Immunohistochemical and ultrastructural changes in rat fat tissue related to the local hCG injection.

OBJECTIVES: Recently, it has been observed that weight loss is accelerated by human chorionic gonadotropin (hCG) hormone preparation used for hypothalamic dysfunction in obesity treatment in both sexes. hCG is also used for in vitro fertilization and in treatment of hypogonadotropic hypogonadism. Our aim was to observe the ultrastructural changes caused by local injections of hCG made for purpose of weight loss and to present them to inform those receiving such therapy. MATERIALS AND METHODS: In our study, 10 obese female, 10 male obese, 10 non-obese female and 10 non-obese male rats were used. In each group, single dose of subcutaneous hCG injection has been applied to 7 rats for 5 weeks in 5 days of the week, and placebo has been applied to the remaining 3 rats. Following the injection, the tissues were evaluated morphologically, immunohistochemically and ultrastructurally. RESULTS: Leptin immunoreactivity was similar in all groups. When the adipose tissue samples were examined under electron microscope, they were observed to exhibit normal structure with organelles located around the nuclei and nucleoli, and no distinctive features were found among the groups. CONCLUSIONS: Administering hCG in addition to diet had no advantage on weight reduction in rats.

Dose-dependent immunohistochemical changes in rat cornea and retina after oral methylphenidate administration.

Methylphenidate hydrochloride (MPH), more commonly known as Ritalin, is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity disorder, one of the most common behavioural disorders of children and young adults. The aim of this study was to investigate dose-dependent immunohistochemical Dopamine 2 receptor (D2) expression and apoptosis in the rat cornea and cornea. In this study, 27 female pre-pubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. They were treated orally with methylphenidate dissolved in saline solution for 5 days per week during 3 months. At the end of the third month, after perfusion fixation, eye tissue was removed. Paraffin sections were collected for immunohistochemical and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling assay studies. In our study, we observed that the cornea D2 receptor reactivity showed a dose-related increase after MPH treatment, especially in basal cells of the epithelium and a dose-dependent decrease in the retinal ganglion cell which was statistically meaningful. Analysis of the cornea thickness results showed no meaningful difference between groups. Apoptotic cell number showed a meaningful increase in the high dose treated group compared to the other groups of the study. The data suggest that Ritalin has degenerative effect on the important functional part of the eye, such as cornea and retina and its activating dopaminergic mechanism via similar neuronal paths, functionally and structurally, to induce morphological changes. As a result, we believe that this morphological changes negatively effecting functional organization of the affected cornea and retina.

Dose-dependent immunohistochemical and ultrastructural changes after oral methylphenidate administration in rat heart tissue.

Methylphenidate, more commonly known as Ritalin, is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity disorder, one of the most common behavioural disorders of children and young adults. Our aims were to investigate dose-dependent immunohistochemical D2 expression and ultrastructural changes of the rat heart tissue, and to demonstrate possible toxicity of the long-term and high dose use of the methylphenidate. In this study, 27 female pre-pubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. They were treated orally with methylphenidate dissolved in saline solution for 5 days/week during 3 months. At the end of the third month, after perfusion fixation, left ventricle of cardiac tissue was removed. Paraffin, semi-thin and thin sections were collected and immunohistochemical, terminal deoxynucleotidyl transferase-mediated Dig-dUTP nick end labelling assay and ultrastructural studies were performed. In conclusion, we believe that Ritalin is dose-related affecting dopaminergic system to increase heart rhythm and contraction. Thus, this drug may cause degenerative ultrastructural changes in mitochondrial path.

Age-related immunohistochemical and ultrastructural changes in rat oculomotor nerve.

