Antenatal glucocorticoids blunt the functioning of the hypothalamic-pituitary-adrenal axis of neonates and disturb some behaviors in juveniles.

Antenatal glucocorticoids are highly effective in preventing respiratory distress of premature babies but can induce physiological and behavioral disturbances in young infants as well as in animals. Therefore, the hypothalamic-pituitary-adrenal (HPA) axis of rat neonates, and the consequences on behavioral development of offspring have been studied after five antenatal injections of dexamethasone (DEX) or vehicle. DEX decreased offspring body weight at birth, and significantly delayed the normal growth for the first 3 weeks of life. This paralleled diminished behavioral performances measured on postnatal day 3 (righting reflex) and postnatal day 10 (grasping test). Circulating levels of adrenocorticotrophin (ACTH) and corticosterone were significantly decreased on postnatal day 1 and this was related to a diminution of HPA axis activity shown by the decrease of central expression of corticotropin releasing hormone (CRH) mRNA, immunoreactive content in paraventricular neurons (PVN) and in the median eminence endings were significantly decreased. On the other hand, expression of another secretagogue of ACTH, arginine vasopressin (AVP), was differently affected in the PVN parvocellular neurons of offspring of the DEX group since AVP mRNA increased whereas immunoreactive content of the PVN parvocellular neurons was lowered. Simultaneously, the co-production of AVP and CRH in PVN neurons was stimulated. This can support the view that antenatal DEX reached the fetus and produced some damage which did not parallel that induced by prenatal stress of the pregnant females, especially the low body weight of offspring. The harmful consequence of antenatal DEX treatment was not restrictively due to the blunting of the HPA axis but also to the low body weight, which disturbed behavioral performances for the first weeks of life and could participate in other disorders in adult life.

Aortic stiffness and left ventricular mass in a rat model of isolated systolic hypertension.

We tested whether cardiac mass can be related to decreased aortic stiffness in an original rat model of isolated systolic hypertension. Increased aortic stiffness was produced by calcium overload of elastic arteries after vitamin D3 plus nicotine treatment. Half of the animals were chronically treated with the angiotensin-converting enzyme inhibitor perindopril (1 mg/kg per day PO). Rats were pithed, and lower body vascular resistance was measured. Blood pressure was then increased by phenylephrine infusion, and carotidofemoral pulse wave velocity was measured. This value together with those for thoracic aorta internal diameter and medial thickness (determined after in situ fixation and histomorphometry) were used to calculate elastic modulus. Vitamin D3 plus nicotine treatment produced parallel increases in cardiac mass and elastic modulus, with a significant correlation between the two. There was no significant change in resistance. Treatment with perindopril reversed the changes in cardiac mass and elastic modulus but had no effect on resistance after calcium overload of the elastic arteries. In this model of isolated systolic hypertension, we showed that cardiac mass is related to arterial elasticity.

Galanin receptor antagonists decrease fat preference in Brattleboro rat.

The Brattleboro rat eats spontaneously 46% of its diet per day in fat when given a choice of carbohydrate, protein and fat. An overexpression of galanin (GAL) has been also observed in the hypothalamic paraventricular nuclei (PVN). This associative correlation has led to a hypothesis of a functional relation between central galanin expression and the preference for a lipid diet. In the present experiments, the effects of two GAL receptor antagonists, C7 and galantide, on fat consumption and central overexpression of GAL were investigated. Both antagonists were injected into either the cerebral ventricles or directly above the PVN, and the diet consumption followed for the subsequent 24h. C7 decreased significantly fat consumption when injected into the ventricles or directly above the PVN. In contrast, galantide must be injected above the PVN to show the same effect. However, the two antagonists did not modify GAL mRNA expression in the PVN when they were injected 2h before sacrifice. These experiments confirm a functional link between the preferential consumption of fat and hypothalamic Galanin; different subtypes of the GAL receptor are probably involved, since both Galanin antagonists were differently efficient in decreasing spontaneous fat selection of the Brattleboro rat.

Increased arterial distensibility induced by the angiotensin-converting enzyme inhibitor, lisinopril, in normotensive rats.

