Etifoxine improves peripheral nerve regeneration and functional recovery.

Peripheral nerves show spontaneous regenerative responses, but recovery after injury or peripheral neuropathies (toxic, diabetic, or chronic inflammatory demyelinating polyneuropathy syndromes) is slow and often incomplete, and at present no efficient treatment is available. Using well-defined peripheral nerve lesion paradigms, we assessed the therapeutic usefulness of etifoxine, recently identified as a ligand of the translocator protein (18 kDa) (TSPO), to promote axonal regeneration, modulate inflammatory responses, and improve functional recovery. We found by histologic analysis that etifoxine therapy promoted the regeneration of axons in and downstream of the lesion after freeze injury and increased axonal growth into a silicone guide tube by a factor of 2 after nerve transection. Etifoxine also stimulated neurite outgrowth in PC12 cells, and the effect was even stronger than for specific TSPO ligands. Etifoxine treatment caused a marked reduction in the number of macrophages after cryolesion within the nerve stumps, which was rapid in the proximal and delayed in the distal nerve stumps. Functional tests revealed accelerated and improved recovery of locomotion, motor coordination, and sensory functions in response to etifoxine. This work demonstrates that etifoxine, a clinically approved drug already used for the treatment of anxiety disorders, is remarkably efficient in promoting acceleration of peripheral nerve regeneration and functional recovery. Its possible mechanism of action is discussed, with reference to the neurosteroid concept. This molecule, which easily enters nerve tissues and regulates multiple functions in a concerted manner, offers promise for the treatment of peripheral nerve injuries and axonal neuropathies.

Gastroprotective effect of Saccharomyces boulardii in a rat model of ibuprofen-induced gastric ulcer.

BACKGROUND/AIMS: Saccharomyces boulardii is a probiotic yeast which has been shown to protect the gastrointestinal microflora from disequilibrium and from associated gastrointestinal disorders. However, no study has explored the potential effect of this probiotic in ulcer models. METHODS: The present study was designed to address this goal using the ibuprofen-induced ulcer rat model. RESULTS: Oral administration of ibuprofen (100 mg/kg) for 6 consecutive days induced ulceration of the gastric mucosa. Oral co-administration of S. boulardii (Biocodex, France) at 1.2, 4 or 12 x 10(10) CFU/kg dose-dependently and significantly reduced the numbers of gastric ulcers and the ulceration surface of the gastric mucosa. At the same time, serum nitrate and nitrite levels were measured before and on the 6th day. Contrary to what we expected, the serum nitrate and nitrite levels did not increase after ibuprofen administration, but this parameter significantly augmented in the groups where ibuprofen was co-administered with the two highest doses of S. boulardii. CONCLUSION: The present findings suggest that S. boulardii offers some potential in the treatment or prevention of ulcers induced by non-steroidal anti-inflammatory drugs, but its mechanism of action needs to be further explored.

Preventive and curative effects of etifoxine in a rat model of brain oedema.

1. The aim of the present study was to test the hypothesis that increasing GABAergic neurotransmission is involved in the prevention or treatment of brain oedema. The study was conducted in the well-established rat triethyltin (TET) model of brain oedema and examined the effects of etifoxine, a compound that increases GABAergic neurotransmission through multiple mechanisms, including neurosteroid synthesis. 2. Daily oral administration of 3 mg/kg per day TET for 5 consecutive days strongly perturbed rat behaviour and induced reproducible cerebral oedema. Coadministration of etifoxine (2 x 25 or 2 x 50 mg/kg per day, p.o.) over the 5 days of TET treatment blocked the development of brain oedema and the increase in brain sodium content induced by TET, as well as reducing the increase in brain chloride content. Moreover, etifoxine inhibited the decrease in bodyweight, the neurological deficit and the altered locomotor activity induced by TET. At a lower dose (2 <--> 10 mg/kg per day, p.o.), etifoxine did not have any preventive effects. 3. To examine the curative effects of etifoxine, it was administered from the 4th day of TET treatment for 5 consecutive days, when brain oedema was already established. In these experiments, etifoxine (2 <--> 50 mg/kg per day, p.o.) significantly reduced cerebral oedema and the outcomes induced by TET treatment. Moreover, etifoxine reduced the mortality in response to TET treatment. 4. In conclusion, because etifoxine has a good safety profile as an anxiolytic, the results of the present study suggest that it is worth further clinical investigation as a neuroprotectant.

