Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Abstract

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of α-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.

Introduction

It has long been suggested that oxygen free radicals contribute to ischemic brain damage [1]. Although many investigators have attempted to prove the pathophysiological role of free radicals in cerebral ischemia, it has not been fully explained by in vivo cerebral ischemia models. Recently, several investigators have demonstrated the protective effects of α-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic brain injury. In the transient forebrain ischemia model of gerbil, PBN reduces the mortality after ischemia [2] and the neuronal damage in the CA1 area of hippocumpus caused by ischemia [3]. However, PBN fails to prevent postischemic CA1 damage in the rat [4]. As for focal cerebral ischemia, Cao and Phillis [5] have demonstrated that PBN significantly reduces cerebral infarction and decreases neurological deficit after ischemia using a rat model of persistent occlusion of the middle cerebral artery (MCA) in rats. In case of transient occlusion of MCA, however, the protective effect of PBN has been demonstrated in only one report as far as we know [6]. In addition, in this report, although there has been significant difference in the neuronal salvaging effect between PBN-treated and untreated animals, each set of data has quite large inter-experimental variation.

Characteristics of cerebral ischemia models are based on similarities with syndromes of human cerebrovascular disease [7] and, above all, the focal ischemia models are the most pertinent to stroke in humans [8]. However, it should be emphasized that each animal model of focal brain ischemia has considerable variations in size and location of infarction. Brint et al. [9] have demonstrated that tandem occlusion model of the MCA/common carotid artery (CCA) in the SHR (spontaneously hypertensive rat) offers a reliable method for producing neocortical infarction with reproducible topography and volume. In normotensive rats, Duverger and MacKenzie [10] have suggested that the MCA occlusion model using the Fischer-344 strain produces a standardized and reproducible infarction. These findings of the above two groups are, however, derived from permanent cerebral ischemia without any reperfusion maneuver.

It has been generally accepted that vascular reperfusion subsequent to a transient occlusion plays an important role in the pathophysiology of cerebral ischemia. Reperfusion is necessary to save brain tissue from ischemic damage on one side, but it may cause so-called reperfusion injury on the other if it is achieved after some critical period of ischemia. Free radicals are considered to be closely related to this type of injury. In the present study, we attempted to evaluate the pathogenetic role of free radicals in a transient ischemia model. The first atempt in this report was to determine a suitable experimental condition under which cerebral infarction with similar size and topography could be always reproduced in the rat of Fisher-344 strain with use of a transient MCA occlusion model. Second, the effects of PBN, a spin trapping agent, MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a newly-developed free radical scavenger [11], [12], [13], and oxypurinol, an inhibitor of xanthine oxidase (XO), were evaluated in this rat model by comparing the infarction size with the ischemic control. The effect of L-nitroarginine (L-NA), a nitric oxide synthase (NOS) inhibitor, was also examined in this model.