1. We investigated possible structural correlates of the beneficial effect of chronic angiotensin-converting enzyme inhibition (ACEI) with lisinopril on the aortic distensibility of normotensive rats. 2. Experiments were performed in young (4-month old), normotensive, Wistar rats which received lisinopril in their drinking water (0.9 or 9 mg kg-1 day-1) for 9 months. 3. Following ACEI treatment, rats were pithed and aortic pulse wave velocity was measured during the progressive rise in mean arterial blood pressure produced by i.v. infusion of the alpha 1-adrenoceptor agonist, phenylephrine. The slope of the regression line relating aortic pulse wave velocity to mean arterial blood pressure was taken as an index of aortic distensibility. Following this, the aorta was fixed in situ at a normotensive pressure level and histomorphometry was performed. We also measured the calcium content of the aortic wall by atomic absorption. 4. The lower dose of lisinopril failed to lower systolic arterial blood pressure (unanaesthetized rat) or mean arterial blood pressure (pithed rat). Chronic ACEI with the higher dose of lisinopril lowered both systolic arterial blood pressure (104 +/- 6 mmHg, controls 133 +/- 4 mmHg, unanaesthetized), and mean arterial blood pressure (27 +/- 1 mmHg, controls 34 +/- 2 mmHg, pithed). 5. Although the lower dose of lisinopril did not lower blood pressure, it did improve aortic distensibility as revealed by a fall in the slope relating aortic pulse wave velocity (Y) to mean arterial blood pressure (X). Values were 5.7 +/- 0.7, 3.8 +/- 0.6 and 2.7 +/- 0.3 in controls, and in low and high ACEI groups, respectively. 6. Lisinopril treatment did not modify the calcium content, the internal and external diameters or the medial thickness of the aorta. Chronic ACEI did, however, increase the thickness of the medial elastic fibres (controls 3.55 +/- 0.05 microm, low dose ACEI 4.05 +/- 0.15 gm (P<0.05), and high dose ACEI4.18 +/- 0.15 microm (P<0.05)).7. In conclusion, we would suggest that ACEI treatment with a low dose of lisinopril can decrease aortic stiffness via a pressure-independent mechanism which possibly involves an effect of ACEI on elastic fibres.

Neuronal Uptake of Monoclonal Anti-Vasopressin Antibodies in vivo and Relationship with the Physiological Status of Vasopressin Neurosecretory Cells.

Abstract Mouse monoclonal anti-vasopressin antibody was injected just above one hypothalamic paraventricular nucleus of the rat brain. Immunocytochemistry and morphometric analysis showed that the antibody was taken up by neurons with the size and stereotaxic distribution of vasopressin-producing neurons. Labelled neurons were counted with an electronic image analyser. When the rats were deprived of drinking water for 48 h before injection, specific uptake was significantly increased in the caudal part of the nucleus. Conversely, rehydration following dehydration or chronic treatment with 1-deamino-8-D-arginine vasopressin significantly decreased neuronal uptake, mainly in the rostral subdivision of the nucleus. By contrast, bilateral adrenalectomy performed 2 weeks before injection did not modify the number of labelled magnocellular neurons, though the accumulation of injected antibody in the external layer of the median eminence indirectly demonstrated the stimulation of antibody uptake by parvocellular vasopressin neurons. The number of labelled neurons was therefore directly related to the physiological state of the vasopressin-producing neurons. Further investigations will have to be performed to prove that the immunological targeting of peptidergic neurons offers a new tool to act in vivo on central neurons.

Immunotargeted lesions of paraventricular CRF and AVP neurons in developing rats reveal the pattern of maturation of these systems and their functional importance.

