Effect of 17ß-estradiol or alendronate on the bone densitometry, bone histomorphometry and bone metabolism of ovariectomized rats

The objective of the present study was to evaluate the effect of 17ß-estradiol or alendronate in preventing bone loss in 3-month-old ovariectomized Wistar rats. One group underwent sham ovariectomy (control, N = 10), and the remaining three underwent double ovariectomy. One ovariectomized group did not receive any treatment (OVX, N = 12). A second received subcutaneous 17ß-estradiol at a dose of 30 µg/kg for 6 weeks (OVX-E, N = 11) and a third, subcutaneous alendronate at a dose of 0.1 mg/kg for 6 weeks (OVX-A, N = 8). Histomorphometry, densitometry, osteocalcin and deoxypyridinoline measurements were applied to all groups. After 6 weeks there was a significant decrease in bone mineral density (BMD) at the trabecular site (distal femur) in OVX rats. Both alendronate and 17ß-estradiol increased the BMD of ovariectomized rats, with the BMD of the OVX-A group being higher than that of the OVX-E group. Histomorphometry of the distal femur showed a decrease in trabecular volume in the untreated group (OVX), and an increase in the two treated groups, principally in the alendronate group. In OVX-A there was a greater increase in trabecular number. An increase in trabecular thickness, however, was seen only in the OVX-E group. There was also a decrease in bone turnover in both OVX-E and OVX-A. The osteocalcin and deoxypyridinoline levels were decreased in both treated groups, mainly in OVX-A. Although both drugs were effective in inhibiting bone loss, alendronate proved to be more effective than estradiol at the doses used in increasing bone mass.

Effects of small doses of ouabain on the arterial blood pressure of anesthetized hypertensive and normotensive rats

Ouabain increases vascular resistance and may induce hypertension by inhibiting the Na+ pump. The effects of 0.18 and 18 µg/kg, and 1.8 mg/kg ouabain pretreatment on the phenylephrine (PHE; 0.1, 0.25 and 0.5 µg, in bolus)-evoked pressor responses were investigated using anesthetized normotensive (control and uninephrectomized) and hypertensive (1K1C and DOCA-salt treated) rats. Treatment with 18 µg/kg ouabain increased systolic and diastolic blood pressure in all groups studied. However, the magnitude of this increase was larger for the hypertensive 1K1C and DOCA-salt rats than for normotensive animals, while the pressor effect of 0.18 µg/kg ouabain was greater only in DOCA-salt rats. A very large dose (1.8 mg/kg) produced toxic effects on the normotensive control but not on uninephrectomized or 1K1C rats. Rat tail vascular beds were perfused to analyze the effects of 10 nM ouabain on the pressor response to PHE. In all animals, 10 nM ouabain increased the PHE pressor response, but this increase was larger in hypertensive DOCA-salt rats than in normotensive and 1K1C rats. Results suggested that a) increases in diastolic blood pressure induced by 18 µg/kg ouabain were larger in hypertensive than normotensive rats; b) in DOCA-salt rats, smaller ouabain doses had a stronger effect than in other groups; c) hypertensive and uninephrectomized rats were less sensitive to toxic doses of ouabain, and d) after treatment with 10 nM ouabain isolated tail vascular beds from DOCA-salt rats were more sensitive to the pressor effect of PHE than those from normotensive and 1K1C hypertensive rats. These data suggest that very small doses of ouabain, which might produce nanomolar plasma concentrations, enhance pressor reactivity in DOCA-salt hypertensive rats, supporting the idea that endogenous ouabain may contribute to the increase and maintenance of vascular tone in hypertension.

A high-fructose diet induces insulin resistance but not blood pressure changes in normotensive rats

Rats fed a high-fructose diet represent an animal model for insulin resistance and hypertension. We recently showed that a high-fructose diet containing vegetable oil but a normal sodium/potassium ratio induced mild insulin resistance with decreased insulin receptor substrate-1 tyrosine phosphorylation in the liver and muscle of normal rats. In the present study, we examined the mean blood pressure, serum lipid levels and insulin sensitivity by estimating in vivo insulin activity using the 15-min intravenous insulin tolerance test (ITT, 0.5 ml of 6 µg insulin, iv) followed by calculation of the rate constant for plasma glucose disappearance (Kitt) in male Wistar-Hannover rats (110-130 g) randomly divided into four diet groups: control, 1:3 sodium/potassium ratio (R Na:K) diet (C 1:3 R Na:K); control, 1:1 sodium/potassium ratio diet (CNa 1:1 R Na:K); high-fructose, 1:3 sodium/potassium ratio diet (F 1:3 R Na:K), and high-fructose, 1:1 sodium/potassium ratio diet (FNa 1:1 R Na:K) for 28 days. The change in R Na:K for the control and high-fructose diets had no effect on insulin sensitivity measured by ITT. In contrast, the 1:1 R Na:K increased blood pressure in rats receiving the control and high-fructose diets from 117 ± 3 and 118 ± 3 mmHg to 141 ± 4 and 132 ± 4 mmHg (P<0.05), respectively. Triacylglycerol levels were higher in both groups treated with a high-fructose diet when compared to controls (C 1:3 R Na:K: 1.2 ± 0.1 mmol/l vs F 1:3 R Na:K: 2.3 ± 0.4 mmol/l and CNa 1:1 R Na:K: 1.2 ± 0.2 mmol/l vs FNa 1:1 R Na:K: 2.6 ± 0.4 mmol/l, P<0.05). These data suggest that fructose alone does not induce hyperinsulinemia or hypertension in rats fed a normal R Na:K diet, whereas an elevation of sodium in the diet may contribute to the elevated blood pressure in this animal model.

