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

Abstract

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.

Introduction

Reactive free radicals, including reactive oxygen species and reactive nitrogen species are implicated in the pathogenesis of central nervous system injuries such as traumatic brain injury (TBI), spinal cord injury, ischemia and chronic neurodegenerative diseases [13], [18], [25]. These highly reactive radicals and oxidants may indiscriminately attack proteins, lipids and DNA, causing oxidative modification and strand breakage [8], [9], [27]. The DNA strand breaks activate the constitutive nuclear enzyme poly(ADP-ribose)polymerase 1 (PARP-1, EC 2.4.2.30), which is implicated in such physiological processes as DNA repair [10], genomic stability [5] and apoptosis [20]. Moreover, PARP-1 has been shown to mediate necrotic cell death in response to excessive DNA damage under pathological conditions [20]. Activated PARP-1 catalyses the addition of long branched chains of poly(ADP-ribose) from its substrate NAD to a set of nuclear proteins including DNA polymerase I and II, Ca²⁺–Mg²⁺-endonuclease, histones, several chromatin-binding proteins and PARP-1 itself [10]. There is now evidence that excess active PARP-1 is a crucial factor in oxidative and excitotoxic cell death [33], [42]. PARP-1 activation leads to NAD depletion, resulting in a loss of ATP as it is used to synthesize new NAD, and finally to cell death [33]. In cultured cells exposed to hydrogen peroxide or peroxynitrite, DNA strand breaks are increased, PARP-1 is activated and energy resources are depleted as indicated by marked drops in NAD and ATP concentrations. These cellular disturbances can be prevented by PARP inhibitors [7], [37], [38], [41]. Multiple and strong experimental evidences have implicated PARP in cerebral ischemia and reperfusion in vivo. Indeed, cerebral ischemia–reperfusion activates PARP [16], [17], and PARP-1 mediates ischemic/reperfusion brain injury in vivo, since PARP-1 null mice and normal rodents treated with various PARP inhibitors, present a reduced infarct size [1], [12], [16], [17], [26], [43], [52]. The findings that both PARP-1 gene disruption and PARP inhibition greatly attenuate ischemic neuronal death in vivo indicate that PARP-1 activation is important in ischemic brain injury. By contrast, only few studies have demonstrated the implication of PARP in traumatic brain injury. PARP inhibition protects hippocampal slices against percussion-induced loss of CA1 pyramidal cells evoked response in vitro [48]. Whalen and co-workers [49], [50] have shown that the motor and cognitive deficits of mice submitted to TBI are less severe when the PARP-1 gene is inactivated. Lastly, it has recently been demonstrated that GPI 6150 (1,11b-dihydro-[²H]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel PARP inhibitor, reduces the lesion area evaluated 24 h after TBI in rats [24]. To date, no published data have shown the effect of the pharmacological inhibition of PARP on the outcomes in the late phase after traumatic brain injury.

The present study was therefore carried out to explore the implication of the peroxynitrite–PARP-1 pathway in the post-traumatic outcomes of focal TBI caused by fluid percussion on rats. We first determined whether TBI triggered the production of peroxynitrite and the activation of PARP-1. We then assessed the role of PARP activation on post-traumatic events by examining the effect of the well-established and commonly used PARP inhibitor 3-aminobenzamide [45], on the neurological deficit and brain lesion volume 7 days after TBI. Lastly, we studied the effect of 3-aminobenzamide on the activation of PARP-1 following TBI.