Correlations between ANP concentrations in atria, plasma and cerebral structures and sodium chloride preference in Wistar rats

We determined whether ANP (atrial natriuretic peptide) concentrations, measured by radioimmunoassay, in the ANPergic cerebral regions involved in regulation of sodium intake and excretion and pituitary gland correlated with differences in sodium preference among 40 Wistar male rats (180-220 g). Sodium preference was measured as mean spontaneous ingestion of 1.5% NaCl solution during a test period of 12 days. The relevant tissues included the olfactory bulb (OB), the posterior and anterior lobes of the pituitary gland (PP and AP, respectively), the median eminence (ME), the medial basal hypothalamus (MBH), and the region anteroventral to the third ventricle (AV3V). We also measured ANP content in the right (RA) and left atrium (LA) and plasma. The concentrations of ANP in the OB and the AP were correlated with sodium ingestion during the preceding 24 h, since an increase of ANP in these structures was associated with a reduced ingestion and vice-versa (OB: r = -0.3649, P<0.05; AP: r = -0.3291, P<0.05). Moreover, the AP exhibited a correlation between ANP concentration and mean NaCl intake (r = -0.4165, P<0.05), but this was not the case for the OB (r = 0.2422). This suggests that differences in sodium preference among individual male rats can be related to variations of AP ANP level. Earlier studies indicated that the OB is involved in the control of NaCl ingestion. Our data suggest that the OB ANP level may play a role mainly in day-to-day variations of sodium ingestion in the individual rat

Effects of acupuncture on behavioral, cardiovascular and hormonal responses in restraint-stressed Wistar rats

Stress is a well-known entity and may be defined as a threat to the homeostasis of a being. In the present study, we evaluated the effects of acupuncture on the physiological responses induced by restraint stress. Acupuncture is an ancient therapeutic technique which is used in the treatment and prevention of diseases. Its proposed mechanisms of action are based on the principle of homeostasis. Adult male Wistar EPM-1 rats were divided into four groups: group I (N = 12), unrestrained rats with cannulas previously implanted into their femoral arteries for blood pressure and heart rate measurements; group II (N = 12), rats that were also cannulated and were submitted to 60-min immobilization; group III (N = 12), same as group II but with acupuncture needles implanted at points SP6, S36, REN17, P6 and DU20 during the immobilization period; group IV (N = 14), same as group III but with needles implanted at points not related to acupuncture (non-acupoints). During the 60-min immobilization period animals were assessed for stress-related behaviors, heart rate, blood pressure and plasma corticosterone, noradrenaline and adrenaline levels. Group III animals showed a significant reduction (60% on average, P<0.02) in restraint-induced behaviors when compared to groups II and IV. Data from cardiovascular and hormonal assessments indicated no differences between group III and group II and IV animals, but tended to be lower (50% reduction on average) in group I animals. We hypothesize that acupuncture at points SP6, S36, REN17, P6 and DU20 has an anxiolytic effect on restraint-induced stress that is not due to a sedative action

Inflammatory response of the spinal cord to multiple episodes of blood-brain barrier disruption and toxic demyelination in Wistar rats

Multiple episodes of blood-brain barrier disruption were induced by sequential intraspinal injections of ethidium bromide. In addition to the barrier disruption, there was toxic demyelination and exposure of myelin components to the immune system. Twenty-seven 3-month-old Wistar rats received 2, 3 or 4 injections of 1 µl of either 0.1% ethidium bromide in normal saline (19 rats) or 0.9% saline (8 rats) at different levels of the spinal cord. The time intervals between the injections ranged from 28 to 42 days. Ten days after the last injection, all rats were perfused with 2.5% glutaraldehyde. The spinal sections were evaluated macroscopically and by light and transmission electron microscopy. All the lesions demonstrated a mononuclear phagocytic infiltrate apparently removing myelin. Lymphocytes were not conspicuous and were found in only 34% of the lesions. No perivascular cuffings were detected. In older lesions (38 days and older) they were found only within Virchow-Robin spaces. This result suggests that multiple blood-brain barrier disruptions with demyelination and exposure of myelin components to the immune system were not sufficient to induce an immune-mediated reaction in the central nervous system.

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.

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.

Aberrant crypt foci and colon cancer: comparison between a short- and medium-term bioassay for colon carcinogenesis using dimethylhydrazine in Wistar rats

