Biomedical subjects

Neil R Sims

Publications and source records attributed to Neil R Sims.

12 recordsLinked to original sources

PubMed2006

Inhibition of nitric oxide synthase with 7-nitroindazole does not modify early metabolic recovery following focal cerebral ischemia in rats.

Nitric oxide has been strongly implicated in the development of tissue infarction in response to focal cerebral ischemia. Nitric oxide and its derivatives can inhibit components of the electron transport chain, providing a likely target for these substances in ischemic and post-ischemic brain. Lactate content is increased during post-ischemic reperfusion in tissue destined to become infarcted, consistent with impairment of mitochondrial respiration. To investigate the possible involvement of nitric oxide in generating these changes, we have tested the effect of 7-nitroindazole, a nitric oxide synthase (NOS) inhibitor, on the content of lactate and other metabolites during early reperfusion following temporary focal ischemia. This treatment inhibited total NOS by approximately 50%. However, the treatment did not significantly affect the marked increases in lactate in post-ischemic brain nor did it alter the recovery of other energy-related metabolites. These findings indicate that inhibition of oxidative metabolism is probably not the primary site of the deleterious effects of nitric oxide and derivatives during early post-ischemic reperfusion.

Adenosine Diphosphate↗

PubMed2006

The metabolism of C-glucose by neurons and astrocytes in brain subregions following focal cerebral ischemia in rats.

To provide insights into the effects of temporary focal ischemia on the function of neurons and astrocytes in vivo, we measured the incorporation of radiolabel from [U-14C]glucose into both glutamate and glutamine in brain subregions at 1 h of reperfusion following occlusion of the middle cerebral artery for 2 or 3 h. Under the experimental conditions used, 14C-glutamate is mainly produced in neurons whereas 14C-glutamine is generated in astrocytes from 14C-glutamate of both neuronal and astrocytic origin. Radiolabel incorporation into both amino acids was greatly decreased. The change in 14C-glutamate accumulation provides strong evidence for substantial reductions in neuronal glucose metabolism. The resulting decrease in delivery of 14C-glutamate from the neurons to astrocytes was probably also the major contributor to the change in 14C-glutamine content. These alterations probably result in part from a marked depression of glycolytic activity in the neurons, as suggested by previous studies assessing deoxyglucose utilization. Alterations in 14C-glucose metabolism were not restricted to tissue that would subsequently become infarcted. Thus, these changes did not inevitably lead to death of the affected cells. The ATP : ADP ratio and phosphocreatine content were essentially preserved during recirculation following 2 h of ischemia and showed at most only moderate losses in some subregions following 3 h of ischemia. This retention of energy reserves despite the decreases in 14C-glucose metabolism in neurons suggests that energy needs were substantially reduced in the post-ischemic brain. Marked increases in tissue lactate accumulation during recirculation, particularly following 3 h of ischemia, provided evidence that impaired pyruvate oxidation probably also contributed to the altered 14C-glucose metabolism. These findings indicate the presence of complex changes in energy metabolism that are likely to greatly influence the responses of neurons and astrocytes to temporary focal ischemia.

Adenosine Diphosphate↗

PubMed2005

(-)-Epigallocatechin gallate as an intervention for the acute treatment of cerebral ischemia.

This study examined the neuroprotective effects and possible hepatotoxicity of (-)-epigallocatechin gallate (EGCG) in a rat model of transient focal cerebral ischemia. Male Sprague-Dawley rats (265-295 g) were treated with either 50 mg kg(-1) of EGCG or saline, i.p., immediately post-ischemia and every day thereafter, in a middle cerebral artery occlusion model of stroke. Sacrifice occurred 72 h post-ischemia and 2,3,5-triphenyltetrazolium chloride staining was used to quantify neuronal infarction. Hepatotoxicity was determined by taking blood samples for plasma alanine aminotransferase (ALT) activity. Spleen, kidney, liver and testes wet weights were also recorded. Total infarct volume was significantly (P<0.05) reduced in the EGCG-treated group as compared to controls. Analysis of the mean infarct area showed a significant (P<0.05) decrease in slices 6 and 7 in the EGCG-treated group. No significant differences were found in organ weights or ALT levels between treatment groups. Our findings, in part, validate and extend previous observations illustrating that 50 mg kg(-1), i.p. EGCG is non-toxic and neuroprotective. However, we also found that EGCG treatment appreciably increased (>50%) the number of animals that developed an intracerebral hemorrhage. We therefore conclude that 50 mg kg(-1) EGCG is not a viable intervention for the acute treatment of cerebral ischemia, as it is likely to increase the risk of intracerebral hemorrhaging.

