Simvastatin in traumatic brain injury: effect on brain edema mechanisms.

OBJECTIVES: Traumatic brain injury causes deleterious brain edema, leading to high mortality and morbidity. Brain edema exacerbates neurologic deficits and may be attributable to the breakdown of endothelial cell junction protein, leukocyte infiltration, and matrix metalloproteinase activation. These all contribute to loss of blood-brain barrier integrity. The pleiotropic effects of statins, hydroxymethylglutaryl-coenzyme A reductase inhibitors, may inhibit posttraumatic brain edema. We therefore investigated the effect of acute simvastatin on neurologic deficits, cerebral edema, and its origins. DESIGN: Randomized laboratory animal study. SETTINGS: University-affiliated research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were subjected to lateral fluid percussion traumatic brain injury. Our preliminary dose-effect study indicated that 37.5 mg/kg simvastatin, administered orally 1 hr and 6 hrs after traumatic brain injury, has the greatest anti-edematous effect. This dose was used to study its effects on brain edema and on its mechanisms. MEASUREMENTS AND MAIN RESULTS: We first assessed the effects of simvastatin 24 hrs after traumatic brain injury on brain edema, brain claudin-5 expression, and the vascular endothelial-cadherin (pTyr731)/total vascular endothelial-cadherin ratio, matrix metalloproteinase-9 activity, intercellular adhesion molecule-1 expression, and polymorphonuclear neutrophil infiltration. We also evaluated blood-brain barrier permeability by measuring Evans blue and fluorescein sodium salt extravasation into the cerebral parenchyma. We then investigated whether simvastatin reduces neurologic deficits, edema, and blood-brain barrier permeability earlier than 24 hrs; these effects were evaluated 6 hrs after traumatic brain injury. The anti-edematous effect of simvastatin 24 hrs after traumatic brain injury was associated with increased claudin-5 and decreased intercellular adhesion molecule-1, polymorphonuclear neutrophil infiltration, and blood-brain barrier permeability, with no effect on matrix metalloproteinase-9 activity or vascular endothelial-cadherin phosphorylation. Earlier, 6-hrs after traumatic brain injury, simvastatin reduced neurologic deficits, cerebral edema, and blood-brain barrier permeability. CONCLUSIONS: Simvastatin could be a new therapy for reducing posttraumatic edema by preventing damage to tight junctions and neutrophil infiltration into the parenchyma, thus preserving blood-brain barrier integrity.

Histological and Behavioral Evaluation after Traumatic Brain Injury in Mice: A Ten Months Follow-Up Study.

Traumatic brain injury (TBI) is a chronic pathology, inducing long-term deficits that remain understudied in pre-clinical studies. In this context, exploration, anxiety-like behavior, cognitive flexibility, and motor coordination were assessed until 5 and 10 months after an experimental TBI in the adult mouse, using two cohorts. In order to differentiate age, surgery, and remote gray and white matter lesions, three groups (unoperated, sham-operated, and TBI) were studied. TBI induced delayed motor coordination deficits at the pole test, 4.5 months after injury, that could be explained by gray and white matter damages in ipsilateral nigrostriatal structures (striatum, internal capsule) that were spreading to new structures between cohorts, at 5 versus 10 months after the injury. Further, TBI induced an enhanced exploratory behavior during stressful situations (active phase during actimetry test, object exploration in an open field), risk-taking behaviors in the elevated plus maze 5 months after injury, and a cognitive inflexibility in the Barnes maze that persisted until 9 months after the injury. These behavioral modifications could be related to the white and gray matter lesions observed in ipsi- and contralateral limbic structures (amygdala, hilus/cornu ammonis 4, hypothalamus, external capsule, corpus callosum, and cingular cortex) that were spreading to new structures between cohorts, at 5 months versus 10 months after the injury. The present study corroborates clinical findings on TBI and provides a relevant rodent chronic model which could help in validating pharmacological strategies against the chronic consequences of TBI.

Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury.

