[Anti-inflammatory modulators in traumatic brain injury]. / Stratégies anti-inflammatoires et traumatisme crânien.

Traumatic brain injury leads to primary and secondary brain injuries. Primary brain injury results from mechanical forces applied to the head at the time of impact. Secondary brain injury occurs at some time after the primary impact. Numerous pathophysiological mechanisms have been postulated to explain the progressive tissue damage produced by secondary injuries. The endogenous neuroinflammatory response after traumatic brain injury contributes to the development of blood-brain barrier breakdown, cerebral oedema and neuronal cell death and this has led to various pharmacological therapies to try to limit this type of damage. Studies employing glutamate receptor antagonist for cerebral protection have yielded promising results in laboratory animals but failed to produce clinically significant improvements. The present review will summarize the mechanisms of post traumatic cerebral inflammation with a special focus on the anti-inflammatory drug targets.

Selective inhibition of inducible nitric oxide synthase reduces neurological deficit but not cerebral edema following traumatic brain injury.

The role of inducible nitric oxide synthase (iNOS) in cerebral edema and neurological deficit following traumatic brain injury (TBI) is not yet clear-cut. Therefore, the aim of this study was to investigate the effect of three different iNOS inhibitors on cerebral edema and functional outcome after TBI. First, the time courses of blood--brain barrier (BBB) breakdown, cerebral edema, and neurological deficit were studied in a rat model of fluid percussion-induced TBI. The permeability of BBB to Evans blue was increased from 1 h to 24 h after TBI. Consistently, a significant increase in brain water content (BWC) was observed at 6 and 24 h post-TBI. A deficit in sensorimotor neurological functions was also observed from 6 h to 7 days with a maximum 24 h after TBI. Second, a single dose of aminoguanidine (AG; 100 mg/kg, i.p.), L-N-iminoethyl-lysine (L-NIL; 20 mg/kg, i.p.), or N-[3-(aminomethyl)benzyl]acetamide (1400W; 20 mg/kg, s.c.) was administered at 6 h post-TBI. Treatment with AG reduced by 71% the increase in BWC evaluated at 24 h, while L-NIL and 1400W had no effect. In contrast, the three iNOS inhibitors reduced the neurological deficit from 30% to 40%. Third, 1400W (20 mg/kg, s.c.) was administered at 5 min, 8 and 16 h post-TBI. Although this treatment paradigm had no effect on cerebral edema evaluated at 24 h, it significantly reduced the neurological deficit and iNOS activity. In conclusion, iNOS contributes to post-TBI neurological deficit but not to cerebral edema. The beneficial effect of iNOS inhibitors is not due to their anti-edematous effect, and the reduction of cerebral edema by AG is unlikely related to iNOS inhibition. The 6 h therapeutic window of iNOS inhibitors could allow their use in the treatment of functional deficit at the acute phase of TBI.

1400W, a potent selective inducible NOS inhibitor, improves histopathological outcome following traumatic brain injury in rats.

There are conflicting data regarding the role of nitric oxide (NO) produced by inducible NO synthase (iNOS) in the pathophysiology of traumatic brain injury (TBI). In this report, we evaluated the effect of a potent selective (iNOS) inhibitor, 1400W, on histopathological outcome following TBI in a rat model of lateral fluid percussion brain injury. First, to design an appropriate treatment protocol, the parallel time courses of iNOS and neuronal NOS (nNOS) gene expression, protein synthesis, and activity were investigated. Early induction of iNOS gene was observed in the cortex of injured rats, from 6 to 72 h with a peak at 24 h. Similarly, iNOS protein was detected from 24 to 72 h and de novo synthesized iNOS was functionally active, as measured by Ca2+-independent NOS activity. The kinetic studies of nNOS showed discrepancies, since nNOS gene expression and protein synthesis were constant in the cortex of injured rats from 24 to 72 h, while Ca2+-dependent constitutive NOS activity was markedly decreased at 24 h, persisting up to 72 h. Second, treatment with 1400W, started as a bolus of 20 mg kg-1 (s.c.) at 18 h post-TBI, followed by s.c.-infusion at a rate of 2.2 mg kg-1 h-1 between 18 and 72 h, reduced by 64% the brain lesion volume at 72 h. However, the same treatment paradigm initiated 24 h post-TBI did not have any effect. In conclusion, administration of a selective iNOS inhibitor, 1400W, even delayed by 18 h improves histopathological outcome supporting a detrimental role for iNOS induction after TBI.