Contribution of transient receptor potential vanilloid subtype 1 to the analgesic and antihyperalgesic activity of nefopam in rodents.

In order to further elucidate the mechanism(s) of action of analgesic and antihyperalgesic nefopam, its interactions with the transient receptor potential vanilloid subtype 1 (TRPV1) were investigated. In sensory neurons of rat embryos, dorsal root ganglion (DRG) in culture, nefopam (3-30 mumol/l) and capsazepine (TRPV1 antagonist, 10 mumol/l) prevented intracellular calcium elevation and calcitonin gene-related peptide release induced by vanilloid agonist capsaicin. Unlike nefopam, capsazepine failed to inhibit these same responses induced by KCl excess. In vivo, nefopam (0.5 and 2 mg/kg, i.v.) and capsazepine (40 mg/kg, i.p.) reduced the licking response due to intraplantar injection of capsaicin in mice. These findings suggest that nefopam exerts its analgesic and antihyperalgesic effects through multiple mechanisms including blockade of TRPV1 in addition to voltage-dependent calcium channels in the DRG.

Modulation of paracetamol and nefopam antinociception by serotonin 5-HT(3) receptor antagonists in mice.

BACKGROUND/AIMS: Post-operative nausea and vomiting are common adverse events that require administration of anti-emetic compounds, such as the serotonin 5-HT(3) receptor antagonists, but these drugs can also reduce the analgesic efficacy of some analgesics (paracetamol, tramadol). METHODS: The present study was designed to explore the effect of 3 serotonin 5-HT(3) receptor antagonists on the antinociceptive efficacy of another frequently used post-operative analgesic, nefopam, in the mouse writhing and formalin tests. RESULTS: Pre-treatment with tropisetron, ondansetron or MDL72222 did not significantly modify nefopam antinociception in both tests. However, paracetamol antinociception was blocked by ondansetron in the formalin test. CONCLUSION: These results provide a rationale for the clinical use of nefopam with anti-emetics during surgery.

Investigation of the anticonvulsive effect of acute immobilization stress in anxious Balb/cByJ mice using GABA A-related mechanistic probes.

RATIONALE: A disordered regulation of neuroactive steroids release in response to acute stress could induce GABAergic dysfunctions underlying anxiety disorders. OBJECTIVES: First, we conducted studies indicating that a short immobilization stress in anxious Balb/cByJ mice produced an anticonvulsive effect. Second, the effects of different positive allosteric modulators (etifoxine, progesterone, clonazepam, and allopregnanolone) of GABA A receptors were compared in a mouse model mimicking the disruption of the acute stress-induced neuroactive steroids release with finasteride (types I and II 5alpha-reductase inhibitor). RESULTS: The acute stress-induced anticonvulsive effect, expressed by the threshold dose of t-butylbicyclophosphorothionate-producing clonic seizures, was time-dependent. The extent of the enhancement of acute stress-induced anticonvulsive effect was lowered in the presence of finasteride. The same effect was observed with PK11195, which behaves as an antagonist of the peripheral benzodiazepine receptor in the dose range used in this study. Picrotoxin reduced the acute stress anticonvulsive effect, proving that this effect operates through the GABA A receptor. Contrary to progesterone (up to 30 mg/kg), etifoxine (50 mg/kg), allopregnanolone (10 mg/kg), and clonazepam (10 microg/kg) inhibited the finasteride effect in stressed animals. The effect of etifoxine was blocked in the presence of finasteride and picrotoxin combined in stressed animals. CONCLUSIONS: These findings support the hypothesis suggesting an involvement of neuroactive steroids in the anticonvulsive effect of restraint stress. The dual and complementary mechanisms of action of etifoxine (directly on the GABA A receptor and indirectly via the neuroactive steroids) may represent a therapeutic benefit in the treatment of various anxiety disorders with abnormal production of neuroactive steroids.

Nefopam and ketoprofen synergy in rodent models of antinociception.

