Iron uptake of the normoxic, anoxic and postanoxic microglial cell line RAW 264.7

Iron is one of the trace elements playing a key role in the normal brain metabolism. An excess of free iron on the other hand is catalyzing the iron-mediated oxygen radical production. Such a condition might be a harmful event leading perhaps to serious tissue damage and degeneration. Therefore, during evolution a complex iron sequestering apparatus developed, minimizing the amount of redox-reactive free iron. However, this system might be severely disturbed under pathophysiological conditions including hypoxia or anoxia. Since little is known about the non-transferrin-mediated iron metabolism of the brain during anoxia/reoxygenation, we tested the ability of the microglial cell line RAW 264.7 to take up iron independently of transferrin under various oxygen concentrations. Microglial cells are thought to be the major player in the maintenance of the extracellular homeostasis in the brain. Therefore, we investigated the iron metabolism of microglial cells employing radiolabeled ferric chloride. We tested the uptake of iron under normoxic, anoxic and postanoxic conditions. Furthermore, the amount of ferritin was measured by immunoblotting. We were able to show that iron enters the microglial cell line in the absence of extracellular transferrin under normoxic, anoxic and postanoxic conditions. Interestingly, the amount of ferritin is decreasing in the early reoxygenation phase. Therefore, we concluded that microglia is able to contribute to the brain iron homeostasis under anoxic and postanoxic conditions.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.     

褪黑素与缺血再灌注损伤

褪黑素(melatonin,MT)具有强效抗氧化作用,在肠、肝脏、心脏、脑等器官的缺血再灌注损伤实验中具有清除自由基、保护线粒体、抗细胞凋亡等保护性作用。本文综合褪黑素应用于缺血再灌注损伤的动物实验的近几年相关文献,总结并分析褪黑素在缺血再灌注损伤动物实验中的保护作用及其相关机制。     

Oxygen radicals in the adult respiratory distress syndrome, in myocardial ischemia and reperfusion injury, and in cerebral vascular damage

Recent work suggests that oxygen radicals may be important mediators of damage in a wide variety of pathologic conditions. In this review we consider the evidence supporting the participation of oxygen radicals in the adult respiratory distress syndrome, in ischemia reperfusion injury in the myocardium, and in cerebral vascular injury in acute hypertension and traumatic brain injury. In the adult respiratory distress syndrome there is active sequestration of polymorphonuclear neutrophils in the pulmonary vascular system. There is evidence that activation of these neutrophils results in the production of oxygen radicals which injure the capillary membrane and increase permeability, leading to progressive hypoxia and decreased lung compliance which are hallmarks of the syndrome. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. The metabolism of arachidonic acid is the source of oxygen free radical production in these conditions. In myocardial ischemia and reperfusion injury, the ischemic myocyte is "primed" for free radical production. With reperfusion and reintroduction of molecular oxygen there is a burst of oxygen radical production resulting in extensive tissue destruction. Myocardial ischemia--reperfusion injury shares in common with the other two syndromes activation of the arachidonic acid cascade and acute inflammation. Thus it would appear that the generation of toxic oxygen species may represent a final common pathway of tissue destruction in several pathophysiologic states.     

Oral administration of Crataegus flavonoids protects against ischemia/reperfusion brain damage in gerbils

Stroke is the third leading cause of death as dementia is a main symptom of Alzheimer's disease. One of the important mechanisms in the pathogeny of stroke is free radical production during the reperfusion period, therefore the effects of a type of natural antioxidant, i.e. Crataegus flavonoids (CF), on brain ischemic insults were investigated in Mongolian gerbil stroke model. Results showed that pretreatment of the animals with CF decreased reactive oxygen species (ROS) production, thiobarbituric acid reactive substances content, and nitrite/nitrate concentration in brain homogenate, increased the brain homogenate-associated antioxidant level in a dose-dependent manner. CF pretreatment increased the amount of biologically available NO by scavenging of superoxide anion produced during reperfusion. At same time, in the process of ischemia/reperfusion brain damage, the content of nitrite/nitrate (the end product of NO) increased, and of NO detected by ESR decreased. Oral pretreatment with CF decreased the nitrite/nitrate content in the brain homogenate and increased the biologically available NO concentration in a dose-dependent manner. The increasing effect of antioxidant on NO might be due to its scavenging effect on superoxide anion, which could react with NO into peroxynitrite. iNOS was implied in delayed neuron death after brain ischemic damage and it was found that pretreatment with CF could decrease the protein level of tumor necrosis factor (TNF)-alpha and nuclear factor-kappa B (NF-kappaB), and increase the mRNA level of NOS estimated by western blotting and RT-PCR. More neurons survived and fewer cells suffered apoptosis in the hippocampal CA1 region of CF treated animal brain. These results suggest that oral administration of this antioxidant increases the antioxidant level in the brain and protects the brain against delayed cell death caused by ischemia/reperfusion injury.     

Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms     

Donepezil provides neuroprotective effects against brain injury and Alzheimer's pathology under conditions of cardiac ischemia/reperfusion injury

Cardiac ischemia/reperfusion (I/R) injury induces brain pathology. Donepezil, a well-known acetylcholine esterase (AChE) inhibitor, has been proven to exert neuroprotective effects against several neurodegenerative diseases. However, the comprehensive mechanism regarding the therapeutic potential of donepezil on the brain under cardiac I/R injury remains obscure. Here, we hypothesized that treatment with donepezil ameliorates brain pathology following cardiac I/R injury by decreasing blood brain barrier (BBB) breakdown, oxidative stress, neuroinflammation, mitochondrial dysfunction, mitochondrial dynamics imbalance, microglial activation, amyloid-beta (Aβ) accumulation, neuronal apoptosis, and dendritic spine loss. Forty-eight adult male Wistar rats were subjected to surgery for cardiac I/R injury. Then, rats were randomly divided into four groups to receive either (1) saline (vehicle group), donepezil 3 mg/kg via intravenously administered (2) before ischemia (pretreatment group), (3) during ischemia (ischemia group), or (4) at the onset of reperfusion (reperfusion group). At the end of cardiac I/R paradigm, the brains were evaluated for BBB breakdown, brain inflammation, oxidative stress, mitochondrial function, mitochondrial dynamics, microglial morphology, Aβ production, neuronal apoptosis, and dendritic spine density. Administration of donepezil at all time points equally showed an attenuation of brain damage in response to cardiac I/R injury, as indicated by increased expression of BBB junction protein, reduced brain inflammation and oxidative stress, improved mitochondrial function and mitochondrial dynamics, and alleviated Aβ accumulation and microglial activation, resulting in protection of neuronal apoptosis and preservation of dendritic spine number. These findings suggest that donepezil potentially protects brain pathology caused by cardiac I/R injury regardless the timing of treatment.     

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.     

Human umbilical vein endothelial cells submitted to hypoxia-reoxygenation in vitro: implication of free radicals, xanthine oxidase, and energy deficiency.

Ischemia-reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)-reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron-chelating agent, xanthine oxidase inhibitors, and energy-supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy-supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O2.(-) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2.(-) and of H2O2 by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 mumol/min/mg prot during the R period. O2.(-) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10(-6) U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia.     

Intermittent Anoxia Reduces Oxygen Free Radicals Formation During Reoxygenation in Rat Hepatocytes

The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.     

When melatonin gets on your nerves: its beneficial actions in experimental models of stroke

This article summarizes the evidence that endogenously produced and exogenously administered melatonin reduces the degree of tissue damage and limits the biobehavioral deficits associated with experimental models of ischemia/reperfusion injury in the brain (i.e., stroke). Melatonin's efficacy in curtailing neural damage under conditions of transitory interruption of the blood supply to the brain has been documented in models of both focal and global ischemia. In these studies many indices have been shown to be improved as a consequence of melatonin treatment. For example, when given at the time of ischemia or reperfusion onset, melatonin reduces neurophysiological deficits, infarct volume, the degree of neural edema, lipid peroxidation, protein carbonyls, DNA damage, neuron and glial loss, and death of the animals. Melatonin's protective actions against these adverse changes are believed to stem from its direct free radical scavenging and indirect antioxidant activities, possibly from its ability to limit free radical generation at the mitochondrial level and because of yet-undefined functions. Considering its high efficacy in overcoming much of the damage associated with ischemia/reperfusion injury, not only in the brain but in other organs as well, its use in clinical trials for the purpose of improving stroke outcome should be seriously considered.     

Oxygen radical mechanisms of brain injury following ischemia and reperfusion.

This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because of the difficulty of measuring radical species in vivo. Breakdown of lipid membranes during ischemia leads to accumulation of free fatty acids. Decreased energy stores during ischemia result in the accumulation of adenine nucleotides. During reperfusion, metabolism of free fatty acids via the cyclooxygenase pathway and metabolism of adenine nucleotides via the xanthine oxidase pathway are the most likely sources of oxygen radicals. Although leukocytes have been found to accumulate in some models of ischemia and reperfusion, their mechanistic role remains in question. Therapeutic strategies aimed at decreasing brain injury have included administration of radical scavengers at the time of reperfusion. Efficacy of traditional oxygen radical scavengers such as superoxide dismutase and catalase may be limited by their inability to cross the blood-brain barrier. Lipid-soluble antioxidants appear more efficacious because of their ability to cross the blood-brain barrier and because of their presence in membrane structures where peroxidative reactions can be halted.     