Pattern of cerebral edema and hemorrhage in a mice model of diffuse brain injury.

This study aims to examine the time course of the brain edema formation in relation with blood-brain barrier (BBB) disruption and cerebral hemorrhage in a murine model of diffuse brain injury. Brain water content increased at 1 h post-injury and persisted up to 7 days. This event was associated with electrolyte imbalance such as Na(+) increase within 24 h. Prominent Evans blue extravasation was also observed from 1 to 6 h post-injury. Concurrently, hemoglobin increased markedly by 1 h, reached a peak at 4 h and declined progressively within a week in association with a rise of parenchyma iron content between 24 h and 7 days. These results suggest that brain edema is vasogenic and that the hemorrhage process is involved in the BBB disruption and edema, both leading to post-traumatic secondary events.

Detrimental role of bradykinin B2 receptor in a murine model of diffuse brain injury.

Inhibition of the bradykinin B2 receptor type (B2R) has been shown to improve neurological outcome in models of focal traumatic brain injury. However, the involvement of B2R in trauma-induced diffuse injury has not yet been explored. This is an important point, since in humans a pattern of diffuse injury is commonly found in severely injured patients and has been associated with a poor neurological outcome and prognosis. Using the non-peptide B2R antagonist LF 16-0687 Ms and B2R null (B2R-/-) mice, we investigated the role of B2R in a model of closed head trauma (CHT). LF 16-0687 Ms given 30 min after injury reduced the neurological deficit by 26% and the cerebral edema by 22% when evaluated 4 h after CHT. Neurological function after CHT was improved in B2R-/- mice compared to B2R+/+ mice, although there was no difference in the development of brain edema. Treatment with LF 16-0687 Ms and B(2)R gene deletion decreased the accumulation of neutrophils at 24 h after CHT (50% and 36%, respectively). In addition, the inducible NO synthase (iNOS) mRNA level increased markedly, and this was reduced by LF 16-0687 Ms. Taken together, these data support a detrimental role of B2R in the development of the neurological deficit and of the inflammatory secondary damage resulting from diffuse traumatic brain injury. Therefore, blockade of bradykinin B2 receptors might represent an attractive therapeutic approach in the pharmacological treatment of traumatic brain injury.

[Neuronal death: potential role of the nuclear enzyme, poly (ADP-ribose) polymerase]. / Mort neuronale: rôle potentiel d'une enzyme nucléaire, la poly(ADP-ribose) polymérase.

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is known as a nuclear enzyme that is activated by DNA strand breaks to participate in DNA repair. It is also called poly(ADP-ribose) synthase (PARS) or poly(ADP-ribose) transferase (PADRT). In physiological conditions, PARP plays an important role in maintaining genomic stability. However, for several pathological situations, which include massive DNA injury (brain ischemia for example), excessive activation of PARP can deplete stores of nicotinamide adenine dinucleotide (NAD+), the PARP substrate, which, with the subsequent ATP depletion, leads to cell death. PARP activation appears to play a major role in neuronal death induced by cerebral ischemia, traumatic brain injury, Parkinson disease and other pathologies. PARP inhibitors (3-aminobenzamide and other compounds) and PARP gene deletion induced dramatic neuroprotection in experimental animals (rats, mice). Accordingly, these data suggest that PARP inhibitors could provide a novel therapeutic approach in a wide range of neurodegenerative disorders including cerebral ischemia and traumatic brain injury.

iNOS contribution to the NMDA-induced excitotoxic lesion in the rat striatum.