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-µm sections at 120-µm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 ± 206, N = 6; 1020 ± 150, N = 6; nonhandled and handled, respectively), paraventricular (584 ± 85, N = 8; 682 ± 62, N = 9) or periventricular (743 ± 118, N = 7; 990 ± 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

Metabolic markers following beta-adrenoceptor agonist infusion in footshock-stressed rats

Stress hormones can alter metabolic functions in adipose tissue and liver, as well as the sensitivity of rat white adipocytes and rat atrial responses to ß-adrenergic agonists. In this study, we examined the effects of three daily footshock stress sessions on the plasma corticosterone, glucose, glycerol and triacylglycerol levels of fed, conscious male rats, and on the plasma glucose, glycerol and triacylglycerol levels of the same rats following iv infusions of ß-adrenergic agonists (isoproterenol: 0.4 nmol kg-1 min-1, noradrenaline: 5.0 µg kg-1 day-1, and BRL 37344 ([±]-[4-(2-[(2-[3-chlorophenyl]-2-hydroxyethyl)amino]propyl)phenoxy]acetic acid), a selective ß3-adrenoceptor agonist: 0.4 nmol kg-1 min-1). Plasma corticosterone levels increased significantly after each stress session, while triacylglycerol levels increased after the first session and glucose increased after the second and third sessions. Glycerol levels were unaltered after stress. These results suggest that repeated footshock stress may induce a metabolic shift from triacylglycerol biosynthesis to glucose release by hepatic tissue, with glycerol serving as one of the substrates in both pathways. Stressed rats were more sensitive to infusion of noradrenaline plus prazosin and to infusion of isoproterenol, with elevated plasma glucose, glycerol and triacylglycerol levels. The higher sensitivity of stressed rats to isoproterenol and noradrenaline was probably related to the permissive effect of plasma corticosterone. Only BRL 37344 increased plasma glycerol levels in stressed rats, probably because ß3-adrenoceptors are not involved in hepatic triacylglycerol synthesis, thus allowing glycerol to accumulate in plasma.

Respiratory mechanics and morphometric changes during anesthesia with ketamine in normal rats

Ketamine is believed to reduce airway and pulmonary tissue resistance. The aim of the present study was to determine the effects of ketamine on the resistive, elastic and viscoelastic/inhomogeneous mechanical properties of the respiratory system, lungs and chest wall, and to relate the mechanical data to findings from histological lung analysis in normal animals. Fifteen adult male Wistar rats were assigned randomly to two groups: control (N = 7) and ketamine (N = 8). All animals were sedated (diazepam, 5 mg, ip) and anesthetized with pentobarbital sodium (20 mg/kg, ip) or ketamine (30 mg/kg, ip). The rats were paralyzed and ventilated mechanically. Ketamine increased lung viscoelastic/inhomogeneous pressure (26%) compared to the control group. Dynamic and static elastances were similar in both groups, but the difference was greater in the ketamine than in the control group. Lung morphometry demonstrated dilation of alveolar ducts and increased areas of alveolar collapse in the ketamine group. In conclusion, ketamine did not act at the airway level but acted at the lung periphery increasing mechanical inhomogeneities possibly resulting from dilation of distal airways and alveolar collapse.

Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 ± 6 and 21 ± 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 ± 2 nmol Pi mg-1 min-1 for AMP and 1.52 ± 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

GLUT4 protein expression in obese and lean 12-month-old rats: insights from different types of data analysis

GLUT4 protein expression in white adipose tissue (WAT) and skeletal muscle (SM) was investigated in 2-month-old, 12-month-old spontaneously obese or 12-month-old calorie-restricted lean Wistar rats, by considering different parameters of analysis, such as tissue and body weight, and total protein yield of the tissue. In WAT, a ~70% decrease was observed in plasma membrane and microsomal GLUT4 protein, expressed as µg protein or g tissue, in both 12-month-old obese and 12-month-old lean rats compared to 2-month-old rats. However, when plasma membrane and microsomal GLUT4 tissue contents were expressed as g body weight, they were the same. In SM, GLUT4 protein content, expressed as µg protein, was similar in 2-month-old and 12-month-old obese rats, whereas it was reduced in 12-month-old obese rats, when expressed as g tissue or g body weight, which may play an important role in insulin resistance. Weight loss did not change the SM GLUT4 content. These results show that altered insulin sensitivity is accompanied by modulation of GLUT4 protein expression. However, the true role of WAT and SM GLUT4 contents in whole-body or tissue insulin sensitivity should be determined considering not only GLUT4 protein expression, but also the strong morphostructural changes in these tissues, which require different types of data analysis.

Dopaminergic modulation of grooming behavior in virgin and pregnant rats

Dopamine receptors are involved in the expression of grooming behavior. The pregnancy-induced increase in self-licking observed in rats is important for mammary gland development and lactation. This study focuses on the role of dopamine receptor subtypes in grooming behavior of virgin and pregnant female rats. General and mammary gland grooming were measured in virgin rats treated with 0.25 mg/kg of the D1-like agonist SKF-81297 and antagonist SKF-83566 and the D2-like agonist lisuride and antagonist sulpiride. The effects of 0.01 and 0.25 mg/kg doses of the same agonists and antagonists were evaluated in pregnant rats as well. In virgin animals both SKF-83566 and sulpiride treatments significantly reduced the time spent in general grooming, while none of the dopamine agonists was able to significantly change any parameter of general grooming. Time spent in grooming directed at the mammary glands was not affected significantly by any of the drug treatments in virgin rats. All drugs tested significantly decreased the frequency of and the time spent with general grooming, while SKF-81297 treatment alone did not significantly reduce the duration of mammary gland grooming in pregnant rats. These data show that in female rats the behavioral effects of D1-like and D2-like dopamine receptor stimulation and blockade differ according to physiological state. The results suggest that dopamine receptors may play specific roles modulating grooming behavior in pregnant rats. Since grooming of the mammary gland during pregnancy may influence lactation, this aspect is relevant for studies regarding the perinatal use of dopamine-related drugs.

Plasma insulin levels are increased by sertraline in rats under oral glucose overload

Recognition and control of depression symptoms are important to increase patient compliance with treatment and to improve the quality of life of diabetic patients. Clinical studies indicate that selective serotonin reuptake inhibitors (SSRI) are better antidepressants for diabetic patients than other drugs. However, preclinical trials have demonstrated that not all SSRI reduce plasma glucose levels. In fact, fluoxetine increases and sertraline decreases glycemia in diabetic and non-diabetic rats. In the present study we evaluated plasma insulin levels during fasting and after glucose overload after treatment with sertraline. Adult male Wistar rats were fasted and treated with saline or 30 mg/kg sertraline and submitted or not to glucose overload (N = 10). Blood was collected and plasma insulin was measured. The mean insulin levels were: fasting group: 25.9 ± 3.86, sertraline + fasting group: 31.10 ± 2.48, overload group: 34.1 ± 3.40, and overload + sertraline group: 43.73 ± 5.14 µU/ml. Insulinemia was significantly increased in the overload + sertraline group. There were no differences between the other groups. No difference in glucose/insulin ratios could be detected between groups. The overload + sertraline group was the only one in which a significant number of individuals exceeded the upper confidence limit of insulin levels. This study demonstrates that sertraline increases glucose-stimulated insulin secretion without any change in peripheral insulin sensitivity.

Comparison of Pavlovian serial conditional discrimination in rats and hamsters in the same experimental situation

The present study compares behavioral changes between two distinct rodent groups, hamsters (Mesocricetus auratus) and Wistar rats, when submitted in the same homogeneous experimental situations to a serial conditional discrimination procedure which involves water deprivation and the processing of temporal variables. Both hamsters and rats acquired serial positive conditional discrimination as indicated by higher frequencies of magazine-oriented behavior during the tone followed by reinforcement (T+) and preceded by the feature stimulus light (L) and during the empty interval, than during the tone alone not followed by reinforcement (T-). Rats' frequencies of magazine-oriented behavior were high during T+ and T-, initially during training, and decreased during T- as the training progressed. However, the hamsters' frequencies of magazine-oriented behavior started very low and increased only during T+ as the training progressed. Comparison of the frequencies of magazine-oriented behavior during the empty interval in relation to the frequencies during the preceding L period showed that rats' frequencies remained very high and hamsters' frequencies increased during training. These results suggest that rats and hamsters have different behavioral strategies for the acquisition of a conditional discrimination. The results of the comparisons made in these experiments support the view of the importance of an ecological psychology approach to the understanding of complex learning in animals.