Combinations of analgesics with different mechanisms of action offer the possibility of efficient analgesia with a decrease in side effects as a result of reduced dosages of one or both compounds. Based on a clinical observation of synergism between nefopam, a centrally acting non-opioid that inhibits monoamines reuptake, and ketoprofen, a non-steroidal anti-inflammatory drug, the objective of this study was to further explore this antinociceptive synergy in four distinct animal models of pain (both drugs were administered subcutaneously). Strong antinociceptive properties were observed in the mouse writhing abdominal test with ED50 values of 2.56+/-0.38 and 1.41+/-0.41 mg/kg for nefopam and ketoprofen, respectively. In the inflammatory phase of the mouse formalin test, both compounds significantly inhibited the licking time of the injected hind-paw with ED50 of 4.32+/-0.17 mg/kg for nefopam and 49.56+/-15.81 mg/kg for ketoprofen. Isobolographic analysis revealed that this drug combination is synergistic in the formalin test and additive in the writhing test. In rat carrageenan-induced tactile allodynia, single administration of nefopam or ketoprofen only partially reduced allodynia. Combination of low analgesic doses of nefopam (10 or 30 mg/kg) with low analgesic doses of ketoprofen (30 or 100 mg/kg) significantly reduced or reversed allodynia, with a more pronounced anti-allodynic effect and a longer duration efficacy. In a rat model of postoperative thermal hyperalgesia induced by incision, co-administration of nefopam at a low analgesic dose (10 mg/kg) with ketoprofen at non-analgesic doses (30 or 100 mg/kg) showed the appearance of a strong anti-hyperalgesic effect, maintained during at least 3 h. In conclusion, co-administration of nefopam with ketoprofen is synergistic, and should allow either to increase their analgesic efficacy and/or to reduce their side effects.

Moclobemide attenuates anoxia and glutamate-induced neuronal damage in vitro independently of interaction with glutamate receptor subtypes.

Recent data suggested the existence of a bidirectional relation between depression and neurodegenerative diseases resulting from cerebral ischemia injury. Glutamate, a major excitatory neurotransmitter, has long been recognised to play a key role in the pathophysiology of anoxia or ischemia, due to its excessive accumulation in the extracellular space and the subsequent activation of its receptors. A characteristic response to glutamate is the increase in cytosolic Na(+) and Ca(2+) levels which is due mainly to influx from the extracellular space, with a consequent cell swelling and oxidative metabolism dysfunction. The present study examined the in vitro effects of the antidepressant and type-A monoamine oxidase inhibitor, moclobemide, in neuronal-astroglial cultures from rat cerebral cortex exposed to anoxia (for 5 and 7 h) or to glutamate (2 mM for 6 h), two in vitro models of brain ischemia. In addition, the affinity of moclobemide for the different glutamate receptor subtypes and an interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and K(+) excess, respectively, were evaluated. Moclobemide (10-100 microM) included in the culture medium during anoxia or with glutamate significantly increased in a concentration-dependent manner the amount of surviving neurons compared to controls. Moclobemide displayed no binding affinity for the different glutamate receptor subtypes (IC(50)>100 microM) and did not block up to 300 microM the entry of Na(+) and of Ca(2+) activated by veratridine and K(+), respectively. These results suggest that the neuroprotective properties of moclobemide imply neither the glutamate neurotransmission nor the Na(+) and Ca(2+) channels.

Lack of interaction between etifoxine and CRF1 and CRF2 receptors in rodents.

Hyperactivity of the corticotropin-releasing factor (CRF) system occurs in some patients with anxiety disorders and depression. Blockade of CRF1 and CRF2 receptors can underlie the anxiolytic effects of drugs. In the present investigation, in vivo and in vitro studies were designed to determine whether the anxiolytic drug etifoxine, known to enhance GABAergic synaptic transmission, behaves also as a CRF1 and CRF2 receptor antagonist. A drug exerting multiple actions may be of clinical interest in the treatment of various different forms of mood disorders. Using two animal models, it was found that etifoxine reversed the excess CRF-induced grooming but not the hypo-locomotion of the rat placed in an open field. Etifoxine attenuated the CRF-induced gastric emptying delay in the mouse. On the other hand, in vitro, binding of etifoxine to CRF1 and CRF2 receptors on rat brain membranes was negligible and functionally, etifoxine did not block the CRF1 and CRF2 activation-induced cAMP production in presence of CRF in human neuroblastoma SH-SY5Y cells. The selective anxiolytic properties of etifoxine appear unrelated to an antagonist activity at the CRF1 and CRF2 receptors. The decrease in CRF activity produced by etifoxine may be related to its GABAergic properties.