1. The aim of this study was to assess whether an excitotoxic insult induced by NMDA may induce an iNOS activity which contributes to the lesion in the rat striatum. 2. For this purpose, rats were perfused with 10 mM NMDA through a microdialysis probe implanted in the left striatum. Microdialysate nitrite content, striatal Ca-independent nitric oxide synthase activity and lesion volume were measured 48 h after NMDA exposure in rats treated with dexamethasone (DXM) (3 mg kg(-1) x 4) or aminoguanidine (AG) (100 mg kg(-1) x 4). 3. A significant increase in microdialysate nitrite content and in the Ca-independent NOS activity was observed 48 h after NMDA infusion. Both these increases were reduced by DXM and AG. The NMDA-induced striatal lesion was also reduced by both treatments. 4. Our results demonstrate that NMDA excitotoxic injury induces a delayed, sustained activation of a Ca-independent NOS activity. This activity is blocked by DXM and AG, strongly suggesting the involvement of iNOS. The fact that AG and DXM reduce the NMDA-elicited lesion suggests that iNOS contributes to the brain damage induced by excitotoxic insult.

Reduction of tyrosine nitration after N(omega)-nitro-L-arginine-methylester treatment of mice with traumatic brain injury.

Oxygen free radicals and nitric oxide (NO) have been proposed to be involved in the cascade of injury elicited by traumatic brain injury. However, the mechanism(s) of injury remain to be explored. Since superoxide generation is triggered by traumatic brain injury, the cytotoxic peroxynitrite could be formed, but it is not known if this actually occurs. Dot blot and immunohistochemistry studies were performed to quantify tyrosine nitration and identify cell types in which such reactions occur in the brain of mice submitted to traumatic brain injury. Nitrotyrosine formation increased from 4 to 24 h after traumatic brain injury and was primarily observed in degenerating neurons, in areas corresponding to the sites of direct impact (frontal cortex) and diffuse impact (frontoparietal cortex and ventromedial hypothalamic nucleus). Furthermore, N omega-nitro-L-arginine-methylester (L-NAME), a NO-synthase inhibitor which has previously been shown to promote neurological recovery in traumatic brain injury, reduced nitrotyrosine formation and the number of nitrotyrosine-positive neurons. These results indicate that traumatic brain injury induces peroxynitrite formation which may contribute to cell damage.

Protective effect of melatonin in a model of traumatic brain injury in mice.

The pineal hormone melatonin has recently been shown to exert neuroprotective activity in a variety of experimental neuropathologies in which free radicals are involved. This neuroprotective effect has been attributed to the antioxidant properties of melatonin. Considering that free radicals also play a deleterious role in traumatic brain injury (TBI), the purpose of the present study was to determine whether melatonin would have a beneficial effect in this pathology. Head injury was induced in mice and the neurological deficit was evaluated at 24 hr by a grip test. In this model, the free radical scavenger, alpha-phenyl-tert-butyl-nitrone (2 x 100 mg/kg, i.p.) given 5 min and repeated at 4 hr after TBI was neuroprotective. Melatonin (1.25 mg/kg, i.p.) given 5 min and repeated at 1, 2, and 3 hr after head trauma also significantly reduced the neurological deficit. This beneficial effect was not due to melatonin-induced hypothermia since repeated treatment with melatonin did not modify the colonic temperature of mice. This study shows that melatonin exerts a beneficial effect on the neurological deficit induced by traumatic brain injury in mice. The mechanisms of this neuroprotection remains to be established, and more particularly, the contribution of the antioxidant activity of melatonin.

Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal cerebral ischaemia in mice.

1. The temporal changes in constitutive NO-synthase (cNOS) and in calcium-independent NO-synthase activities were studied in mice subjected to 2 h of transient focal cerebral ischaemia. The changes in brain nitrites/nitrates (NOx) content were also studied. 2. NOS activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Brain NOx contents were investigated by the Griess colourimetric method. 3. cNOS activity in the infarcted cortical area was significantly reduced after 6 h of reperfusion and this activity remained attenuated for up to 10 days after ischaemia. A calcium-independent NOS activity began to increase 48 h after reperfusion, reached a maximum at 7 days and returned to baseline at 10 days. 4. There was a significant increase of brain NOx content beginning after 3 days of reperfusion. This increase was maximal at 7 days and returned to baseline at 10 days. 5. Thus, ischaemia followed by recirculation leads to a rapid, prolonged drop in cNOS activity in the infarcted cortex. There is also a substantial appearance of calcium-independent NOS activity in the later phase of transient ischaemia, leading to an important increase of NOx production.