BACKGROUND: Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models. RESULTS: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), beta-microtubulin and S100beta were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen); GAPDH > 18S rRNA > S100beta > beta-microtubulin > beta-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100beta > 18S rRNA > beta-actin > beta-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100beta > beta-microtubulin > beta-actin. CONCLUSION: This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that beta-actin and beta-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed.

Combination therapy with fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, and simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on experimental traumatic brain injury.

We and others have demonstrated that fibrates [peroxisome proliferator-activated receptor (PPAR)alpha agonists] and statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) exerted neuroprotective and pleiotropic effects in experimental models of traumatic brain injury (TBI). Because the combination of statins and fibrates synergistically enhanced PPARalpha activation, we hypothesized that the combination of both drugs may exert more important and/or prolonged beneficial effects in TBI than each alone. In this study, we examined the combination of fenofibrate with simvastatin, administered 1 and 6 h after injury, on the consequences of TBI. First, our dose-effect study demonstrated that the most efficient dose of simvastatin (37.5 mg/kg) reduced post-traumatic neurological deficits and brain edema. Then, the effects of the combination of fenofibrate (50 mg/kg) and simvastatin (37.5 mg/kg), given p.o. at 1 and 6 h after TBI, were evaluated on the TBI consequences in the early and late phase after injury. The combination exerted more sustained neurological recovery-promoting and antiedematous effects than monotherapies, and it synergistically decreased the post-traumatic brain lesion. Furthermore, a delayed treatment given p.o. at 3 and 8 h after TBI with the combination was still efficient on neurological deficits induced by TBI, but it failed to reduce the brain edema at 48 h. The present data represent the first demonstration that the combination of fenofibrate and simvastatin exerts prolonged and synergistic neuroprotective effects than each drug alone. Thus, these results may have important therapeutic significance for the treatment of TBI.

Reduction of hemorrhagic transformation by PJ34, a poly(ADP-ribose)polymerase inhibitor, after permanent focal cerebral ischemia in mice.

Hemorrhagic transformation is an aggravating event that occurs in 15 to 43% of patients suffering from ischemic stroke. This phenomenon due to blood-brain barrier breakdown appears to be mediated in part by matrix metalloproteinases (MMPs) among which MMP-2 and MMP-9 could be particularly involved. Recent experimental studies demonstrated that post-ischemic MMP-9 overexpression is regulated by poly(ADP-ribose)polymerase (PARP). In this context, our study aimed to evaluate the effect of PJ34 (N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetamide), a potent PARP inhibitor, on MMP-2 and MMP-9 levels and on hemorrhagic transformations in a model of permanent focal cerebral ischemia in mice. PJ34 (6.25-12.5 mg/kg, i.p.) was given at the time of ischemia onset and 4 h later. Hemorrhagic transformations, divided into microscopic and macroscopic hemorrhages, were counted 48 h after ischemia on 12 coronal brain slices. Microscopic and macroscopic hemorrhages were respectively reduced by 38% and 69% with 6.25 mg/kg PJ34. The anti-hemorrhagic effect of PJ34 was associated with a 57% decrease in MMP-9 overexpression assessed by gelatin zymography. No increase in MMP-2 activity was observed after ischemia in our model. The vascular protection achieved by PJ34 was associated with a reduction in the motor deficit (P<0.05) and in infarct volume (-31%, P<0.01). In conclusion, our study demonstrates for the first time that PJ34 reduces hemorrhagic transformations after cerebral ischemia. Thus this PARP inhibitor exhibits both anti-hemorrhagic and neuroprotective effects that may be of valuable interest for the treatment of stroke.

Neurological recovery-promoting, anti-inflammatory, and anti-oxidative effects afforded by fenofibrate, a PPAR alpha agonist, in traumatic brain injury.

