Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

Dipeptidyl peptidase IV in the hypothalamus and hippocampus of monosodium glutamate obese and food-deprived rats

Proline-specific dipeptidyl peptidases are emerging as a protease family with important roles in the regulation of signaling by peptide hormones related to energy balance. The treatment of neonatal rats with monosodium glutamate (MSG) is known to produce a selective damage on the arcuate nucleus with development of obesity. This study investigates the relationship among dipeptidyl peptidase IV (DPPIV) hydrolyzing activity, CD26 protein, fasting, and MSG model of obesity in 2 areas of the central nervous system. Dipeptidyl peptidase IV and CD26 were, respectively, evaluated by fluorometry, and enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction in soluble (SF) and membrane-bound (MF) fractions from the hypothalamus and hippocampus of MSG-treated and normal rats, submitted or not to food deprivation (FD). Dipeptidyl peptidase IV in both areas was distinguished kinetically as insensitive (DI) and sensitive (DS) to diprotin A. Compared with the controls, MSG and/or FD decreased the activity of DPPIV-DI in the SF and MF from the hypothalamus, as well as the activity of DPPIV-DS in the SF from the hypothalamus and in the MF from the hippocampus. Monosodium glutamate and/or FD increased the activity of DPPIV-DI in the MF from the hippocampus. The monoclonal protein expression of membrane CD26 by enzyme-linked immunosorbent assay decreased in the hypothalamus and increased in the hippocampus of MSG and/or FD relative to the controls. The existence of DPPIV-like activity with different sensitivities to diprotin A and the identity of insensitive with CD26 were demonstrated for the first time in the central nervous system. Data also demonstrated the involvement of DPPIV-DI/CD26 hydrolyzing activity in the energy balance probably through the regulation of neuropeptide Y and beta-endorphin levels in the hypothalamus and hippocampus. (C) 2011 Elsevier Inc. All rights reserved.

Neutral Aminopeptidase and Dipeptidyl Peptidase IV Activities in Plasma of Monosodium Glutamate Obese and Food-deprived Rats

Biometric parameters, glycemia and activity levels of plasma neutral aminopeptidase (APN) and dipeptidyl peptidase IV (DPPIV) were measured in monosodium glutamate obese and food-deprived rats (MSG-FD), to analyze the involvement of these enzymes in such situations. Plasma APN was distinguished as sensitive (PSA) (K(m) = 7.8 x 10(-5) mol/l) and predominantly insensitive (APM) (K(m) = 21.6 x 10(-5) mol/l) to puromycin, whereas DPPIV was sensitive (DPPIV-DS) (K(m) = 0.24 x 10(-5) mol/l) and predominantly insensitive (DPPIV-DI) (K(m) = 7.04 x 10(-5) mol/l) to diprotin A. Although unchanged in the MSG and food-deprived animals, APM activity levels were closely correlated with body mass, Lee index, and mass of retroperitoneal fat pad in the food deprived, but not in the MSG animals. DPPIV-DI activity levels decreased by 33% and were correlated with body mass, Lee index, and mass of periepididymal fat pad in the food-deprived MSG rats. These data suggest that APM and DPPIV-DI are respectively related to the downregulation of somatostatin in food-deprived rats, and to the recovery of energy balance in MSG obese rats during food deprivation.

Fibroelastic Components of the Vesicourethral Junction of Rats in the Aging Process

The vesicourethral junction comprising the vesical trigone, is relevant in setting and positioning of the urinary bladder, along with the vesical neck, fixed by lateral ligaments of the bladder and tendinous arch of the pelvis fascia. Namely, the puboprostatic ligament (men) and the pubovesical (women). The circular set elastic fibers in this junction are important and valuable in the elasticity and plasticity of the area, allowing quick expansion and withdrawal with the flow of urine, and associated to smooth muscle tissue and nerve control form an important collective to maintain urinary continence. The objective of the present study is to describe the elastic system in the vesicouretral junction in relation to aging and its involvement in the states of urinary continence and incontinence, as well as the study of the vesicouretral junction in various age groups while evaluating with electron transmission microscopy. To carry out the study, 12 Wistar rats were used, divided into groups: neonate (4 animals), adult group (4 animals) and aged group (4 animals). Electron transmission microscopy with use of tanic acid technique associated to glutaraldehyde fixation, satisfactorily showed the extreme structural differences between mature elaunin and oxytalan fibers present between intercelular spaces and bundles of collagen fibers. The phases of elastogenesis in neonate animals and degradation of the elastic system of older animals were also evaluated.

APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (it) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD. FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity. (c) 2010 Elsevier B.V. All rights reserved.

Short- and long-term anxiogenic effects induced by a single injection of subconvulsant doses of pilocarpine in rats: investigation of the putative role of hippocampal pathways

Behavioral consequences of convulsive episodes are well documented, but less attention was paid to changes that occur in response to subconvulsant doses of drugs. We investigated short- and long-term effects of a single systemic injection of a subconvulsant dose of pilocarpine on the behavior of rats as evaluated in the elevated plus maze. Pilocarpine induced an anxiogenic-like profile 24 h later, and this effect persisted for up to 3 months (% of time spent on open arms at 24 h, control = 35.47 +/- 3.23; pilocarpine 150 = 8.2 +/- 2.6; 3 months, control = 31.9 +/- 5.5; pilocarpine 150 = 9.3 +/- 4.9). Temporary inactivation of fimbria-fornix with lidocaine 4% promoted an anxiolytic-like effect per se, suggesting a tonic control of this pathway on the modulation of anxiety-related behaviors. Lidocaine also reduced the anxiogenic-like profile of animals tested 1 month after pilocarpine treatment (% of time spent on open arms, saline + phosphate-buffered saline (PBS) = 31.7 + 3.7; saline + lidocaine = 54.4 + 4.7; pilocarpine + PBS = 10.3 + 4.1; pilocarpine + lidocaine = 40.1 + 9.1). To determine whether the anxiogenic-like effect was mediated by septal region or by direct hippocampal projections to the diencephalon, the neural transmission of post-commissural fornix was blocked, and a similar reduction in the anxiogenic-like effect of pilocarpine was observed. Our findings suggest that a single systemic injection of pilocarpine may induce long-lasting anxiogenic-like behavior in rats, an effect that appears to be mediated, in part, through a direct path from hippocampus to medial hypothalamic sites involved in fear responses.

Postnatal fluoxetine treatment affects the development of serotonergic neurons in rats

The goal of the present study was to investigate morphological changes in the serotonergic neurons/terminals in the dorsal (DR) and median (MnR) raphe nuclei and on the hippocampal dentate gyrus (DG) in neonatal rats treated from the 1st to the 21st postnatal day with fluoxetine (10 mg/kg sc, daily) or drug vehicle (0 9% saline 1 ml/kg). The results show that postnatal chronic treatment with fluoxetine promoted. (1) a smaller body weight increase during the pre-weaning period; (2) smaller number of 5-HT neurons in the DR, (3) smaller 5-HT neuronal cell bodies (area, perimeter and diameter) in the DR and the MnR and (4) diminished serotonergic terminals in the DG. These data suggest that the development of the serotonergic system was impaired and that early exposure to fluoxetine damaged the morphology of 5-HT neurons in young adult rats While these findings are consistent with other work, more studies are needed to better clarify the effects of postnatal chronic treatment with fluoxetine on the serotonergic system and, consequently, on the functions modulated by serotonin (C) 2010 Elsevier Ireland Ltd All rights reserved

L-NAME-treatment alters ectonucleotidase activities in kidney membranes of rats

Aims: To investigate the effect of N omega-Nitro-L-arginine methyl ester CL-NAME) treatment, known to induce a sustained elevation of blood pressure, on ectonucleotidase activities in kidney membranes of rats. Main methods: L-NAME (30 mg/kg/day) was administered to Wistar rats for 14 days in the drinking water. Enzyme activities were determined colorimetrically and their gene expression patterns were analyzed by semi-quantitative RT-PCR. The metabolism of ATP and the accumulation of adenosine were evaluated by HPLC in kidney membranes from control and hypertensive rats. PKC phosphorylation state was investigated by Western blot. Key findings: We observed an increase in systolic blood pressure from 115 +/- 12 mmHg (control group) to 152 18 mmHg (L-NAME-treated group). Furthermore, the hydrolysis of ATP, ADP, AMP, and p-Nph-5`TMP was also increased (17%, 35%, 27%, 20%, respectively) as was the gene expression of NTPDase2, NTPDase3 and NPP3 in kidneys of hypertensive animals. Phospho-PKC was increased in hypertensive rats. Significance: The general increase in ATP hydrolysis and in ecto-5`-nucleotidase activity suggests a rise in renal adenosine levels and in renal autoregulatory responses in order to protect the kidney against the threat presented by hypertension. (C) 2010 Elsevier Inc. All rights reserved.

