Spontaneous resurgence of conditioned fear weeks after successful extinction in brain injured mice.

Mild traumatic brain injury (TBI) is a major risk factor for post-traumatic stress disorder (PTSD), and both disorders share common symptoms and neurobiological defects. Relapse after successful treatment, known as long-term fear resurgence, is common in PTSD patients and a major therapeutic hurdle. We induced a mild focal TBI by controlled cortical impact (CCI) in male C57BL/6 J mice and used fear conditioning to assess PTSD-like behaviors and concomitant alterations in the fear circuitry. We found for the first time that mild TBI, and to a lesser extent sham (craniotomy), mice displayed a spontaneous resurgence of conditioned fear when tested for fear extinction memory recall, despite having effectively acquired and extinguished conditioned fear 6 weeks earlier in the same context. Other characteristic symptoms of PTSD are risk-taking behaviors and cognitive deficits. CCI mice displayed risk-taking behaviors, behavioral inflexibility and reductions in processing speed compared to naïve mice. In conjunction with these changes there were alterations in amygdala morphology 3 months post-trauma, and decreased myelin basic protein density at the primary lesion site and in distant secondary sites such as the hippocampus, thalamus, and amygdala, compared to sham mice. Furthermore, activity-dependent brain-derived neurotrophic factor (BDNF) transcripts were decreased in the prefrontal cortex, a key region for fear extinction consolidation, following fear extinction training in both TBI and, to a lesser extent, sham mice. This study shows for the first time that a mild brain injury can generate a spontaneous resurgence of conditioned fear associated with defective BDNF signalling in the prefrontal cortex, PTSD-like behaviors, and have enduring effects on the brain.

Oxidative stress implication in a new phenotype of amyotrophic quadricipital syndrome with cardiac involvement due to lamin A/C mutation.

This study aimed at evaluating OS in an amyotrophic quadricipital syndrome with cardiac impairment in a family of 80 members with a mutation in lamin A/C gene. Twelve patients had cardiac involvement (5 cardiac and skeletal muscles impairment). OS was evaluated in blood samples (thiobarbituric acid-reactive substances (TBARS), carbonylated proteins (PCO)) 6 "affected patients" with phenotypic and genotypic abnormalities without heart failure and 3 "healthy carrier" patients. OS was higher in affected patients than in healthy, as shown by the higher TBARS and PCO values. Patients with cardiac and peripheral myopathy exhibited a higher OS than patients with only cardiac disease (TBARS: 1.73 +/- 0.05 vs. 1.51 +/- 0.04 mmol/l (p = 0.051), PCO: 2.73 +/- 0.34 vs. 0.90 +/- 0.10 nmol/mg protein (p = 0.47)), and with healthy carriers patients (TBARS: 1.73 +/- 0.05 vs. 1.16 +/- 0.14 mmol/l (p = 0.05), PCO: 2.73 +/- 0.34 vs. 0.90 +/- 0.20 nmol/mg protein (p = 0.47)). OS may thus contribute to the degenerative process of this laminopathy. ROS production occurs, prior to heart failure symptoms. We suggest that the extent activation may also promote the variable phenotypic expression of the disease.

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.

Anti-inflammatory effects of PJ34, a poly(ADP-ribose) polymerase inhibitor, in transient focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Activation of poly(ADP-ribose) polymerase (PARP) is deleterious during cerebral ischemia. We assessed the influence of PARP activation induced by cerebral ischemia on the synthesis of proinflammatory mediators including the cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) and the adhesion molecules, E-selectin and intercellular adhesion molecule-1 (ICAM-1). EXPERIMENTAL APPROACH: Ischemia was induced by intravascular occlusion of the left middle cerebral artery for 1 h in male Swiss mice anaesthetized with ketamine and xylazine. The PARP inhibitor PJ34 (1.25-25 mg kg(-1)) was administered intraperitoneally 15 min before and 4 hours after, the onset of ischemia. Animals were killed 6 h or 24 h after ischemia and cerebral tissue removed for analysis. KEY RESULTS: Ischemia increased TNF-alpha protein in cerebral tissue at 6 and 24 h after ischemia. All doses of PJ34 blocked the increase in TNF-alpha at 6 h and 25 mg kg(-1) PJ34 had a sustained effect for up to 24 h. Quantitative real time polymerase chain reaction showed that PJ34 (25 mg kg(-1)) reduced the increase in TNF-alpha mRNA by 70% at 6 h. PJ34 also prevented the increase in mRNAs encoding IL-6 (-41%), E-selectin (-81%) and ICAM-1 (-54%). PJ34 (25 mg kg(-1)) reduced the infarct volume (-26%) and improved neurological deficit, 24 h after ischemia. CONCLUSIONS AND IMPLICATIONS: PJ34 inhibited the increase in the mRNAs of four inflammatory mediators, caused by cerebral ischemia. The contribution of this effect of PJ34 to neuroprotection remains to be clarified.

