Renal effects of angiotensin II receptor subtype 1 and 2-selective ligands injected into the paraventricular nucleus of conscious rats

We determined the effects of losartan and CGP42112A (selective ligands of the AT1 and AT2 angiotensin receptors, respectively) and salarasin (a relatively nonselective angiotensin receptor antagonist) on urinary volume and urinary sodium and potassium excretion induced by administration of angiotensin II (ANG II) into the paraventricular nucleus (PVN) of conscious rats. Both the AT1 and AT2 ligands and salarasin administered in the presence of ANG II elicited a concentration-dependent inhibition of urine excretion, but losartan inhibited only 75% of this response. The IC50 for salarasin, CGP42112A, and losartan was 0.01, 0.05, and 6 nM, respectively. Previous treatment with saralasin, CGP42112A and losartan competitively antagonized the natriuretic responses to PVN administration of ANG II, and the IC50 values were 0.09, 0.48, and 10 nM, respectively. The maximum response to losartan was 65% of that obtained with saralasin. Pretreatment with saralasin, losartan, and CGP42112A injected into the PVN caused shifts to the right of the concentration-response curves, but the losartan concentrations were disproportionately greater compared with salarasin or CGP42112A. The IC50 values were 0.06, 0.5, and 7.0 for salarasin, CGP42112A, and losartan, respectively. These results suggest that both AT1 and AT2 receptor subtypes in the PVN are involved in ANG II-related urine, sodium, and potassium excretion, and that the inhibitory responses to AT2 blockade are predominant. Copyright (C) 1999 Elsevier Science B.V.

Ultrastructural alterations of choroid plexuses of lateral ventricles of rats (rattus norvegicus) submitted to experimental chronic alcoholism

Adult male rats (Wistar lineage) were alcoholized with sugar cane liquor diluted at 30° GL during 300 days and sacrificed every 60 days in 5 stages. Samples of choroid plexuses of lateral ventricles were collected and examined at transmission electronic microscope to detect possible ultrastructural alterations and to raise possible pathological correlations. Gradual changes were observed in these animals during all the experiment: dilatation and enlargement of cisternae of Golgi complex, dilatation of RER, presence of digestive vacuoles and a large amount of pinocytic vesicles as well as vesicles with electronlucent content throughout cytoplasm, as well as an enlargement of intercellular space between basolateral interdigitation of the cells and of the connective tissue. The changes observed in the epithelium and connective tissue of choroid plexuses specially in 240 and 300 days of treatment are presumably due to a disturbance in hydroelectrolitic homeostasis, contributing to several morpho-functional disturbs of central nervous system. No changes were observed in the control group animals.

Subcutaneous and testicular metastasis from prostatic adenocarcinoma with neuroendocrine differentiation

The pathological finding of testicular metastasis in cases of disseminated prostatic adenocarcinoma is rare, but was more frequently reported in the past, when bilateral castration was performed more often. The existence of skin and subcutaneous metastasis adds a worse prognosis, because generally it is sign of advanced disease with an average survival time of less than one year. The synchronous occurrence of such metastasis has not been described previously, neither their association to neuroendocrine differentiation. The presence of such differentiation of prostatic adenocarcinoma represents a very unfavorable prognostic factor, as suggested in recent literature. Herein, we discuss the case of a 53 year old man, who presented with macroscopic hematuria and frequency associated to several painless subcutaneous nodules in left axilla and shoulder, as well as in the lower abdominal wall. The right testis was painful, endured and on rectal examination, the prostate was diffusely enlarged. Serum PSA was elevated, reaching 1760 ng/ml and prostatic biopsy disclosed a Gleason 10 prostatic adenocarcinoma with neuroendocrine differentiation. The same pathological pattern was detected in the right testis and in all subcutaneous nodules, documented by positive staining of chromogranin, a marker of neuroendocrine cells. He was submitted to a prostate tunnelization and maximal androgen blockade plus adjuvant chemotherapy, nevertheless, he died 5 months latter.