We previously demonstrated that fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, reduced the neurological deficit, the edema and the cerebral lesion induced by traumatic brain injury (TBI). In order to elucidate these beneficial effects, in the present study, we investigated, in the same TBI model, fenofibrate's effects on the inflammation and oxidative stress. Male Sprague Dawley rats were randomized in four groups: non-operated, sham-operated, TBI + vehicle, TBI + fenofibrate. TBI was induced by lateral fluid percussion of the temporoparietal cortex. Rats were given fenofibrate (50 mg/kg) or its vehicle (water containing 0.2% methylcellulose), p.o. 1 and 6 h after brain injury. A neurological assessment was done 24 h after TBI, then rats were killed and the brain COX2, MMP9 expression, GSx, GSSG levels were determined. The same schedule of treatment was used to evaluate the effect of fenofibrate on immunohistochemistry of 3NT, 4HNE and iNOS at 24 h post-injury. Our results showed that fenofibrate promotes neurological recovery by exerting anti-inflammatory effect evidenced by a decrease in iNOS, COX2 and MMP9 expression. In addition, fenofibrate showed anti-oxidant effect demonstrated by a reduction of markers of oxidative stress: loss of glutathione, glutathione oxidation ratio, 3NT and 4HNE staining. Our data suggest that PPARalpha activation could mediate pleiotropic effects and strengthen that it could be a promising therapeutic strategy for TBI.

Acute systemic inflammation induces central mitochondrial damage and mnesic deficit in adult Swiss mice.

The aim of this study was to investigate how the brain is affected during systemic inflammation. For this purpose, Swiss mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 250microg/mouse) to mimic aspects of systemic infection. Spatial learning in Y-maze test demonstrated a differential learning profile during the training test between control and LPS-treated mice, with an alteration in the latter group. We show that systemic LPS-induced inflammation and oxidative injury as assessed by reactive oxygen species (ROS) and nitrites/nitrates (NOx) production associated with reduced glutathione (GSH) depletion, cyclooxygenase-2 (COX-2) expression, and lipid peroxidation. LPS also induced a loss in mitochondrial integrity as shown by a significant decrease in membrane potential and impairment in mitochondrial redox activity. Thus, peripheral inflammation by producing brain inflammation and oxidative injury causes mnesic deficits. It remains to determine whether such events can induce neuronal dysfunction/degeneration and, with time, lead to cholinergic deficiency, amyloid deposits and cognitive impairments as they occur in Alzheimer's disease.

Neuroinflammation, myelin and behavior: Temporal patterns following mild traumatic brain injury in mice.

Traumatic brain injury (TBI) results in white matter injury (WMI) that is associated with neurological deficits. Neuroinflammation originating from microglial activation may participate in WMI and associated disorders. To date, there is little information on the time courses of these events after mild TBI. Therefore we investigated (i) neuroinflammation, (ii) WMI and (iii) behavioral disorders between 6 hours and 3 months after mild TBI. For that purpose, we used experimental mild TBI in mice induced by a controlled cortical impact. (i) For neuroinflammation, IL-1b protein as well as microglial phenotypes, by gene expression for 12 microglial activation markers on isolated CD11b+ cells from brains, were studied after TBI. IL-1b protein was increased at 6 hours and 1 day. TBI induced a mixed population of microglial phenotypes with both pro-inflammatory, anti-inflammatory and immunomodulatory markers from 6 hours to 3 days post-injury. At 7 days, microglial activation was completely resolved. (ii) Three myelin proteins were assessed after TBI on ipsi- and contralateral corpus callosum, as this structure is enriched in white matter. TBI led to an increase in 2',3'-cyclic-nucleotide 3'-phosphodiesterase, a marker of immature and mature oligodendrocyte, at 2 days post-injury; a bilateral demyelination, evaluated by myelin basic protein, from 7 days to 3 months post-injury; and an increase in myelin oligodendrocyte glycoprotein at 6 hours and 3 days post-injury. Transmission electron microscopy study revealed various myelin sheath abnormalities within the corpus callosum at 3 months post-TBI. (iii) TBI led to sensorimotor deficits at 3 days post-TBI, and late cognitive flexibility disorder evidenced by the reversal learning task of the Barnes maze 3 months after injury. These data give an overall invaluable overview of time course of neuroinflammation that could be involved in demyelination and late cognitive disorder over a time-scale of 3 months in a model of mild TBI. This model could help to validate a pharmacological strategy to prevent post-traumatic WMI and behavioral disorders following mild TBI.