Pituitary ACTH secretion in the rat is controlled by a number of hypothalamic secretagogues, like CRF and AVP and by inhibitory feedback provided by glucocorticoids. During development, little is known about the precise regulation of ACTH release by hypothalamic neuropeptides and glucocorticoids. We used immunotargeted chemical PVN lesions to investigate the role of CRF and AVP neurons of the hypothalamic paraventricular nucleus (PVN) in the control of ACTH secretion in neonatal rats under basal conditions and 5 days after adrenalectomy (ADX). Neonates aged day (d) 4 or d14 were injected over the PVN with ricin A toxin associated with either non-specific antibodies (IgG/Tx), or monoclonal antibodies directed towards CRF (CRF/Tx) or AVP (AVP/Tx). Rats from each group received either sham surgery (SHAM) or were adrenalectomized (ADX). Pups were sacrificed 5 days after PVN treatment and adrenal surgery (d9 or 19). Plasma ACTH and corticosterone (B) levels were measured by RIAs. Changes in CRF and AVP expression in the PVN and other brain regions were determined by immunohistochemistry (ICC) and in situ hybridization. Injection of the toxin associated with IgGs did not have non specific effects on body weight gain, neuropeptide expression or plasma ACTH and B secretion compared to intact, uninjected rats. Lesions of CRF or AVP neurons greatly reduced peptide expression and mRNA levels in the PVN and median eminence at both ages. However, the specificity of the lesion was greater in older than in young pups. At both ages, we observed a dissociation between the morphological effects of the lesions and hormonal responses. In d14-19 pups, CRF and AVP lesions prevented ADX-induced changes in mRNA levels and peptide expression but did not reduce ACTH secretion under basal or stimulated (post ADX) conditions. However, CRF and AVP lesions increased the expression of CRF in the central amygdala and the bed nucleus of the stria terminalis. Lesions with AVP also stimulated CRF expression in the PVN. Thus, these compensatory changes could take over some of the hypophysiotropic actions of the damaged PVN neurons. In young pups (d4-9), we did not observe the typical increase in CRF and AVP mRNA levels and peptide expression found after ADX in older pups or adults. Lesions of the CRF neurons also affected the AVP system and reciprocally. We suggest that this could be explained by a high degree of colocalization of CRF and AVP observed in parvocellular and small, immature magnocellular neurons in young pups. The lesions did not affect basal or ADX-induced ACTH secretion, suggesting that during the early neonatal period, the pituitary is the major site of glucocorticoid inhibitory feedback on ACTH secretion and that the hypothalamus does not exert a tonic control over basal pituitary secretion. These results unravel ontogenetical differences in the regulation of ACTH secretion by hypothalamic CRF and AVP. During the first 10 days of life, within the adrenal stress hyporesponsive period, hypothalamic CRF and AVP neurons are not sensitive to glucocorticoid feedback and basal ACTH secretion appears to be relatively independent from hypothalamic input. After the second week of life, maturation of glucocorticoid receptors, neuronal phenotype and connections of the PVN to other brain structures (bed nucleus of the stria terminalis, central amygdala) allows for the full expression of corticosterone effect on hypothalamic neurons and for compensatory changes to occur following lesions. These results emphasize the extraordinary capacity of the developing central nervous system to adapt to changes in functionning of some neuronal areas critical for homeostatic balance and the important potential role of intra-hypothalamic and extrahypothalamic relationships in maintaining control over ACTH and glucocorticoid production during development.

Quantitative analysis of corticotropin-releasing factor and arginine vasopressin mRNA in the hypothalamus during chronic morphine treatment in rats: an in situ hybridization study.

The content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN) increases during chronic morphine treatment. Because these experiments cannot distinguish between increased synthesis or reduced release, the present study measured changes in CRF and AVP mRNAs in the PVN by in situ hybridization. Concomitantly, changes in noradrenaline turnover in the PVN and changes in plasma corticosterone release were determined. Male rats were implanted with placebo (naive) or morphine pellets for 7 days. On day 7, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg, i.p.). Acute morphine injection did not change the total size of the labelled area for CRF mRNA in the PVN of naive or morphine-pelleted rats, indicating that the number of CRF-containing neurones was unchanged. On the other hand, in rats chronically treated with morphine, the intensity of labelling for CRF mRNA was significantly reduced, suggesting a decrease in the synthesis of CRF. In placebo rats, injection of saline or morphine did not affect the surface hybridized for AVP mRNA. By contrast, in the morphine-group injected with saline, there was a significant reduction in the number of labelled neurones, measured by the size of labelled area. Similarly, there was a decrease in intensity of AVP mRNA expression in the parvocellular and magnocellular neurones of the PVN in the morphine-group injected with saline, suggesting a decreased synthesis of AVP in these neurones. In parallel with the decrease in the expression of CRF and AVP mRNAs in the PVN, there was a pronounced decrease in noradrenaline turnover and in the release of corticosterone in the morphine-pelleted rats. In conclusion, present results show that, in addition to modifications in corticosterone secretion and in noradrenaline turnover, chronic morphine administration produces a reduction in the synthesis of CRF and AVP.

