Involvement of the caudal raphe nuclei in the feeding behavior of rats

Involvement of the caudal raphe nuclei (raphe pallidus, RPa; raphe magnus, RMg, and raphe obscurus, ROb) in feeding behavior of adult rats was studied by measuring c-Fos protein expression, in animals submitted to the "meal-feeding" model of food restriction in which the rats were fed ad libitum only from 7:00 to 9:00 h, for 15 days. The experimental groups submitted to chronic fasting, named 'search for food' (SF), 'ingestion of food' (IF) and 'satiety of food' (SaF) were scheduled after a previous study in which the body weight and the general and feeding behaviors were evaluated by daily monitoring. Acute, 48-h fasting (AF) was used as control. In the chronic group, the animals presented a 16% reduction in body weight in the first week, followed by a continuous, slow rise in weight over the subsequent days. Entrainment of the sleep-wake cycle to the schedule of food presentation was also observed. The RPa was the most Fos immunopositive nucleus in the chronic fasting group, followed by the RMg. The ANOVA and Tukey test (P<0.05) confirmed these results. The IF group was significantly different from the other three groups, as also was the number of labeled cells in the RPa in SF and IF groups. Nevertheless, no significant difference was observed between RMg and RPa, or RMg and ROb in the SaF and AF. However, it is interesting to observe that the groups in which the animals were more active, searching for or ingesting food, presented a larger number of labeled cells. These results suggest a different involvement of the caudal raphe nuclei in the somatic and autonomic events of feeding behavior, corroborating the functions reported for them earlier.

Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation.

Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

The cardiopulmonary reflexes of spontaneously hypertensive rats are normalized after regression of left ventricular hypertrophy and hypertension

Cardiopulmonary reflexes are activated via changes in cardiac filling pressure (volume-sensitive reflex) and chemical stimulation (chemosensitive reflex). The sensitivity of the cardiopulmonary reflexes to these stimuli is impaired in the spontaneously hypertensive rat (SHR) and other models of hypertension and is thought to be associated with cardiac hypertrophy. The present study investigated whether the sensitivity of the cardiopulmonary reflexes in SHR is restored when cardiac hypertrophy and hypertension are reduced by enalapril treatment. Untreated SHR and WKY rats were fed a normal diet. Another groups of rats were treated with enalapril (10 mg kg-1 day-1, mixed in the diet; SHRE or WKYE) for one month. After treatment, the volume-sensitive reflex was evaluated in each group by determining the decrease in magnitude of the efferent renal sympathetic nerve activity (RSNA) produced by acute isotonic saline volume expansion. Chemoreflex sensitivity was evaluated by examining the bradycardia response elicited by phenyldiguanide administration. Cardiac hypertrophy was determined from the left ventricular/body weight (LV/BW) ratio. Volume expansion produced an attenuated renal sympathoinhibitory response in SHR as compared to WKY rats. As compared to the levels observed in normotensive WKY rats, however, enalapril treatment restored the volume expansion-induced decrease in RSNA in SHRE. SHR with established hypertension had a higher LV/BW ratio (45%) as compared to normotensive WKY rats. With enalapril treatment, the LV/BW ratio was reduced to 19% in SHRE. Finally, the reflex-induced bradycardia response produced by phenyldiguanide was significantly attenuated in SHR compared to WKY rats. Unlike the effects on the volume reflex, the sensitivity of the cardiac chemosensitive reflex to phenyldiguanide was not restored by enalapril treatment in SHRE. Taken together, these results indicate that the impairment of the volume-sensitive, but not the chemosensitive, reflex can be restored by treatment of SHR with enalapril. It is possible that by augmenting the gain of the volume-sensitive reflex control of RSNA, enalapril contributed to the reversal of cardiac hypertrophy and normalization of arterial blood pressure in SHR.

Effects of exercise training on autonomic and myocardial dysfunction in streptozotocin-diabetic rats

Several investigators have demonstrated that diabetes is associated with autonomic and myocardial dysfunction. Exercise training is an efficient non-pharmacological treatment for cardiac and metabolic diseases. The aim of the present study was to investigate the effects of exercise training on hemodynamic and autonomic diabetic dysfunction. After 1 week of diabetes induction (streptozotocin, 50 mg/kg, iv), male Wistar rats (222 ± 5 g, N = 18) were submitted to exercise training for 10 weeks on a treadmill. Arterial pressure signals were obtained and processed with a data acquisition system. Autonomic function and intrinsic heart rate were studied by injecting methylatropine and propranolol. Left ventricular function was assessed in hearts perfused in vitro by the Langendorff technique. Diabetes (D) bradycardia and hypotension (D: 279 ± 9 bpm and 91 ± 4 mmHg vs 315 ± 11 bpm and 111 ± 4 mmHg in controls, C) were attenuated by training (TD: 305 ± 7 bpm and 100 ± 4 mmHg). Vagal tonus was decreased in the diabetic groups and sympathetic tonus was similar in all animals. Intrinsic heart rate was lower in D (284 ± 11 bpm) compared to C and TD (390 ± 8 and 342 ± 14 bpm, respectively). Peak systolic pressure developed at different pressures was similar for all groups, but +dP/dt max was decreased and -dP/dt max was increased in D. In conclusion, exercise training reversed hypotension and bradycardia and improved myocardial function in diabetic rats. These changes represent an adaptive response to the demands of training, supporting a positive role of physical activity in the management of diabetes.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and ß-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the ß-receptor second messenger) were not affected by IIH.