Morphological aspects of rabbit masseter muscle after cervical sympathectomy

The objective of this paper was to study the effect of sympathetic innervation on morphological and histochemical aspects of skeletal muscle tissue. Rabbit masseter muscle was studied using histochemical and immunohistochemical methods for periods of up to 18 months post-sympathectomy. The morphological and enzymatic characteristics of control masseter muscles were similar on both the left and right sides. The main features were muscle fibres with a mosaic pattern and a predominance of type IIa fibres, followed by type I. Type IIb fibres showed very low frequency. Sympathectomized animals showed varying degrees of metabolic and morphological alterations, especially 18 months after sympathectomy. The first five groups showed a higher frequency of type I fibres, whilst the oldest group showed a higher frequency of type lib fibres. In the oldest group, a significant variation in fibre diameter was observed. Many fibres showed small diameter, atrophy, hypertrophy, splitting, and necrosis. Areas with fibrosis were observed. Thus cervical sympathectomy induced morphological alterations in the masseter muscles. These alterations were, in part, similar to both denervation and myopathy. These findings indicate that sympathetic innervation contributes to the maintenance of the morphological and metabolic features of masseter muscle fibres.

Avaliação da população de neurônios mioentéricos NADPH-diaforase positivos do corpo do estômago de ratos com diabetes crônico induzido pela estreptozootocina

The aim of this study was to analyze the morphoquantitative features of the nitrergic myenteric neurons from the body of the stomach of diabetic rats. The body of the stomach of five normoglicemic rats and of five diabetic rats were prepared as whole-mounts stained by the histochemical technique of NADPH-diaphorase. Decreased body weight and increased daily ingestion of water, fast glicemia and glycated hemoglobin were verified in diabetic animals. According to the data obtained, significant difference in the density of nitrergic neurons was not observed between the two groups, but the areas of the neuronal cell body profiles in the diabetic rats were significantly larger. Results showed that the streptozotocin that induced diabetes does not accelerate the death of the nitrergic neurons, but increases the expression of these cells.

Morphometric and quantitative evaluation of the NADH-diaphorase positive myenteric neurons of the jejunum of streptozotocin-diabetic rats supplemented with acetyl-L-carnitine

In this study we investigated the effect of the acetyl-L-carnitine (ALC) supplementation on the myenteric neurons of the jejunum of rats made diabetic at the age of 105 days by streptozotocin (35 mg/kg body weight). Four groups were used: non-diabetic (C), non-diabetic supplemented with ALC (CC), diabetic (D), diabetic supplemented with ALC (DC). After 15 weeks of diabetes induction the blood was collected by cardiac puncture to evaluate glycaemia and glycated haemoglobin. Next the animals were killed and the jejunum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The neuronal counts were made in 80 microscopic fields, in tissue samples of five animals of each group. The profiles of the cell bodies of 1000 neurons per group were analysed. Diabetes induced a significant increase in the area of the cell body and decrease in the number of NADH-diaphorase positive myoenteric neurons. ALC suplementation to the diabetic group promoted smaller hypertrophic effects and less neuronal loss than in the myoenteric neurons of the diabetic rats, and in addition diminished the body weight decrease and reduced the fasting glycaemia. © 2005 Blackwell Verlag.

Inclusion complex of S(-) bupivacaine and 2-hydroxypropyl- β-cyclodextrin: Study of morphology and cytotoxicity

Local anesthetics (LA) belong to a class of pharmacological compounds that attenuate or eliminate pain by binding to the sodium channel of excitable membranes, blocking the influx of sodium ions and the propagation of the nerve impulse. S (-) bupivacaine (S(-) bvc) is a local anesthetic of amino-amide type, widely used in surgery and obstetrics for sustained peripheral and central nerve blockade. This article focuses on the characterization of an inclusion complex of S(-) bvc in 2-hydroxypropyl-β-cyclodextrin (HP-β-CD). Differential scanning calorimetry, scanning electron microscopy and X-Ray diffraction analysis showed structural changes in the complex. In preliminary toxicity studies, the cell viability tests revealed that the inclusion complex decreased the toxic effect (p<0.001) produced by S(-) bvc. These results suggest that the S(-) bvc:HP-β-CD inclusion complex represents a promising agent for the treatment of regional pain.

