Free radical spin trap α-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury

doi:10.1016/S0891-5849(01)00700-6

Free Radical Biology and Medicine

Volume 31, Issue 10, 15 November 2001, Pages 1191-1197

https://doi.org/10.1016/S0891-5849(01)00700-6 Get rights and content

Abstract

It has been documented that α-phenyl-N-tert-butyl-nitron (PBN) possesses a potent neuroprotective effect when administered after transient focal cerebral ischemia. However, contradicting results were reported regarding its effect in transient global ischemia. To further elucidate the mechanism of PBN action, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global ischemia in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of reperfusion. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neocortex and thalamus. Close to 100% damage was observed in the stratum and hippocampal CA1, CA3, and dentate gyrus subregions. Postischemic PBN treatment significantly enhanced animal survival and reduced damage in the neocortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neocortex. PBN suppressed the activation of caspase-3 in both structures. It is concluded that PBN is a potent neuroprotectant against both focal and global ischemia; besides its function as a free radical scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.

Introduction

Cerebral ischemia and reperfusion enhance production of reactive oxygen species (ROS), which can cause membrane damage or enzyme inactivation due to lipid peroxidation and/or protein oxidation [1], [2]. Recent studies have shown that enhanced ROS production triggers a secondary mitochondrial dysfunction and formation of a mitochondrial permeability transition (MPT). Once MPT has formed, apoptosis-inducing factors and cytochrome c are released from mitochondria into the cytoplasm, which triggers programmed cell death pathways involving caspase activation and, eventually, lead to DNA fragmentation. Spin trapping agents have been shown to reduce ischemic brain damage induced by transient or permanent focal ischemia in rats and in primates [3], [4], [5], [6], [7] even when it was administered 3 h after reperfusion. In previous studies reported by Folbergrová et al. and Kuroda et al. PBN, given 1 h after the start of recirculation following a 2-h period of transient focal ischemia, ameliorated the secondary decrease of bioenergetic state and of ADP-stimulated mitochondrial respiration observed after 4 h of reperfusion [4], [8]. These results suggest that PBN acts at the mitochondrial level. In addition to its effect on preventing secondary mitochondrial dysfunction, nitrone-based free radical trap is also known to suppress oxidative stress through suppression of the action of the inflammatory cytokine cascade [9], [10].

Although PBN possesses a potent neuroprotective effect against focal ischemia, its effect in global ischemia has not been established. Cao et al. reported that PBN reduced hippocampal CA1 damage in gerbils after 5 min of global ischemia [11]. When the study was repeated in rats subjected to 12 or 15 min of global ischemia, the neuroprotective effect of PBN was minimal, if any [12]. It is not clear whether the discrepancy of the above-mentioned studies is due to the differences between animal species (rat vs. gerbil and primate) or the underlying mechanisms involved in the two types of ischemia models (focal vs. global). To further elucidate the mechanisms of PBN action on global ischemia, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 after 30 min of global ischemia in vehicle- and PBN-treated rats.

Section snippets

Surgical procedures

Male Wistar rats (Simonsen Laboratory, Gilrey, CA, USA), weighing 290–320 g, were fasted overnight with free access to tap water. Animal operative procedures were identical to those described previously [13]. Briefly, anesthesia was induced by inhalation of 3.5% halothane in a mixture of N₂O and O₂ (70:30). The animals were intubated and connected to a rodent ventilator. Anesthesia was maintained with 1.5% halothane in N₂O and O₂ (70:30) during the operation. The common carotid arteries were

Physiological parameters

Physiological parameters were measured 10 min before the induction of ischemia. Mean blood glucose concentrations were 4–5 mmol/l, PaCO₂ was maintained close to 40 mm Hg, PaO₂ was maintained close to 100 mm Hg, arterial pH was maintained close to 7.40, and arterial blood pressure was maintained close to 100–120 mmHg. Head and core temperatures were recorded at 10 min before induction of ischemia, 10 min after induction of ischemia, and at the end of 30 min ischemia, and both were maintained at

Discussion

Global ischemia of 30 min duration induced high mortality and severe brain damage. All vehicle-treated animals died after 1–2 d of reperfusion. Histopathological examination under light microscope showed close to 100% neuronal death in the striatum, the hippocampal CA1, CA3, CA4, and dentate gyrus subregions. Severe edema, microinfarct foci, and more than 50% damaged neurons were observed in the cingulate and neocortical areas and in the thalamus. These findings are in accordance with our

Acknowledgements

The present study was supported by research grants to from Hawaii Community Foundation (991881, 20001740), American Heart Association (99-51251Z), and Research Centers in Minority Institutions via NIH (RR3061-16).

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Original contributionFree radical spin trap α-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury

Abstract

Introduction

Section snippets

Surgical procedures

Physiological parameters

Discussion

Acknowledgements

Brain Res.

Neurobiol. Dis.

Life Sci.

Biochim. Biophys. Acta

Neurosci. Lett.

Brain Res.

Exp. Neurol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Cell

Chem. Biol.

J. Mol. Cell Cardiol.

J. Neurol. Sci.

Brain Res.

Free radical damage to protein and DNAmechanisms involved and relevant observations on brain undergoing oxidative stress

Ann. Neurol.

Reactive oxygen species and the central nervous system

J. Neurochem.

Neuroprotective effects of a novel nitrone, NXY-059, after transient focal ischemia in the rat

J. Cereb. Blood Flow Metab.

NXY-059, a free radical-trapping agent, substantially lessens the functional disability resulting from cerebral ischemia in a primate species

Stroke

Delayed treatment with the spin trap α -phenyl-N-tert-butyl nitrone (PBN) reduces infarct size following transient middle cerebral artery occlusion in rats

Acta Physiol. Scand.

N-tert-butyl-α -phenyl nitrone improves recovery of brain energy state in rats following transient focal ischemia

Proc. Natl. Acad. Sci.

Effects of the spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) in transient forebrain ischaemia in the rat

Acta Physiol. Scand.

The mitochondrial death/life regulator in apoptosis and necrosis

Ann. Rev. Physiol.

Original contribution
Free radical spin trap α-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury