Masamitsu Kawauchi

Detection of Lipid Peroxidation and Hydroxyl Radicals in Brain Contusion of Rats

To examine the relationship between the free radicals and brain tissue damage, we investigated the intensity of brain hydroxyl (OH) radical generation and lipid peroxidation in the rat contusion injury model. A unilateral contusion was induced by a weight-drop method. All rats were decapitated six hours after the injury, and brain samples were taken from three portions (core, peripheral, and distal) to examine the specific gravity as an indicator of brain edema, generation of OH using an electron paramagnetic resonance spectrometer (EPR), and malondialdehyde (MDA) and 4-hydroxyalkenals production. Analysis of the specific gravity revealed cerebral edema on the ipsilateral side in the injured group. The signal intensity of EPR in the core and peripheral portions in the contusion group was significantly higher than that in the distal portion of the contusion group and that of all portions in the control animals. No significant difference was observed between the core and peripheral portions of the contusion group. The MDA and 4-hydroxyalkenals production was significantly higher in the core and peripheral portions than in the distal portion of the contusion group and that of all portions of the control group. The degree of posttraumatic brain edema was closely correlated with the increase of DMPO-OH adduct, MDA, and 4-hydroxyalkenals. These results support the current concept that free radical production following traumatic brain injury may induce lipid peroxidation and may be the direct cause of edema formation.

Effects of lecithinized superoxide dismutase on neuronal cell loss in CA3 hippocampus after traumatic brain injury in rats

BACKGROUND The protective effect of excitatory amino acid antagonists for CA3 hippocampal neuronal loss has been well documentated. From a clinical point of view, however, alternative therapies should also be explored because excitatory amino acid antagonists have relatively deleterious side effects. Administration of lecithinized superoxide dismutase (PC-SOD) has recently been demonstrated to reduce brain edema after traumatic brain injury (TBI) in the cerebral cortex. In this study, we investigated the effectiveness of PC-SOD on CA3 hippocampal cell loss by examining hematoxylin and eosin-stained sections. METHODS Rats were divided at random into three groups. The first group received 1 mL of saline (contusion + saline group, n = 5). Rats of the second group were treated with 3000 IU/kg of PC-SOD (contusion + SOD 1 group, n = 5), while the third group received 5000 IU/kg of PC-SOD (contusion + SOD 2 group, n = 5). All agents were administered intraperitoneally 1 minute after traumatic insult and every 24 hours until 2 or 3 days post-TBI. Animals were sacrificed 3 or 7 days after contusion injury. RESULTS PC-SOD prevented CA3 neuronal loss 3 days after TBI, and increased the number of surviving CA3 neurons 7 days after TBI. CONCLUSION Our findings suggest that PC-SOD may serve as a pharmacological agent in the treatment of neuronal loss after TBI.

Induction of Cu,Zn-superoxide dismutase after cortical contusion injury during hypothermia

To determine the effect of hypothermia on superoxide injury after cerebral contusion, the induction of Cu,Zn-superoxide dismutase was examined 6 h after contusion in rats using Northern blotting. Cu,Zn-superoxide dismutase gene expression increased at the periphery of the contusion, which may indicate the severity of the superoxide stimulus. This increase was preserved after contusion under hypothermia, which may show that superoxide injury is still severe although brain edema is decreased.

Effects of lecithinized SOD on sequential change in SOD activity after cerebral contusion in rats.

To analyze the effect of lecithinized superoxide dismutase (SOD) on superoxide accumulation after traumatic brain injury (TBI) in rats, we studied the SOD activity by NBT-reducing method and the expression of Cu,Zn-SOD mRNA by Northern blot analysis. As determined by the specific gravity method, the administration of lecithinized SOD decreased brain edema in the periphery of the lesion at 6 hr after contusion. SOD activity, without lecithinized SOD administration, increased at the peripheral portion at 30 min after contusion, but decreased to normal level at 6 hr after TBI. By administration of lecithinized SOD, the increase of SOD activity was preserved until 6 hr after TBI. The expression of Cu,Zn-SOD mRNA increased in the core lesion, peripheral portion, and contralateral hemisphere until 6 hr after TBI, then was suppressed in all three areas by lecithinized SOD. These results support the hypothesis that superoxide anions may play an important role in the development of brain edema after TBI, and that leciyhinized SOD appears to prevent brain edema through a protective effect against superoxide anions.

Prediction and evaluation of brainstem function by auditory brainstem responses in patients with uncal herniation

Serial measurements of auditory brainstem-evoked responses (BERs) were conducted in 15 patients with supratentorial mass lesions. Significant prolongation of the latency of wave V BERs, which originates in the inferior colliculus, occurred when the intracranial pressure (ICP) approached 30 mmHg. In four of five patients whose BERs were measured before pupillary changes, a significant lengthening of wave V latency was observed prior to clinical manifestation of uncal herniation. These results suggest that immediate medical or surgical decompression of ICP should be performed when ICP approaches 30 mmHg with significant prolongation of wave V latency.

