Time course of oxidative damage in different brain regions following transient cerebral ischemia in gerbils

These include rapid decreases in ATP, calcium release from intracellular stores, loss of ion homeostasis, excitotoxicity, activation of enzymes (phospholipases, proteases, protein kinases, nitric oxide synthases, endonucleases), arachidonic acid release and metabolism, mitochondrial dysfunction, acidosis and edema (Macdonald and Stoodley, 1998; Lee et al., 1999). Many of these changes are associated with increased reactive oxygen species (ROS) production that can occur both during ischemia and at reperfusion. The role of oxidative stress becomes much greater in the case where cerebral blood flow is restored, because reflow to previous ischemic brain results in an increase in oxygen level, and consequently causes severe oxidative injury to the tissue by massive production of ROS (Chan, 1996).

However, reperfusion is necessary to salvage the compromised ischemic tissue.

Oxidative stress is one of the most important factors that exacerbate brain damage by reperfusion. A large body of experimental research clearly shows that ischemia-reperfusion injury involves oxidatively damaging events (Cao et al., 1988;Kitagawa et al., 1990;Facchinetti et al., 1998). The brain is particularly vulnerable to oxidative injury because of its high rate of oxidative metabolic activity, intense production of reactive oxygen metabolites, high content of polyunsaturated fatty acids, relatively low antioxidant capacity, low repair mechanism activity and non-replicating nature of its neuronal cells (Evans, 1993).

Forebrain ischemia-reperfusion in gerbil is a model for human cerebral ischemia resulting from transient cardiac arrest. Certain brain regions, such as the striatum, neocortex and particularly the hippocampus, are more susceptible to ischemic damage (Kindy et al., 1992). In the hippocampus, the cornu Ammonis 1 (CA1) pyramidal neurons undergo selective delayed death several days after the injury (Kirino, 1982;Nitatori et al., 1995;Rao et al., 2000). Several lines of evidence indicate that oxidative stress contributes to delayed neuronal death after global cerebral ischemia (Kitagawa et al., 1990;Hall et al., 1993;Oostveen et al., 1998), suggesting that ROS formation may cooperate in a series of molecular events that link ischemic injury to neuronal cell death. Thus, elucidation of the extent and the role of oxidative stress in the brain after ischemia-reperfusion are of great importance. A better understanding of the timing and factors that influence ROS formation is required for effective antioxidant intervention and for enlarging our knowledge of the pathophysiological mechanisms of cerebral ischemia. The temporal profile of histopathological changes in the gerbil brain following global ischemia has been extensively characterized, showing no neuronal loss up to 2-3 days of reperfusion but an extensive delayed neuronal loss at 5-7 days of reperfusion in the hippocampal CA1 region (Kirino, 1982;Rao et al., 2000;Martínez et al., 2001; Candelario-Jalil et al., unpublished data). Although ROS have been postulated to play an important role in the progression of reperfusion injury, the time course of oxidative damage following transient forebrain ischemia has been poorly characterized. In the present study, we have examined the time course of oxidative injury in different brain regions following transient global cerebral ischemia in gerbils. Further, the antioxidant capacity in each brain area was studied at different sampling times after the ischemic insult in view that oxidative stress may result not only from an increase in free radical production but also from a decrease in cellular antioxidant mechanisms. To our knowledge, the time course of the activity of glutathionerelated enzymes as well as the content of both GSH and GSSG following transient forebrain ischemia in gerbils had not been previously characterized.

Studies were performed in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by National Institutes of Health (Bethesda, MD, USA). The experimental protocol was approved by our institutional animal care and use committee. Male Mongolian gerbils (Meriones unguiculatus; Hoe Gerk jirds strain) weighing 60-75 g at the time of surgery were used in this study. These animals were housed five per cage, exposed to a 12-h light/dark cycle, and had free access to food and water throughout the study period. The gerbils were anesthetized with chloral hydrate (300 mg/kg, i.p.). In the supine position, a midline ventral incision was made in the neck. Both common carotid arteries were exposed, separated carefully from the vagus nerve, and occluded for 5 minutes with microaneurysmal clips, which consistently resulted in delayed neuronal death in the CA1 region of the hippocampus (Kirino, 1982;Martínez et al., 2001). Blood flow during the occlusion an