Evaluation of two experimental models of hepatic encephalopathy in rats

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Effect of an isotonic rehydration sports drink and exercise on urolithiasis in rats

The objective of the present study was to evaluate the role of physical exercise as well as the influence of hydration with an isotonic sports drink on renal function in male Wistar rats. Four groups were studied over a period of 42 days: 1) control (N = 9); 2) physical exercise (Exe, N = 7); 3) isotonic drink (Drink, N = 8); 4) physical exercise + isotonic drink (Exe + Drink, N = 8). Physical exercise consisted of running on a motor-driven treadmill for 1 h/day, at 20 m/min, 5 days a week. The isotonic sports drink was a commercial solution used by athletes for rehydration after physical activity, 2 ml administered by gavage twice a day. Urine cultures were performed in all animals. Twenty-four-hour urine samples were collected in metabolic cages at the beginning and at the end of the protocol period. Urinary and plasma parameters (sodium, potassium, urea, creatinine, calcium) did not differ among groups. However, an amorphous material was observed in the bladders of animals in the Exe + Drink and Drink groups. Characterization of the material by Western blot revealed the presence of Tamm-Horsfall protein and angiotensin converting enzyme. Physical exercise and the isotonic drink did not change the plasma or urinary parameters measured. However, the isotonic drink induced the formation of intravesical matrix, suggesting a potential lithogenic risk.

Maternal aggression in Wistar rats: effect of 5-HT2A/2C receptor agonist and antagonist microinjected into the dorsal periaqueductal gray matter and medial septum

The objective of the present study was to assess the role of the 5-HT2A/2C receptor at two specific brain sites, i.e., the dorsal periaqueductal gray matter (DPAG) and the medial septal (MS) area, in maternal aggressive behavior after the microinjection of either a 5-HT2A/2C receptor agonist or antagonist. Female Wistar rats were microinjected on the 7th postpartum day with the selective agonist alpha-methyl-5-hydroxytryptamine maleate (5-HT2A/2C) or the antagonist 5-HT2A/2C, ketanserin. The agonist was injected into the DPAG at 0.2 (N = 9), 0.5 (N = 10), and 1.0 µg/0.2 µl (N = 9), and the antagonist was injected at 1.0 µg/0.2 µl (N = 9). The agonist was injected into the medial septal area (MS) at 0.2 (N = 9), 0.5 (N = 7), and 1.0 µg/0.2 µl (N = 6) and the antagonist was injected at 1.0 µg/0.2 µl (N = 5). For the control, saline was injected into the DPAG (N = 7) and the MS (N = 12). Both areas are related to aggressive behavior and contain a high density of 5-HT receptors. Non-aggressive behaviors such as horizontal locomotion (walking) and social investigation and aggressive behaviors such as lateral threat (aggressive posture), attacks (frontal and lateral), and biting the intruder were analyzed when a male intruder was placed into the female resident's cage. For each brain area studied, the frequency of the behaviors was compared among the various treatments by analysis of variance. The results showed a decrease in maternal aggressive behavior (number of bites directed at the intruder) after microinjection of the agonist at 0.2 and 1.0 µg/0.2 µl (1.6 ± 0.7 and 0.9 ± 0.3) into the DPAG compared to the saline group (5.5 ± 1.1). There was no dose-response relationship with the agonist. The present findings suggest that the 5-HT2A/2C receptor agonist has an inhibitory effect on maternal aggressive behavior when microinjected into the DPAG and no effect when microinjected into the MS. Ketanserin (1.0 µg/0.2 µl) decreased locomotion when microinjected into the DPAG and MS, but did not affect aggressive behavior. We interpret these findings as evidence for a specific role of 5-HT2A/2C receptors in the DPAG in the inhibition of female aggressive behavior, dissociated from those on motor activity.

Preoperative cobalt60 irradiation delays the healing of rectal anastomoses in rats

The healing of colorectal anastomoses after irradiation therapy continues to be a major concern. The authors evaluated the healing of rectal anastomoses in a rat model after a preoperative 500-cGy dose of cobalt60 irradiation. Thirty-six male Wistar rats were divided into two equal groups: control (group A), and irradiation group (group B). Group B received a single 500-cGy dose of irradiation, and a rectal resection and end-to-end anastomosis was performed in both groups on the 7th day after irradiation. Parameters of the healing process included bursting pressure and collagen content on the 5th, 7th, and 14th days after surgery. In the irradiation group, the mean bursting pressure on the 5th, 7th, and 14th days was 116, 218, and 273 mmHg, respectively. The collagen content assessed by histomorphometry was 9.0, 20.8, and 32%, respectively. In contrast, the control group had a mean bursting pressure of 175, 225 and 263 mmHg, and a collagen content of 17.8, 28.1, and 32.1%, respectively. The adverse effect of irradiation on healing was detectable only on the 5th postoperative day, as demonstrated by lower bursting pressure (P < 0.013) and collagen content (P < 0.008). However, there was no failure of anastomotic healing such as leakage or dehiscence due to irradiation. We conclude that a single preoperative 500-cGy dose of irradiation delays the healing of rectal anastomosis in rats.