Effect of naloxone-precipitated morphine withdrawal on CRH and vasopressin mRNA expression in the rat hypothalamic paraventricular nucleus.

Morphine withdrawal is characterized by an increase in the hypothalamus-pituitary-adrenocortical (HPA) axis activity. Here, by means of in situ hybridization, the changes in CRH and vasopressin (AVP) mRNAs have been analysed within the rat hypothalamic paraventricular nucleus (PVN) during morphine dependence and after naloxone-precipitated morphine withdrawal. CRH and AVP mRNA expression were analysed 30 min following administration of saline or naloxone to control groups and to morphine dependent rats. The data for in situ hybridization analysis of PVN neurons show that there were no changes in the total size of labelled area for CRH or AVP mRNA during morphine withdrawal, indicating that dependence on morphine does not involve alterations in the number of neurons expressing CRH or AVP mRNA. However, levels of mRNA encoding for CRH were decreased in the PVN during morphine dependence and withdrawal. By contrast, injection of saline or naloxone to morphine dependent rats did not affect the intensity of AVM mRNA expression. All these findings are discussed in term of cellular events that couple morphine dependence-increased HPA axis activity with changes in gene expression in selective neurons of the PVN.

[Endothelial morphometry and perforating keratoplasties]. / Morphométrie endothéliale et kératoplasties perforantes.

50 corneal grafts were studied by specular microscopy. The endothelial cell morphology of the grafts was analysed by using a computerised image analysis system. The endothelial cell density decreased rapidly during the first three post-operative months (17%). It continued to decrease for the first year (52%, after which cell loss occurred at a slower rate (3% of the sample cell count per year). The coefficient of variation in cell area remained constant (27%). The percentage of endothelial cell loss might be closely associated with the reestablishment of the hexagonal cellular pattern. These results suggest that the endothelium of the graft is in a state of transition during the first year after surgery.

[The co-localization of neurohypophysis peptides and the corticotropin-releasing factor in the rat brain. Contribution of morphometric analyses]. / La colocalisation des peptides neurohypophysaires et de la corticolibérine dans le cerveau de rat. Apport des méthodes d'analyse d'images.

Using adjacent serial brain sections, a morphometric method has been developed for analysing the coexistence of the neurophysial hormones, vasopressin (VP) and oxytocin (OT), with their specific neurophysins (N). A significative correlation was found between the immunoreactive areas stained with (1) anti-VP and anti-N-VP sera, and between the immunoreactive areas detected with anti-OT and anti-N-OT antibodies. Besides, the immunoreactive areas stained with (1) anti-VP and anti-OT antibodies, (2) anti-N-OT and anti-VP antibodies, (3) anti-N-OT and anti-N-VP antibodies or (4) anti-OT and anti-N-VP antibodies were totally independent. A different method projecting the microscope images on a reference grid with a camera lucida permitted to quantify the coexistence of an ovine corticotropin-releasing factor-related-peptide (41-CRF) with OT in the paraventricular neurons of the Brattloro rat brain. In these animals, the same method applied after total hypophysectomy demonstrated that the neurons synthezising simultaneously 41-CRF and OT projected their axons to the neurohypophysis; the same operation increased the relative number of neurons containing 41-CRF only; it can be supposed that they originated the infundibular terminals.

[Postnatal changes in the content of neurohypophyseal enzymes in the Brattleboro rat]. / Evolution postnatale du contenu en hormones neurohypophysaires chez le Rat Brattleboro.