Aberrant crypt foci (ACF) in the colon of carcinogen-treated rodents are considered to be the earliest hallmark of colon carcinogenesis. In the present study the relationship between a short-term (4 weeks) and medium-term (30 weeks) assay was assessed in a model of colon carcinogenesis induced by dimethylhydrazine (DMH) in the rat. Six-week-old male Wistar rats were given subcutaneous injections of DMH (40 mg/kg) twice a week for 2 weeks and killed at the end of the 4th or 30th week. ACF were scored for number, distribution pattern along the colon and crypt multiplicity in 0.1% methylene-blue whole-mount preparations. ACF were distinguished from normal crypts by their larger size and elliptical shape. The incidence, distribution and morphology of colon tumors were recorded. The majority of ACF were present in the middle and distal colon of DMH-treated rats and their number increased with time. By the 4th week, 91.5% ACF were composed of one or two crypts and 8.5% had three or more crypts, while by the 30th week 46.9% ACF had three or more crypts. Thus, a progression of ACF consisting of multiple crypts was observed from the 4th to the 30th week. Nine well-differentiated adenocarcinomas were found in 10 rats by the 30th week. Seven tumors were located in the distal colon and two in the middle colon. No tumor was found in the proximal colon. The present data indicate that induction of ACF by DMH in the short-term (4 weeks) assay was correlated with development of well-differentiated adenocarcinomas in the medium-term (30 weeks) assay.

Effect of the acupoints ST-36 (Zusanli) and SP-6 (Sanyinjiao) on intestinal myoelectric activity of Wistar rats

Despite its ancient use as a therapeutic tool to treat several ailments, acupuncture still faces the challenge of scrutiny by Western science both in terms of its efficacy and in terms of the characterization of its effects and mechanisms of actions underlying these effects. We investigated under well-controlled and carefully characterized conditions the influence of electrical stimulation of acupuncture points ST-36 (Zusanli) and SP-6 (Sanyinjiao) on the myoelectric activity of the small intestine of 38 adult male Wistar rats. Electrical recordings obtained by means of four electrodes chronically implanted in the small intestine were used to assess the effects of acupuncture (electroacupuncture stimulation set at 2 Hz, intermittent stimulation, 1 V, for 30 min). Immobilization of the animals was associated with a consistent decrease (-8 ± 7%) in the myoelectric activity of the small intestine as measured by means of the root mean square. Conversely, acupuncture was able to significantly increase (overshoot) this activity compared to baseline (+44 ± 7%). In contrast, immobilized animals subjected to sham acupuncture had only modest (nonsignificant) increases in myoelectric activity (+9 ± 6%). Using carefully controlled conditions we confirmed previous noncontrolled studies on the ability of acupuncture to alter intestinal motility. The characterization of the topographic and temporal profiles of the effects observed here represents a basis for future dissection of the physiological and pharmacological systems underlying these effects.

Low ethanol consumption increases insulin sensitivity in Wistar rats

Several human studies suggest that light-to-moderate alcohol consumption is associated with enhanced insulin sensitivity, but these studies are not free of conflicting results. To determine if ethanol-enhanced insulin sensitivity could be demonstrated in an animal model, male Wistar rats were fed a standard chow diet and received drinking water without (control) or with different ethanol concentrations (0.5, 1.5, 3, 4.5 and 7%, v/v) for 4 weeks ad libitum. Then, an intravenous insulin tolerance test (IVITT) was performed to determine insulin sensitivity. Among the ethanol groups, only the 3% ethanol group showed an increase in insulin sensitivity based on the increase of the plasma glucose disappearance rate in the IVITT (30%, P<0.05). In addition, an intravenous glucose tolerance test (IVGTT) was performed in control and 3% ethanol animals. Insulin sensitivity was confirmed in 3% ethanol rats based on the reduction of insulin secretion in the IVGTT (35%, P<0.05), despite the same glucose profile. Additionally, the 3% ethanol treatment did not impair body weight gain or plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the present study established that 3% ethanol in the drinking water for 4 weeks in normal rats is a model of increased insulin sensitivity, which can be used for further investigations of the mechanisms involved.

Morphological changes induced by testosterone in the mammary glands of female Wistar rats

Increased levels of androgens in postmenopausal women are considered to be a risk factor for breast cancer. Testosterone, alone or in combination with estrogen, induces epithelial dysplasia and mammary tumors in Noble rats. Since this model of hormone-induced neoplasia has not been reported in other rat strains, we studied the effect of testosterone on the mammary gland morphology of female Wistar rats. Sixty adult, non-castrated, female Wistar rats were implanted in the dorsum midline with a silicone tube containing 50 mg testosterone (testosterone propionate in 30 animals and non-esterified testosterone in the remaining 30 animals) and 20 additional animals were implanted with empty tubes and used as control. Five animals per group were killed 30, 60, 90, 120, 150, and 180 days after implantation, and the mammary glands were dissected, fixed and embedded in paraffin. Histological sections were then stained with hematoxylin and eosin and picrosyrius red for collagen visualization. Morphological and morphometric analysis demonstrated ductal proliferation and acinotubular differentiation with secretory activity in all treated animals, peaking at 90 days of androgen exposure. After 90 days the proliferation of acinar epithelial cells was evident, but there was a progressive reduction of secretory differentiation and an increase in intralobular collagen fibers. There was no morphological evidence of dysplastic changes or other pre-neoplastic lesions. Testosterone treatment applied to adult, non-castrated female Wistar rats induced a mammary gland hyperplasia resembling the lactating differentiation, with progressive reduction in secretory differentiation.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

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.

