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.

Detection of vascular cell adhesion molecule-1 expression with USPIO-enhanced molecular MRI in a mouse model of cerebral ischemia.

Vascular damage plays a critical role after stroke, leading notably to edema, hemorrhages and stroke recurrence. Tools to characterize the vascular lesion are thus a real medical need. In this context, the specific nanoparticular contrast agent P03011, an USPIO (ultrasmall superparamagnetic iron oxide) conjugated to a peptide that targets VCAM-1 (vascular cell adhesion molecule-1), was developed to detect this major component of the vascular inflammatory response. This study aimed to make the proof of concept of the capacity of this targeted USPIO to detect VCAM-1 with MRI after cerebral ischemia in mouse. The time course of VCAM-1 expression was first examined by immunohistochemistry in our model of cerebral ischemia-reperfusion. Secondly, P03011 or nontargeted USPIO P03007 were injected 5 h after ischemia (100 µmol iron kg⁻¹; i.v.) and in vivo and ex vivo MRI were performed 24 h after ischemia onset. Double labeling immunofluorescence was then performed on brain slices in order to detect both USPIO and VCAM-1. VCAM-1 expression was significantly up-regulated 24 h after ischemia in our model. In animals receiving P03011, both in vivo and ex vivo MRI performed 24 h after ischemia onset showed hypointense foci which could correspond to iron particles. Histological analysis showed a co-localization of the targeted USPIO and VCAM-1. This study demonstrates that VCAM-1 detection is possible with the USPIO P03011 in a model of cerebral ischemia. This kind of contrast agent could be an interesting clinical tool to characterize ischemic lesions in terms of vascular damage.

Plasma concentrations of arginine and related amino acids following traumatic brain injury: Proline as a promising biomarker of brain damage severity.

The aim of this study was to find a plasma biomarker, in relation with nitric oxide (NO), as a sign of brain damage severity following traumatic brain injury (TBI). We first investigated the post-traumatic evolution of the plasma concentrations of NO via the level of NO end-products metabolites (nitrite plus nitrate, NO(x)), that of l-arginine (Arg) and amino acids involved in its metabolism as well as the time course of neurological score in a rat model of lateral fluid percussion brain injury. First, the level of NO(x) was increased in plasma at 24 and 48 h post-TBI with a marked increase at 72 h. In contrast, this elevation was neither accompanied by a modification of plasma concentrations of Arg, nor of amino acids involved in NO and Arg metabolism, l-ornithine (Orn), l-glutamate (Glu), and l-glutamine (Gln). Second, TBI induced a fall of plasma l-proline (Pro) concentrations. The time course of post-TBI neurological deficit showed also a decrease of neurological score at 24, 48, and 72 h. Furthermore, there is a weak negative correlation between the neurological score and the plasma level of NO(x) (r=-0.305; P<0.05), while a marked positive correlation has been found between the neurological score and the plasma level of Pro (r=0.563; P<0.001). In conclusion, the plasma concentrations of Pro could be a promising marker of post-traumatic neurological deficit.

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.

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.

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.

Apoptotic mitochondrial pathway in neurones and astrocytes after neonatal hypoxia-ischaemia in the rat brain.

Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute ischaemia. Recent studies have provided evidence that caspase-3 is an important downstream event after hypoxia-ischaemia in the immature brain, but a minor event in the adult brain. Our investigations have focused on cell populations that expressed apoptotic effectors in the enzymatic death pathway including cytochrome c, caspase-9 and caspase-3. Expression, activation and cellular localization of these proteins were studied using cleavage of fluorogenic substrate and immunohistochemistry in neonatal rat brain after unilateral focal ischaemia. Caspase-3 enzyme activity was elevated in brain homogenate between 6 and 48 h after reperfusion. This activation was preceded by that of caspase-9, between 3 and 24 h. Apoptotic cell death was finally accomplished by poly-ADP-ribose polymerase cleavage, an endogenous caspase-3 substrate. In addition, immunodetection demonstrated that cytochrome c and activated caspase-9 and caspase-3 were expressed not only in the neurones, the primarily affected cells, but also within the astrocytes, which constituted a dense network delineating the infarct. These results suggested that glial injury may promote the formation of cystic lesions such as those observed clinically in the newborn brain.

[NO synthases: new pharmacological targets in cerebrovascular accident?]. / Les NO-synthases: de nouvelles cibles pharmacologiques dans l'accident vasculaire cérébral?