NMDA receptor activation inhibits alpha-secretase and promotes neuronal amyloid-beta production.

Acute brain injuries have been identified as a risk factor for developing Alzheimer's disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-beta (Abeta) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Abeta. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-beta precursor protein (KPI-APP) followed by a shift from alpha-secretase to beta-secretase-mediated APP processing. This shift is a result of the inhibition of the alpha-secretase candidate tumor necrosis factor-alpha converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Abeta secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Abeta production and represent a causal risk factor for developing AD.

Autoradiographic analysis of mouse brain kinin B1 and B2 receptors after closed head trauma and ability of Anatibant mesylate to cross the blood-brain barrier.

The potent non-peptide B2 receptor (R) antagonist, Anatibant mesylate (Ms) (LF 16-0687 Ms), reduces brain edema and improves neurological function recovery in various focal and diffuse models of traumatic brain injury in rodents. In the present study, alteration of kinin B1 and B2R after closed head trauma (CHT) and in vivo binding properties of Anatibant Ms (3 mg/kg, s.c.) injected 30 min after CHT were studied in mice by autoradiography using the radioligands [125I]HPP-Hoe 140 (B2R), and [125I]HPP-des-Arg10-Hoe 140 (B1R). Whereas B1R is barely detected in most brain regions, B2R is extensively distributed, displaying the highest densities in the hindbrain. CHT was associated with a slight increase of B1R and a decrease of B2R (10-50%) in several brain regions. Anatibant Ms (Ki = 22 pM) displaced the B2R radioligand from its binding sites in several areas of the forebrain, basal ganglia and hindbrain. Displacement was achieved in 1 h and persisted at 4 h post-injection. The inhibition did not exceed 50% of the total specific binding in non-injured mice. After CHT, the displacement by Anatibant Ms was higher and almost complete in the cortex, caudate putamen, thalamus, hippocampus, medial geniculate nucleus, ventral tegmental area, and raphe. Evans blue extravasation in brain tissue at 4 h after CHT was abolished by Anatibant Ms. It appeared that Anatibant Ms penetrated into the brain in sufficient amounts, particularly after disruption of the blood-brain barrier, to account for its B2R-mediated neuro- and vascular protective effects. The diminished binding of B2R after CHT may reflect the occupancy or internalization of B2R following the endogenous production of bradykinin (BK).

Impairment of lymphocyte function in head-injured rats: effects of standard and immune-enhancing diets for enteral nutrition.

BACKGROUND: The metabolic response to head injury (HI) is characterized by a dysimmunity which may be a risk factor of a septic state. The use of immune enhancing diets (IEDs) could be a promising approach to improve immune functions. The aim of the study was to investigate the consequences of HI on lymphocyte function and to determine the effects of an enteral IED comparatively to a standard enteral nutrition. METHOD: A rat model of HI by fluid percussion was used. Twenty-five male Sprague-Dawley rats were randomized into 4 groups: rats receiving standard chow diet ad libitum (AL), rats sustaining HI and receiving standard chow diet and enteral saline (HI), rats receiving the enteral standard diet Sondalis HP (HIS), and rats receiving the IED Crucial (HIC). The two enteral diets were infused continuously during 4 days after the HI and were isocaloric, isonitrogenous and isovolumic. RESULTS: HI induced a thymus atrophy (HI vs. AL, P<0.05), and an impairment in lymphocyte CD25 receptor density responsiveness to stimulation. The IED blunted thymus atrophy and allowed to preserve the stimulation of blood and Peyer patches lymphocytes (HIC: Stimulated vs. Basal, P<0.05). CONCLUSION: IED seems more adapted for preserving lymphocyte function than standard diet in HI patients.

Induction of type 2 iodothyronine deiodinase in astrocytes after transient focal cerebral ischemia in the rat.

