Michèle Brochu

Induction of intrauterine growth restriction with a low-sodium diet fed to pregnant rats

OBJECTIVE A low-sodium diet fed to female rats before mating through parturition leads to pups of lower weight. We characterized the effect of low dietary sodium during the last week of gestation (after fetal organogenesis) on fetal and maternal homeostasis. STUDY DESIGN Pregnant Sprague-Dawley rats were randomly assigned to a control group or to a group fed a low-sodium diet from gestational days 15 through 22. Systolic blood pressures were measured throughout pregnancy. On day 22 plasma volume was measured and blood samples were taken for electrolyte and hormonal measurements. Fetal and placental weights were also determined. RESULTS Plasma renin activity and aldosterone level were significantly higher in the experimental group than in the control group. Plasma volume was significantly lower in pregnant rats receiving a low-sodium than in those receiving a control diet. Rats receiving a low-sodium diet had pups of lower weight and length (4.45 +/- 0.22 g, 3.90 +/- 0.06 cm) than pups of the control group (5.21 +/- 0.12 g, 4.10 +/- 0.02 cm). Pups born to mothers with low-sodium diets recuperated from intrauterine growth restriction by 14 days after birth. CONCLUSION These data indicate that a low-sodium diet given to pregnant rats for the last 7 days of gestation leads to reduced plasma volume expansion and fetal growth restriction. This could prove to be a simple animal model for studying the relationship between maternal plasma volume and fetal growth.

Intra-uterine growth restriction and the programming of left ventricular remodelling in female rats

Epidemiological studies link intra‐uterine growth restriction (IUGR) with increased incidence of hypertension and cardiac disease in adulthood. Our rat model of IUGR supports this contention and provides evidence for the programming of susceptibility for hypertension in all offspring. Moreover, in the female offspring only, gross anatomical changes (cardiac ventricle to body ratios) and increased left cardiac ventricular atrial natriuretic peptide (ANP) mRNA levels provide evidence for programming of cardiac disease in this gender. The aim of the current study was to measure changes in cardiac tissue that support remodelling that could be implicated in the initiation of hypertrophy. Adult female rats from our IUGR model and age‐ and sex‐matched controls were killed at 12 weeks of age. Left cardiac ventricles were removed and used for monitoring changes in several key genes, Na+,K+‐ATPase β1 protein expression, cardiomyocyte morphology and contractility as well as citrate synthase and aconitase activities. When compared to controls, female offspring of our IUGR rat model exhibit higher expression (mRNA) of ANP and the atrial isoform of the myosin light chain, lower levels of Na+,K+‐ATPase β1 protein, increased cardiomyocyte depth and volume, increased sarcomere length, diminished cardiomyocyte contractility and lower aconitase activity. Female offspring of our IUGR rat model exhibit changes as adults that are consistent with the onset of cardiac remodelling. The decrease in aconitase activity suggests that oxidative stress may be implicated in this response.

Early-Life Stress Is Associated with Gender-Based Vulnerability to Epileptogenesis in Rat Pups

During development, the risk of developing mesial temporal lobe epilepsy (MTLE) increases when the developing brain is exposed to more than one insult in early life. Early life insults include abnormalities of cortical development, hypoxic-ischemic injury and prolonged febrile seizures. To study epileptogenesis, we have developed a two-hit model of MTLE characterized by two early-life insults: a freeze lesion-induced cortical malformation at post-natal day 1 (P1), and a prolonged hyperthermic seizure (HS) at P10. As early life stressors lead to sexual dimorphism in both acute response and long-term outcome, we hypothesized that our model could lead to gender-based differences in acute stress response and long-term risk of developing MTLE. Male and female pups underwent a freeze-lesion induced cortical microgyrus at P1 and were exposed to HS at P10. Animals were monitored by video-EEG from P90 to P120. Pre and post-procedure plasma corticosterone levels were used to measure stress response at P1 and P10. To confirm the role of sex steroids, androgenized female pups received daily testosterone injections to the mother pre-natally and post-natally for nine days while undergoing both insults. We demonstrated that after both insults females did not develop MTLE while all males did. This correlated with a rise in corticosterone levels at P1 following the lesion in males only. Interestingly, all androgenized females showed a similar rise in corticosterone at P1, and also developed MTLE. Moreover, we found that the cortical lesion significantly decreased the latency to generalized convulsion during hyperthermia at P10 in both genders. The cortical dysplasia volumes at adulthood were also similar between male and female individuals. Our data demonstrate sexual dimorphism in long-term vulnerability to develop epilepsy in the lesion + hyperthermia animal model of MTLE and suggest that the response to early-life stress at P1 contributes significantly to epileptogenesis in a gender-specific manner.

