The Effects of Active Oxygen Free Radical Scavengers on Iron‐Induced Seizures and Neural Membrane Peroxidation
Authors: Yokoi Isao , Kabuto Hideaki , Mori Akitane , Ogawa Norio 
Published: August 2009

Purpose: Intracortical injection of iron ions has been shown to induce recurrent seizures and epileptic discharges in the electrocorticograms (ECoGs) of rats and cats and has been used to model posttraumatic epilepsy. It has been proposed that active oxygen free radicals (AOFRs), especially hydroxyl radicals, participate in the mechanisms that produce epileptic discharges. After intracranial hemorrhage, red blood cells break down and release iron ions from hemoglobin. AOFRs are generated in brain tissue by iron‐mediated reactions, and they peroxidate lipids in the neural membranes, to produce thiobarbituric acid‐reactive substances (TBARS). This finally resulted in the formation of a posttraumatic epileptic focus. In this study, we observed the effects of some AOFRs scavengers on the peroxidation of neural membranes and the occurrence of epileptic discharges induced by the injection of irons into the rat cortex.

Methods: Male Sprague‐Dawley rats weighing 250–350 g were anesthetized with pentobarbital (PTB) and the brain perfused with ice‐cold saline. Brain tissue was homogenized with 20 volumes of ice‐cold Tris‐HCl buffer (20 mM, pH 7.4). Peroxidation was initiated by adding 250 μ1 of the homogenate to 250 PL of FeCl3, solution (final concentration, 50 mM) with and without antioxidants. After incubation for 20 min at 37°C, 1 ml of the thiobarbituric acid reagent (0.375% TBA, 15% trichloroacetic acid, 250 mM HCl, and 0.1 mM EDTA) was added. Then the mixture was heated at 100°C for 10 min, and cooled. The amount of, TBARS was determined spectrophotometrically by using malondialdehyde as the standard. In another study, rats were immobilized with succinylcholine chloride under artificial ventilation with tracheal intubation. Four electrodes were placed epidurally, and the electrocorticogram (ECoG) was recorded from four unipolar leads. A burr‐hole was made over the left sensorimotor cortex, 1 mm posterior to bregma and 1 mm lateral to the midline, for intracortical injection of FeCl3 (2.0 mm helow the exposed dura). The saline solution of FeCl3 (100 mM 5 μl) was injected at a rate of 1 μl/min.

Results: Epigallocatechin (EGC) and epigallocatechin‐3‐O‐gallate (EGCG) inhibited TBARS production dose‐dependently and median effective dose (ED50) values were 75 and 100 μM, respectively. a‐Tocopheryl‐L‐ascorbate‐2‐O‐phosphate diester potassium salt (EPC) and Trolox also inhibited TBARS production dose‐dependently with an ED, of 75 and 100 μM, respectively. However, ascorbic acid (vitamin C) increased TBARS production dose‐dependently. The occurrence of epileptic discharges induced by the injection of FeCl3 into the cortex was prevented or delayed by the intraperitoneal injection of 200 mg/kg of these four antioxidants 60 min before the injection of FeCl3, whereas the injection of vitamin C worsened the FeCl3‐induced changes in the ECoG.

Conclusions: EGC and EGCG, which were isolated from green tea leaves, are known to be AOFRs scavengers based on electron spin resonance spectrometry with a spin trap. Although α‐tocopherol (vitamin E) does not dissolve in water, Trolox is synthesized as a water‐soluble form of vitamin E. Because EPC is synthesized from vitamin E and C connected by a phosphate, it can dissolve in both water and lipids. These two compounds are also known to scavenge AOFRs. In this study, the occurrence of FeCl3,‐induced epileptic discharges was prevented by antioxidants that have antimembrane peroxidation activity. These data indicate that these compounds may be clinically useful not only in preventing the focus formation in posttraumatic epilepsy, but also in treating and attenuating free radical‐induced degenerative disorders.