Neonatal handling induces anovulatory estrous cycles in rats

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

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.

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.

Gender differences in vascular expression of endothelin and ET A/ET B receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

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.

Effect of intraperitoneally administered hydrolyzed whey protein on blood pressure and renal sodium handling in awake spontaneously hypertensive rats

The present study evaluated the acute effect of the intraperitoneal (ip) administration of a whey protein hydrolysate (WPH) on systolic arterial blood pressure (SBP) and renal sodium handling by conscious spontaneously hypertensive rats (SHR). The ip administration of WPH in a volume of 1 ml dose-dependently lowered the SBP in SHR 2 h after administration at doses of 0.5 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 176.6 ± 4.9 mmHg, N = 8, P = 0.001) and 1.0 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 163.8 ± 5.9 mmHg, N = 8, P = 0.0018). Creatinine clearance decreased significantly (P = 0.0084) in the WPH-treated group (326 ± 67 µL min-1 100 g body weight-1) compared to 0.15 M NaCl-treated (890 ± 26 µL min-1 100 g body weight-1) and captopril-treated (903 ± 72 µL min-1 100 g body weight-1) rats. The ip administration of 1.0 g WPH/kg also decreased fractional sodium excretion to 0.021 ± 0.019% compared to 0.126 ± 0.041 and 0.66 ± 0.015% in 0.15 M NaCl and captopril-treated rats, respectively (P = 0.033). Similarly, the fractional potassium excretion in WPH-treated rats (0.25 ± 0.05%) was significantly lower (P = 0.0063) than in control (0.91 ± 0.15%) and captopril-treated rats (1.24 ± 0.30%), respectively. The present study shows a decreased SBP in SHR after the administration of WPH associated with a rise in tubule sodium reabsorption despite an angiotensin I-converting enzyme (ACE)-inhibiting in vitro activity (IC50 = 0.68 mg/mL). The present findings suggest a pathway involving ACE inhibition but measurements of plasma ACE activity and angiotensin II levels are needed to support this suggestion.

Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+) handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c), sarcolemmal Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), ryanodine receptor (RyR2), and phospholamban (PLB) mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control) or high-fat diet (obese) for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05) but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

Gentle handling temporarily increases c-Fos in the substantia nigra pars compacta

Dopaminergic neurotransmission is involved in the regulation of sleep. In particular, the nigrostriatal pathway is an important center of sleep regulation. We hypothesized that dopaminergic neurons located in substantia nigra pars compacta (SNpc) could be activated by gentle handling, a method to obtain sleep deprivation (SD). Adult male C57/BL6J mice (N = 5/group) were distributed into non-SD (NSD) or SD groups. SD animals were subjected to SD once for 1 or 3 h by gentle handling. Two experiments were performed. The first determined the activation of SNpc neurons after SD, and the second examined the same parameters after pharmacologically induced dopaminergic depletion using intraperitoneal reserpine (2 mg/kg). After 1 or 3 h, SD and NSD mice were subjected to motor evaluation using the open field test. Immediately after the behavioral test, the mice were perfused intracardially to fix the brain and for immunohistochemical analysis of c-Fos protein expression within the SNpc. The open field test indicated that SD for 1 or 3 h did not modify motor behavior. However, c-Fos protein expression was increased after 1 h of SD compared with the NSD and 3-h SD groups. These immunohistochemistry data indicate that these periods of SD are not able to produce dopaminergic supersensitivity. Nevertheless, the increased expression of c-Fos within the SNpc suggests that dopaminergic nigral activation was triggered by SD earlier than motor responsiveness. Dopamine-depleted mice (experiment 2) exhibited a similar increase of c-Fos expression compared to control animals indicating that dopamine neurons are still activated in the 1-h SD group despite the exhaustion of dopamine. This finding suggests that this range (2-5-fold) of neuronal activation may serve as a marker of SD.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score ≥ ±2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.

Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

Relationship between pulmonary function and indoor air pollution from coal combustion among adult residents in an inner-city area of southwest China

Few studies evaluate the amount of particulate matter less than 2.5 mm in diameter (PM2.5) in relation to a change in lung function among adults in a population. The aim of this study was to assess the association of coal as a domestic energy source to pulmonary function in an adult population in inner-city areas of Zunyi city in China where coal use is common. In a cross-sectional study of 104 households, pulmonary function measurements were assessed and compared in 110 coal users and 121 non-coal users (≥18 years old) who were all nonsmokers. Several sociodemographic factors were assessed by questionnaire, and ventilatory function measurements including forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), the FEV1/FVC ratio, and peak expiratory flow rate (PEFR) were compared between the 2 groups. The amount of PM2.5 was also measured in all residences. There was a significant increase in the relative concentration of PM2.5 in the indoor kitchens and living rooms of the coal-exposed group compared to the non-coal-exposed group. In multivariate analysis, current exposure to coal smoke was associated with a 31.7% decrease in FVC, a 42.0% decrease in FEV1, a 7.46% decrease in the FEV1/FVC ratio, and a 23.1% decrease in PEFR in adult residents. The slope of lung function decrease for Chinese adults is approximately a 2-L decrease in FVC, a 3-L decrease in FEV1, and an 8 L/s decrease in PEFR per count per minute of PM2.5 exposure. These results demonstrate the harmful effects of indoor air pollution from coal smoke on the lung function of adult residents and emphasize the need for public health efforts to decrease exposure to coal smoke.

STATISTICAL ANALYSYS OF THE SCFE OF A BRAZILAN MINERAL COAL

The influence of some process variables on the productivity of the fractions (liquid yield times fraction percent) obtained from SCFE of a Brazilian mineral coal using isopropanol and ethanol as primary solvents is analyzed using statistical techniques. A full factorial 23 experimental design was adopted to investigate the effects of process variables (temperature, pressure and cosolvent concentration) on the extraction products. The extracts were analyzed by the Preparative Liquid Chromatography-8 fractions method (PLC-8), a reliable, non destructive solvent fractionation method, especially developed for coal-derived liquids. Empirical statistical modeling was carried out in order to reproduce the experimental data. Correlations obtained were always greater than 0.98. Four specific process criteria were used to allow process optimization. Results obtained show that it is not possible to maximize both extract productivity and purity (through the minimization of heavy fraction content) simultaneously by manipulating the mentioned process variables.