Long-term time course of regional changes in cholinergic indices following transient ischemia in the spontaneously hypertensive rat brain

Using an animal model of forebrain ischemia in spontaneously hypertensive rats (SHR) by 3-h bilateral carotid occlusion, and various indices of the cerebral cholinergic system were assessed for periods up to 24 weeks. The lesions observed histologically in the hippocampus of SHR 2 weeks after ischemia were less severe than those in the frontal cortex. Marked elevation of acetylcholine concentration was transiently observed in the frontal cortex, hippocampus and thalamus + midbrain at 2 weeks, and in the striatum at 1-4 weeks after ischemia. Choline acetyltransferase activity remained unchanged in all regions throughout the experimental period except for a minimal decrease in the frontal cortex at 4 weeks. Choline esterase (ChE) activity was slightly decreased in the frontal cortex at 2-4 weeks after ischemia but recovered by 8 weeks. A decrease in the hippocampus was seen at 8 weeks. The B(max) for the M1-receptor was significantly reduced by 2 weeks in the frontal cortex and by 4 weeks in the hippocampus. Low B(max) values in both regions persisted through week 24. These delayed hippocampal changes in the ChE activity and M1-receptor in SHR were similar to those of the very much delayed changes in M1-receptor previously reported in the gerbil model for transient ischemia. In contrast, Wistar-Kyoto rats (WKY), used as normotensive controls, exhibited no histological or biochemical changes for up to 24 weeks. The difference between SHR and WKY may depend on the more severe cerebral blood flow depletion during carotid ligation in the former. The chronic state of SHR after the transient ischemia may be a useful pathophysiological model for human cerebral infarctions with hypertension.

Superoxide scavenging activity in the extracellular space of the brain in forming edema.

We carried out a time course study of cerebral superoxide scavenging activity using a modified microdialysis technique. Twelve cats were divided into two groups; six were the reperfusion injury models, and six were cold injury models. In the reperfusion injury model, dialysates were collected during 60 minutes of middle cerebral artery occlusion and at 300 minutes during reperfusion. In the cold injury model, dialysates were collected 240 minutes after the injury. Regional cerebral blood flow on the injured side decreased during occlusion in the reperfusion injury model and 60 minutes after injury in the cold injury model. In the reperfusion model, superoxide scavenging activity, as determined with electron spin resonance, increased in the first 30 minutes and decreased 300 and 330 minutes after occlusion. In the dialysate, albumin increased 180 minutes after cold injury, which may show the progress of vasogenic edema. An increase in water content was observed on the injured side of both models, and a correlation between water content and superoxide scavenging activity was found in the reperfusion injury model. By this technique, a method of detecting the alteration of superoxide scavenging activity in the extracellular space of the brain was established.

Effects of hypothermia and rewarming on evoked potentials during transient focal cerebral ischemia in cats

Abstract We examined the effects of mild to moderate hypothermia and the influence of rewarming on electrophysiological function using somatosensory evoked potentials (SEPs) in transient focal ischemia in the brain. Nineteen cats underwent 60 min of left middle cerebral artery occlusion under normothermic (36 ° -37 ° C, n = 6) or hypothermic (30 ° -31 ° C, n = 13) conditions followed by 300 min of reperfusion with slow (120 min, n = 6) or rapid (30 min, n = 7) rewarming. Whole-body hypothermia was induced during ischemia and the first 180 min of reperfusion. SEPs and regional cerebral blood flow were measured before and during ischemia and during reperfusion. The specific gravity of gray and white matter was examined as the indicator of edema. During rewarming, SEP amplitudes recovered gradually. After rewarming, SEPs in the normothermic and rapid rewarming groups remained depressed (20%-40% of pre-occlusion values); however, recovery of SEPs was significantly enhanced in the slow rewarming group (p < 0.05). Hypothermia followed by slow rewarming reduced edema in gray and white matter. Rapid rewarming did not reduce edema in the white matter. The recovery of SEPs correlated with the extent of brain edema in transient focal ischemia. Rapid rewarming reduced the protective effect of hypothermia. [Neurol Res 2002; 24: 621-626]

Expression of Cu,Zn-superoxide dismutase mRNA after cold and contusion injury in the rat brain

We analyzed the expression of Cu,Zn-superoxide dismutase (SOD) mRNA in both contusion and cold injury. Twenty-three rats were divided into 3 groups: a control group, a contusion group, and a cold injury group. Six hours after the injury, the rats were decapitated and the gray matter was resected from 3 portions: the core of the injured cortex, its periphery, and a distal portion on the non-lesion side. Based on the specific gravity of each sample, almost the same degree of edema developed in both injury groups. The mRNA expression in the cold injury group, however, significantly decreased in all portions. The extremely low temperature associated with cold injury is a possible cause of the decrease in Cu,Zn-SOD mRNA.

Endogenous Superoxide Dismutase Activity in Reperfusion Injuries

To elucidate the relationship between reperfusion injuries and free radicals, we monitored the endogenous superoxide dismutase (SOD) activity by intracerebral microdialysis. Six cats underwent a transient occlusion of the middle cerebral artery for 60 minutes after microdialysis probes were implanted bilaterally into the white matter under the ectosylvian gyrus. Dialysates were collected at 30 minute intervals over the course of 5 hours after reperfusion. The SOD activity of the dialysates was measured with electron spin resonance spectrometry. Regional cerebral blood flow was measured simultaneously and the water content of the white matter was assayed at the end of the experiment. After reperfusion, SOD activity increased significantly in the first 30 minutes compared with the preoperative value, and decreased over 4-4.5 hours and 4.5-5 hours in the occluded side. The water content in the occluded side was significantly higher than that in the contralateral side. The highest SOD activity during reperfusion and the water content in the occluded side seemed to correlate, although not significantly. A leakage of intracellular SOD or a reactive increase of SOD activity in response to the reperfusion injury are possible mechanisms of increase in extracellular SOD.