Effect of D-alpha-tocopherol on tubular nephron acidification by rats with induced diabetes mellitus

The objective of the present study was to determine if treatment of diabetic rats with D-alpha-tocopherol could prevent the changes in glomerular and tubular function commonly observed in this disease. Sixty male Wistar rats divided into four groups were studied: control (C), control treated with D-alpha-tocopherol (C + T), diabetic (D), and diabetic treated with D-alpha-tocopherol (D + T). Treatment with D-alpha-tocopherol (40 mg/kg every other day, ip) was started three days after diabetes induction with streptozotocin (60 mg/kg, ip). Renal function studies and microperfusion measurements were performed 30 days after diabetes induction and the kidneys were removed for morphometric analyses. Data are reported as means ± SEM. Glomerular filtration rate increased in D rats but decreased in D + T rats (C: 6.43 ± 0.21; D: 7.74 ± 0.45; D + T: 3.86 ± 0.18 ml min-1 kg-1). Alterations of tubular acidification observed in bicarbonate absorption flux (JHCO3) and in acidification half-time (t/2) in group D were reversed in group D + T (JHCO3, C: 2.30 ± 0.10; D: 3.28 ± 0.22; D + T: 1.87 ± 0.08 nmol cm-2 s-1; t/2, C: 4.75 ± 0.20; D: 3.52 ± 0.15; D + T: 5.92 ± 0.19 s). Glomerular area was significantly increased in D, while D + T rats exhibited values similar to C, suggesting that the vitamin prevented the hypertrophic effect of hyperglycemia (C: 8334.21 ± 112.05; D: 10,217.55 ± 100.66; D + T: 8478.21 ± 119.81µm²). These results suggest that D-alpha-tocopherol is able to protect rats, at least in part, from the harmful effects of diabetes on renal function.

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 µg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

Routine post-weaning handling of rats prevents isolation rearing-induced deficit in prepulse inhibition

Rats reared under isolation conditions from weaning present a number of behavioral changes compared to animals reared under social conditions (group housing). These changes include deficits in prepulse inhibition (PPI) of the startle reflex to a loud sound. PPI refers to the reduction of the magnitude of the startle reflex when a relatively weak stimulus (the prepulse) precedes by an appropriate time interval the intense startle-elicing stimulus (the pulse). PPI is useful for studying sensorimotor integration. The present study evaluated the effect of handling on the impairment of PPI induced by isolation-rearing. Male Wistar rats (N = 11-15/group) were housed in groups (5 per cage and handled three times a week) or isolated (housed individually) since weaning (21 days) for 10 weeks when they reach approximately 150 g. The isolated rats were divided into "minimally handled" animals (handled once a week for cleaning purposes only) or "handled" animals (handled three times a week). This handling consisted of grasping the rat by the tail and moving it to a clean cage (approximately 5 s). A statistically significant reduction (52%) in the PPI test was found only in the isolated group with minimal handling while no difference was seen between grouped animals and isolated handled animals. These results indicate that isolation rearing causes disruption in the PPI at adult age, which serves as an index of attention deficit. This change in the sensory processing of information induced by post-weaning isolation can be prevented by handling during the development of the animal.

Delayed Schwann cell and oligodendrocyte remyelination after ethidium bromide injection in the brainstem of Wistar rats submitted to streptozotocin diabetogenic treatment

Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. Since Schwann cell and oligodendrocyte remyelination in the central nervous system is a well-known event in the ethidium bromide (EB) demyelinating model, the aim of this investigation was to determine the behavior of both cell types after local EB injection into the brainstem of streptozotocin diabetic rats. Adult male Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 µL 0.1% (w/v) EB or 0.9% saline solution into the cisterna pontis. Ten microliters of 0.1% EB was also injected into non-diabetic rats. The animals were anesthetized and perfused through the heart 7 to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy. The final balance of myelin repair in diabetic and non-diabetic rats at 31 days was compared using a semi-quantitative method. Diabetic rats presented delayed macrophage activity and lesser remyelination compared to non-diabetic rats. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, first appearing at 11 days in non-diabetic rats and by 15 days in diabetic rats. Results indicate that short-term streptozotocin-induced diabetes hindered both oligodendrocyte and Schwann cell remyelination (mean remyelination scores of 2.57 ± 0.77 for oligodendrocytes and 0.67 ± 0.5 for Schwann cells) compared to non-diabetic rats (3.27 ± 0.85 and 1.38 ± 0.81, respectively).