Morphine treatment during pregnancy modulates behavioral selection in lactating rats

Previous studies have demonstrated that treatment of postpartum female rats with morphine inhibits maternal behavior and stimulates foraging. Exposure to drugs of abuse may result in a progressive enhancement of their reinforcing effects. Puerperal treatment with morphine leads to reverse tolerance to this drug. The present study investigated whether repeated morphine treatment during late pregnancy may influence the effects of different morphine dosages on behavioral selection in lactating rats. Females were simultaneously exposed to pups and insects, and the choice between taking care of the pups and hunting insects was observed. Female Wistar rats were treated with morphine (3.5 mg/kg/day, subcutaneous [s.c.]) or saline for 5 days beginning on pregnancy day 17. On day 5 of lactation, animals were acutely challenged with morphine (0.5, 1.0, or 1.5 mg/kg, s.c.; MM0.5, MM1.0, and MM1.5 groups, respectively) or saline (MS group) and tested for predatory hunting and maternal behavior. Control groups were pretreated with saline and challenged with morphine (SM0.5, SM1.0, and SM1.5 groups) or saline (SS group). Animals treated with morphine during late pregnancy and acutely challenged with 1.0 mg/kg morphine (MM1.0 group) exhibited significantly decreased maternal behavior and enhanced hunting. This effect was not evident with the 0.5 mg/kg dose. The 1.5 mg/kg morphine dose decreased maternal behavior and increased hunting in both the MM1.5 group and in animals challenged with morphine after previous saline treatment (SM1.5 group). These results provide evidence of plasticity of the opioidergic role in behavioral selection during lactation. (C) 2010 Elsevier Inc. All rights reserved.

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H(2)O(2)) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3 beta/GSK-3 beta ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H(2)O(2) (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3 beta phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H(2)O(2) levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.

Prolonged consumption of soy or fish-oil-enriched diets differentially affects the pattern of hypothalamic neuronal activation induced by refeeding in rats

We used c-Fos immunoreactivity to estimate neuronal activation in hypothalamic feeding-regulatory areas of 3-month-old rats fed control or oil-enriched diets (soy or fish) since weaning. While no diet effect was observed in c-Fos immunoreactivity of 24-h fasted animals, the acute response to refeeding was modified by both hyperlipidic diets but with different patterns. Upon refeeding, control-diet rats had significantly increased c-Fos immunoreactivity only in the paraventricular hypothalamic nucleus (PVH, 142%). In soy-diet rats, refeeding with the soy diet increased c-Fos immunoreactivity in dorsomedial hypothalamic nucleus (DMH, 271%) and lateral hypothalamic area (LH, 303%). Refeeding fish-diet rats with the fish diet increased c-Fos immunoreactivity in PVH (161%), DMH (177%), VMH (81%), and ARC (127%). Compared to the fish-diet, c-Fos immunoreactivity was increased in LH by the soy-diet while it was decreased in ventromedial hypothalamic nucleus (VMH) and arcuate hypothalamic nucleus (ARC). Based on the known roles of the activated nuclei, it is suggested that, unlike the fish-diet, the soy-diet induced a potentially obesogenic profile, with high LH and low VMH/PVH activation after refeeding.

