Valérie C. Besson

Fenofibrate, a peroxisome proliferator-activated receptor α 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.

Deleterious poly(ADP-ribose)polymerase-1 pathway activation in traumatic brain injury in rat

Traumatic brain injury produces nitric oxide and reactive oxygen species. Peroxynitrite, resulting from the combination of nitric oxide and superoxide anions, triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1. As excessive activation of this enzyme induces cell death, we examined the production of nitrosative stress, the activation of poly(ADP-ribose)polymerase-1, and the role of this enzyme in the outcomes of traumatic brain injury produced by fluid percussion in rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 30 min post-injury, and that persisted for 72 h. The poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide, at 10 and 30 mg/kg, significantly improved the neurological deficit, with a 60% reduction in the brain lesion volume and inhibition of poly(ADP-ribose)polymerase-1 activation. Thus, poly(ADP-ribose)polymerase-1 is involved in the neurological consequences of traumatic brain injury and may be a promising therapeutic target in clinical treatment of acute brain trauma.

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.

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)α 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 PPARα 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.

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.

Drug targets for traumatic brain injury from poly(ADP-ribose)polymerase pathway modulation

The deleterious pathophysiological cascade induced after traumatic brain injury (TBI) is initiated by an excitotoxic process triggered by excessive glutamate release. Activation of the glutamatergic N‐methyl‐D‐aspartate receptor, by increasing calcium influx, activates nitric oxide (NO) synthases leading to a toxic production of NO. Moreover, after TBI, free radicals are highly produced and participate to a deleterious oxidative stress. Evidence has showed that the major toxic effect of NO comes from its combination with superoxide anion leading to peroxynitrite formation, a highly reactive and oxidant compound. Indeed, peroxynitrite mediates nitrosative stress and is a potent inducer of cell death through its reaction with lipids, proteins and DNA. Particularly DNA damage, caused by both oxidative and nitrosative stresses, results in activation of poly(ADP‐ribose) polymerase (PARP), a nuclear enzyme implicated in DNA repair. In response to excessive DNA damage, massive PARP activation leads to energetic depletion and finally to cell death. Since 10 years, accumulating data have showed that inactivation of PARP, either pharmacologically or using PARP null mice, induces neuroprotection in experimental models of TBI. Thus TBI generating NO, oxidative and nitrosative stresses promotes PARP activation contributing in post‐traumatic motor, cognitive and histological sequelae. The mechanisms by which PARP inhibitors provide protection might not entirely be related to the preservation of cellular energy stores, but might also include other PARP‐mediated mechanisms that needed to be explored in a TBI context. Ten years of experimental research provided rational basis for the development of PARP inhibitors as treatment for TBI.

Long-term histological and behavioural characterisation of a collagenase-induced model of intracerebral haemorrhage in rats

Although intracerebral haemorrhage (ICH) entails the highest rates of mortality and disability of all stroke subtypes, efficient neuroprotective therapy is still needed. As functional recovery is a major endpoint in clinical trials, preclinical studies must demonstrate the potential of drugs to improve the sensorimotor and cognitive function of animals. In addition, behavioural studies should be performed on the long-term in order to truly mimic clinical needs. The aim of our study was to characterise a model of intracerebral haemorrhage using both histology and long-term behaviour. ICH was induced in rats by an intrastriatal injection of collagenase. Histology was performed 24h, 7 days and 2 months after ICH. Among a set of sensorimotor tests, we discriminate those able to reveal long-term deficits (up to 2 months) after cerebral haemorrhage. Our five behavioural tests (a neurological score, an adhesive removal test, two beam-walking tests and ipsilateral circling induced by dexamphetamine) proved to be effective in revealing sensorimotor deficits up to 35 days or more after cerebral haemorrhage. In conclusion, these behavioural tests appear of particular interest to screen protective agents that may exhibit benefits in patients who suffer ICH.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.

Opportunities for the repurposing of PARP inhibitors for the therapy of non-oncological diseases

The recent clinical availability of the PARP inhibitor olaparib (Lynparza) opens the door for potential therapeutic repurposing for non‐oncological indications. Considering (a) the preclinical efficacy data with PARP inhibitors in non‐oncological diseases and (b) the risk–benefit ratio of treating patients with a compound that inhibits an enzyme that has physiological roles in the regulation of DNA repair, we have selected indications, where (a) the severity of the disease is high, (b) the available therapeutic options are limited, and (c) the duration of PARP inhibitor administration could be short, to provide first‐line options for therapeutic repurposing. These indications are as follows: acute ischaemic stroke; traumatic brain injury; septic shock; acute pancreatitis; and severe asthma and severe acute lung injury. In addition, chronic, devastating diseases, where alternative therapeutic options cannot halt disease development (e.g. Parkinsons disease, progressive multiple sclerosis or severe fibrotic diseases), should also be considered. We present a preclinical and clinical action plan for the repurposing of PARP inhibitors.

Deleterious activation of poly(ADP-ribose)polymerase-1 in brain after in vivo oxidative stress.

Oxidative stress has been shown to be implicated in the pathogenesis of central nervous system injuries such as cerebral ischemia and trauma, and chronic neurodegenerative diseases. In vitro studies show that oxidative stress, particularly peroxynitrite, could trigger DNA strand breaks, which lead to the activation of repairing enzymes including Poly(ADP-ribose) Polymerase-1 (PARP-1). As excessive activation of this enzyme induces cell death, we examined whether such a cascade also occurs in vivo in a model of oxidative stress in rat brain. For this purpose, the mitochondrial toxin malonate, which promotes free radical production, was infused into the left striatum of rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 1 h after malonate, and that they persisted for 24 h. The PARP inhibitor, 3-aminobenzamide, significantly reduced the lesion and inhibited PARP-1 activation induced by malonate. These results demonstrate that oxidative stress induced in vivo in the central nervous system leads to the activation of poly(ADP-ribose)polymerase-1, which contributes to neuronal cell death.