This study investigated the expression of deiodinases of thyroid hormones in the rat brain after transient occlusion of the middle cerebral artery. The activity of type 2 deiodinase (D2), which catalyzes the deiodination of thyroxine into the more active thyroid hormone 3,5,3'-triiodothyronine, was strongly increased by cerebral ischemia at 6 and 24 hours in the striatum and at 24 hours in the cerebral cortex. The activity of type 3 deiodinase, which catalyzes the inactivation of thyroid hormones, was not affected by ischemia. In situ hybridization showed, as soon as 6 hours, an upregulation of the expression of D2 mRNA in the ipsilateral striatum, which disappeared at 24 hours. In the ipsilateral cortex, the induction of D2 mRNA started at 6 hours, was increased at 24 hours and finally declined at 72 hours. These results were confirmed by reverse transcription-PCR for D2 mRNA in the striatum and cerebral cortex. The upregulation of D2 mRNA after ischemia was mainly localized in astrocytic cell bodies. These results show that D2 is rapidly induced in astrocytes after ischemic stroke. Future work will include the exploration of the role of the upregulation of this enzyme, responsible for local 3,5,3'-triiodothyronine production as a neuroprotective mechanism in the brain.

Antioxidant strategies in the treatment of stroke.

Excessive production of free radicals is known to lead to cell injury in a variety of diseases, such as cerebral ischemia. In this review, we describe some of the numerous studies that have examined this oxidative stress and the efficiency of antioxidant strategies in focal cerebral ischemia. Besides using genetically modified mice, these strategies can be divided into three groups: (1) inhibition of free radical production, (2) scavenging of free radicals, and (3) increase of free radical degradation by using agents mimicking the enzymatic activity of endogenous antioxidants. Finally, the clinical trials that have tested or are currently testing the efficiency of antioxidants in patients suffering from stroke are reviewed. The results presented here lead us to consider that antioxidants are very promising drugs for the treatment of ischemic stroke.

Characterization of the alteration of nutritional state in brain injury induced by fluid percussion in rats.

OBJECTIVE: Patients suffering from traumatic brain injury (TBI) undergo rapid weight loss with negative nitrogen balance and enhanced whole-body protein breakdown, with protein wasting causing morbidity and increased mortality. Many experimental models of TBI have been used to evaluate strategies to improve the outcome of these patients, but nutritional status has not been considered in experiments published to date, although this may have great importance and influence the results obtained with TBI models. This study characterized the hypercatabolism level and nutritional status of TBI rats. DESIGN: Twenty-four male Wistar rats were randomized into three groups. Rats from the TBI group were anesthetized and fluid percussion was applied. The pair-fed (PF) group was healthy but was pair-fed to the TBI group. The ad libitum (AL) group was healthy and fed ad libitum. The study was performed over 10 days post-TBI. MEASUREMENTS AND RESULTS: TBI in rats was characterized by remarkable long-lasting anorexia, renal failure (creatinine clearance: AL 1.8+/-0.2 and PF 1.5+/-0.1 vs. TBI 0.9+/-0.1 l/24 hour), anorexia (appetite depressed throughout the study), increased myofibrillar proteolysis (3-methylhistidine/creatinine ratio (day 2: AL 36+/-1 and PF 38+/-2 vs. TBI 54+/-5 micromol/mmol), and intestinal atrophy (ileum: AL 29.3+/-2.5 and PF 28.7+/-1.1 vs. TBI 22.5+/-1.4 mg/cm). In addition, anorexia led to muscular atrophy and decreased nitrogen balance. The metabolic alterations described above can increase morbidity and mortality. CONCLUSIONS: TBI by fluid percussion in rats is a model reproducing the metabolic and nutritional alterations observed in clinical practice and is suitable for further studies exploring the efficacy of optimized nutritional support.

Characterization of a rat model to study acute neuroinflammation on histopathological, biochemical and functional outcomes.