The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain.

The peripheral benzodiazepine receptors (PBR) might be involved in certain pathophysiological events, such as anxiety, by stimulating the production of neuroactive steroids in the brain. A recent electrophysiological study has revealed an interaction between PK11195, a PBR ligand and the anxiolytic compound etifoxine at micromolar concentrations. The present work was aimed at further characterizing the etifoxine-PBR interaction. In membrane preparations from intact male rat forebrain, etifoxine uncompetitively inhibited the binding of [(3)H]PK11195 with an IC(50) = 18.3 +/- 1.2 microM, a value consistent with etifoxine plasma and brain concentrations measured after an anxiolytic-like dose (50 mg/kg). In vivo, that etifoxine dose was associated with increased concentrations of pregnenolone, progesterone, 5alpha-dihydroprogesterone and allopregnanolone in plasma and brain of sham-operated animals. In adrenalectomized and castrated rats, etifoxine enhanced the brain levels of these steroids, suggesting a stimulation of their local synthesis and/or a decrease of their disappearance rate, independently of peripheral sources. Finasteride, an inhibitor of 5alpha-reductase that converts progesterone into its 5alpha-reduced metabolites like allopregnanolone, attenuated the anti-conflict effect of etifoxine even though brain allopregnanolone contents were drastically reduced. These results indicate that following activation of the PBR in the brain, an increased cerebral production of allopregnanolone, a potent positive modulator of the GABA(A) receptor function, may partially contribute to the anxiolytic-like effects of etifoxine.

The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit.

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) potentiates GABA(A) receptor function in cultured neurons (Neuropharmacology 39 (2000) 1523). However, the molecular mechanisms underlying these effects are not known. In this study, we have determined the influence of GABA(A) receptor subunit composition on the effects of etifoxine, using recombinant murine GABA(A) receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric beta receptors were reduced by micromolar concentrations of etifoxine, showing that beta subunits possess a binding site for this modulator. In oocytes expressing alpha(1)beta(x) GABA(A) receptors (x=1, 2 or 3), etifoxine evoked a chloride current in the absence of GABA and enhanced GABA (EC10)-activated currents, in a dose-dependent manner. Potentiating effects were also observed with alpha(2)beta(x), beta(x)gamma(2s) or alpha(1)beta(x)gamma(2s) combinations. The extent of potentiation was clearly beta-subunit-dependent, being more pronounced at receptors containing a beta(2) or a beta(3) subunit than at receptors incorporating a beta(1) subunit. The mutation of Asn 289 in the channel domain of beta(2) to a serine (the homologous residue in beta(1)) did not significantly depress the effects of etifoxine at alpha(1)beta(2) receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABA(A) receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

Nefopam blocks voltage-sensitive sodium channels and modulates glutamatergic transmission in rodents.

In order to specify the nature of interactions between the analgesic compound nefopam and the glutamatergic system, we examined the effects of nefopam on binding of specific ligands on the three main subtypes ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), or quisqualic acid (QA) and kainic acid (KA) in rat brain membrane preparations. Functionally, we investigated the effects of nefopam against the seizures induced by agonists of these excitatory glutamate receptors in mice. Since the synaptic release of glutamate mainly depends upon the activation of membrane voltage-sensitive sodium channels (VSSCs), the nature of interactions between nefopam and these ionic channels was studied by evaluating the effects of nefopam on binding of 3H-batrachotoxinin, a specific ligand of the VSSCs in rat brain membrane preparations. The functional counterpart of the binding of nefopam on VSSCs was evaluated by its effects on the 22Na uptake-stimulated by veratridine on human neuroblastoma cells and in the maximal electroshock test in mice. Nefopam showed no affinity for the subtypes of ionotropic glutamate receptors up to 100 microM. On the other hand, nefopam was effective against NMDA, QA and KA induced clonic seizures in mice. Nefopam displaced 3H-batrachotoxinin and inhibited the uptake of 22Na in the micromolar range and it protected mice against electroshock induced seizures. Nefopam may block the VSSCs activity: consequently, at the presynaptic level, this effect led to a reduction of glutamate release and at the postsynaptic level, it led to a decrease of the neuronal excitability following activation of the glutamate receptors.

