Original Contributions
Superoxide anion production during reperfusion is reduced by an antineutrophil antibody after prolonged cerebral ischemia

https://doi.org/10.1016/S0891-5849(98)00215-9Get rights and content

Abstract

Neutrophils may be involved in the pathophysiology of reperfusion injury following cerebral ischemia. One potential mechanism of reperfusion injury by neutrophils is through production of the superoxide anion. We hypothesized that, due to progressive endothelial damage during ischemia, neutrophil activation would be more prominent after longer periods of ischemia prior to reperfusion. Thus, neutrophils would contribute more to pathological processes such as superoxide anion formation after longer than after shorter periods of ischemia. A reversible middle cerebral artery occlusion model in rats was employed and superoxide anion concentration was measured with a cytochrome c coated electrode placed on the cortical penumbral region. Occlusion times were varied from 60 min to 2 h, and neutrophils were inhibited with an antiCD18 antibody administered prior to occlusion. Neutrophil accumulation and reduction with antibody treatment was confirmed immunohistochemically. Superoxide anion (O2•−) concentration was detected during the hours following 60 min of occlusion, and increased further with 2 h of occlusion. Treatment with the antiCD18 antibody had no effect on O2•− concentration during reperfusion in the 60–90 min occlusion groups, but O2•− concentration was significantly lower in the antiCD18 antibody treated group than in the control group during reperfusion after 120 min of ischemia. The antibody also reduced cortical neutrophil accumulation in the 120 min ischemia group. These results indicate for the first time that superoxide production by neutrophils becomes more important with longer periods of ischemia, and other quantitatively less important sources of superoxide predominate with shorter periods of ischemia. This phenomenon may explain some of the variation seen between different models of ischemia with different durations of ischemia when targeting reactive oxygen species, and supports an approach to combination therapy to extend the therapeutic window and reduce the deleterious effects of reperfusion.

Introduction

In patients with ischemic stroke, restoration of blood flow to ischemic brain tissue can prevent brain tissue injury, but at the risk of hemorrhage and the development of cerebral edema, especially following prolonged periods of occlusion [1]. Recruitment of activated neutrophils by injured tissue during reperfusion may exacerbate the injury and increase the risk of reperfusion, but studies of the effect of neutrophil inhibition in models of ischemia and reperfusion using ischemia of a single duration have had mixed results [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. One important toxic secretory product of neutrophils is superoxide anion (O2•−), for which there is evidence of a role in progression of the ischemic injury [13]. We evaluated the contribution of neutrophils to superoxide formation seen during reperfusion after ischemia of various durations in a rat model of reversible middle cerebral artery (MCA) occlusion [14].

Using a cytochrome c coated platinized carbon electrode implanted on the pial surface of the ischemic hemisphere, we have confirmed that O2•− concentration in cerebral tissue is increased during reperfusion after MCA occlusion. Because of likely progressive endothelial injury and expression of neutrophil adhesion molecules after prolonged ischemia [12], [13], [14], [15], we hypothesized that most O2•− produced in the brain during reperfusion following prolonged ischemia is due to the activity of neutrophils recruited from the systemic circulation. We tested this hypothesis by applying in vivo electrochemical measurement of O2•− at the pial surface in a reversible MCA occlusion model of cerebral ischemia. Rats were treated with an antibody that inhibits recruitment of neutrophils. We examined in detail whether the antibody treatment effect was dependent on ischemia duration.

Section snippets

Materials and methods

Normotensive Sprague-Dawley rats of either sex (∼350 g) were anesthetized by 2% Halothane in O2 and room air (30:70) inhalation and then intubated and ventilated with a fixed volume ventilator using the same gas mixture. The femoral vein and artery were cannulated for blood pressure measurement, blood gas determinations, and for drug delivery. Rectal temperature was monitored and maintained at 38.0°C ± 0.5°C. A craniotomy was placed to expose an area of the superior and lateral cortex.

Results

There was no significant difference in MABP, PaO2, PaCO2, pH, and rectal temperatures between groups.

In 4 out of 6 animals, an increase in the O2•− specific electrode current was detectable during reperfusion following cerebral ischemia of 120 min, while there was a smaller increase in O2•− specific electrode current in the 60–90 min ischemia groups Fig. 1, Fig. 2 . There was no significant difference in the increase of the superoxide concentration (as indicated by the mean net CC-PACE charge

Discussion

These results demonstrate a differential effect on brain superoxide levels after reduction of neutrophil recruitment that depends on the duration of ischemia prior to reperfusion, which suggests that neutrophils are a more important source of O2•− production with prolonged ischemia prior to reperfusion. Matsuo et al. [7], demonstrated that inhibition of neutrophils reduced ascorbyl radical concentrations to baseline levels during reperfusion in a rat model of cerebral ischemia of 1 h duration

Acknowledgements

Acknowledgements — We thank Ms. H. Charmaine Rea for her technical assistance, and Prof. Ralph N. Adams and Dr. Arvin Oke for their invaluable expertise regarding the electrochemical methods.

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    This work was supported by the American Heart Association, Texas Affiliate, Inc.

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