Original Contributions
Production of hydroxyl free radical by brain tissues in hyperglycemic rats subjected to transient forebrain ischemia

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Abstract

Preischemic hyperglycemia is known to aggravate brain damage resulting from transient ischemia. In the present study, we explored whether this aggravation is preceded by an enhanced formation of reactive oxygen species (ROS) during the early reperfusion period. To that end, normo- and hyperglycemic rats were subjected to 15 min of forebrain ischemia and allowed recovery periods of 5, 15, and 60 min. Sodium salicylate was injected intraperitoneally in a dose of 100 mg/kg, and tissues were sampled during recirculation to allow analyses of salicylic acid (SA) and its hydroxylation products, 2,3- and 2,5-dihydroxybenzoate (DHBA). Tissue sampled from thalamus and caudoputamen in normoglycemic animals failed to show an increase in 2,3- or 2,5-DHBA after 5 and 15 min of recirculation. However, such an increase was observed in the neocortex after 60 min of recirculation, with a suggested increase in the hippocampus as well. Hyperglycemia had three effects. First, it increased 2,5-DHBA in the thalamus and caudoputamen to values exceeding normoglycemic ones after 15 min of recirculation. Second, it increased basal values of 2,5- and total DHBA in the neocortex. Third, it increased the 60-min values for 2,5- and total DHBA in the hippocampus. These results hint that, at least in part, hyperglycemia may aggravate damage by enhancing basal- and ischemia-triggered production of ROS.

Introduction

Transient cerebral ischemia is known to give rise to postischemic production of ROS. Such production was first demonstrated by Cao et al. [1], who assessed the hydroxylation of intraperitoneally administered SA in gerbils subjected to 2, 5, or 15 min of forebrain ischemia. The results (see also [2]) demonstrated that enhanced hydroxylation of the salicylate molecule, yielding the 2,3- and 2,5-metabolites, was observed after 5 and 15 (but not 2) min of ischemia, the rate of hydroxylation being larger in the hippocampus than in the neocortex. These results were confirmed in rats in which microdialysis techniques were used to assess ROS production by spin trapping [3], [4].

Additional aspects on this subject have been published. Thus the time course of changes in ROS production has been studied by Dirnagl et al. [5], who recorded continuously the chemiluminescence associated with ROS production before, during, and after transient ischemia. Further, the microdialysis techniques of Piantadosi and Zhang [6] allowed them to assess a mitochondrial origin of the ROS produced.

It is widely assumed that ROS formed during reperfusion after transient ischemia contribute to the reperfusion damage incurred [7], [8], [9]. Indirect evidence supporting this notion was reported by Kil et al. [10], who noted that hyperthermia (39°C) enhanced and hypothermia (30°C) attenuated the postischemic production of 2,3-DHBA.

In the present study, we used the salicylate technique reported by Cao et al. [1] to explore whether preischemic hyperglycemia, which is known to aggravate ischemic damage in animals subjected to transient ischemia (for reviews, see [11], [12]), leads to an exaggerated production of ROS during recirculation. To that end, rats were injected intraperitoneally with SA (100 mg/kg) and subjected to 15 min of forebrain ischemia, tissues being sampled after 5, 15, and 60 min of recirculation for analyses of salicylic acid as well as of 2,3- and 2,5-DHBA.

Section snippets

Operative procedures

Male Wistar rats of an SPF strain (Møllegaard’s Breeding Center, Copenhagen, Denmark), weighing 290–340 g, were used. The animals were not fed the night before the day of the operation, with free access to water. Anesthesia was induced by inhalation of 3.5% halothane in a mixture of N2O and O2 (70:30). After intubation with a polyethylene tubing (Intramedic PE 240, Clay-Adams, New York, NY, USA), the animals were connected to a mechanical ventilator. Anesthesia was maintained with 1.0%

Results

The plasma glucose concentrations and physiologic variables were measured 10 min before ischemia. Plasma glucose was approximately 20 mM in hyperglycemic animals and approximately 6 mM in normoglycemic ones. The level of PCO2 was kept close to 35 mmHg, PO2 was kept to above 100 mmHg, and pH was kept to 7.40. Both head and body temperatures were controlled close to 37°C, and mean arterial blood pressure was kept to 120 mmHg. There were no statistically significant differences in physiologic

Discussion

A pathogenic role for free radical-induced lipid peroxidative processes in cerebral ischemic damage was first proposed by Demopoulos et al. [18]. Since then, many studies have been carried out that demonstrate the importance of the free radical-induced damage (for data and reviews see [18], [19], [20], [21], [22], [23], [24]. Such experiments give strong support to the contention that free radicals play an important role in ischemia–reperfusion brain damage. Furthermore, free radical scavengers

Acknowledgements

This study was supported by the U.S. Public Health Service via the National Institute of Health (Grant 5 R01NS07838), the Juvenile Diabetes Foundation International, and the Queen Emma Foundation in Hawaii.

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