Reduction of the neurological deficit in mice with traumatic brain injury by nitric oxide synthase inhibitors.

This study investigates the effect of the NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), on the neurological deficit 24 h after a moderate closed head injury in mice. Low doses of L-NAME or 7-NI given soon after the injury significantly reduced the neurological deficit compared to the vehicle-treated group. L-Arginine (300 mg/kg) did not alter the neurological deficit, but reversed the protective effects of both L-NAME and 7-NI when given at the same time. Both L-NAME and 7-NI had dose-related effects. The neuroprotective effects of L-NAME and 7-NI occurred when the drugs were given 5, 30, or 60 min after brain injury, but not when treatment was begun 2 h after brain injury, suggesting a short therapeutic window for both drugs. These results suggest that NO synthesis by neuronal NO synthase plays an important role in the early neurotoxic cascade leading to neurological deficit following traumatic brain injury.

Reduction of the neurological deficit in mice with traumatic brain injury by nitric oxide synthase inhibitors.

This study investigates the effect of the NO synthase inhibitors, NG-nitro L-arginine methyl ester (L-NAME) and 7-nitro indazole (7-NI), on the neurological deficit 24 h after a moderate closed head injury in mice. Low doses of L-NAME or 7-NI given soon after the injury significantly reduced the neurological deficit compared to the vehicle-treated group. L-Arginine (300 mg/kg) did not alter the neurological deficit, but reversed the protective effects of both L-NAME and 7-NI when given at the same time. Both L-NAME and 7-NI had dose-related effects. The neuroprotective effects of L-NAME and 7-NI occurred when the drugs were given 5, 30, or 60 min after brain injury, but not when treatment was begun 2 h after brain injury, suggesting a short therapeutic window for both drugs. These results suggest that NO synthesis by neuronal NO synthase plays an important role in the early neurotoxic cascade leading to neurological deficit following traumatic brain injury.

Blockers of NMDA-operated channels decrease glutamate and aspartate extracellular accumulation in striatum during forebrain ischaemia in rats.

Brain microdialysis was used to study changes in the glutamate and aspartate extracellular concentrations in the striatum of conscious rats submitted to 30 minutes cerebral ischaemia, using the four-vessel occlusion model. Perfusion of the N-methyl-D-aspartate (NMDA) receptor channel blockers, dizocilpine (MK-801; 75 microM) and Mg2+ (2.5 mM), inhibited the ischaemia-induced accumulation of glutamate and aspartate. The AMPA/kainate receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamylbenzo (F) quinoxaline (NBQX; 15 microM and 450 microM) had no effect on glutamate and aspartate levels during ischaemia. On the other hand, omission of Ca2+ from the perfusing solution did not alter the increases in glutamate and aspartate induced by ischaemia. These results suggest that the glutamate and aspartate accumulation in four-vessel occlusion ischaemia is mediated by activation of NMDA receptors in a Ca2+ independent manner.

Nitric oxide: an endogenous anticonvulsant substance.

In the present study, we examine the involvement of the L-arginine-nitric oxide pathway in seizure activity termination. Convulsions were induced reproducibly by intracerebroventricular administration of N-methyl-D-aspartate to conscious mice. The duration of the seizure activity was increased by inhibition of the NO-pathway or by intracerebroventricular injection of methylene blue, an inhibitor of guanylate cyclase activity. This increased duration in seizure activity was reversed by co-administration of L-arginine or by intracerebroventricular injection of guanosine 3':5' cyclic monophosphate (cGMP). These results suggest that nitric oxide produced in response to NMDA receptor activation leads to an increase in cGMP which induces the seizure activity termination.

Muscarinic binding of pial vessels and arachnoid membrane.