The hemolytic component of cancer anemia: effects of osmotic and metabolic stress on the erythrocytes of rats bearing multifocal inoculations of the Walker 256 tumor

Cancer anemia is classified as an anemia of chronic diseases, although it is sometimes the first symptom of cancer. Cancer anemia includes a hemolytic component, important in the terminal stage when even transfused cells are rapidly destroyed. The presence of a chronic component and the terminal complications of the illness limit studies of the hemolytic component. A multifocal model of tumor growth was used here to simulate the terminal metastatic dissemination stage (several simultaneous inoculations of Walker 256 cells). The hemolytic component of anemia began 3-4 days after inoculation in 100% of the rats and progressed rapidly thereafter: Hb levels dropped from 14.9 ± 0.02 to 8.7 ± 0.06 from days 7 to 11 (~5 times the physiologically normal rate in rats) in the absence of bleeding. The development of anemia was correlated (r2 = 0.86) with the development of other systemic effects such as anorexia. There was a significant decrease in the osmotic fragility of circulating erythrocytes: the NaCl concentration causing 50% lysis was reduced from 4.52 ± 0.06 to 4.10 ± 0.01 (P<0.01) on day 7, indicating a reduction in erythrocyte volume. However, with mild metabolic stress (4-h incubation at 37oC), the erythrocytes showed a greater increase in osmotic fragility than the controls, suggesting marked alteration of erythrocyte homeostasis. These effects may be due to primary plasma membrane alterations (transport and/or permeability) and/or may be secondary to metabolic changes. This multifocal model is adequate for studying the hemolytic component of cancer anemia since it is rapid, highly reproducible and causes minimal animal suffering.

Dietary sodium intake induced myenteric neuron hypertrophy in Wistar rats

In the present study we investigated the effect of salt intake on myenteric neuron size of the colon of adult male Wistar rats. The animals were placed on either a high-salt (HS; 8%; 12 animals) or a low-salt diet (LS; 0.15%; 12 animals) for 15 or 52 weeks and blood pressure was measured. The sizes of myenteric neurons of the distal colon from both groups were measured. No difference in neuron size was observed between the HS and LS groups after 15 weeks. After 52 weeks on HS, neuron size was increased (P<0.005) when compared with the LS group. The rats also presented hypertension, which was significantly different at 52 weeks (142 ± 11 vs 119 ± 7 mmHg). These results suggest that a long time on an HS diet can significantly increase myenteric nerve cell size.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

Chronic intrathecal cannulation enhances nociceptive responses in rats

The influence of a chronically implanted spinal cannula on the nociceptive response induced by mechanical, chemical or thermal stimuli was evaluated. The hyperalgesia in response to mechanical stimulation induced by carrageenin or prostaglandin E2 (PGE2) was significantly increased in cannulated (Cn) rats, compared with naive (Nv) or sham-operated (Sh) rats. Only Cn animals presented an enhanced nociceptive response in the first phase of the formalin test when low doses were used (0.3 and 1%). The withdrawal latency to thermal stimulation of a paw inflamed by carrageenin was significantly reduced in Cn rats but not in Nv or Sh rats. In contrast to Nv and Sh rats, injection in Cn animals of a standard non-steroid anti-inflammatory drug, indomethacin, either intraperitoneally or into the spinal cord via an implanted cannula or by direct puncture of the intrathecal space significantly blocked the intensity of the hyperalgesia induced by PGE2. Cannulated animals treated with indomethacin also showed a significant inhibition of second phase formalin-induced paw flinches. Histopathological analysis of the spinal cord showed an increased frequency of mononuclear inflammatory cells in the Cn groups. Thus, the presence of a chronically implanted cannula seems to cause nociceptive spinal sensitization to mechanical, chemical and thermal stimulation, which can be blocked by indomethacin, thus suggesting that it may result from the spinal release of prostaglandins due to an ongoing mild inflammation.