Modulation by nitric oxide of cerebral neutrophil accumulation after transient focal ischemia in rats.

A beneficial role of nitric oxide (NO) after cerebral ischemia has been previously attributed to its vascular effects. Recent data indicate a regulatory role for NO in initial leukocyte-endothelial interactions in the cerebral microcirculation under basal and ischemic conditions. In this study, the authors tested the hypothesis that endogenous NO production during and/or after transient focal cerebral ischemia can also be neuroprotective by limiting the process of neutrophil infiltration and its deleterious consequences. Male Sprague-Dawley rats were subjected to 2 hours occlusion of the left middle cerebral artery and the left common carotid artery. The effect of NG-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg, intraperitoneally), an NO synthase inhibitor, was examined at 48 hours after ischemia on both infarct size and myeloperoxidase activity, an index of neutrophil infiltration. L-NAME given 5 minutes after the onset of ischemia increased the cortical infarct volume by 34% and increased cortical myeloperoxidase activity by 60%, whereas administration of L-NAME at 1, 7, and 22 hours of reperfusion had no effect. Such exacerbations of infarction and myeloperoxidase activity produced when L-NAME was given 5 minutes after the onset of ischemia were not observed in rats rendered neutropenic by vinblastine. These results suggest that after transient focal ischemia, early NO production exerts a neuroprotective effect by modulating neutrophil infiltration.

Early endonuclease activation following reversible focal ischemia in the rat brain.

The structural changes that occur in chromatin DNA after ischemic brain injury are poorly understood. The presence of oligonucleosome fragments that are recognized as the characteristic DNA ladder has been demonstrated in global and focal ischemia, associated or not with random DNA fragmentation. Using pulsed-field gel electrophoresis, which improves DNA separation, we have now detected initial stages of DNA fragmentation that occur already 6 h after reversible focal cerebral ischemia in rats. This result confirms that internucleosomal DNA fragmentation precedes random DNA fragmentation in vulnerable striatal and cortical neurons following reversible focal cerebral ischemia.

Short therapeutic window for MK-801 in transient focal cerebral ischemia in normotensive rats.

The present study investigates the role of N-methyl-D-aspartate (NMDA) receptors in a model of transient focal cerebral ischemia in normotensive rats. The left middle cerebral artery and both common carotid arteries were occluded for 60 min. Preliminary studies indicated that this gave reproducible infarctions of the cortex and striatum. These infarctions were the result of severe ischemia followed by complete reperfusion after clamp removal, as showed by striatal tissue Po2 monitoring. Microdialysis indicated that glutamate concentration increased immediately after occlusion and returned to the baseline value 40 min after clamp removal. MK-801 (1 mg kg-1 i.v.), an antagonist of the NMDA glutamatergic receptor, reduced the cortical infarct volume by 29% (p < 0.001) and the striatal infarct volume by 14% (p < 0.05) when given just prior to ischemia, but had no neuroprotective activity when given 30 min after the onset of ischemia. This short therapeutic window for MK-801 suggests that NMDA receptors play only a transient role in reversible focal ischemia in rats.

Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis.

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alternations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385-388). We now show that a significant number of striatal and cortical neurons, exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

Antisense oligodeoxynucleotide to inducible nitric oxide synthase protects against transient focal cerebral ischemia-induced brain injury.

Nitric oxide (NO) has been suspected to mediate brain damage during ischemia. Here the authors studied the effects of an antisense oligodeoxynucleotide (ODN) directed against the inducible isoform of NO synthase (iNOS) in a model of transient focal cerebral ischemia in rats. Treatment consisted of seven intracerebroventricular injections of a phosphodiester/phosphorothioate chimera ODN (3 nmol each) at 12-hour intervals, and was initiated 12 hours before a 2-hour occlusion of the left middle cerebral artery and common carotid artery. Outcomes were measured three days after ischemia. When compared with animals treated with vehicle or an appropriate random non-sense control ODN sequence, the antisense treatment reduced the lesion volume by 30% and significantly improved recovery of sensorimotor functions, as assessed on a neuroscore. This effect was associated with a decrease in iNOS expression, as assessed by Western blot, a 39% reduction in iNOS enzymatic activity evaluated as Ca2+-independent NOS activity, and a 37% reduction in nitrotyrosine formation, reflecting protein nitration by NO-derived peroxynitrite. These findings provide new evidence that inhibition of iNOS may be of interest for the treatment of stroke.

alpha-Phenyl-N-tert-butylnitrone attenuates excitotoxicity in rat striatum by preventing hydroxyl radical accumulation.