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4ºC, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(³H-methyl)-methionine was used. The O-methylated derivatives were oxidized to ³H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18%) and hippocampal dopamine (about 20%) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39%) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20%) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

Effect of antipyrine on the gastric emptying of liquid in rats

Antipyrine (At) and dipyrone (Dp) delay gastric emptying (GE) in rats. The objective of the present study was to assess the effects of intravenous (iv) and intracerebroventricular (icv) administration of At and Dp on the GE of liquid by rats. GE was assessed in male Wistar rats (5-10 in each group) 10 min after the icv or iv drug injection by measuring percent gastric retention (%GR) of a saline test meal labeled with phenol red 10 min after administration by gavage. The At iv group was significantly higher (64.4 ± 2.6%) compared to control (33.4 ± 1.5%) but did not differ from the Dp group (54.3 ± 3.8%). After icv administration of At, %GR (34.2 ± 2%) did not differ from control (32.6 ± 1.9%), but was significantly higher after Dp (54.5 ± 2.3%). Subdiaphragmatic vagotomy significantly reduced %GR in the At group (30.2 ± 0.7%) compared to the sham group, but was significantly higher than in the controls (23.0 ± 0.5%). In the animals treated with At iv, baclofen significantly reduced %GR (28.3 ± 2.4%) compared to vehicle-treated animals (55.2 ± 3.2%). The same occurred in the animals treated iv with vehicle and icv with baclofen. Although vagotomy and baclofen reduced %GR per se, the reduction was twice more marked in the animals treated with At. The results suggest that At administered iv, but not icv, delays GE of liquid in rats with the participation, at least in part, of the vagus nerve and that this phenomenon is blocked by the activation of GABA B receptors in the central nervous system.

Behavioral and physiological methods for early quantitative assessment of spinal cord injury and prognosis in rats

Methods for reliable evaluation of spinal cord (SC) injury in rats at short periods (2 and 24 h) after lesion were tested to characterize the mechanisms implicated in primary SC damage. We measured the physiological changes occurring after several procedures for producing SC injury, with particular emphasis on sensorimotor functions. Segmental and suprasegmental reflexes were tested in 39 male Wistar rats weighing 250-300 g divided into three control groups that were subjected to a) anesthesia, b) dissection of soft prevertebral tissue, and c) laminectomy of the vertebral segments between T10 and L1. In the lesion group the SC was completely transected, hemisected or subjected to vertebral compression. All animals were evaluated 2 and 24 h after the experimental procedure by the hind limb motility index, Bohlman motor score, open-field, hot-plate, tail flick, and paw compression tests. The locomotion scale proved to be less sensitive than the sensorimotor tests. A reduction in exploratory movements was detected in the animals 24 h after the procedures. The hot-plate was the most sensitive test for detecting sensorimotor deficiencies following light, moderate or severe SC injury. The most sensitive and simplest test of reflex function was the hot-plate. The hemisection model promoted reproducible moderate SC injury which allowed us to quantify the resulting behavior and analyze the evolution of the lesion and its consequences during the first 24 h after injury. We conclude that hemisection permitted the quantitation of behavioral responses for evaluation of the development of deficits after lesions. Hind limb evaluation scores and spontaneous exploration events provided a sensitive index of immediate injury effects after SC lesion at 2 and 24 h. Taken together, locomotion scales, open-field, and hot-plate tests represent reproducible, quantitatively sensitive methods for detecting functional deficiencies within short periods of time, indicating their potential for the study of cellular mechanisms of primary injury and repair after traumatic SC injury.

Mineralocorticoid modulation of central angiotensin-induced neuronal activity, water intake and sodium appetite

Central angiotensin II (AngII) stimulates water and salt solution intake. Pretreatment with low-dose mineralocorticoid (DOCA) enhances this AngII-induced intake of salt solutions (the synergy theory) in Wistar and Sprague Dawley rats but not in Fischer rats. This response is mediated via the AT-1 receptor. Electrophysiological experiments using iontophoretic application of AngII and the AT-1 receptor-specific non-peptide antagonist losartan showed excitation of neurons in the preoptic/medial septum region of urethane-anesthetized male Wistar rats. DOCA pretreatment further enhances this neuronal excitation in response to AngII and reduces the responses to losartan. This generated the hypothesis that DOCA-enhanced AngII-induced neuronal excitation is the neural support for the synergy theory. AT-2 receptors modulate these intake responses depending on sodium in the diet, and diuretic-induced dehydration during pregnancy produces a higher salt intake in the offspring. AngII-induced salt and water intakes were tested in offspring from Sprague Dawley mothers with only 1.8% NaCl to drink in which half were treated with furosemide. The important observations were a) the AT-1 antagonist alone suppressed intakes in offspring from mothers not treated with furosemide, b) both AT-1 and AT-2 antagonists suppressed intakes in offspring from furosemide-treated mothers, and c) combined administration of AT-1 and AT-2 antagonists greatly suppressed water intake in offspring from mothers not treated with furosemide. These results suggest that AT-1 and AT-2 receptors have variable properties (receptor number and/or second messengers). Furthermore, the activity and function of these central AngII receptors depend on the background mineralocorticoid levels. The exact mechanism of this influence, however, remains to be determined.