Natriuretic Peptide Receptor-C Attenuates Hypertension in Spontaneously Hypertensive Rats: Role of Nitroxidative Stress and Gi Proteins

C-Atrial natriuretic peptide (ANP)4–23, a ring deleted analog of ANP that specifically interacts with natriuretic peptide receptor-C (NPR-C), has been shown to decrease the enhanced expression of Gi&agr; proteins implicated in the pathogenesis of hypertension. In the present study, we investigated whether in vivo treatment of spontaneously hypertensive rats (SHRs) with C-ANP4–23 could attenuate the development of high blood pressure (BP) and explored the underlying mechanisms responsible for this response. Intraperitoneal injection of C-ANP4–23 at the concentration of 2 or 10 nmol/kg body weight to prehypertensive SHRs attenuated the development of high BP, and at 8 weeks it was decreased by ≈20 and 50 mm Hg, respectively; however, this treatment did not affect BP in Wistar-Kyoto rats. C-ANP4–23 treatment of adult SHRs for 2 weeks also attenuated high BP, heart rate, and restored the impaired vasorelaxation toward control levels. In addition, the enhanced levels of superoxide anion (O2−), peroxynitrite, NADPH oxidase activity, and the enhanced expression of Gi&agr; proteins, NOX4, p47phox, nitrotyrosine, and decreased levels of endothelial nitric oxide synthase (eNOS or NOS3) and NO in SHRs were attenuated by C-ANP4–23 treatment; however, the altered levels of NPR-A/NPR-C were not affected by this treatment. In conclusion, these results indicate that NPR-C activation by C-ANP4–23 attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Gi&agr; proteins and nitroxidative stress and not through eNOS/cGMP pathway and suggest that NPR-C ligand may have the potential to be used as therapeutic agent in the treatment of cardiovascular complications including hypertension.

Effects of nifedipine and Bay K 8644 on myotropic responses in aortic rings of pregnant rats

The hypothesis that Ca2+ channel function is altered during pregnancy was tested by comparing responses to potassium chloride (KCl) and phenylephrine in aortic rings of virgin and term-pregnant rats under the influence of nifedipine and Bay K 8644. Maximum response to KCl was progressively reduced by increasing nifedipine concentrations (1.0-100 nM) in both groups of tissues. Nifedipine produced a smaller inhibition of KCl-induced contraction in aortic rings of pregnant than of virgin rats. It exerted little inhibition on the concentration-response curve to phenylephrine. The Ca2+ channel antagonist (100 nM) reduced the maximum response to the alpha-adrenoceptor agonist in rings from virgin rats, but had no effect in pregnant rats. Bay K 8644, a Ca2+ channel activator, potentiated the responses to low concentrations of both phenylephrine and KCl in the tissues of both virgin and pregnant rats, but did not affect maximum responses. It also induced concentration-dependent contractions in rings of virgin but not of pregnant rats. The effects of Bay K 8644 were markedly potentiated by precontracting the aorta with 10mM KCl. Nevertheless tissues from pregnant rats were still less responsive to Bay K 8644. However, when the strips were precontracted to the same level by different concentrations of KCl, the concentration-response curves to Bay K 8644 were identical in both groups. [3H]Nitrendipine binding to membrane preparations of the thoracic aorta was similar in virgin and pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly sensitive and rapid radioimmunoassay for aldosterone in plasma and cell culture medium.