Various in vitro experiments have indicated that oxygen-derived free radicals may contribute to excitotoxic neuronal death. In the present study we induced excitotoxicity in rat striatum by perfusing glutamate at a high concentration through a microdialysis probe. We observed an increased formation of hydroxyl radicals (.OH) during the perfusion of the excitotoxin and an extensive striatal lesion 24 h after the insult. The spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), attenuated both hydroxyl radical levels and the volume of the lesion. This result suggests that the neuroprotection may be due to a free radical scavenging mechanism. It also implies that PBN may be used in pathological situations involving excitotoxicity such as stroke, brain trauma, and chronic neurologic diseases.

The neuroprotective effect of a nitric oxide inhibitor in a rat model of focal cerebral ischaemia.

Recent data showed that glutamate toxicity in primary cortical cultures is mediated by nitric oxide. In order to investigate the effect of inhibition of NO synthase on focal cerebral ischaemia in rats, we studied the histological consequences of a middle cerebral artery (MCA) occlusion after post-operative treatment with NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase. We found a significant reduction of cortical (-43%) and striatal (-25%) necrotic volumes induced by MCA occlusion, indicating that NO synthesis plays an important role in the neurotoxic cascade leading to neuronal damage after focal cerebral ischaemia in rats.

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.

Dose- and time-dependence of L-NAME neuroprotection in transient focal cerebral ischaemia in rats.

1. In this study the effect of the dose and administration time of NG-nitro-L-arginine methyl ester (L-NAME), an NO-synthase inhibitor, in a model of transient focal cerebral ischaemia in rats was investigated. 2. Two injections of L-NAME were given, of 1, 3 and 10 mg kg-1, 5 min and 3 h after the onset of ischaemia. None of the doses gave any striatal neuroprotection, but 1 and 3 mg kg-1 L-NAME reduced the infarcted volume in the cortex (by 26%, P < 0.01 for 1 mg kg-1 and 21%, P < 0.05 for 3 mg kg-1), whereas 10 mg kg-1 had no neuroprotective effect. 3. Single injections of L-NAME 1 mg kg-1, given 5 min or 3 h after ischaemia onset, had similar neutoprotective effects on the cortical infarction as did the repeated injections. 4. L-NAME 1 mg kg-1 given 3, 6 or 9 h after ischaemia induction reduced the cortical infarct volume by 19% (P < 0.01) when given 3 h after ischaemia, by 21% (P < 0.01) when given at 6 h, and by 16% (P < 0.05) when given at 9 h, but had no neuroprotective activity when given 12 h after ischaemia. 5. Thus a low dose of L-NAME is neuroprotective in a model of transient focal ischaemia, with a wide therapeutic window, much larger than that found or MK-801.

Loss of NADPH-diaphorase containing neurones after reversible focal ischaemia in rats delayed by L-NAME.

In the present study, NADPH-diaphorase histochemistry was used to assess the temporal evolution of the number of nitric oxide (NO)-synthase containing neurones after reversible focal cerebral ischaemia in rats. The number of NADPH-diaphorase containing neurones was reduced by 50% and 90% respectively 6 and 24 h after ischaemia. L-NAME, a NO-synthase inhibitor, prevented the loss of NADPH-diaphorase containing neurones observed 6 h after ischemia but not 24 h after ischaemia, suggesting that in the early phase, nitric oxide is involved in this phenomenon.

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.

Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury.

1. The aim of this study was to investigate the effect of N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium-independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats. 2. Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg(-1)) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium-independent NOS) were evaluated 3 days after ischaemia. 3. 1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction. 4. 1400W attenuated the calcium-independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity. 5. These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke.

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.

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.

Striatal dopamine participates in glutamate-induced hydroxyl radical generation.

Using a microdialysis technique we showed that the exposure of the rat striatum to glutamate yields hydroxyl radicals and results in striatal damage. We postulated that dopamine release is enhanced by glutamate perfusion and that the enzymatic metabolism of dopamine may account for this hydroxyl radical formation. The inhibition of monoamine oxidases by i.p. co-administration of clorgy-line and deprenyl reduced hydroxyl radical production induced by glutamate perfusion, but significantly increased the striatal damage. Our results suggest that the enzymatic metabolism of dopamine participates in glutamate-induced hydroxyl radical generation but that other by-products of dopamine may be responsible for the aggravation of the striatal injury.