Elsevier

Brain Research

Volume 958, Issue 1, 20 December 2002, Pages 166-175
Brain Research

Research report
Changes in oxidative stress, iNOS activity and neutrophil infiltration in severe transient focal cerebral ischemia in rats

https://doi.org/10.1016/S0006-8993(02)03685-5Get rights and content

Abstract

Oxidative stress, inducible nitric oxide synthase (iNOS) and neutrophils all contribute to post-ischemic brain damage. This study has determined the time courses of these three phenomena after ischemia in parallel with histological and functional outcomes. Ischemia was produced in rats by occluding the left middle cerebral artery and both common carotid arteries for 20 min. Regional cerebral blood flow (rCBF) rapidly decreased to 20% of its preischemic value during occlusion and stabilized at 60% following reperfusion. The striatal infarction was maximal 15 h after reperfusion (50±3 mm3), whereas the cortical infarction reached its maximum at 48 h (183±10 mm3). This drastic decrease in rCBF followed by incomplete reperfusion and massive infarction is, thus, extremely severe. The cortical infarction was strongly correlated with the neurologic deficit and loss of body weight. Oxidative stress, evaluated by the decrease in glutathione concentrations, appeared in the striatum at 6 h after reperfusion and in the cortex at 15 h. Calcium-independent NOS activity, considered as inducible NOS activity, was significantly enhanced at 24 h in the striatum and at 48 h in the cortex. Myeloperoxidase activity, a marker of neutrophil infiltration, was significantly increased at 48 h in both the striatum and cortex. These time courses show that the delayed iNOS activity and neutrophil infiltration that occur after the maturation of infarction in severe ischemia may not contribute to ischemic brain damage. By contrast, early oxidative stress may well be implicated in cerebral injury.

Introduction

Cerebral ischemia results in a time-dependent cascade of molecular events including the rapid depletion of intracellular energy stores, anaerobic glycolysis, lactic acidosis, membrane depolarization, glutamate excitotoxicity, intracellular calcium overload, activation of calcium-stimulated enzymes (phospholipases, proteases, protein kinases, nitric oxide synthase or NOS, endonucleases), mitochondrial dysfunction, free radical production, activation of the immune system (neutrophils, monocytes/macrophages), overexpression of genes and neuronal death [41], [42], [64]. The present study focussed on three of these changes that are interdependent, free radical production, NOS activity and neutrophil infiltration.

Free radicals are believed to be implicated in stroke [10], [11], [12], [44], [59], [69]. Mitochondria generate basal amounts of superoxide anions under physiological conditions and these are rapidly scavenged by endogenous antioxidant systems (catalase, superoxide dismutase, glutathione peroxidase). Reperfusion after ischemia provides oxygen as a substrate for numerous enzyme oxidation reactions that produce free radicals (superoxide anions, hydroxyl radicals and hydrogen peroxide) to such extent that antioxidant systems are overwhelmed [12], [16], [47]. This oxidative stress results in oxidative damage, including lipid peroxidation, protein oxidation and DNA damage, which can lead to cell death [20], [24], [25], [44], [59]. The only neuroprotective treatment studied to date in phase III in stroke patients is the antioxidant ebselen [54], [73]. Beside the proven benefits of reperfusion therapies with rtPA (recombinant tissue plasminogen activator), there is indeed a lack of neuroprotective agents in clinical trials [4].

Nitric oxide (NO) also plays a complex role in cerebral ischemia [7], [37], [61], [70]. NO is a physiological messenger in the CNS and is synthesized by three types of NOS: the constitutive calcium/calmodulin-dependent neuronal (nNOS) and endothelial (eNOS) isoforms, and an inducible calcium-independent isoform (iNOS) [2]. NO can be neuroprotective or neurotoxic during ischemia depending on the NOS involved. Endothelial NOS produces NO with beneficial effects (vasodilation, inhibition of platelet aggregation and of polymorphonuclear neutrophil (PMN) adhesion) [33], whereas nNOS and iNOS appear to be deleterious. These deleterious effects were demonstrated using knockout mice [28], [32], [38], selective NOS inhibitors [18], [34], [52], [56], [75], [77], [78] and antisense [46], [57]. NO promotes neuronal cell death mainly by its reaction with superoxide anions to generate the highly reactive, cytotoxic substance peroxynitrite that can oxidize proteins, lipids and DNA [65].

Neutrophils are also implicated in post-ischemic brain damage [39], [63], [72]. PMN infiltrate into the cerebral parenchyma after ischemia reperfusion [5], [48], [76]. Their deleterious actions have been demonstrated in animals rendered neutropenic with antineoplastic agents like vinblastine [30], or anti-neutrophil antibodies [48]. Similar results have been obtained by inhibiting neutrophil adhesion with dextran sulfate [74], anti-adhesion molecule antibodies [49] and mice lacking one of the genes encoding adhesion molecules [14], [15], [60].

Multiple interactions may occur between neutrophils, free radicals and NO. In vitro studies indicated that oxidative stress activates the transcription of the nuclear factor kappa B, which in turn triggers the synthesis of iNOS [3], [29], [55] and adhesion molecules (VCAM-1, ICAM-1, E-selectin) [3], [13], [31], [55]. Neutrophils are also a potential source of NO [36], [37], [44] and free radicals [43], [59], [63], [69].

The present study was done on rats with transient focal cerebral ischemia produced by occluding the left middle cerebral artery (MCA) and both common carotid arteries (CCAs) for 20 min. We first verified that artery occlusion decreased cerebral blood flow and was followed by reperfusion. We then monitored the temporal changes in infarct volume, the neurologic deficit and the body weight loss. We focussed on the three deleterious events described above and assessed the time courses of (1) oxidative stress by measuring total tissue glutathione, the main endogenous antioxidant agent, (2) calcium-dependent and -independent NOS activities by a radioenzymatic assay, and (3) neutrophil infiltration by measuring the activity of myeloperoxidase (MPO), an enzyme marker of PMN.

Section snippets

Materials

All chemicals were purchased from Sigma Chemical Corporation (Saint Quentin Fallavier, France), except as otherwise stated.

Animals

All experiments were performed on male Sprague–Dawley rats (weighing 300 to 330 g, Iffa Credo, L’Arbresle, France) in accordance with National Institutes of Health and French Department of Agriculture guidelines (license number 01352).

Induction of transient focal cerebral ischemia

Rats were anesthetized with chloral hydrate (400 mg/kg, i.p., Prolabo, Fontenay-sous-Bois, France) and allowed to breathe spontaneously. The

Experiment 1: Change in regional cerebral blood flow

Occlusion of the MCA and both CCAs caused a sustained decrease in rCBF monitored at the left cortical surface (Fig. 1). The rCBF was decreased to 17±4% of the preischemic value (P<0.001) 1 min after occlusion of the three arteries, and remained unchanged throughout the entire occlusion period. The rCBF immediately increased when the MCA microclip and CCA clamps were removed, and had returned to the preischemic value 5 min later (86±13%). The rCBF had stabilized at approximately 60% of the

Discussion

The transient focal cerebral ischemia used in the present study caused the rapid maturation of infarction together with a neurologic deficit and a loss of body weight. There was also early decreases in GSx concentration and cNOS activity, while iNOS activity and neutrophil infiltration were delayed.

We monitored rCBF on the left cortical surface using laser-Doppler flowmetry to assess the severity of ischemia and determine whether reperfusion had occurred. Occlusion of the three arteries (MCA

Acknowledgements

We thank Owen Parkes for checking the English text and Doctor Marcel Debray for statistical analysis.

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