Neuroinflammation is one of the events occurring after acute brain injuries. The aim of the present report was to characterize a rat model to study acute neuroinflammation on the histopathological, biochemical and functional outcomes. Lipopolysaccharide (LPS), known as a strong immunostimulant, was directly injected into the hippocampus. The spatiotemporal evolution of inducible NOS (iNOS) and cell death was studied from 6 h to 7 days. A perfect time course correlation was observed between iNOS immunoreactivity and iNOS activity showing an acute, expansive and transient iNOS induction in the hippocampus with a peak at 24 h. It was associated with a marked increase in NO metabolite (NO(x)) levels, and a high level of myeloperoxidase (MPO) activity. This inflammation precedes a massive cellular loss including at least neurons and astrocytes, and a drop of constitutive NOS activity, restrictive to the ipsilateral hippocampus from 48 h after LPS injection. Moreover, sensorimotor function impairment occurred from 24 h to 7 days with a maximum at 24 h post-LPS injection. Therefore, we characterized an in vivo model of acute neuroinflammation and neurodegeneration, in relation with a neurological deficit, which may be a powerful tool for mechanistic studies and for further evaluation of the potential neuroprotective agents.

Beneficial effects of PJ34 and INO-1001, two novel water-soluble poly(ADP-ribose) polymerase inhibitors, on the consequences of traumatic brain injury in rat.

Traumatic brain injury produces peroxynitrite, a powerful oxidant which triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1 (PARP-1). We previously demonstrated that 3-aminobenzamide, a PARP inhibitor, is neuroprotective in a model of traumatic brain injury induced by fluid percussion in rat, suggesting that PARP-1 could be a therapeutic target. In order to confirm this hypothesis, we investigated the effects of PJ34 and INO-1001, two PARP inhibitors from structural classes other than benzamide, on the post-traumatic consequences. Pre- and post-treatments with PJ34 (30 mg/kg/day) and INO-1001 (10 mg/kg/day) decrease the neurological deficit at 3 days post-injury and this deficit is still reduced at 7 days. These neurological recovery-promoting effects are associated with the inhibition of PARP-1 activation caused by trauma, as demonstrated by abolishment of immunostaining of poly(ADP-ribose). Thus, the present work strengthens strongly the concept that PARP-1 inhibition may be a suitable approach for the treatment of brain trauma.

Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, exerts neuroprotective effects in traumatic brain injury.

Peroxisome proliferator-activated receptor alpha (PPARalpha) has been demonstrated to reduce inflammation in various inflammatory diseases. As traumatic brain injury (TBI) caused a neuroinflammatory response, we examined the effect of fenofibrate, a PPARalpha agonist, on the post-traumatic consequences caused by lateral fluid percussion of brain in rats. The effects of fenofibrate (50 and 100mg/kg) were evaluated on the consequences of TBI in the early phase (6 and 24h) and the late phase (7 days) after TBI. Neurological deficit, brain lesion, cerebral oedema and ICAM-1 expression were evaluated. Treatment with fenofibrate (given p.o. at 1 and 6h after TBI) decreases the neurological deficit induced by TBI at 24h. Furthermore, fenofibrate reduces brain oedema and ICAM-1 expression at 24h post-TBI. Rats given fenofibrate at 1, 6, 24, 48 and 72h after TBI show neurological recovery associated with a reduction of the brain lesion at 7 days post-TBI. The present data represents the first demonstration that fenofibrate, a PPARalpha agonist, exerts neuroprotective effects in TBI. The activation of receptor PPARalpha could be beneficial by counteracting the deleterious inflammatory response following TBI. This suggests that PPARalpha activation could be a new and promising therapeutic strategy for the treatment of brain trauma.

Time course of oxidative stress, lesion and edema after intrastriatal injection of malonate in rat: effect of alpha-phenyl-N-tert-butylnitrone.

