Volume expansion enhances plasma endothelin-1.

We hypothesized that acute volume expansion by saline infusion triggers the release of endothelin-1. Bolus intravenous saline infusion (8 mL/min) in six groups of conscious Wistar rats and spontaneously hypertensive rats did not change mean arterial pressure or heart rate (n = 8 to 12). At 1 min after infusion, the plasma endothelin-1 level was significantly increased in Wistar rats and in spontaneously hypertensive rats by 42% and 61%, respectively (unpaired data). In 12 Wistar rats, the endothelin-1 level increased from 0.68 +/- 0.13 to 1.19 +/- 0.17 fmol/mL (mean +/- SEM, P <.0001, paired data). Thus, acute volume load by rapid saline infusion increases plasma endothelin-1 levels. Vasoconstriction induced by endothelin-1 may counteract enhanced circumferential stretch created by volume expansion.

The place preference task: a new tool for studying the relation between behavior and place cell activity in rats

This study describes a task that combines random searching with goal directed navigation. The testing was conducted on a circular elevated open field (80 cm in diameter), with an unmarked target area (20 cm in diameter) in the center of 1 of the 4 quadrants. Whenever the rat entered the target area, the computerized tracking system released a pellet to a random point on the open field. Rats were able to learn the task under light and in total darkness, and on a stable or a rotating arena. Visual information was important in light, but idiothetic information became crucial in darkness. Learning of a new position was quicker under light than in total darkness on a rotating arena. The place preference task should make it possible to study place cells (PCs) when the rats use an allothetic (room frame) or idiothetic (arena frame) representation of space and to compare the behavioral response with the PCs' activity.

Disposition of venlafaxine enantiomers in rats with hepatic encephalopathy after chronic drug treatment.

Portacaval shunted (PCS) rats, a model of hepatic encephalopathy, and control animals were administered racemic venlafaxine for 14 days (10 mg/kg). The levels of the S- and R-enantiomers and the S/R-enantiomer ratios of venlafaxine and its metabolites were assessed by an enantiomer-selective chromatographic assay in serum, brain parenchyma, and brain dialysate of both groups. Higher levels of the S- and R-enantiomers of venlafaxine were found in serum and brain of PCS vs. normal rats (median values of S- and R-venlafaxine in serum: 290 and 201 nM in PCS; 97 and 66 nM in normal rats; median values of S- and R-venlafaxine in cortex: 956 and 939 nM in PCS; 357 and 318 nM in normal rats). Interestingly, similar S/R-venlafaxine ratios were observed in PCS and normal rats both in serum (S/R = 1.4) and brain compartments (S/R = l.0-1.1). These findings may have clinical relevance for the safety of venlafaxine in chronic hepatic encephalopathy.

Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae endocarditis in rats.

Cpl-1, a pneumococcal phage lytic enzyme, was tested in rats with experimental endocarditis due to Streptococcus pneumoniae WB4. High-dose regimen Cpl-1 eliminated pneumococci from blood within 30 min and decreased bacterial titers in vegetations (>4 log10 CFU/g) within 2 h. Rapid bacterial lysis induced by Cpl-1 treatment increased cytokine secretion noticeably.

Hypotensive effect of the active fragment derived from factor XII is mediated by an activation of the plasma kallikrein-kinin system.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

Mechanisms of the anti-obesity effects of oxytocin in diet-induced obese rats.

Apart from its role during labor and lactation, oxytocin is involved in several other functions. Interestingly, oxytocin- and oxytocin receptor-deficient mice develop late-onset obesity with normal food intake, suggesting that the hormone might exert a series of beneficial metabolic effects. This was recently confirmed by data showing that central oxytocin infusion causes weight loss in diet-induced obese mice. The aim of the present study was to unravel the mechanisms underlying such beneficial effects of oxytocin. Chronic central oxytocin infusion was carried out in high fat diet-induced obese rats. Its impact on body weight, lipid metabolism and insulin sensitivity was determined. We observed a dose-dependent decrease in body weight gain, increased adipose tissue lipolysis and fatty acid β-oxidation, as well as reduced glucose intolerance and insulin resistance. The additional observation that plasma oxytocin levels increased upon central infusion suggested that the hormone might affect adipose tissue metabolism by direct action. This was demonstrated using in vitro, ex vivo, as well as in vivo experiments. With regard to its mechanism of action in adipose tissue, oxytocin increased the expression of stearoyl-coenzyme A desaturase 1, as well as the tissue content of the phospholipid precursor, N-oleoyl-phosphatidylethanolamine, the biosynthetic precursor of the oleic acid-derived PPAR-alpha activator, oleoylethanolamide. Because PPAR-alpha regulates fatty acid β-oxidation, we hypothesized that this transcription factor might mediate the oxytocin effects. This was substantiated by the observation that, in contrast to its effects in wild-type mice, oxytocin infusion failed to induce weight loss and fat oxidation in PPAR-alpha-deficient animals. Altogether, these results suggest that oxytocin administration could represent a promising therapeutic approach for the treatment of human obesity and type 2 diabetes.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

Transitory glutathione deficit during brain development induces cognitive impairment in juvenile and adult rats: relevance to schizophrenia.