Neuroprotective Effect of 6-Paradol in Focal Cerebral Ischemia Involves the Attenuation of Neuroinflammatory Responses in Activated Microglia

Paradols are non-pungent and biotransformed metabolites of shogaols and reduce inflammatory responses as well as oxidative stress as shogaols. Recently, shogaol has been noted to possess therapeutic potential against several central nervous system (CNS) disorders, including cerebral ischemia, by reducing neuroinflammation in microglia. Therefore, paradol could be used to improve neuroinflammation-associated CNS disorders. Here, we synthesized paradol derivatives (2- to 10-paradols). Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity. Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α). To pursue whether the beneficial in vitro effects of 6-paradol leads towards in vivo therapeutic effects on transient focal cerebral ischemia characterized by neuroinflammation, we employed middle cerebral artery occlusion (MCAO)/reperfusion (M/R). Administration of 6-paradol immediately after reperfusion significantly reduced brain damage in M/R-challenged mice as assessed by brain infarction, neurological deficit, and neural cell survival and death. Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge. Collectively, this study provides evidences that 6-paradol effectively protects brain after cerebral ischemia, likely by attenuating neuroinflammation in microglia, suggesting it as a potential therapeutic agent to treat cerebral ischemia.     

Nitric oxide during ischemia attenuates oxidant stress and cell death during ischemia and reperfusion in cardiomyocytes

Nitric oxide (NO) has been implicated as a cardioprotective agent during ischemia/reperfusion (I/R), but the mechanism of protection is unknown. Oxidant stress contributes to cell death in I/R, so we tested whether NO protects by attenuating oxidant stress. Cardiomyocytes and murine embryonic fibroblasts were administered NO (10-1200 nM) during simulated ischemia, and cell death was assessed during reperfusion without NO. In each case, NO abrogated cell death during reperfusion. Cells overexpressing endothelial NO synthase (NOS) exhibited a similar protection, which was abolished by the NOS inhibitor N(omega)-nitro-l-arginine methyl ester. Protection was not mediated by guanylate cyclase or the mitochondrial K(ATP) channel, as inhibitors of these systems failed to abolish protection. NO did not prevent decreases in mitochondrial potential, but cells protected with NO demonstrated recovery of potential at reperfusion. Measurements using C11-BODIPY reveal that NO attenuates lipid peroxidation during ischemia and reperfusion. Measurements of oxidant stress using the ratiometric redox sensor HSP-FRET demonstrate that NO attenuates protein oxidation during ischemia. These findings reveal that physiological levels of NO during ischemia can attenuate oxidant stress both during ischemia and during reperfusion. This response is associated with a remarkable attenuation of cell death, suggesting that ischemic cell death may be a regulated event.     

Tenoxicam Exerts a Neuroprotective Action after Cerebral Ischemia in Rats

In this study we investigated the effects of Tenoxicam, a type 2 cyclooxygenase (COX-2) inhibitor, on brain damage induced by ischemia-reperfusion. Male Wistar rats (18-month old average) were anesthetized and submitted to ischemia occlusion of both common carotid arteries (BCAO) for 45 min. After 24 h of reperfusion, rats were decapitated and hippocampi removed for further assays. Animals were divided into sham-operated, ischemia, ischemia + Tenoxicam 2.5 mg/kg, and ischemia + Tenoxicam 10 mg/kg groups. Tenoxicam was administered intraperitoneally immediately after BCAO. Histological analyses show that ischemia produced significant striatal as well as hippocampal lesions which were reversed by the Tenoxicam treatment. Tenoxicam also significantly reduced, to control levels, the increased myeloperoxidase activity in hippocampus homogenates observed after ischemia. However, nitrite concentrations showed only a tendency to decrease in the ischemia + Tenoxicam groups, as compared to that of ischemia alone. On the other hand, hippocampal glutamate and aspartate levels were not altered by Tenoxicam. In conclusion, we showed that ischemia is certainly related to inflammation and to increased free radical production, and selective COX-2 inhibitors might be neuroprotective agents of potential benefit in the treatment of cerebral brain ischemia.     