Role of the histamine system in nefopam-induced antinociception in mice.

The present study explored the role of the histaminergic system in nefopam analgesia based on the structural relationship between nefopam and diphenhydramine. In vitro binding assays revealed that nefopam possesses moderate affinity for histamine H1 and H2 receptor subtypes, with IC50 of 0.8 and 6.9 microM, respectively, but no affinity for histamine H(3) receptor subtype until 100 microM. Subcutaneous nefopam administration dose-dependently inhibited pain in acetic acid-induced writhing (1-30 mg/kg) and formalin (1-10 mg/kg) tests in the mouse. Pretreatment with the histamine-depleting agent alpha-fluoromethylhistidine (alpha-FMH, 50 mg/kg), the histamine H1 receptor antagonist pyrilamine (3 or 10 mg/kg), or the histamine H2 receptor antagonists cimetidine (100 mg/kg) and zolantidine (10 or 30 mg/kg) did not significantly modify nefopam antinociception in both tests. The histamine H3 receptor agonist R(-)alpha-methylhistamine (RAMH, 10 mg/kg) did not significantly modify the nefopam analgesic activity in the writhing test. At 25 mg/kg, RAMH inhibited nefopam antinociception at 3 mg/kg, but not at 10 mg/kg in the formalin test. However, pretreatment with the histamine H3 receptor antagonist thioperamide (25 mg/kg) inhibited nefopam antinociception in the writhing test, but not in the formalin test. In conclusion, nefopam analgesic activity is not mediated by histamine H1 or H2 receptors, but can be slightly modulated by histamine H3 receptors in mouse pain tests.

Effects of stress and etifoxine on pentobarbital-induced loss of righting reflex in Balb/cByJ and C57BL/6J mice.

We hypothesized that functional changes in the GABAergic system induced by stress would differ between two inbred mouse strains BALB/cByJ and C57BL/6J. We compared the effects of restraint stress and of the anxiolytic drug etifoxine (EFX) on the duration of pentobarbital-induced loss of righting reflex (hypnotic effect) in the two strains. Naive BALB/cByJ mice were less sensitive than naive C57BL/6J mice to the hypnotic effect of pentobarbital. C57BL/6J mice exhibited a shortening in the duration of pentobarbital-induced hypnosis following stress whereas stress had no effect in BALB/cByJ mice. EFX reversed the shortening of pentobarbital-induced hypnosis elicited by stress in C57BL/6J and shortened the duration of pentobarbital-induced hypnosis after stress in BALB/cByJ mice. Alterations in the GABAergic function in BALB/cByJ mice could be corrected by EFX, an enhancer of GABAergic transmission.

Effects of etifoxine on stress-induced hyperthermia, freezing behavior and colonic motor activation in rats.

Anxiety disorders are often associated with autonomic symptoms, including heart palpitations, sweating, elevation of body temperature and alterations of gastrointestinal motility. Some of the alterations observed in animals exposed to stress are analogous to changes in a number of physiological and endocrine parameters observed in anxious patients. With the purpose to guide further clinical studies in subtypes of anxious patients, etifoxine, a nonbenzodiazepine anxiolytic compound, was evaluated in two rat models of anxiety with measures of physiological manifestations: stress-induced hyperthermia (SIH) and conditioned-fear-stress-induced freezing behavior and activation of colonic motility. The sequential handling of animals induced a rise in body temperature attenuated by etifoxine (50 mg/kg IP). The emotional stress induced by fear to receive electric foot shocks is accompanied by freezing behavior and an increase of the frequency of ceco-colonic spike bursts: both parameters were reduced by etifoxine (25-50 mg/kg IP), independently of changes in pain perception and memory-related processes. In response to a stressful event, the stimulation of the corticotropin-releasing hormone (CRH) system is probably involved in the observed modifications of body temperature and colonic motility. It is hypothesized that stress-induced CRH activation is attenuated by the enhancement of the inhibitory GABAergic system activity associated with etifoxine. These findings will guide future evaluation of etifoxine in the treatment of selected anxious patients with altered autonomic symptomatology.