Biodistribution of 99m technetium- labeled creatinine in healthy rats

The distribution of creatinine, one of the toxic guanidine compounds, in various tissues has not been studied in detail by using radiolabeled creatinine. Our objective was to investigate the biodistribution of creatinine labeled with 99m technetium (99mTc) by the stannous (II) chloride method in healthy male Wistar rats. Quality controls were carried out by radio thin layer chromatography, high-performance liquid chromatography, and paper electrophoresis. The labeling yield was 85 ± 2% under optimum conditions (pH 7 and 100 µg stannous chloride). Rats (N = 12) were injected intravenously with 99mTc-creatinine and their blood and visceral organs were evaluated for 99mTc-creatinine uptake as percent of the injected dose per gram wet weight of each tissue (%ID/g). The lowest amount of uptake was detected in the brain and testis. When the rate of uptake was evaluated, only the kidney showed increasing rates of uptake of 99mTc-creatinine throughout the study. Kidneys showed the highest amount of uptake throughout the study (P < 0.001 compared to all other organs), followed by liver, spleen and lung tissue.

Effect of 5-HT1B receptor agonists injected into the prefrontal cortex on maternal aggression in rats

Serotonin (5-HT1B) receptors play an essential role in the inhibition of aggressive behavior in rodents. CP-94,253, a 5-HT1B receptor agonist, can reduce aggression in male mice when administered directly into the ventro-orbitofrontal (VO) prefrontal cortex (PFC). The objective of the current study was to assess the effects of two selective 5-HT1B receptor agonists (CP-94,253 and CP-93,129), microinjected into the VO PFC, on maternal aggressive behavior after social instigation in rats. CP-94,253 (0.56 µg/0.2 µL, N = 8, and 1.0 µg/0.2 µL, N = 8) or CP-93,129 (1.0 µg/0.2 µL, N = 9) was microinjected into the VO PFC of Wistar rats on the 9th day postpartum and 15 min thereafter the aggressive behavior by the resident female against a male intruder was recorded for 10 min. The frequency and duration of aggressive and non-aggressive behaviors were analyzed using ANOVA and post hoc tests. CP-93,129 significantly decreased maternal aggression. The frequency of lateral attacks, bites and pinnings was reduced compared to control, while the non-aggressive behaviors and maternal care were largely unaffected by this treatment. CP-94,253 had no significant effects on aggressive or non-aggressive behaviors when microinjected into the same area of female rats. CP-93,129, a specific 5-HT1B receptor agonist, administered into the VO PFC reduced maternal aggressive behavior, while the CP-94,253 agonist did not significantly affect this behavior after social instigation in female rats. We conclude that only the 5-HT1B receptor agonist CP-93,129 administered into the VO PFC decreased aggression in female rats postpartum after social instigation.

Effect of 4-aminoantipyrine on gastric compliance and liquid emptying in rats

Dipyrone (Dp) delays gastric emptying (GE) in rats. There is no information about whether 4-aminoantipyrine (AA), one of its metabolites, has the same effect. The objectives of the present study were to assess the effects of AA and Dp on GE when administered intravenously (iv) and intracerebroventricularly (icv) (240 µmol/kg and 4 µmol/animal, respectively) and on gastric compliance when administered iv (240 µmol/kg). GE was determined in male Wistar rats weighing 250-300 g (5-10 per group) after icv or iv injection of the drug by measuring percent gastric retention (GR) of a saline meal labeled with phenol red 10 min after administration by gavage. Gastric compliance was estimated in anesthetized rats (10-11 per group), with the construction of volume-pressure curves during intragastric infusion of a saline meal. Compliance was significantly greater in animals receiving Dp (mean ± SEM = 0.26 ± 0.009 mL/mmHg) and AA (0.24 ± 0.012 mL/mmHg) than in controls (0.19 ± 0.009 mL/mmHg). AA and Dp administered iv significantly increased GR (64.4 ± 2.5 and 54.3 ± 3.8%, respectively) compared to control (34 ± 2.2%), a phenomenon observed only with Dp after icv administration. Subdiaphragmatic vagotomy reduced the effect of AA (GR = 31.4 ± 1.5%) compared to sham-treated animals. Baclofen, a GABA B receptor agonist, administered icv significantly reduced the effect of AA (GR = 28.1 ± 1.3%). We conclude that Dp and AA increased gastric compliance and AA delayed GE, with the participation of the vagus nerve, through a pathway that does not involve a direct action of the drug on the central nervous system.