staining was also detected in adrenal medullary cells. PR staining of weak intensity was present in a low proportion of cells in the zona fasciculata and reticularis of all mare groups. Weak PR staining was also found in a high proportion of adrenal medullary cells. In contrast to staining in the adrenal cortex, which was always located within the cell nuclei, medullary staining for both ERalpha and PR was observed only in the cell cytoplasm.

Conclusion

The present results show the presence of ERalpha in the adrenal cortex, indicating oestradiol may have a direct effect on mare adrenal function. However, further studies are needed to confirm the presence of PR as staining in the present study was only weak and/or minor. Also, any possible effect of oestradiol treatment on the levels of steroid receptors cannot be determined by the present study, as treatment time was of a too short duration.