In cerebral ischemia, the disappointment related to anti-glutamate strategies in clinical trials has led to examine new targets for the treatment of stroke. In vitro studies demonstrated that overactivation of glutamate receptors leads to nitric oxide (NO) production that contributes to the excitotoxic neuronal death. The role of NO was then studied in in vivo models of cerebral ischemia. In the early phase after ischemia, NO is produced by the constitutive endothelial and neuronal isoforms of NO-synthase (NOS 3 and NOS 1) while in the later phase, the inducible NOS (NOS 2) is responsible for the delayed production of NO. NOS 3 appears beneficial via vasodilatation and inhibition of leukocyte adhesion and platelet aggregation. By contrast NOS 1 and NOS 2 were demonstrated deleterious in cerebral ischemia. This was shown by three distinct strategies: selective inhibitors, mutant mice deficient in NOS 1 or NOS 2, and antisenses directed to one of these isoforms. Moreover it is now thought that NO-induced neuronal death is mainly mediated through the formation of peroxynitrite anions resulting from the reaction between NO and superoxyde anion. Peroxynitrites indeed damage lipids, proteins and nucleic acids. DNA strand breaks in turn activate poly(ADP-ribose) polymerase (PARP). Overactivation of this enzyme in pathological conditions such as cerebral ischemia seems deleterious by depleting ATP stores. Thus inhibition of the NO-peroxynitrites-PARP pathway may lead to neuroprotective therapeutics in stroke.

[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.

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.

Influence of anesthesia protocol in experimental traumatic brain injury.

Most pharmacologic studies on brain trauma in animals are performed while the animals are under general anesthesia, which can interfere with brain metabolism and modify the experimental results. This study investigates the effects of three anesthetic drugs (halothane 2% and 4%, propofol at 10 mg/kg, and chloral hydrate at 400 mg/kg) on the traumatic brain injury-induced neurologic deficit in mice. Trauma was induced with a weight-drop device. For each drug, animals were divided into four groups; the first did not receive either anesthesia or trauma, the second received anesthesia but no trauma, the third received a trauma without anesthesia, and the fourth received anesthesia before the trauma. A neurologic examination using two different scorings (string and grip test) was performed 1 hour and 24 hours after the trauma. Mortality after trauma was increased for halothane 4% (48% versus 20% in unanesthetized mice), propofol (80% versus 30%), and chloral hydrate (70% versus 44%). Halothane 2% did not increase the mortality in traumatized mice. Halothane 2% or 4% anesthesia did not modify the string score after the trauma. Grip score after the trauma was better in mice anesthetized with halothane at either 2% or 4%. Mice injured under anesthesia with chloral hydrate had worse grip and string scores (P < .05) than unanesthetized mice. These results lead us to question the influence of anesthesia on the results obtained in experimental neuropharmacologic studies, particularly when there are discrepancies between two studies on the same pharmacologic treatment, which differ in their anesthesia protocols.

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.

Poly(ADP-ribose) synthase inhibition reduces ischemic injury and inflammation in neonatal rat brain.

Poly(ADP-ribose) synthase (PARS), an abundant nuclear protein, has been described as an important candidate for mediation of neurotoxicity by nitric oxide. However, in cerebral ischemia, excessive PARS activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARS on the (a) degree of cerebral injury, (b) process of inflammatory responses, and (c) functional outcomes in a neonatal rat model of focal ischemia. We demonstrate that administration of 3-aminobenzamide, a PARS inhibitor, leads to a significant reduction of infarct volume: 63 +/- 2 (untreated) versus 28 +/- 4 mm(3) (treated). The neuroprotective effects currently observed 48 h postischemia hold up at 7 and 17 days of survival time and attenuate neurological dysfunction. Inhibition of PARS activity, demonstrated by a reduction in poly(ADP-ribose) polymer formation, also reduces neutrophil recruitment and levels of nitrotyrosine, an indicator of peroxynitrite generation. Taken together, our results demonstrate that PARS inhibition reduces ischemic damage and local inflammation associated with reperfusion and may be of interest for the treatment of neonatal stroke.

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.

ARNT2, a transcription factor for brain neuron survival?

The processes responsible for the limited ability to divide and long survival of neurons are not well understood but may involve aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a recently identified protein, apparently belonging to the basic helix-loop-helix superfamily of transcription factors, which is expressed almost exclusively in brain during the whole lifetime. In agreement, we show, in the rat, that ARNT2 immunoreactivity could be observed only within nuclei of brain neurons and of dividing and neuronal PC12 cells, a localization consistent with a role in transcription regulation. Cell death elicited either by focal ischaemia in brain or oxidative stress in PC12 cells was largely preceded by an almost complete suppression of ARNT2 expression. In contrast, when PC12 cell cycle progression was impaired, ARNT2 expression was enhanced. Finally, the downregulation of ARNT2 levels induced by antisense oligonucleotides prevented PC12 cell proliferation and induced apoptosis. These observations support the hypothesis that ARNT2 is a neuronal transcription factor, regulating cell cycle progression and preventing cell death, whose sustained expression might ensure brain neuron survival.