Molecular Mechanisms of Ischemia–Reperfusion Injury in Brain: Pivotal Role of the Mitochondrial Membrane Potential in Reactive Oxygen Species Generation

Stroke and circulatory arrest cause interferences in blood flow to the brain that result in considerable tissue damage. The primary method to reduce or prevent neurologic damage to patients suffering from brain ischemia is prompt restoration of blood flow to the ischemic tissue. However, paradoxically, restoration of blood flow causes additional damage and exacerbates neurocognitive deficits among patients who suffer a brain ischemic event. Mitochondria play a critical role in reperfusion injury by producing excessive reactive oxygen species (ROS) thereby damaging cellular components, and initiating cell death. In this review, we summarize our current understanding of the mechanisms of mitochondrial ROS generation during reperfusion, and specifically, the role the mitochondrial membrane potential plays in the pathology of cerebral ischemia/reperfusion. Additionally, we propose a temporal model of ROS generation in which posttranslational modifications of key oxidative phosphorylation (OxPhos) proteins caused by ischemia induce a hyperactive state upon reintroduction of oxygen. Hyperactive OxPhos generates high mitochondrial membrane potentials, a condition known to generate excessive ROS. Such a state would lead to a “burst” of ROS upon reperfusion, thereby causing structural and functional damage to the mitochondria and inducing cell death signaling that eventually culminate in tissue damage. Finally, we propose that strategies aimed at modulating this maladaptive hyperpolarization of the mitochondrial membrane potential may be a novel therapeutic intervention and present specific studies demonstrating the cytoprotective effect of this treatment modality.     

Brain Mitochondrial DNA Is Not Damaged by Prolonged Cardiac Arrest or Reperfusion

Postischemic reperfusion is known to cause iron-mediated peroxidation of polyunsaturated fatty acids in membranes, including mitochondrial membranes, in the brain cortex. Consequently, we tested the hypothesis that this radical-mediated damage would extend to DNA. Mitochondrial DNA (mtDNA) was chosen because of its presence at a known site of free radical formation, its sensitivity and ease of assay, and its known lack of any repair systems. In model experiments we utilized endonuclease III or piperidine to amplify topological form conversions in mtDNA damaged by in vitro reactions with hydroxyl radical. We then applied the amplified detection assays to dog brain mtDNA isolated after 2 or 8 h of reperfusion following a 20-min cardiac arrest. We found that ischemia and reperfusion caused no topological form conversions in mtDNA. Similarly, nucleotide incorporation by a gap-filling reaction showed no sensitivity to digestion of the mtDNA by exonuclease III, an enzyme known to remove blocked 3' termini at the site of radical-generated nicks. Furthermore, the recovery of mtDNA was similar in all experimental groups, suggesting that putatively damaged forms had not been removed by rapid degradation. Thus, despite mitochondrial membrane damage, brain mtDNA does not accumulate oxygen radical damage during postischemic brain reperfusion.     

LPS-induced protein oxidation and proteolysis in BV-2 microglial cells

Exposure of proteins to oxidants leads to increased oxidation followed by preferential degradation by the proteasomal system. The role of the biological oxidant production in microglial BV-2 cells in the oxidation and turnover of endogenous proteins was measured. It could be demonstrated, that BV-2 cells are relatively resistant to fluxes of oxidants, but nevertheless protein oxidation occurs due to activation by LPS. This protein oxidation is followed by an enhanced degradation of endogenous proteins. Using PBN, a free radical scavenger and antioxidant, we could demonstrate the involvement of free radicals in the increased proteolysis in BV-2 cells after LPS-treatment. A slight but significant up-regulation of the proteasomal system after LPS activation takes place, indicating the importance of his proteolytic system in the maintenance of the protein pool of microglial cells.     

Structural alterations in synaptosomal membrane-associated proteins and lipids by transient middle cerebral artery occlusion in the cat

We have previously reported that ischemia reperfusion injury results from free radical generation following transient global ischemia, and that this radical induced damage is evident in the synaptosomal membrane of the gerbil. [Hall et al, (1995) Neuroscience 64: 81–89] In the present study we have extended these observations to transient focal ischemia in the cat. We prepared synaptosomal membranes from frontal, parietal-temporal, and occipital regions of the cat cerebral cortex with reperfusion times of 1 and 3 hours following 1 hour right middle cerebral artery occlusion. The membranes were selectively labeled with protein and lipid specific paramagnetic spin labels and analyzed using electron paramagnetic resonance spectrometry. There were significant motional changes of both the protein and lipid specific spin labels in the parietal-temporal and occipital regions with 1 hour reperfusion; but, both parameters returned to control values by 3 hours reperfusion. No significant changes were observed in the normally perfused frontal pole at either reperfusion time. These results support the argument that free radicals play a critical role in cell damage at early reperfusion times following ischemia.