A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke

Abstract

Oxidative stress is a major source of injury from cerebral ischemia and reperfusion. We hypothesized that a catalytic antioxidant AEOL 10150 [manganese (III) meso-tetrakis (di-N-ethylimidazole) porphyrin] would attenuate changes in brain gene expression in a mouse model of transient middle cerebral artery occlusion (MCAO). C57BL/6J mice were subjected to either sham surgery or 60 min of right MCAO. AEOL 10150 or phosphate-buffered saline was given intravenously 5 min after onset of reperfusion (n = 6 per group). Six hours later, parenchyma within the MCA distribution was harvested. RNA from the six brains in each group was pooled and mRNA expression determined using an Affymetrix murine MG_U74A v. 2.0 expression microarray. Each experiment was performed three times. The largest changes in expression occurred in stress response and inflammatory genes such as heat shock protein, interleukin-6, and macrophage inflammatory protein-2. Treatment with AEOL 10150 attenuated only the increase in expression of inflammatory genes. This suggests that AEOL 10150 protects brain by attenuating the immune response to ischemia and reperfusion.

Introduction

Cerebral ischemia is characterized by a severe reduction of blood flow to brain tissue. If blood flow is not rapidly restored, neuronal death ensues. Paradoxically, reperfusion can cause additional brain injury from excessive production of reactive oxygen species (ROS). Endogenous antioxidants are the primary defense against ROS-mediated injury; however, there is insufficient antioxidant capacity to prevent reperfusion-induced oxidative injury in the brain. Furthermore, the mechanisms by which ROS and antioxidants reduce reperfusion-induced brain injury remains poorly understood.

To study the role of superoxide and hydrogen peroxide in different diseases, we have synthesized a series of metalloporphyrin-based catalytic antioxidants that have potent in vitro superoxide dismutase and catalase activity [1]. These metalloporphyrins can pharmacologically augment natural antioxidant defenses and have been shown to attenuate pulmonary fibrosis [2], paraquat toxicity [3], and seizure-induced cell death [4]. Recently, metalloporphyrins have been shown to cause a major reduction of histologic injury resulting from middle cerebral artery occlusion (MCAO), even when given to rats as late as 6 h after onset of reperfusion [5]. Although the antioxidant properties of metalloporphyrins are well understood in vitro, their mechanism of protection in vivo is poorly understood. Metalloporphyrin catalytic antioxidants could attenuate stroke by affecting transcription (gene expression), translation (protein expression) or post-translational modifications to proteins.

This study uses microarray analysis to determine which changes in gene expression are associated with metalloporphyrin catalytic antioxidant administration (also see companion paper by Sheng et al. [5a]) after MCAO. Recently there have been advances that have implicated changes in gene expression as crucial events in determining the extent of injury after ischemia and reperfusion. Mice deficient in neurotrophin-4 (NT4), brain-derived neurotrophic factor (BDNF) [6], interferon regulatory factor-1 (IRF-1) [7], matrix metalloproteinase-9 (MMP-9) [8], and intercellular adhesion molecule-1 (ICAM-1) [9] have shown reductions in brain injury after ischemia and reperfusion. Experiments with transgenic mice have shown that overexpression of genes such as Bcl-2 [10] and heat shock protein 70 [11] reduced infarct size. These experiments suggest that there are several classes of genes whose expression may contribute to the pathogenesis of ischemia and reperfusion: stress response genes, growth factors, matrix metalloproteinases, and genes involved in inflammation.

To elucidate which of these classes of genes catalytic antioxidant metalloporphyrins might be affecting, we used the Affymetrix murine genome U74A (MG_U74A) gene chips to study gene expression changes 6 h after transient MCAO in the presence or absence of the catalytic antioxidant manganese (III) meso-tetrakis (di-N-ethylimidazole) porphyrin (AEOL 10150). We hypothesized general mechanisms by which AEOL 10150 could attenuate ischemic injury. First, if ROS were the primary source of injury after ischemia and reperfusion, one would expect that AEOL 10150 treatment would attenuate all changes in gene expression that occur during reperfusion. Second, if ROS constituted only part of the injury response, one would expect that AEOL 10150 treatment would attenuate only selected classes of genes.