Glutathione (GSH) metabolism dysfunction is one risk factor in schizophrenia. A transitory brain GSH deficit was induced in Wistar (WIS) and mutant (ODS; lacking ascorbic acid synthesis) rats using BSO (l-buthionine-(S,R)-sulfoximine) from post-natal days 5-16. When GSH was re-established to physiological levels, juvenile BSO-ODS rats were impaired in the water maze task. Long after treatment cessation, adult BSO-WIS/-ODS rats showed impaired place discrimination in the homing board with distributed visual or olfactory cues. Their accuracy was restored when a single cue marked the trained position. Similarly, more working memory errors were made by adult BSO-WIS in the radial maze when several olfactory cues were present. These results reveal that BSO rats did not suffer simple sensory impairment. They were selectively impaired in spatial memory when the task required the integration of multimodal or olfactory cues. These results, in part, resemble some of the reported olfactory discrimination and cognitive impairment in schizophrenia.

Spaced training facilitates long-term retention of place navigation in adult but not in adolescent rats

Young and adult Long Evans rats were tested in the water maze according to two different procedures: half of the subjects were given one session of four trials a day for 6 days, whereas the other subjects had the same amount of training massed in 1 day. For both conditions, a 14-day retention interval was then introduced to test long-term memory. This was followed by a four-trial reversal session. All groups showed a significant learning curve, but escape latencies were shorter for the adult than for the young rats, without differential effect of the training procedure. A first probe trial (PT1) confirmed similar accurate short-term retention in all the groups. But unimpaired long-term memory was only seen in the adult rats trained with the spaced procedure. The young rats trained over 1 day also showed some retention of the platform location after 14 days, but not the other two groups. Reversal acquisition of the new platform location was rapid in the four groups. These results indicate that although accurate short-term spatial memory in the water maze is seen after a 1-day massed training in both age groups, unimpaired long-term retention is only observed in adult rats trained with 24-h inter-session intervals.

The PPARbeta/delta agonist GW0742 relaxes pulmonary vessels and limits right heart hypertrophy in rats with hypoxia-induced pulmonary hypertension.

BACKGROUND: Pulmonary vascular diseases are increasingly recognised as important clinical conditions. Pulmonary hypertension associated with a range of aetiologies is difficult to treat and associated with progressive morbidity and mortality. Current therapies for pulmonary hypertension include phosphodiesterase type 5 inhibitors, endothelin receptor antagonists, or prostacyclin mimetics. However, none of these provide a cure and the clinical benefits of these drugs individually decline over time. There is, therefore, an urgent need to identify new treatment strategies for pulmonary hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the PPARbeta/delta agonist GW0742 induces vasorelaxation in systemic and pulmonary vessels. Using tissue from genetically modified mice, we show that the dilator effects of GW0742 are independent of the target receptor PPARbeta/delta or cell surface prostacyclin (IP) receptors. In aortic tissue, vascular relaxant effects of GW0742 were not associated with increases in cGMP, cAMP or hyperpolarisation, but were attributed to inhibition of RhoA activity. In a rat model of hypoxia-induced pulmonary hypertension, daily oral dosing of animals with GW0742 (30 mg/kg) for 3 weeks significantly reduced the associated right heart hypertrophy and right ventricular systolic pressure. GW0742 had no effect on vascular remodelling induced by hypoxia in this model. CONCLUSIONS/SIGNIFICANCE: These observations are the first to show a therapeutic benefit of 'PPARbeta/delta' agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.

Intrahippocampal cholinergic grafts in aged rats compensate impairments in a radial maze and in a place learning task

Age-related cognitive impairments were studied in rats kept in semi-enriched conditions during their whole life, and tested during ontogeny and adult life in various classical spatial tasks. In addition, the effect of intrahippocampal grafts of fetal septal-diagonal band tissue, rich in cholinergic neurons, was studied in some of these subjects. The rats received bilateral cell suspensions when aged 23-24 months. Starting 4 weeks after grafting, they were trained during 5 weeks in an 8-arm maze made of connected plexiglass tunnels. No age-related impairment was detected during the first eight trials, when the maze shape was that of a classical radial maze in which the rats had already been trained when young. The older rats were impaired when the task was made more difficult by rendering two arms parallel to each other. They developed an important neglect of one of the parallel tunnels resulting in a high amount of errors before completion of the task. In addition, the old rats developed a systematic response pattern of visits to adjacent arms in a sequence, which was not observed in the younger subjects. None of these behaviours were observed in the old rats with a septal transplant. Sixteen weeks after grafting, another experiment was conducted in a homing hole board task. Rats were allowed to escape from a large circular arena through one hole out of many, and to reach home via a flexible tube under the table. The escape hole was at a fixed position according to distant room cues, and olfactory cues were made irrelevant by rotating the table between the trials. An additional cue was placed on the escape position. No age-related difference in escape was observed during training. During a probe trial with no hole connected and no proximal cue present, the old untreated rats were less clearly focussed on the training sector than were either the younger or the grafted old subjects. Taken together, these experiments indicate that enriched housing conditions and spatial training during adult life do not protect against all age-related deterioration in spatial ability. However, it might be that the considerable improvement observed in the grafted subjects results from an interaction between the graft treatment and the housing conditions.