The muscarinic sites in arachnoid and pial vessels were compared by analysis of the binding of quinuclidinyl benzilate (QNB) to membrane preparations. Saturation analysis indicated that the process was saturable, high affinity, and related to protein concentration in both structures. Although the affinities in the two structures [KD = 0.039 (arachnoid) and 0.097 nM (pial vessels)] were similar, the arachnoid had approximately 10-fold more binding sites (Bmax = 2,100 fmol/mg of protein) than the pial vessels (Bmax = 250 fmol/mg of protein). This difference was found in both bovine and porcine fractions. Pharmacological analysis of [3H]QNB displacement by muscarinic and nonmuscarinic ligands gave the typical pattern of muscarinic receptors in both structures. Inhibition of binding to pial vessels by the M1 antagonist pirenzepine revealed only one low-affinity site (Ki = 7.8 x 10(-7) M), whereas, the arachnoid had a small proportion (21%) of high-affinity sites (Ki = 2.2 x 10(-9) M) associated with low-affinity sites (Ki = 5.50 x 10(-7) M). It is concluded that muscarinic-mediated effects that do not involve the M1 subtype are induced in bovine pial vessels by a relatively low concentration of binding sites. The high content of muscarinic binding sites and their diversity in the arachnoid suggest a functional role for muscarinic cholinergic receptors in this structure.

Cortical blood flow increases induced by stimulation of the substantia innominata in the unanesthetized rat.

The possible implication of projections from the substantia innominata (SI) to the cerebral cortex in the control of local cortical blood flow (CoBF) was studied in adult Fischer rats. Local blood flow (by helium clearance) and tissue gas partial pressures (pO2, pCO2) as metabolic indices, were measured in the frontal and parietal cortices in unanesthetized animals via chronically implanted probes connected to a mass spectrometer. Stimulating electrodes, also implanted chronically, were placed in the region of the SI. Out of 37 correctly located sites, 28 gave rise to cerebrovascular responses without significant hypertension or agitation. Both frontal (+114%) and parietal CoBF (+28%) increased significantly during ipsilateral 50 microA stimulation, but did not further significantly increase at 100 microA. Contralateral stimulation induced only small, non-significant effects. SI stimulation simultaneously increased cortical pO2 and decreased cortical pCO2, significantly more so in the frontal compared to the parietal cortex, and ipsilaterally compared to contralaterally. Both the CoBF and the tissue gas changes induced by SI stimulation were strongly potentiated by infusion of 0.15 mg/kg/h of the cholinomimetic agent physostigmine. The electrocorticogram (ECoG) was not systematically activated during the SI stimulation. The evidence presented favors a role for the cholinergic projections of the SI in control of CoBF (particularly frontal cortex), especially since the flow changes observed showed no obvious dependence on changes in local pCO2 or on paCO2, and could not be attributed to hypertension or behavioral changes.

Effect of the muscarinic agonist carbachol on pial arteries in vivo after endothelial damage by air embolism.

Reactions of pial arteries to the muscarinic agonist carbachol were tested in vivo in chloralose anesthetized cats before and after endothelial damage. Moderate endothelial damage was induced by arterial air embolism and verified by electron microscopy for the vessels tested. The experiments had three phases; First, the normal reactivity of pial arteries to carbachol (10(-7) to 10(-5) M) was tested using the microapplication technique, then, after air embolism, the reactivity was reinvestigated at the same vessel. Finally, pial arteries were taken out for scanning electron microscopy. The results show carbachol (10(-6) and 10(-5) M) induced significant dilations under control conditions, also after repetition at the same vessel. After air embolism, the reactions to carbachol were abolished. Morphologic data revealed that whereas control pial arteries showed intact endothelium, the embolized vessels revealed various degrees of endothelial alterations. All showed flattening of endothelial nuclei, to a greater or lesser degree, and in many cases, the endothelium had a wrinkled appearance; several arteries showed severe degradation of the intercellular junctions. It is concluded that (a) carbachol-induced muscarinic vasodilatation of pial arteries in vivo can be abolished after a morphologically verified endothelial lesion--thus confirming in vitro studies in larger arteries and (b) disturbed vascular function does not require rubbing of the endothelium, but occurs already with moderate endothelial damage.