The development of the neurohypophysial content of vasopressin and oxytocin in the Brattleboro rats is investigated. During the early development stages, no alteration of the oxytocin content could be observed owing to the reduced synthesis of vasopressin in the heterozygous rats (Di/+) or to the lack of this hormone in the homozygous rats (Di/Di).

Dietary preferences of Brattleboro rats correlated with an overexpression of galanin in the hypothalamus.

Galanin (GAL) is a neuropeptide cosynthesized with vasopressin (AVP) in neurons of the hypothalamo-neurohypophysial system. It increases food intake when injected into the brain and elicits an overconsumption of fat. The Brattleboro rat (DI) is genetically unable to produce AVP; the AVP-deficient-producing neurons of the hypothalamo-neurohypophysial system of DI rats are chronically stimulated and DI rats suffer from diabetes insipidus. We studied the central expression of GAL and the dietary preferences in the DI rat. GAL was overexpressed in the hypothalamus of the DI rat. GAL mRNA was higher by 1.8-fold in the supraoptic (P < 0.05) and by four-fold in the paraventricular nuclei (P < 0.001) of male and female DI rats compared with those of control Long Evans (LE) rats. However, GAL mRNA was lower in the arcuate nuclei of DI rats and equal to that of LE rats in the dorsomedian nuclei. We also measured a high preference for a lipid diet (45% of the daily consumption) when DI rats ate from a choice of the three macronutrients. Chronic infusion with deamino-8D-AVP (agonist of AVP V2 receptors) prevented the diabetes insipidus and the chronic stimulation of the hypothalamo-neurohypophysial system of the DI rats. However, the treatment did not suppress the overexpression of GAL, nor did it affect the rats' preference for a lipid diet. We conclude that the DI rat provides a novel animal model in which a spontaneous dietary preference correlates with the overexpression of one of the hypothalamic peptides, GAL.

Comparative immunocytochemical localization of corticotropin releasing factor (CRF-41) and neurohypophysial peptides in the brain of Brattleboro and Long-Evans rats.

The localization of CRF-41 related peptide was studied in the brain and posterior pituitary of the homozygous rats for the inherited diabetes insipidus (Brattleboro strain, DI) and of the Long-Evans rats (LE) as control. It was compared to the distribution of vasopressin (AVP), oxytocin (OXY) and OXY-neurophysin (N I). In both strains, CRF-41 was identified in two morphologically distinct systems: one was a hypothalamoneurohypophysial system simultaneously containing CRF-41, OXY and N I; the other was a hypothalamoinfundibular system carrying CRF-41 only. CRF containing neurons were located in the periventricular area of the anterior hypothalamus, in the retrochiasmatic part of the supraoptic nuclei (SON) and, for some of them, in the antechiasmatic part of SON. CRF immunostainings were enhanced by colchicine treatment in LE rats and by DDAVP therapy in DI rats.

A new technique for study of impact of arterial elasticity on left ventricular mass in rats.

We investigated possible links between left ventricular mass and central arterial elasticity in the adult spontaneously hypertensive rat (SHR) and in a subgroup of SHR in which blood pressure was normalized by chronic antihypertensive drug treatment; results were compared with those of age-matched normotensive Wistar-Kyoto rats. Two indexes of arterial elasticity, based on the measurement of aortic pressure pulse wave velocity, were used. First, the slope relating carotidofemoral pulse wave velocity to blood pressure in the phenylephrine-infused pithed preparation was used as a pressure-independent index of wall elasticity. Second, to account for hypertension- and treatment-induced aortic remodeling, elastic modulus was determined from the pulse wave velocity recorded when blood pressure reached that measured in awake animals before anesthesia and pithing, together with values for wall thickness and lumen diameter evaluated by histomorphometric analysis after in situ fixation at the same pressure. In control SHR, regression analysis of variance revealed significant correlations between left ventricular mass and both wave velocity/pressure slope and elastic modulus. Chronic antihypertensive treatment normalized all three parameters. In conclusion, this new technique provides experimental evidence of a link between left ventricular mass and central arterial elasticity.