During ageing process, multiple changes occur on nervous tissue composed of cells and extracellular matrix. Changes on nervous tissues are usually known as degenerative changes on axon structure and connective tissue covering the nerve such as a decrease in the number of fibre or general structural changes. For this purpose, we have studied age-dependent ultrastructural changes in the rat oculomotor nerve with electron microscopy and also demonstrated collagen structure of the neural sheaths with immunohistochemical techniques. This study was conducted in Gazi University Faculty of Medicine, Department of Anatomy with a total of nine Wistar albino rats. We observed strong collagen type I immunoreactivity in endoneurium and slight to moderate reactivity in fibroblast cytoplasm in 3-month- and 12-month-old groups and mild reactivity in 24-month-old group. Collagen type IV immunoreactivity was stronger in endoneurium and perineurium in the 3-month- and 12-month-old groups compared with collagen type I and fibroblast cytoplasm showed a very strong reactivity. On the other hand, in the 24-month-old group, there was slight reactivity in endoneurium and a strong reactivity in perineurium. NGF staining showed moderate to strong reactivity on Schwann cells of the 3-month-old group. The immunoreactivity decreased in the 12-month- and 24-month-old groups. In the 3-month-old rat group, Schwann cell cytoplasm, mitochondrial structure and neurofilaments were normal. In the 12-month-old group, there were no changes in organelle distribution, mitochondrial structure and neurofilaments, but there was an increase in the connective tissue. An inconsiderable number of degenerated myelinated nerves were observed. We detected an important decrease in the collagen type I immunoreactivity, which could suggest that the endoneurium, perineurium and epineurium are less resistant to the age-related collagen loss and that the peripheral nerve is protected by a weaker barrier in the old group. The collagen type IV immunoreactivity was significantly decreased with age. NGF synthesis decreases with age because of Schwann cell structural degeneration or for different reasons. Thus, this could explain the diminished capacity of regeneration and damage of the myelination of the peripheral nerve.

Branching patterns of rabbit oculomotor and trochlear nerves demonstrated by Sihler's stain technique.

A modified Sihler's stain technique was used to visualize the branching patterns of oculomotor and trochlear nerves. The levator palpebrae, superior rectus, inferior rectus, medial rectus, inferior oblique, superior oblique and tensor trochlea muscles were isolated from the eyes of normal rabbits and processed using modified Sihler's technique. The distributions and terminal ramifications of the oculomotor and trochlear nerves were observed. Two distinct divisions and terminal branches of the oculomotor nerve were shown in detail together with the trochlear nerve distribution. The application of Sihler's technique enables researchers to trace nerve branching within relatively transparent muscles, whereas the nerve fibers are counterstained and clearly visible. This technique could be useful for detailed studies of the motor control of extraocular muscles.

Morphological characteristics of styloid process evaluated by computerized axial tomography.

Morphological characteristics of styloid process and ossified stylohyoid ligament and their overall relationships to age and sex were studied by using computerized axial tomography images. The styloid process and ossified stylohyoid ligaments were classified into seven groups according to their shapes and lengths. The styloid process of a length of 25-40 mm, was the most frequently encountered. The elongated styloid process was mostly seen in males. There was no overall correlation between the types of SP and sex. The progressive increase in length with age was not seen in our study.

Prolonged kallikrein inhibition does not affect the basal growth and secretory capacity of rat adrenal cortex, but enhances mineralo- and glucocorticoid response to ACTH and handling stress.

The effects on the pituitary-adrenocortical functions of the prolonged (7-day) blockade of endogenous bradykinin (BK) synthesis, obtained by the administration of the kallikrein inhibitor (K-I) cyclohexylacetyl-Phe-Arg-Ser-Val-Gln amide, were investigated in the rat. K-I treatment did not cause significant changes in the (i) body and adrenal weights; (ii) basal plasma levels of ACTH, aldosterone and corticosterone; and (iii) average volume of adrenocortical cells and their basal secretory capacity. Conversely, K-I administration induced a significant magnification of the in vivo mineralo- and glucocorticoid responses to the intraperitoneal (i.p.) bolus injection of ACTH. Moreover, K-I-treated rats, but not control ones, displayed a moderate and short-term adrenal secretory response to the mild stress evoked by the placebo i.p. injection. Collectively, these findings rule out the possibility that endogenous BK plays a relevant role in the control of adrenocortical function under basal conditions. However, they suggest that endogenous BK may be involved in quenching exceedingly high adrenocortical responses to ACTH and stresses.

Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus- pituitary-adrenal axis.