Cardiovascular responses produced by central injection of hydrogen peroxide in conscious rats

Reactive oxygen species (ROS) have been shown to modulate neuronal synaptic transmission and may play a role on the autonomic control of the cardiovascular system. In this study we investigated the effects produced by hydrogen peroxide (H 2O 2) injected alone or combined with the anti-oxidant agent N-acetil-l-cysteine (NAC) or catalase into the fourth brain ventricle (4th V) on mean arterial pressure and heart rate of conscious rats. Moreover the involvement of the autonomic nervous system on the cardiovascular responses to H 2O 2 into the 4th V was also investigated. Male Holtzman rats (280-320 g) with a stainless steel cannula implanted into the 4th V and polyethylene cannulas inserted into the femoral artery and vein were used. Injections of H 2O 2 (0.5, 1.0 and 1.5 μmol/0.2 μL, n = 6) into the 4th V produced transient (for 10 min) dose-dependent pressor responses. The 1.0 and 1.5 μmol doses of H 2O 2 also produced a long lasting bradycardia (at least 24 h with the high dose of H 2O 2). Prior injection of N-acetyl-l-cysteine (250 nmol/1 μL/rat) into the 4th V blockade the pressor response and attenuated the bradycardic response to H 2O 2 (1 μmol/0.5 μL/rat, n = 7) into the 4th V. Intravenous (i.v.) atropine methyl bromide (1.0 mg/kg, n = 11) abolished the bradycardia but did not affect the pressor response to H 2O 2. Prazosin hydrochloride (1.0 mg/kg, n = 6) i.v. abolished the pressor response but did not affect the bradycardia. The increase in the catalase activity (500 UEA/1 μL/rat injected into the 4th V) also abolished both, pressor and bradycardic responses to H 2O 2. The results suggest that increased ROS availability into 4th V simultaneously activate sympathetic and parasympathetic outflow inducing pressor and bradycardic responses. © 2006 Elsevier Inc. All rights reserved.

Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (T b) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1- yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase T b of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither T b nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the T b response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on T b. Copyright © 2007 Society for Neuroscience.

Evaluation of intracellular calcium in golden retriever muscular dystrophy

The present study had the objective of evaluating calcium accumulations in muscle fibers and their correlation with the canine muscular dystrophy. After the deaths of the animals (13 dystrophic and 3 non-dystrophic), samples of the skeletal muscles were collected. The material was stained with hematoxylin-eosin, Gomori's modified trichrome and alizarin red S technique (pH 4.3). The histopathological changes were analyzed and the proportions of calcium-positive (CPF) and negative muscle fibers were evaluated. Histopathological changes such as muscle fiber diameter changes, necrosis, hyalinization, presence of inflammatory infiltrate and fatty atrophy were identified in all the dystrophic muscles. Statistically significant differences in numbers of CPF between dystrophic muscles and non-dystrophics were observed for the masseter (6%), brachial biceps (5%) and triceps, sartorius and femoral biceps (4%) muscles. The identifying calcium is of interest as a parameter for helping in diagnostic screening.

Subclinical muscle injuries in dogs infected with Leishmania (Leishmania) infantum chagasi

Although canine visceral leishmaniasis (CVL) has been extensively studied, muscular damage due to Leishmania (Leishmania) infantum chagasi infection remains to be fully established. The aim of this study was to describe the electromyographic and histological changes, as well as search for the presence of amastigote forms of Leishmania spp, CD3+ T-lymphocytes, macrophages and IgG in skeletal muscles of dogs with visceral leishmaniasis (VL). Four muscles (triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius) from a total of 17 naturally infected and six healthy dogs were used in this study. Electromyographic alterations such as fibrillation potentials, positive sharp waves and complex repetitive discharges were observed in, at least, three muscles from all infected dogs. Myocyte necrosis and degeneration were the most frequent muscular injury seen, followed by inflammatory reaction, fibrosis and variation in muscle fibers size. Immunohistochemistry in muscle samples revealed amastigote forms in 4/17 (23. 53%), IgG in 12/17 (70. 58%), CD3+ T-lymphocytes in 16/17 (94. 12%) and macrophages in 17/17 (100%) dogs. Statistically positive correlation was observed between: inflammatory infiltrate (p=0. 0305) and CD3+ immunoreaction (p=0. 0307) in relation to the number of amastigote forms; inflammatory infiltrate (p=0. 0101) and macrophage immunoreaction (p=0. 0127) in relation to the amount of CD3+; and inflammatory infiltrate (p=0. 0044) and degeneration/necrosis (p<0. 0001) in relation to the presence of macrophages. Our results suggest that different mechanisms contribute to the development of myocytotoxicity, including celular and humoral immune responses and direct muscular injury by the parasite. Nevertheless, the catabolic nature of the disease can probably interact with other factors, but cannot be incriminated as the only responsible for myositis.