Nefopam potentiates morphine antinociception in allodynia and hyperalgesia in the rat.

The objective of this study was to resolve discrepancies regarding the possible antinociceptive synergy between morphine and nefopam in animal models of pain. Firstly, we have examined the antinociceptive activity of nefopam, a nonopioid antinociceptive compound that inhibits monoamine reuptake, in pain models of allodynia and hyperalgesia induced by carrageenan injection, or skin and muscle incision of the rat hind paw. Single subcutaneous administration of nefopam at 30 mg/kg blocked carrageenan- and incision-induced thermal hyperalgesia, and weakly but significantly diminished carrageenan-induced tactile allodynia. A weaker dose of nefopam (10 mg/kg) only reduced carrageenan-induced tactile allodynia and incision-induced thermal hyperalgesia. Secondly, we assessed the usefulness of the coadministration of nefopam with morphine. Combination of a nonanalgesic dose of nefopam (10 mg/kg) with a nonanalgesic dose of morphine (0.3 or 1.0 mg/kg) completely inhibited carrageenan- or incision-induced thermal hyperalgesia, respectively. In carrageenan-induced tactile allodynia, coadministration of weak analgesic doses of nefopam (10 and 30 mg/kg) with a nonanalgesic dose (1 mg/kg) or moderately analgesic dose (3 mg/kg) of morphine significantly reduced or reversed allodynia, respectively. In conclusion, coadministration of nefopam with morphine enhances the analgesic potency of morphine, indicating a morphine sparing effect of nefopam.

Differential effects of etifoxine on anxiety-like behaviour and convulsions in BALB/cByJ and C57BL/6J mice: any relation to overexpression of central GABAA receptor beta2 subunits?

Dysfunction of GABAergic transmission related to abnormal expression of GABA(A) receptor subunits in specific brain regions underlies some pathological anxiety states. Besides involvement of the benzodiazepine recognition site of GABA(A) receptor in the expression of anxiety-like behaviour, the roles of the ß(2)/ß(3) subunits are not well characterized. To address this issue, the experimental design of this study utilized the GABAergic compound etifoxine (with a preferential effectiveness after binding to a specific site at ß(2)/ß(3) subunits) tested in two inbred mouse strains: BALB/cByJ and C57BL/6J mice using three behavioural paradigms (light/dark box, elevated plus maze and restraint stress-induced small intestinal transit inhibition) and the t-butylbicyclophosphorothionate-induced convulsions model. Etifoxine plasma and brain levels and ß(2)/ß(3) mRNAs and protein expression levels in various brain regions were compared between the two strains. The two mouse strains differed markedly in basal anxiety level. Etifoxine exhibited more pronounced anxiolytic and anticonvulsant effects in the BALB/cByJ mice compared to the C57BL/6J mice. The etifoxine brain/plasma ratios of the two strains were not different. Beta2 subunit mRNA and protein expression levels were around 25 and 10% higher respectively in the anterodorsal nucleus of the thalamus and the CA3 field of hippocampus of BALB/cByJ mice compared to C57BL/6J mice. Beta3 subunit mRNA and protein expression levels did not differ between the two strains. Based on these results, it is suggested that overexpression of GABA(A) receptor ß(2) subunit in BALB/cByJ mice relative to C57BL/6j mice contributes to the dysfunction in GABA(A) transmission in regions of brain known to regulate responses to stress. The dysregulated GABA(A) function in BALB/cByJ mice may be corrected by the administration of etifoxine.

The anxiolytic etifoxine protects against convulsant and anxiogenic aspects of the alcohol withdrawal syndrome in mice.