A radioimmunoassay for aldosterone was developed using a sensitive and specific antibody and 125Iodinated-aldosterone. This assay could be used for direct determination of aldosterone in cell culture medium or after extraction of aldosterone from plasma by solid phase procedure using C18 Sep-pak cartridges. The very low cross-reactivity of the antibody with cortisol and corticosterone (0.005% and 0.04% respectively) would allow the direct determination of aldosterone in cell culture medium without any prior extraction step. Since the incubation is performed at room temperature for 1 h and then, at 4 degrees C for 15 min, the results can be obtained in less than 3 h. The assay was linear from 0.5 fmol/tube to 1500 fmol/tube with an ED50 at 30 fmol/tube. The accuracy of the assay estimated using spiked plasma samples with a known amount of aldosterone give a coefficient of correlation of 0.97 (n = 10) between the aldosterone concentrations found and expected levels. The within-assay variability for plasma aldosterone varied from 4.7 to 11.1% and the between-assay variability ranged from 13.9 to 14.2%. The coefficient of correlation between plasma aldosterone measured by this new assay or by a current assay was 0.8 (n = 43). In summary, the combination of a shortened incubation time with a simple solid phase extraction for aldosterone in serum samples represents the major advantage of the present assay over the current methodology which usually requires a chromatographic separation of the mineralocorticoid prior to radioimmunoassay. Therefore this assay would be useful in experimental studies as well as in clinical laboratory.

Decreased Response to Vasopressin in the Mesenteric Resistance Arteries of Pregnant Rats: Effects of Nifedipine and Bay K 8644

Objective: The purpose of this study was to investigate the contribution of potential-operated calcium (POC) channels in the mechanisms of the blunted effects of vasoconstrictors on mesenteric resistance arteries during normal pregnancy. Methods: Mesenteric resistance arteries of virgin and term pregnant rats were set up under optimum passive tension in wire myograph systems. Cumulative concentration-response curves of arginine8-vasopressin (AVP) were measured in the absence and presence of nifedipine or Bay K 8644, a blocker and an activator, respectively, of POC channels. Binding studies were performed on membrane preparations of the mesenteric vascular bed of both groups of rats using saturation with [3H]nitrendipine. Results: The maximal response to AVP was statistically similar in the two groups of arteries. Pregnancy shifted the AVP concentration-response curves to the right. nifedipine (1 μmol/L) similarly reduced the maximum response to AVP in arteries of both groups, but produced a larger increase in EC50, the concentration inducing 50% maximum response, in resistance arteries of virgin versus pregnant rats. Bay K 8644 did not affect the maximum tension reached with AVP. However, it increased the effects of small concentrations of AVP in arteries of both groups. This was more important in tissues of virgin than pregnant rats. Binding of [3H]interdipine to membrane preparations of mesenteric vessels was not modified by pregnancy. Conclusion: Our results suggest a reduced functional influence of POC channels in the myotropic effects of AVP on mesenteric resistance arteries in pregnancy. This decreased influence of POC channels may contribute to resistance of the vasculature to vasopressor agents during pregnancy.

Modulation of body fluids and angiotensin II receptors in a rat model of intra‐uterine growth restriction

We previously reported that sodium restriction during pregnancy reduces plasma volume expansion and promotes intra‐uterine growth restriction (IUGR) in rats while it activates the renin–angiotensin–aldosterone system (RAAS). In the present study, we proceeded to determine whether expression of the two angiotensin II (ANGII) receptor subtypes (AT1 and AT2) change in relation to maternal water–electrolyte homeostasis and fetal growth. To this end, pregnant (gestation day 15) and non‐pregnant Sprague‐Dawley rats were randomly assigned to two groups fed either normal, or Na+‐restricted diets for 7 days. At the end of the treatment period, plasma aldosterone and renin activity as well as plasma and urine electrolytes were measured. Determinations for AT1 and AT2 mRNA and protein were made by RNase protection assay and photoaffinity labelling, respectively, using a number of tissues implicated in volume regulation and fetal growth. In non‐pregnant rats, Na+ restriction decreases Na+ excretion without altering plasma volume, plasma Na+ concentration or the expression of AT1 and AT2 mRNA or protein in the tissues examined. In normally fed pregnant rats when compared to non‐pregnant controls, AT1 mRNA increases in the hypothalamus as well as pituitary and declines in uterine arteries, while AT1 protein decreases in the kidney and AT2 mRNA declines in the adrenal cortex. In pregnant rats, Na+ restriction induces a decrease in plasma Na+, an increase in plasma urea, as well as a decline in renal urea and creatinine clearance rates. Protein levels for both AT1 and AT2 in the pituitary and AT2 mRNA in the adrenal cortex are lower in the Na+‐restricted pregnant group when compared to normally fed pregnant animals. Na+ restriction also induces a decrease in AT1 protein in the placenta. In conclusion, these results suggest that pregnancy may increase sensitivity to Na+ depletion by the tissue‐specific modulation of ANGII receptors. Finally, these receptors may be implicated in the IUGR response to low Na+.