The aim of this study was to characterize the model of oxidative stress consisting in the infection of malonate (3 mumol), an inhibitor of mitochondrial complex II, in the rat striatum. The striatal concentrations of both the reduced and oxidized forms of glutathione (the major endogenous antioxidant) were determined at various times after malonate injection (1-4 h) in order to evaluate the evolution of oxidative stress. The progression of lesion size and edema was also determined up to 24 h after malonate administration. Finally, the effect of alpha-phenyl-N-tert-butylnitrone (PBN), an antioxidant nitrone, was studied. The levels of reduced glutathione (GSH) progressively decreased after malonate injection up to 40% of those of sham animals at 4 h. An increase in the concentrations of oxidized glutathione (GSSG) was also observed as early as 1 h after malonate administration which was maintained up to 4 h. The size of the lesion was maximal within 2 h of malonate injection, whereas edema continued to increase between 2 and 24 h. Injection of PBN at 100 mg/kg i.p. 30 min before and 2 h after malonate administration abolished the GSSG increase caused by malonate but did not modify the drop in GSH. This moderate antioxidant effect of PBN was associated with a slight decrease of the lesion area at two levels (10.7 and 10.2 mm anterior to the interaural line), but the lesion volume remained unchanged. By contrast, PBN reduced edema by 30%. Taken together, these results show that malonate induced a severe oxidative stress leading to the rapid development of the lesion. PBN demonstrates anti-edematous properties that are not sufficient to reduce the lesion.

Caspase inhibition after neonatal ischemia in the rat brain.

Caspase-3 has been identified as a key protease in the execution of apoptosis and appears to be an important downstream event after hypoxia-ischemia in the immature brain. The efficacy of a pan-caspase inhibitor, boc-aspartyl-(Ome)-fluoromethyl-ketone (BAF), was tested in a model of unilateral focal ischemia with reperfusion in 7-day-old rats. The BAF inhibitor was given intraperitoneally 5 minutes before reperfusion via the carotid artery. This procedure reduced the activity of caspase-3 by 79% but did not induce a significant reduction in infarct volume (23.8 +/- 7.5% versus 30.1 +/- 6.4%). Animals were distributed in two populations. One population exhibited an infarct, whereas the other appeared to be fully protected. BAF-treated animals exhibiting an infarct mostly displayed necrotic cell death, whereas apoptotic nuclei were observed in untreated or vehicle-treated animals. Repeated dose of BAF (5 minutes before and 9 hours after reperfusion) did not also provide benefit after neonatal ischemia, although a general trend to reduce lesion was observed (20.5 +/- 3.7% versus 34.4 +/- 5.9%). These findings raise critical questions about the use of peptide ketone apoptotic inhibitors in improving histopathologic outcomes after neonatal stroke.

LF 16-0687 Ms, a bradykinin B2 receptor antagonist, reduces ischemic brain injury in a murine model of transient focal cerebral ischemia.

1. Bradykinin promotes neuronal damage and brain edema through the activation of the B(2) receptor. The neuroprotective effect of LF 16-0687 Ms, a B(2) receptor antagonist, has been described when given prior to induction of transient focal cerebral ischemia in rat, but there are no data regarding the consequence of a treatment when given after injury. Therefore, in a murine model of transient middle cerebral artery occlusion (MCAO), we evaluated the effect of LF 16-0687 Ms given prior to and/or after the onset of ischemia on neurological deficit, infarct volume and inflammatory responses including cerebral edema, blood-brain barrier (BBB) disruption and neutrophil accumulation. 2. LF 16-0687 Ms (1, 2 and 4 mg kg(-1)) administered 0.5 h before and, 1.25 and 6 h after MCAO, decreased the infarct volume by a maximum of 33% and significantly improved the neurological recovery. 3. When given at 0.25 and 6.25 h after MCAO, LF 16-0687 Ms (1.5, 3 and 6 mg kg(-1)) decreased the infarct volume by a maximum of 25% and improved the neurological score. 4. Post-treatment with LF 16-0687 Ms (1.5 mg kg(-1)) significantly decreased brain edema (-28%), BBB disruption (-60%) and neutrophil accumulation (-65%) induced by ischemia. Physiological parameters were not modified by LF 16-0687 Ms. 5. These data emphasize the role of bradykinin B(2) receptor in the development of infarct lesion, neurological deficit and inflammatory responses resulting from transient focal cerebral ischemia. Therefore, B(2) receptor antagonist might represent a new therapeutic approach in the pharmacological treatment of stroke.