Alanine Transaminase↗

PubMed2005

Astrocytic function assessed from 1-14C-acetate metabolism after temporary focal cerebral ischemia in rats.

Astrocytes play many roles essential for normal brain activity. The ability of these cells to recover after temporary focal cerebral ischemia is likely to be one important determinant of the extent of brain dysfunction and tissue damage. We have assessed astrocytic function based on the incorporation of radiolabel from 1-14C-acetate into glutamine at 1 hour of recirculation after middle cerebral artery occlusion for 2 or 3 hours in rats. There were marked differences in the response between subregions within the tissue subjected to ischemia, but the overall pattern of changes was similar after each ischemic period. The striatum, which forms part of the severely ischemic focal tissue during arterial occlusion, showed a large (44% to 68%) decrease in glutamine labeling compared with equivalent tissue from the contralateral hemisphere. In contrast, 14C-glutamine content was not significantly altered in perifocal tissue in the cerebral cortex, which was subjected to more moderate ischemia. Cortical focal tissue also was not significantly affected, but the response was much more variable between rats. In these brain subregions, the extent of recovery of the 14C-acetate metabolism after ischemia was not a good predictor of the likelihood of subsequent infarct development. Interestingly, a similar pattern of responses persisted when recirculation was extended to 4 hours. These results indicate that many astrocytes, particularly in the cortex, remain viable and capable of at least some complex oxidative metabolism during the first few hours of recirculation.

Acetates↗

PubMed2004

Highly selective and prolonged depletion of mitochondrial glutathione in astrocytes markedly increases sensitivity to peroxynitrite.

Glutathione, a major endogenous antioxidant, is found in two intracellular pools in the cytoplasm and the mitochondria. To investigate the importance of the smaller mitochondrial pool, we developed conditions based on treatment with ethacrynic acid that produced near-complete and highly selective depletion of mitochondrial glutathione in cultured astrocytes. Recovery of mitochondrial glutathione was only partial over several hours, suggesting slow net uptake from the cytoplasm. Glutathione depletion alone did not significantly affect mitochondrial membrane potential, ATP content, or cell viability when assessed after 24 hr, although the activities of respiratory chain complexes were altered. However, these astrocytes showed a greatly enhanced sensitivity to 3-morpholinosydnonimine, a peroxynitrite generator. Treatment with 200 microm 3-morpholinosydnonimine produced decreases within 3 hr in mitochondrial membrane potential and ATP content and caused the release of lactate dehydrogenase, contrasting with preservation of these properties in control cells. These properties deteriorated further by 24 hr in the glutathione-depleted cells and were associated with morphological changes indicative of necrotic cell death. This treatment enhanced the alterations in activities of the respiratory chain complexes observed with glutathione depletion alone. Cell viability was markedly improved by cyclosporin A, suggesting a role for the mitochondrial permeability transition in the astrocytic death. These studies provide the most direct evidence available for any cell type on the roles of mitochondrial glutathione. They demonstrate the critical importance of this metabolite pool in protecting against peroxynitrite-induced damage in astrocytes and indicate a key contribution in determining the activities of respiratory chain components.

Adenosine Triphosphate↗

PubMed2004

Glutathione monoethyl ester provides neuroprotection in a rat model of stroke.

Oxidative stress plays an important role in the development of tissue damage following transient focal cerebral ischaemia. Glutathione is a central component in the antioxidant defence of cells. We have previously shown a close association between mitochondrial glutathione loss and cell death following middle cerebral artery (MCA) occlusion. Glutathione monoethyl ester increases cellular glutathione and is particularly effective in increasing the mitochondrial pool. In the present investigation, we infused glutathione monoethyl ester into the third ventricle during 2 h of MCA occlusion and 48 h of reperfusion. Infarct size was reduced from 46% of the total ischaemic hemisphere in saline-treated animals to 16% following ester treatment. Thus, glutathione monoethyl ester provides neuroprotection following transient focal cerebral ischaemia.

Animals↗

PubMed2004

Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia.

