Mechanisms Of Ischemic Brain Injury Research Articles

Astrocyte Mitochondrial Mechanisms of Ischemic Brain Injury and Neuroprotection

Research on ischemic brain injury has established a central role of mitochondria in neuron death. Astrocytes are also damaged by ischemia, although the participation of mitochondria in their injury is ill defined. As astrocytes are responsible for neuronal metabolic and trophic support, astrocyte dysfunction will compromise postischemic neuronal survival. Ischemic alterations to astrocyte energy metabolism and the uptake and metabolism of the excitatory amino acid transmitter glutamate may be particularly important. Despite the significance of ischemic astrocyte injury, little is known of the mechanisms responsible for astrocyte death and dysfunction. This review focuses on differences between astrocyte and neuronal metabolism and mitochondrial function, and on neuronal-glial interactions. The potential for astrocyte mitochondria to serve as targets of neuroprotective interventions is also discussed.

Protection by Pyruvate against Transient Forebrain Ischemia in Rats

Pyruvate has a remarkable protective effect against zinc neurotoxicity. Because zinc neurotoxicity is likely one of the key mechanisms of ischemic brain injury, the neuroprotective effect of pyruvate was tested in a rat model of transient forebrain ischemia. Control experiments in mouse cortical culture showed that pyruvate almost completely blocked zinc toxicity but did not attenuate calcium-overload neuronal death. Adult rats subjected to 12 min forebrain ischemia exhibited widespread zinc accumulation and neuronal death throughout hippocampus and cortex 72 hr after reperfusion. However, rats injected intraperitoneally with sodium pyruvate (500-1000 mg/kg) within 1 hr after 12 min forebrain ischemia showed almost no neuronal death. In addition, the mortality was markedly decreased in the pyruvate-protected groups (3.8%) compared with the NaCl-injected control group (58.1%). The neuroprotective effect persisted even at 30 d after the insult. The spectacular protection without noticeable side effects makes pyruvate a promising neuroprotectant in human ischemic stroke.

Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice.

Cyclooxygenase-2 (COX-2), a prostanoid-synthesizing enzyme that contributes to the toxicity associated with inflammation, has recently emerged as a promising therapeutic target for several illnesses, ranging from osteoarthritis to Alzheimer's disease. Although COX-2 has also been linked to ischemic stroke, its role in the mechanisms of ischemic brain injury remains controversial. We demonstrate that COX-2-deficient mice have a significant reduction in the brain injury produced by occlusion of the middle cerebral artery. The protection can be attributed to attenuation of glutamate neurotoxicity, a critical factor in the initiation of ischemic brain injury, and to abrogation of the deleterious effects of postischemic inflammation, a process contributing to the secondary progression of the damage. Thus, COX-2 is involved in pathogenic events occurring in both the early and late stages of cerebral ischemia and may be a valuable therapeutic target for treatment of human stroke.

What animal models have taught us about the treatment of acute stroke and brain protection.

Stroke research has progressed in leaps and bounds in the past decades. A driving force is the increasing availability of new research tools in this field (eg, animal stroke models). Animal stroke models have been extensively applied to advance our understanding of the mechanisms of ischemic brain injury and to develop novel therapeutic strategies for reducing brain damage after a stroke. Animal stroke models have been useful in characterizing the molecular cascades of injury processes. These "injury pathways" are also the targets of therapeutic interventions. The major achievements made in the past 2 decades applying animal stroke models include 1) the identification of the mediator role of excitotoxin and oxygen free radicals in ischemic brain injury; 2) the confirmation of apoptosis as a major mechanism of ischemic cell death; 3) the characterization of postischemic gene expression; 4) the delineation of postischemic inflammatory reaction; 5) the application of transgenic mice to confirm the roles of purported mediators in ischemic brain injury; 6) development of novel magnetic resonance imaging sequences for early noninvasive detection of ischemic brain lesions; and, 7) the development of novel therapeutic strategies based on preclinical findings derived from animal stroke models.

A reproducible model of thromboembolic stroke in mice.