Change in the function of gamma-aminobutyric acid(A) (GABA(A)) receptors attributable to alterations in receptor subunit composition is one of main molecular mechanisms with those affecting the glutamatergic system which accompany prolonged alcohol (ethanol) intake. These changes explain in part the central nervous system hyperexcitability consequently to ethanol administration cessation. Hyperexcitability associated with ethanol withdrawal is expressed by physical signs, such as tremors, convulsions, and heightened anxiety in animal models as well as in humans. The present work investigated the effects of anxiolytic compound etifoxine on ethanol-withdrawal paradigms in a mouse model. The benzodiazepine diazepam was chosen as reference compound. Ethanol was given to NMRI mice by a liquid diet at 3% for 8 days, then at 4% for 7 days. Under these conditions, ethanol blood level ranged between 0.5 and 2 g/L for a daily ethanol intake varying from 24 to 30 g/kg. These parameters permitted the emergence of ethanol-withdrawal symptoms once ethanol administration was terminated. Etifoxine (12.5-25 mg/kg) and diazepam (1-4 mg/kg) injected intraperitoneally 3h 30 min after ethanol removal, decreased the severity in handling-induced tremors and convulsions in the period of 4-6h after withdrawal from chronic ethanol treatment. In addition when administered at 30 and 15 min, respectively, before the light and dark box test, etifoxine (50mg/kg) and diazepam (1mg/kg) inhibited enhanced aversive response 8h after ethanol withdrawal. Etifoxine at 25 and 50 mg/kg doses was without effects on spontaneous locomotor activity and did not exhibit ataxic effects on the rota rod in animals not treated with ethanol. These findings demonstrate that the GABAergic compound etifoxine selectively reduces the physical signs and anxiety-like behavior associated with ethanol withdrawal in a mouse model and may hold promise in the treatment of ethanol-withdrawal syndrome in humans.

Beneficial effects of citrulline malate on skeletal muscle function in endotoxemic rat.

Although citrulline malate (CM; CAS 54940-97-5, Stimol) is used against fatigue states, its anti-asthenic effect remains poorly documented. The objective of this double-blind study was to evaluate the effect of oral ingestion of CM on a rat model of asthenia, using in situ (31)Phosphorus magnetic resonance spectroscopy ((31)P-MRS). Muscle weakness was induced by intraperitoneal injections of Klebsiella pneumoniae endotoxin (lipopolysaccharides at 3 mg/kg) at t(0) and t(0)+24 h. For each animal, muscle function was investigated strictly non-invasively before (t(0)-24 h) and during (t(0)+48 h) endotoxemia, through a standardized rest-stimulation-recovery protocol. The transcutaneous electrical stimulation protocol consisted of 5.7 min of repeated isometric contractions at a frequency of 3.3 Hz, and force production was measured with an ergometer. CM supplementation in endotoxemic animals prevented the basal phosphocreatine/ATP ratio reduction and normalized the intracellular pH (pH(i)) time-course during muscular activity as a sign of an effect at the muscle energetics level. In addition, CM treatment avoided the endotoxemia-induced decline in developed force. These results demonstrate the efficiency of CM for limiting skeletal muscle dysfunction in rats treated with bacterial endotoxin.

Endotoxemia does not limit energy supply in exercising rat skeletal muscle.

Although depletion in high-energy phosphorylated compounds and mitochondrial impairment have been reported in septic skeletal muscle at rest, their impact on energy metabolism has not been documented during exercise. In this study we aimed to investigate strictly gastrocnemius muscle function non-invasively, using magnetic resonance techniques in endotoxemic rats. Endotoxemia was induced by injecting animals intraperitoneally at t(0) and t(0) + 24 h with Klebsiella pneumoniae lipopolysaccharides (at 3 mg kg(-1)). Investigations were performed at t(0) + 48 h during a transcutaneous electrical stimulation protocol consisting of 5.7 min of repeated isometric contractions at a frequency of 3.3 HZ. Endotoxin treatment produced a depletion in basal phosphocreatine content and a pronounced reduction in oxidative adenosine triphosphate (ATP) synthesis capacity, whereas the resting ATP concentration remained unchanged. During the stimulation period, endotoxemia caused a decrease in force-generating capacity that was fully accounted for by the loss of muscle mass. It further induced an acceleration of glycolytic ATP production and an increased accumulation of adenosine diphosphate (ADP, an important mitochondrial regulator) that allowed a near-normal rate of oxidative ATP synthesis. Finally, endotoxemia did not affect the total rate of ATP production or the ATP cost of contraction throughout the whole stimulation period. These data demonstrate that, in an acute septic phase, metabolic alterations in resting muscle do not impact energy supply in exercising muscle, likely as a result of adaptive mechanisms.