Anti-hyperalgesic effect of electroacupuncture in a model of post-incisional pain in rats

Electroacupuncture has been proposed to be a low cost and practical method that allows effective pain management with minimal collateral effects. In this study we have examined the effect of electroacupuncture against the hyperalgesia developed in a model of post-incisional pain in rats. A 1-cm longitudinal incision was made through the skin and fascia of the plantar region of the animal hind paw. Mechanical hyperalgesia in the incision was evaluated 135 min after the surgery with von Frey filaments. The tension threshold was reduced from 75 g (upper limit of the test) to 1.36 ± 0.36 g (mean ± SEM) in control rats. It is shown that a 15-min period of electroacupuncture applied 120 min after surgery to the Zusanli (ST36) and Sanyinjiao (SP6) points, but not to non-acupoints, produces a significant and long-lasting reduction of the mechanical hyperalgesia induced by the surgical incision of the plantar surface of the ipsilateral hind paw. The tension threshold was reduced from 75 to 27.6 ± 4.2 g in animals soon after the end of electroacupuncture. The mechanical threshold in this group was about 64% less than in control. Electroacupuncture was ineffective in rats treated 10 min earlier with naloxone (1 mg/kg, ip), thus confirming the involvement of opioid mechanisms in the antinociceptive effects of such procedure. The results indicate that post-incisional pain is a useful model for studying the anti-hyperalgesic properties of electroacupuncture in laboratory animals.

Effects of adrenalectomy and adrenal enucleation on liquid gastric emptying in rats

The effects of adrenalectomy and adrenal enucleation on liquid gastric emptying were studied in male Wistar rats that were adrenalectomized, adrenal enucleated (AE) or sham operated (SH). The animals in the first group had free access to a 1% NaCl solution (ADS), while the animals in the second and third groups were divided into two subgroups, which ingested either tap water (AEW, SHW) or 1% NaCl solution (AES, SHS). The gastric emptying study was performed on the 16th post-operative day. Three test meals labeled with phenol red (6 mg/dl) were used (0.9% NaCl, 1.8% NaCl and 5% glucose). Percent gastric retention was determined 10 min after orogastric infusion of the NaCl test meals and 15 min after the glucose meal. Gastric retention of the ADS subgroup was significantly lower (P<0.01) (median = 19.8% vs 25.5% for SHW, vs 31.9% for SHS, vs 25.7% for AEW, and vs 27.1% for AES) for the 0.9% NaCl test meal and for the 1.8% NaCl test meal (33.5% for ADS vs 47.5% for AEW and 50.6% for AES). When 5% glucose was used as a test meal, gastric retention was similar for all subgroups. These results suggest that ablation of the adrenal cortex results in increased gastric emptying of an isosmolar NaCl meal.

Inhibition of gastric emptying and intestinal transit in anesthetized rats by a Tityus serrulatus scorpion toxin

The effects of a fraction (T1) of Tityus serrulatus scorpion venom prepared by gel filtration on gastric emptying and small intestinal transit were investigated in male Wistar rats. Fasted animals were anesthetized with urethane, submitted to tracheal intubation and right jugular vein cannulation. Scorpion toxin (250 µg/kg) or saline was injected iv and 1 h later a bolus of saline (1.0 ml/100 g) labeled with 99m technetium-phytate (10 MBq) was administered by gavage. After 15 min, animals were sacrificed and the radioactivity remaining in the stomach was determined. Intestinal transit was evaluated by instillation of a technetium-labeled saline bolus (1.0 ml) through a cannula previously implanted in the duodenum. After 60 min, the progression of the marker throughout 7 consecutive gut segments was estimated by the geometric center method. Gastric retention of the liquid test meal in rats injected with scorpion toxin (median: 88%; range: 52-95%) was significantly higher (P<0.02) than in controls (54%; 21-76%), an effect which was not modified by gastric secretion blockade with ranitidine. The progression of the isotope marker throughout the small intestine was significantly slower (P<0.05) in rats treated with toxin (1.2; 1.0-2.5) than in control animals (2.3; 1.0-3.2). Inhibition of both gastric emptying and intestinal transit in rats injected with scorpion toxin suggests an increased resistance to aboral flow, which might be caused by abnormal neurotransmitter release or by the local effects of venom on smooth muscle cells.

Pup age and aggressive behavior in lactating rats

High levels of aggressive behaviors against intruders in the nest area are displayed by female rats during the first 10 days after delivery, declining thereafter to very low levels, even though lactation continues. Cross-fostering experiments were undertaken to test the hypothesis that pup age may affect aggression in lactating rats. The behavior of females on the 8th day after delivery when raising fostered 8-day-old pups was compared to that of females on the 8th postpartum day raising older pups (18 days old) for the last 5 days, and females on the 18th day after delivery raising fostered 18-day-old pups were compared to females in the same postpartum period nursing younger pups (8 days of age at the time of the maternal aggression test) for 5 days. Pup retrieval activity and plasma prolactin level were also analyzed. Females on the 8th postpartum day nursing 18-day-old pups were less aggressive than females in the same postpartum period, but with 8-day-old pups. Likewise, females on the 18th postpartum day nursing younger pups were more aggressive and presented higher levels of prolactin than females nursing older pups. Thus, pup development can alter the natural decline of maternal aggressive behavior.