Subconjunctival Injection of XG-102, a c-Jun N-Terminal Kinase Inhibitor Peptide, in the Treatment of Endotoxin-Induced Uveitis in Rats.

Abstract Purpose: XG-102, a TAT-coupled dextrogyre peptide inhibiting the c-Jun N-terminal kinase, was shown efficient in the treatment of experimental uveitis. Preclinical studies are now performed to determine optimal XG-102 dose and route of administration in endotoxin-induced uveitis (EIU) in rats with the purpose of clinical study design. METHODS: EIU was induced in Lewis rats by lipopolysaccharides (LPS) injection. XG-102 was administered at the time of LPS challenge by intravenous (IV; 3.2, 35 or 355 μg/injection), intravitreal (IVT; 0.08, 0.2 or 2.2 μg/eye), or subconjunctival (SCJ; 0.2, 1.8 or 22 μg/eye) routes. Controls received either the vehicle (saline) or dexamethasone phosphate injections. Efficacy was assessed by clinical scoring, infiltrating cells count, and expression of inflammatory mediators [inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant-1 (CINC-1)]. The effect of XG-102 on phosphorylation of c-Jun was evaluated by Western blot. RESULTS: XG-102 demonstrated a dose-dependent anti-inflammatory effect in EIU after IV and SCJ administrations. Respective doses of 35 and 1.8 μg were efficient as compared with the vehicle-injected controls, but only the highest doses, respectively 355 and 22 μg, were as efficient as dexamethasone phosphate. After IVT injections, the anti-inflammatory effect of XG-102 was clinically evaluated similar to the corticoid's effect with all the tested doses. Regardless of the administration route, the lowest efficient doses of XG-102 significantly decreased the ration of phospho c-Jun/total c-Jun, reduced cells infiltration in the treated eyes, and significantly downregulated iNOS and CINC-1 expression in the retina. CONCLUSION: These results confirm that XG-102 peptide has potential for treating intraocular inflammation. SCJ injection appears as a good compromise to provide a therapeutic effect while limiting side effects.

Calcium-sensing receptors modulate renin release in vivo and in vitro in the rat.

OBJECTIVES: Calcium-sensing receptors (CaSRs) have been localized in the juxtaglomerular apparatus where they may contribute to the regulation of renin release. In the present study, we investigated the in-vitro and in-vivo effects of the calcimimetic R-568 on renin release. METHODS: In vitro, the effect of calcimimetics on renin release was assessed by incubating freshly isolated rat juxtaglomerular cells with or without R-568 (1 and 10 mumol/l) in serum-free medium in the presence or absence of forskolin or CaCl2. In vivo, we measured the impact of R-568 (20 ng/min intravenously) on the acute changes in plasma renin activity (PRA) induced by either a 90 min infusion of the angiotensin-converting enzyme inhibitor captopril, or the beta-receptor agonist isoproterenol, or of a vehicle in or after a furosemide challenge in conscious Wistar rats. RESULTS: In vitro, R-568 dose-dependently blunted renin release, but also reduced the increase in renin due to forskolin (P < 0.01). Both isoproterenol and enalapril increased in vivo PRA to 3.1 +/- 0.3 and 3.7 +/- 0.5 ng Ang I/ml per h, respectively (P < 0.01), compared with vehicle (1.5 +/- 0.2 ng Ang I/ml per h). R-568 significantly reduced PRA to 2.1 +/- 0.1 ng/ml per h in isoproterenol-treated rats and to 1.6 +/- 0.2 ng/ml per h in enalapril-treated rats (P < 0.05). In low-salt treated animals, acute infusion of furosemide increased PRA from 8.7 +/- 3.2 to 18.6 +/- 2.3, whereas R-568 partially blunted this rise to 11.2 +/- 1.5 (P = 0.02). In vivo, R-568 significantly lowered serum calcium and PTH1-84, but the drug-induced changes in PRA were independent of the changes in calcium and parathyroid hormone. CONCLUSION: After the recent discovery of CaSRs in juxtaglomerular cells of mice, our results confirm the presence of such receptors in rats and demonstrate that these receptors modulate renin release both in vitro and in vivo. This suggests that CaSRs play a role as a regulatory pathway of renin release.

Endothelin-1-induced spreading depression in rats is associated with a microarea of selective neuronal necrosis.

Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Leão and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1-induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 muM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ET(A) receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ET(A) receptor.