Secretin, glucagon, gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH) belong, together with vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase (AC)-activating polypeptide, to a family of peptides (the VIP-secretin-glucagon family), which also includes growth hormone-releasing hormone and exendins. All the members of this peptide family possess a remarkable amino-acid sequence homology, and bind to G-protein-coupled receptors, whose signaling mechanism primarily involves AC/protein kinase A and phospholipase C/protein kinase C cascades. VIP and pituitary AC-activating polypeptide play a role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and in this review we survey findings that also other members of the VIP-secretin-glucagon family may have the same function. Secretin and secretin receptors are expressed in the hypothalamus and pituitary gland, and secretin inhibits adrenocorticotropic hormone (ACTH) release. No evidence is available for the presence of secretin receptors in adrenal glands, but secretin selectively depresses the glucocorticoid response to ACTH of dispersed zona fasciculata-reticularis (ZF/R) cells. Glucagon and glucagon-like peptide-1 are contained in the hypothalamus, and all the components of the HPA axis are provided with glucagon and glucagons-like-1 receptors. These peptides exert a short-term inhibitory effect on stress-induced pituitary ACTH release and depress the ZF/R cell response to ACTH by inhibiting the AC/protein kinase A cascade; they also stimulate hypothalamic arginine-vasopressin release. GIP receptors are present in the ZF/R of the normal adrenals, and are particularly abundant in some types of adrenocortical adenomas and hyperplasias. GIP, through the activation of the AC/protein kinase A cascade, evokes a sizeable glucocorticoid secretagogue effect, leading to the identification of a food/GIP-dependent Cushing's syndrome. PTH and PTH-related protein are expressed in the hypothalamus and pituitary gland, and PTH and PTH-related protein receptors in all the components of the HPA axis. Both peptides enhance ACTH and arginine-vasopressin release, as well as stimulate aldosterone and glucocorticoid secretion of dispersed zona glomerulosa and ZF/R cells, respectively. The involvement of growth hormone-releasing hormone and exendins in the functional regulation of the HPA axis has not yet been extensively investigated.

Blockade of angiotensin II type 1 receptor and not of endothelin receptor prevents hypertension and cardiovascular disease in transgenic (mREN2)27 rats via adrenocortical steroid-independent mechanisms.

We investigated the role of angiotensin II (Ang II) and endothelin-1 (ET-1) in transgenic (mREN2)27 rats, a model of the monogenic renin-dependent form of severe hypertension and cardiovascular disease. Four-week-old heterozygous male transgenic (mREN2)27 rats (n=24) were matched according to body weight (BW) and blood pressure (BP) and randomly allocated to receive a placebo (group P), the mixed endothelin type A and B receptor antagonist bosentan (100 mg/kg BW PO, group B), the Ang II type 1-specific receptor antagonist irbesartan (50 mg/kg BW PO, group I), or the endothelin type A-selective antagonist BMS-182874 (52 mg/kg BW PO, group BMS). After 4 weeks of treatment, during which BW and BP were measured weekly, animals were euthanized, and the heart, left ventricle, right ventricle, adrenal gland, brain, and kidney were weighed. The plasma levels of adrenocortical steroids were measured by high-performance liquid chromatography. The tension responses of ET-free segments of the thoracic aorta to 5 x 10(-6) mmol/L phenylephrine, 60 mmol/L KCl, and cumulative doses of ET-1 were assessed. The density of ET-1 receptor subtypes in the aorta and vascular structural changes in the mesenteric arterioles (100 to 200 microm ID) were also measured with autoradiography and myography, respectively. Compared with all other groups, group I rats showed significantly (P<0.001) lower systolic BP (group I, 161+/-8 mm Hg; group P, 269+/-23 mm Hg; group B, 275+/-17 mm Hg; and group BMS, 254+/-21 mm Hg), left ventricular weight (2.28+/-0.15 versus 3. 71+/-0.26, 3.38+/-0.27, and 3.96+/-0.51 mg/g BW, respectively), tension responses to vasoconstrictors, and normalized media thickness of the mesenteric arterioles (22.3+/-0.6 versus 25.3+/-0.5, 25.5+/-0.7, and 24.1+/-1.5 microm, respectively). Compared with levels in group P (78+/-25 pmol/mL), plasma aldosterone levels were significantly decreased in group B (51+/-11 pmol/mL) and group I (40+/-16 pmol/mL). Thus, endogenous ET-1 and Ang II contribute to the regulation of aldosterone, but only Ang II is crucial for the development of hypertension and related target organ damage via the Ang II type 1 receptor. Endogenous Ang II does not appear to enhance cardiovascular production of ET-1 in this model of hypertension within the time span of our experiment.

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland.

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [125I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22-52), a putative ADM-receptor antagonist, and CGRP(8-37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K+-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22-52) and CGRP(8-37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca2+-channel activator BAYK-8644, and blocked K+- and BAYK-8644-evoked rise in the intracellular Ca2+ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22-52) and CGRP(8-37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22-52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca2+ channel-mediated Ca2+ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.