Cardiovascular and respiratory responses to microinjection of L-glutamate into the caudal pressor area in conscious and anesthetized rats

The role of the caudal pressor area (CPA) in the maintenance of vasomotor tonus in anesthetized and decerebrate animals has been clearly established. In conscious animals, however, the participation of CPA in the cardiovascular control remains to be fully elucidated. In the present study, unilateral L-glutamate (L-Glu) (10 and/or 20 nmol/70 nl) microinjection into CPA, in conscious male Wistar rats (250-280 g) caused a significant increase in mean arterial blood pressure (MAP; control: 112 ± 1.9 mmHg; after 20 nmol L-Glu: 139 ± 4.5 mmHg, N = 12, P<0.05) and respiratory rate (control: 81 ± 3.5 breaths/min; after 10 nmol L-Glu: 92 ± 3 breaths/min, P<0.05; after 20 nmol L-Glu: 104 ± 5 breaths/min, N = 6, P<0.05). The subsequent anesthesia with urethane caused a significant increase in basal respiratory frequency (conscious: 81 ± 3.5 breaths/min; under urethane: 107 ± 1.3 breaths/min, N = 6, P<0.05). Anesthesia also significantly attenuated L-Glu-evoked pressor (conscious: deltaMAP = +27 mmHg; anesthetized: deltaMAP = +18 mmHg, P<0.05) and respiratory responses. These results suggest that glutamatergic receptors in the CPA are involved in cardiovascular and respiratory modulation in conscious rats.

Influence of the test situation on pup retrieval behavior of normal and undernourished lactating rats

Undernutrition of dams and pups disrupts the retrieval efficiency of mothers. However, if the mothers are assessed in their home cages, they spend more time with their litters. In the present study the effect of test conditions on pup retrieval behavior of mothers receiving a 25% (well-nourished group) and 8% casein diet (undernourished group) was examined. In agreement with previous studies, undernourished mothers spent more time with their litters than well-nourished dams as lactation proceeded. Pup retrieval behavior varied with test conditions. In the first experiment, the maternal behavior of dams was assessed by the standard procedure (pups were separated from their mother and scattered over the floor of the home cage). The mother was then returned and the number of retrieved pups was recorded. From day 3 to 8, the retrieval efficiency of undernourished dams decreased, while the retrieval efficiency of well-nourished mothers did not vary. In the second experiment, mothers were subjected to a single retrieval test (on day 9 of lactation) using the procedure described for experiment 1. No difference between well-nourished and undernourished mothers was observed. In the third experiment, seven-day-old pups were separated from the mothers and returned individually to a clean home cage. Dietary treatment did not affect the retrieval efficiency. However, undernourished dams reconstructed the nest more slowly than did well-nourished dams. Taken together, these results suggest that pup retrieval behavior of the undernourished mother is not impaired by dietary restriction when the maternal environment is disturbed minimally.

Ethidium bromide-induced demyelination of the sciatic nerve of adult Wistar rats

Peripheral nerve ultrastructure was assessed after single or multiple local injections of the intercalating dye ethidium bromide. Thirty-four adult Wistar rats of both sexes were divided into five groups and maintained in a controlled environment with rat chow and water ad libitum throughout the experiment. The experimental animals were injected with 1 µl of 0.1% ethidium bromide in 0.9% saline into the central third of the left sciatic nerve 1 (group 1), 2 (group 2), 4 (group 3), 6 (group 4) or 8 (group 5) times. In groups 2 to 5 the injections were made at 28-day intervals. Control animals received the same amount of 0.9% saline. The animals were killed at different times after injection: group 1 at 7 days (2 rats) and 15 days (2 rats); for groups 2, 3, 4 and 5, all rats were killed 10 days after the last injection and the lesions were investigated by light and transmission electron microscopy. In the acute lesions, intoxicated Schwann cells showed a vacuolated cytoplasm and separation of the sheaths from the axon. Myelin sheaths underwent progressive vesiculation and subsequent segmental demyelination. Myelin debris were withdrawn by macrophages and remyelination by Schwann cells was prominent. With the increase in the number of injections collagen fibers also increased in number and progressively enveloped smaller numbers of remyelinated axons composing new fascicles. Wallerian degeneration of fibers apparently not affected by ethidium bromide was more intense in the nerves from groups 4 and 5. The peripheral nerve repairs itself after demyelinating challenges with a profusion of collagen fibers and new fasciculations. This experimental model is valid to mimic recurrent demyelinating neuropathies.