Glutathione is a central component in the antioxidant defences of cells. We have recently reported an early and selective loss of total (reduced plus oxidised) glutathione from mitochondria isolated from rat brain following occlusion of the middle cerebral artery. This mitochondrial glutathione depletion showed an apparent association with the tissue damage that developed during subsequent reperfusion, suggesting that it could be an important determinant of susceptibility to cell loss. In the present study, we have investigated whether in vivo treatment with glutathione ethyl ester can modulate mitochondrial glutathione in the brain and whether this treatment can influence the response to focal ischemia. In further support of our previous findings, middle cerebral artery occlusion caused a duration-dependent partial loss of mitochondrial glutathione. Bilateral injections of glutathione ethyl ester immediately prior to induction of unilateral focal ischemia resulted in a substantial increase in glutathione in mitochondria from the striatum of both the non-ischemic hemisphere (190% of saline-treated controls) and the ischemic hemisphere (240% of controls) at 2h after arterial occlusion. Total tissue glutathione was not affected by the ester treatment at this time. A smaller increase in mitochondrial glutathione was observed at 3h of occlusion in the non-ischemic striatum following ester treatment but at this time point glutathione was not significantly altered in mitochondria from the ischemic hemisphere. Pre-ischemic treatment with glutathione ester did not significantly change the volume of tissue infarction assessed at 48 h following ischemia for 2 or 3h. These studies demonstrate that glutathione ethyl ester is a highly effective modulator of the mitochondrial glutathione pool in the intact brain and provides a useful means for further investigating the role of this antioxidant in the development of tissue damage in ischemia and other brain disorders.

Animals↗

PubMed2004

Increased mitochondrial permeability in response to intrastriatal N-methyl-D-aspartate: detection based on accumulation of radiolabel from [3H]deoxyglucose.

Induction of the mitochondrial permeability transition has been proposed as an important contributor to cell loss in several neurological disorders, but the evidence that this change can develop in cells in the intact mature brain is largely indirect. In this study, we have tested whether an intrastriatal injection of N-methyl-D-aspartate results in increases in inner membrane permeability that can be detected from mitochondrial accumulation of metabolites of 3H-deoxyglucose previously taken up by brain cells. An increase in incorporation of deoxyglucose metabolites was found in mitochondria prepared from the striatum but not from cerebral cortex distant from the injection site. This change developed more than 8 h after treatment with N-methyl-D-aspartate and is consistent with the induction of the permeability transition as a late change in the progression to irreversible neuronal damage in response to this excitotoxic insult. At earlier times, the restricted permeability of the inner mitochondrial membrane was apparently preserved, at least sufficiently to prevent significant diffusion of metabolites between the cytoplasm and the matrix.

Animals↗

PubMed2003

Losses of NG2 and NeuN immunoreactivity but not astrocytic markers during early reperfusion following severe focal cerebral ischemia.

The ability of glia to recover essential functions following a period of focal cerebral ischemia is likely to be one important factor influencing the severity of tissue damage that subsequently develops. In this study, we have compared changes in immunoreactivity of markers specific for astrocytes, NG2-positive glia and neurons in tissue subregions during early reperfusion following 3 h of middle cerebral artery occlusion to provide insights into possible differential susceptibility of these cell populations. Under the conditions used, infarction ultimately encompasses most of the perfusion territory of the occluded artery. Nonetheless, alterations in immunoreactivity during the first 3 h of recirculation were restricted to brain regions that had been subjected to severe ischemia. In the striatum, cellular immunoreactivity for NG2 and neuronal markers, NeuN and microtubule-associated protein 2, was greatly reduced by 1 h of reperfusion and declined further at 3 h. NG2 labeling of blood vessels in the striatum appeared post-ischemically, mimicking expression of this protein during development. Less severe changes were seen in the neuronal markers in overlying cerebral cortex. In contrast to the losses of other cellular proteins, immunoreactivity for the astrocytic marker, glial fibrillary acidic protein, was preserved in all tissue that had been subjected to severe ischemia and labeling of another astrocytic protein, glutamine synthetase, was increased by 3 h of reperfusion. These findings provide the first evidence of marked sensitivity of NG2-immunoreactivity to severe ischemia and suggest a greater initial resistance of astrocytes compared with neurons and NG2-positive glia to ischemia-reperfusion damage.

Animals↗

PubMed2003

Fluorocitrate-mediated astroglial dysfunction causes seizures.