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).

Opposite lipemic response of Wistar rats and C57BL/6 mice to dietary glucose or fructose supplementation

The metabolic effects of carbohydrate supplementation in mice have not been extensively studied. In rats, glucose- and fructose-rich diets induce hypertriacylglycerolemia. In the present study, we compared the metabolic responses to two monosaccharide supplementations in two murine models. Adult male Wistar rats (N = 80) and C57BL/6 mice (N = 60), after 3 weeks on a standardized diet, were submitted to dietary supplementation by gavage with glucose (G) or fructose (F) solutions (500 g/L), 8 g/kg body weight for 21 days. Glycemia was significantly higher in rats after fructose treatment (F: 7.9 vs 9.3 mM) and in mice (G: 6.5 vs 10 and F: 6.6 vs 8.9 mM) after both carbohydrate treatments. Triacylglycerolemia increased significantly 1.5 times in rats after G or F supplementation. Total cholesterol did not change with G treatment in rats, but did decrease after F supplementation (1.5 vs 1.4 mM, P < 0.05). Both supplementations in rats induced insulin resistance, as suggested by the higher Homeostasis Model Assessment Index. In contrast, mice showed significant decreases in triacylglycerol (G: 1.8 vs 1.4 and F: 1.9 vs 1.4 mM, P < 0.01) and total cholesterol levels (G and F: 2.7 vs 2.5 mM, P < 0.05) after both monosaccharide supplementations. Wistar rats and C57BL/6 mice, although belonging to the same family (Muridae), presented opposite responses to glucose and fructose supplementation regarding serum triacylglycerol, free fatty acids, and insulin levels after monosaccharide treatment. Thus, while Wistar rats developed features of plurimetabolic syndrome, C57BL/6 mice presented changes in serum biochemical profile considered to be healthier for the cardiovascular system.

Effect of gallium-arsenide laser, gallium-aluminum-arsenide laser and healing ointment on cutaneous wound healing in Wistar rats

This study determined the effects of gallium-aluminum-arsenide laser (GaAlAs), gallium-arsenide laser (GaAs) and Dersani® healing ointment on skin wounds in Wistar rats. The parameters analyzed were: type I and III collagen fiber concentrations as well as the rate of wound closure. Five wounds, 12 mm in diameter, were made on the animals’ backs. The depth of the surgical incision was controlled by removing the epithelial tissue until the dorsal muscular fascia was exposed. The animals were anesthetized with ketamine and xylazine via intraperitoneal injection. The rats were randomly divided into five groups of 6 animals each, according to the treatment received. Group 1 (L4): GaAs laser (4 J/cm²); group 2 (L30): GaAlAs laser (30 J/cm²); group 3 (L60): GaAlAs laser (60 J/cm²); group 4 (D): Dersani® ointment; group 5 (control): 0.9% saline. The applications were made daily over a period of 20 days. Tissue fragments were stained with picrosirius to distinguish type I collagen from type III collagen. The collagen fibers were photo-documented and analyzed using the Quantum software based on the primary color spectrum (red, yellow and blue). Significant results for wound closing rate were obtained for group 1 (L4), 7.37 mm/day. The highest concentration of type III collagen fibers was observed in group 2 (L30; 37.80 ± 7.10%), which differed from control (29.86 ± 5.15%) on the 20th day of treatment. The type I collagen fibers of group 1 (L4; 2.67 ± 2.23%) and group 2 (L30; 2.87 ± 2.40%) differed significantly from control (1.77 ± 2.97%) on the 20th day of the experiment.

Motor behavioral abnormalities and histopathological findings of Wistar rats inoculated with HTLV-1-infected MT2 cells

The objective of the present study was to describe motor behavioral changes in association with histopathological and hematological findings in Wistar rats inoculated intravenously with human T-cell lymphotropic virus type 1 (HTLV-1)-infected MT2 cells. Twenty-five 4-month-old male rats were inoculated with HTLV-1-infected MT2 cells and 13 control rats were inoculated with normal human lymphocytes. The behavior of the rats was observed before and 5, 10, 15, and 20 months after inoculation during a 30-min/rat testing time for 5 consecutive days. During each of 4 periods, a subset of rats was randomly chosen to be sacrificed in order to harvest the spinal cord for histopathological analysis and to obtain blood for serological and molecular studies. Behavioral analyses of the HTLV-1-inoculated rats showed a significant decrease of climbing, walking and freezing, and an increase of scratching, sniffing, biting, licking, and resting/sleeping. Two of the 25 HTLV-1-inoculated rats (8%) developed spastic paraparesis as a major behavioral change. The histopathological changes were few and mild, but in some cases there was diffuse lymphocyte infiltration. The minor and major behavioral changes occurred after 10-20 months of evolution. The long-term observation of Wistar rats inoculated with HTLV-1-infected MT2 cells showed major (spastic paraparesis) and minor motor abnormalities in association with the degree of HTLV-1-induced myelopathy.