Alterações eletromiográficas e histopatológicas da musculatura de cães naturalmente infectados por Leishmania infantum

The aim of this study was to evaluate the electromyographic and histopathological changes in skeletal muscles of dogs naturally infected by L. infantum. Twenty five mixed breed adult dogs with parasitological, molecular and serological diagnosis were selected. The evaluated muscles were: triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius. One dog had locomotor clinical signs with hind limbs paresis associated with severe muscle atrophy. Twenty-three (92%) had some type of muscular change, and in 22 (88%) such changes were directly identified by electromyography. Even without any clinical signs of the disease, 10 (40%) dogs had electromyographic and histopathological changes. Leishmania antigens were detected in muscles of four (16%) dogs. The electromyographic evaluation indicated the occurrence of chronic polymyositis in 13 (52%) dogs, the presence of both acute and chronic muscle inflammation four (16%), acute myopathy in two (8%) and absence of electromyographic abnormalities in three (12%) dogs. The most frequently observed histopathological changes were degeneration and necrosis of myofibers and inflammatory infiltration observed in 12 (48%) dogs. Other changes were decreased diameter of muscle fibers in 15 (60%) and peri or endomysial fibrosis in 14 (56%) animals. The changes observed in the present study showed that even in the absence of clinical signs, most dogs infected by Leishmania infantum have chronic polymyositis.

Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. © 2012 Elsevier Ltd.

Control of breathing and blood pressure by parafacial neurons in conscious rats

New Findings: • What is the central question of this study? The main purpose of the present manuscript was to investigate the cardiorespiratory responses to hypoxia or hypercapnia in conscious rats submitted to neuronal blockade of the parafacial region. We clearly showed that the integrity of parafacial region is important for the respiratory responses elicited by peripheral and central chemoreflex activation in freely behavior rats. • What is the main finding and its importance? Since the parafacial region is part of the respiratory rhythm generator, they are essential for postnatal survival, which is probably due to their contribution to chemoreception in conscious rats. The retrotrapezoid nucleus (RTN), located in the parafacial region, contains glutamatergic neurons that express the transcriptor factor Phox2b and that are suggested to be central respiratory chemoreceptors. Studies in anaesthetized animals or in vitro have suggested that RTN neurons are important in the control of breathing by influencing respiratory rate, inspiratory amplitude and active expiration. However, the contribution of these neurons to cardiorespiratory control in conscious rats is not clear. Male Holtzman rats (280-300 g, n= 6-8) with bilateral stainless-steel cannulae implanted into the RTN were used. In conscious rats, the microinjection of the ionotropic glutamatergic agonist NMDA (5 pmol in 50 nl) into the RTN increased respiratory frequency (by 42%), tidal volume (by 21%), ventilation (by 68%), peak expiratory flow (by 24%) and mean arterial pressure (MAP, increased by 16 ± 4, versus saline, 3 ± 2 mmHg). Bilateral inhibition of the RTN neurons with the GABAA agonist muscimol (100 pmol in 50 nl) reduced resting ventilation (52 ± 34, versus saline, 250 ± 56 ml min-1 kg-1 with absolute values) and attenuated the respiratory response to hypercapnia and hypoxia. Muscimol injected into the RTN slightly reduced resting MAP (decreased by 13 ± 7, versus saline, increased by 3 ± 2 mmHg), without changing the effects of hypercapnia or hypoxia on MAP and heart rate. The results suggest that RTN neurons activate facilitatory mechanisms important to the control of ventilation in resting, hypoxic or hypercapnic conditions in conscious rats. © 2012 The Authors. Experimental Physiology © 2012 The Physiological Society.

Myenteric denervation in gastric carcinogenesis: Differential modulation of nitric oxide and annexin-A1

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