Natriuretic Peptide Receptor-C Attenuates Hypertension in Spontaneously Hypertensive RatsNovelty and Significance: Role of Nitroxidative Stress and Gi Proteins

C-Atrial natriuretic peptide (ANP)4–23, a ring deleted analog of ANP that specifically interacts with natriuretic peptide receptor-C (NPR-C), has been shown to decrease the enhanced expression of Gi&agr; proteins implicated in the pathogenesis of hypertension. In the present study, we investigated whether in vivo treatment of spontaneously hypertensive rats (SHRs) with C-ANP4–23 could attenuate the development of high blood pressure (BP) and explored the underlying mechanisms responsible for this response. Intraperitoneal injection of C-ANP4–23 at the concentration of 2 or 10 nmol/kg body weight to prehypertensive SHRs attenuated the development of high BP, and at 8 weeks it was decreased by ≈20 and 50 mm Hg, respectively; however, this treatment did not affect BP in Wistar-Kyoto rats. C-ANP4–23 treatment of adult SHRs for 2 weeks also attenuated high BP, heart rate, and restored the impaired vasorelaxation toward control levels. In addition, the enhanced levels of superoxide anion (O2−), peroxynitrite, NADPH oxidase activity, and the enhanced expression of Gi&agr; proteins, NOX4, p47phox, nitrotyrosine, and decreased levels of endothelial nitric oxide synthase (eNOS or NOS3) and NO in SHRs were attenuated by C-ANP4–23 treatment; however, the altered levels of NPR-A/NPR-C were not affected by this treatment. In conclusion, these results indicate that NPR-C activation by C-ANP4–23 attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Gi&agr; proteins and nitroxidative stress and not through eNOS/cGMP pathway and suggest that NPR-C ligand may have the potential to be used as therapeutic agent in the treatment of cardiovascular complications including hypertension.

Fetal adrenal gland alterations in a rat model of adverse intrauterine environment

By feeding a low-sodium diet to dams over the last third of gestation, we have developed an animal model of intrauterine growth restriction (IUGR). Given that fetal adrenal development and maturation occur during late gestation in rats, the aim of this study was to evaluate the expression of proteins and enzymes involved in steroidogenesis and catecholamine synthesis in adrenal glands from IUGR fetuses. A gene microarray was performed to investigate for alteration in the pathways participating in hormone production. Results show that increased aldosterone serum concentrations in IUGR fetuses were associated with higher mRNA adrenal levels of angiotensin II receptor type 1 (AT(1)R) and cytochrome P450 aldosterone synthase in response to decreased serum sodium content. Conversely, reduced serum corticosterone concentrations in these fetuses appear to result from alterations in gene expression involved in cholesterol metabolism, such as the augmented apolipoprotein E levels, and in steroidogenesis, like the decreased levels of cytochrome P45011beta-hydroxylase. Furthermore, increased AT(2)R expression and the presence of hypoxia and oxidative stress may, in turn, explain the higher adrenal mRNA levels of enzymes involved in catecholamine synthesis. Despite this increase, catecholamine adrenal content was reduced in males and was similar in females compared with sex-matched controls, suggesting higher catecholamine secretion. This could be associated with the induction of genes involved in inflammation-related, acute-phase response in IUGR fetuses. All of these alterations could have long-lasting health effects and may, hence, be implicated in the pathogenesis of increased blood pressure and cardiac hypertrophy observed in IUGR adult animals from this model.