Forebrain projections to brainstem nuclei involved in the control of mandibular movements in rats

Mandibular movements occur through the triggering of trigeminal motoneurons. Aberrant movements by orofacial muscles are characteristic of orofacial motor disorders, such as nocturnal bruxism (clenching or grinding of the dentition during sleep). Previous studies have suggested that autonomic changes occur during bruxism episodes. Although it is known that emotional responses increase jaw movement, the brain pathways linking forebrain limbic nuclei and the trigeminal motor nucleus remain unclear. Here we show that neurons in the lateral hypothalamic area, in the central nucleus of the amygdala, and in the parasubthalamic nucleus, project to the trigeminal motor nucleus or to reticular regions around the motor nucleus (Regio h) and in the mesencephalic trigeminal nucleus. We observed orexin co-expression in neurons projecting from the lateral hypothalamic area to the trigeminal motor nucleus. In the central nucleus of the amygdala, neurons projecting to the trigeminal motor nucleus are innervated by corticotrophin-releasing factor immunoreactive fibers. We also observed that the mesencephalic trigeminal nucleus receives dense innervation from orexin and corticotrophin-releasing factor immunoreactive fibers. Therefore, forebrain nuclei related to autonomic control and stress responses might influence the activity of trigeminal motor neurons and consequently play a role in the physiopathology of nocturnal bruxism.

Role of spinal microglia in myositis-induced central sensitisation: An immunohistochemical and behavioural study in rats

There is increasing evidence that spinal glial cells play an important role in chronic pain states. However, so far no data on the role of microglia in muscle pain are available. The aim of the present study was to investigate the involvement of spinal microglial cells in chronic muscle pain. In a rat model of chronic muscle inflammation (injection of complete Freunds adjuvant into the gastrocnemius-soleus muscle) alterations of microglia were visualized with quantitative OX-42 immunohistochemistry in the dorsal horn of the segments L4 and L5 12 days after induction of inflammation. In behavioural experiments the influence of chronic intrathecally applied minocycline - a specific microglia inhibitor - or an antibody against tumour necrosis factor-alpha (TNF-alpha: a cytokine released from microglia) on pain-related behaviour was investigated after 1, 3, 6, and 12 days. The immunhistochemical data show that in the deep laminae of the spinal dorsal horn microglial cells reacted with morphological changes to the muscle inflammation. Following inflammation, the mean boundary length surrounding the OX-42 immunostained area was significantly shorter. This indicates that microglial cells were activated by the myositis and withdrew their processes. Chronic intrathecal administration of minocycline or anti TNF-alpha with an osmotic mini-pump largely normalised the inflammation-induced changes in spontaneous exploratory behaviour and attenuated the hypersensitivity to mechanical stimulation. Both the immunohistochemical and behavioural data show that spinal microglial cells are involved in nociceptive processes in the cause of a chronic muscle inflammation. (C) 2008 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

Ectonucleotidase activities are altered in serum and platelets of L-NAME-treated rats

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects: thus, ADP is the most important platelet agonist and recruiting agent, while adenosine, all end product Of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5`-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonuclectidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days Plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In Serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis Was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%, respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation. (C) 2008 Elsevier Inc. All rights reserved.

Effects of exercise on the morphology of the myenteric neurons of the duodenum of Wistar rats during the ageing process

This study aimed to evaluate the effects of regular physical activity on the morphology of the myenteric plexus of the duodenum in rats during the ageing process. To this end, 45 Wistar rats were divided into three groups: C (sedentary - 6 months old), S (sedentary - 12 months old) and T (trained - 12 months old). The animals of group S were given with a physical activity programme consisting of a 10-min-treadmill workout once a week. The animals of group T were submitted to the physical activity programme five times a week. Their duodenums were collected and submitted to the techniques of nicotinamide adenine dinucleotide (NADH)-diaphorase enzyme histochemistry for whole-mount preparations and transmission electron microscopy. No differences in the constitution of the myenteric plexuses were found when the sedentary and trained groups were compared with the control group. The ultrastructural features were similar for the three groups. However, it was verified that the physical activity of the trained animals resulted in a similar myenteric neuron morphology to that of the adult animals (6 months old), thereby confirming its beneficial effect, as the sedentary animals had larger alterations in the collagen fibrils and the basal membrane that occur through ageing. The quantitative analysis showed that the NADH-diaphorase positive neurons decreased with ageing and increased with physical activity (P > 0.05). No significant alteration (P > 0.05) in the neuronal profile area of the NADH-diaphorase positive neurons has been observed with ageing.