A role for astroglia in epileptogenesis has been hypothesised but is not established. Low doses of fluorocitrate specifically and reversibly disrupt astroglial metabolism by blocking aconitase, an enzyme integral to the tricarboxylic acid cycle. We used cerebral cortex injections of fluorocitrate, at a dose that we demonstrated to inhibit astroglial metabolism selectively, to determine whether astroglial disturbances lead to seizures. Rats were halothane-anesthetized, and 0.8 nmol of sodium fluorocitrate was injected into the cerebral cortex. Extradural electroencephalogram (EEG) electrodes were implanted, after which the anesthesia was ceased and the animals were observed. In all experiments, 14 of 15 fluorocitrate-treated animals exhibited epileptiform EEG discharges, with some animals exhibiting convulsive seizures. Discharges commenced as early as 30 min postfluorocitrate injection. Intraperitoneal octanol, but not halothane by inhalation, given to test the possible participation of gap junctions in EEG discharge generation, blocked or delayed the occurrence of discharges after fluorocitrate. These results indicate that focal cerebrocortical astroglial dysfunction leads to focal epileptiform discharges and sometimes to convulsive seizures and that the process possibly depends on effects mediated by gap junctions.

Animals↗

PubMed2002

Mitochondrial contributions to tissue damage in stroke.

Tissue infarction, involving death of essentially all cells within a part of the brain, is a common pathology resulting from stroke and an important determinant of the long-term consequences of this disorder. The cell death that leads to infarct formation is likely to be the result of multiple interacting pathological processes. A range of factors, including the severity of the ischemic insult and whether this is permanent or reversed, determine which mechanisms predominate. Although evaluating mitochondrial properties in intact brain is difficult, evidence for several potentially deleterious responses to cerebral ischemia or post-ischemic reperfusion have been obtained from investigations using animal models of stroke. Marked changes in ATP and related energy metabolites develop quickly in response to occlusion of a cerebral artery, as expected from limitations in the delivery of oxygen and glucose. However, these alterations are often only partially reversed on reperfusion despite improved substrate delivery. Ischemia-induced decreases in the mitochondrial capacity for respiratory activity probably contribute to the ongoing impairment of energy metabolism during reperfusion and possibly also to the magnitude of changes seen during ischemia. Conditions during reperfusion are likely to be conducive to the induction of the permeability transition in mitochondria. There are as yet no well-characterized techniques to identify this change in the intact brain. However, the protective effects of some agents that block formation of the transition pore are consistent with both the induction of the permeability transition during early recirculation and a role for this in the development of tissue damage. Release of cytochrome c into the cytoplasm of cells has been observed with both permanent and reversed ischemia and could trigger the death of some cells by apoptosis, a process which probably contributes to the expansion of the ischemic lesion. Mitochondria are also likely to contribute to the widely-accepted role of nitric oxide in the development of ischemic damage. These organelles are a probable target for the deleterious effects of this substance and can also act as a source of superoxide for reaction with the nitric oxide to produce the damaging species, peroxynitrite. Further characterization of these mitochondrial responses should help to elucidate the mechanisms of cell death due to cerebral ischemia and possibly point to novel sites for therapeutic interventions in stroke.

Animals↗

PubMed2002

The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions.

Glutathione is a key cellular antioxidant that is contained in both cytoplasmic and mitochondrial compartments. Previous investigations indicate that depletion of the mitochondrial pool of glutathione can greatly reduce cell viability. In the present investigation, the effect of focal cerebral ischemia on total (reduced plus oxidized) glutathione in mitochondria was assessed using a rat model of middle cerebral artery occlusion. Total glutathione was substantially decreased in mitochondria prepared from severely ischemic focal tissue in both the cerebral cortex and striatum at 2 h of vessel occlusion and persisted for at least the first 3 h of reperfusion. The loss of mitochondrial glutathione was not associated with decreases of the total tissue glutathione content and was not due to the formation of mixed disulfides with mitochondrial proteins. Thus, an imbalance between uptake and release from the mitochondria in the ischemic tissue provides the most likely explanation for the loss. Decreases in glutathione also developed in mitochondria from the moderately ischemic perifocal tissue when the period of arterial occlusion was extended to 3 h. The presence of mitochondrial glutathione depletion during ischemia showed an apparent close association with the subsequent development of tissue infarction. These findings are consistent with a role for the glutathione depletion in determining the susceptibility of brain tissue to focal ischemia.

Animals↗