AN experimental mouse model of thromboembolic stroke is presented, in which standardized fibrin-rich emboli (150 microm diameter, 1.5 mm or 4 mm length) are injected into the internal carotid artery. Injection of six or eight 1.5 mm clots (corresponding to 0.16 or 0.21 microl clot material) led to a variable decrease in laser Doppler flow (LDF), but did not result in reproducible infarcts of the middle cerebral artery (MCA) territory, as determined by triphenyltetrazolium chloride (TTC) staining 24 h after embolization. Injection of ten 1.5 mm clots (0.27 microl) or four 4 mm clots (0.28 microl), however, caused a persistent LDF decline to about 20-30% of baseline and led to reproducible infarcts covering the entire MCA territory. The method establishes a clinically relevant, reproducible stroke model for the study of molecular mechanisms of ischemic brain injury in genetically engineered mice.

The Effect of Age on Susceptibility to Hypoxic-Ischemic Brain Damage

YAGER, J.Y. AND J.A. THORNHILL. The effect of age on susceptibility to hypoxic-ischemic brain damage. NEUROSCI BIOBEHAV REV 21 (2) 167–174, 1997.—Stroke occurs in all age groups, ranging from the new-born to the elderly. Our current understanding of the mechanisms of ischemic brain injury suggests that, despite age, the underlying cascade of events includes the rapid depletion of energy reserves, lactate accumulation, release of excitatory amino acids, high intracellular concentrations of Ca 2+, and the production of oxygen free radicals. The extent to which these events affect brain injury, however, is profoundly influenced by age. Hyperglycemia for example, markedly enhances hypoxic-ischemic brain damage in adults, but has a protective effect in new-born rats. Insulin-induced hypoglycemia, on the other hand, protects the adult brain, but may be detrimental to the new-born. Substrate utilization of ketone bodies is markedly enhanced in the new-born, and has now been shown also to protect the brain. The immature brain is generally believed to be more resistant to the damaging effects of cerebrovascular compromise compared to the more mature brain. However, recent experiments suggest that the correlation between brain damage and age is not linear. To further clarify the effects of age and development on hypoxic-ischemic brain damage, we developed a model whereby rats of increasing age received identical cerebrovascular insults. Neuropathologic assessment at 7 days of recovery showed that brain damage was most severe in the 1- and 3-week-old animals followed by those that were 6 months. The 6- and 9-week-old groups had significantly less injury than the other three age groups. Hippocampal damage was most severe in the 3-week and 6-month-old rats compared to all other age groups. These findings contrast previously held beliefs regarding the enhanced tolerance of the immature brain to hypoxic-ischemic damage and demonstrate that the immature brain is, in fact, less resistant to hypoxic-ischemic brain damage than its adult counterpart. The results emphasize the need for a greater understanding of the effects of ontogeny on hypoxic-ischemic brain damage, particularly as it pertains to the development of therapeutic interventions. © 1997 Elsevier Science Ltd. All rights reserved.

Parallel antioxidant and antiexcitotoxic therapy improves outcome after incomplete global cerebral ischemia in dogs.

We have previously shown that incomplete global cerebral ischemia complicated by dense acidosis produces a profound secondary deterioration of energy metabolism and cerebral blood flow. Antioxidant treatment only partially averts this deterioration, suggesting that parallel or sequential mechanisms are involved in cerebral ischemic injury. We tested the hypothesis that a novel competitive N-methyl-D-aspartate (NMDA) receptor antagonist GPI 3000 (GPI) ameliorates metabolic injury and that the effectiveness of the iron-chelator and antioxidant deferoxamine (DFO) is augmented by combined therapy with GPI after incomplete global cerebral ischemia. Anesthetized dogs were treated with 30 minutes of global incomplete cerebral ischemia. Preischemic plasma glucose was raised to approximately 500 mg/dL to exaggerate lactic acidosis. Brain ATP, phosphocreatine, and pH were measured by 31P MR spectroscopy for 180 minutes of reperfusion. Neurophysiological outcomes were assessed by evoked potential monitoring. Five groups were treated with either saline; 75 mg/kg DFO preischemia plus 75 mg/kg at reperfusion onset, followed by 27.5 mg/kg per hour for the remainder of reperfusion (DFO group); 25 mg/kg GPI pretreatment, followed by 5 mg/kg per hour (GPI-pre group); 25 mg/kg GPI at reperfusion, followed by 5 mg/kg per hour (GPI-post group); or DFO and GPI-pre at the same doses (Combined group). Ischemic cerebral blood flow (microspheres: 5 to 8 mL/min per 100 g) was similar among the groups. End-ischemic pHi was also similar; 5.9 in saline, 6.1 in DFO, 6.2 in GPI-pre, 6.2 in Combined, and 6.1 in GPI-post groups. Progressive hypoperfusion was observed in all groups except Combined during reperfusion. Metabolic recovery was improved relative to saline in all drug-treated groups. Phosphocreatine recovery was improved in Combined compared with DFO and GPI-pre groups. Somatosensory evoked potential recovery was not observed in the saline group and incomplete in all treatment groups. At 60 and 90 minutes of reperfusion, DFO, GPI-pre, and Combined groups demonstrated improved recovery relative to the saline group. Pretreatment and posttreatment with GPI ameliorated postischemic metabolic failure, suggesting that NMDA-mediated mechanisms are more important in global cerebral ischemia complicated by dense, acidosis than early studies indicated. Combined treatment with GPI and DFO improved cerebral blood flow during reperfusion and one indicator of energy recovery. These data support the hypothesis that parallel therapy aimed at antioxidant and antiexcitotoxic mechanisms of ischemic brain injury augment recovery compared with the individual agents.

Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia.

We investigated whether inducible nitric oxide synthase (iNOS) is expressed after transient cerebral ischemia and, if so, we sought to define the temporal profile and cellular localization of the expression and the role of iNOS in the mechanism of ischemic brain injury. The middle cerebral artery in rats was occluded for 2 hours by an intraluminal filament. The occurrence of transient ischemia and reperfusion was confirmed by laser-Doppler flowmetry (n = 5). iNOS message in the ischemic neocortex was determined by reverse-transcription polymerase chain reaction. iNOS enzymatic activity was assessed by citrulline assay. The cellular localization of iNOS expression was determined by immunohistochemistry. iNOS mRNA was maximally expressed in postischemic brain at 12 hours and was not present at 4 days (n = 3 per time point). iNOS mRNA was not observed in the contralateral cerebral cortex. iNOS enzymatic activity developed in the postischemic brain between 12 and 24 hours (P < .05) and subsided at 4 days (n = 4 to 8 per time point). iNOS immunoreactivity in the ischemic region was restricted to the wall of capillaries and of larger blood vessels at 12 to 24 hours. In regions of early necrosis, inflammatory cells were iNOS positive. Treatment with the iNOS inhibitor aminoguanidine (n = 5; 100 mg/kg IP, BID for 4 days), starting 6 hours after ischemia, reduced infarct size in neocortex by 36 +/- 7% in comparison with vehicle-treated controls (n = 5) (P < .05). Transient focal ischemia leads to iNOS expression in postischemic brain. However, the spatial and temporal patterns of expression differ from those occurring in permanent ischemia: iNOS is induced earlier and predominantly in vascular cells rather than in neutrophils. Thus, the temporal profile and localization of postischemic iNOS expression depend on the nature of the ischemic insult. The finding that aminoguanidine reduces infarct size adds further support to the hypothesis that postischemic iNOS expression contributes to ischemic brain damage.

Calcium transients in brain ischemia: role in neuronal injury.

The involvement of calcium ions in mechanisms of ischemic brain injury has been suggested for several years. Our understanding of the role of intracellular Ca2+ as a trigger of acute neurotoxicity and in the induction of long lasting processes leading to necrotic and/or apoptotic postischemic delayed neuronal death or of compensatory, neuroprotective mechanisms has increased considerably. Still many questions concerning the generation of Ca+ signal such as the nature of the main routes of ischemic Ca2+ influx to neurones, involvement of intracellular Ca2+ stores and Ca2+ buffers, spatial and temporal relations between ischemia-induced increases in intracellular Ca2+ concentration and neurotoxicity remain open. Some conclusions from experiments in cultured neurones concerning glutamate-evoked destabilization of Ca2+ homeostasis and neurotoxicity may be not relevant to in vivo ischemic conditions. This review, apart from emphasising generally proposed mechanisms of Ca2+ transients and toxicity in ischemic neurones, will discuss some of these controversial issues.

Deferoxamine reduces early metabolic failure associated with severe cerebral ischemic acidosis in dogs.

Postischemic metabolic injury may be mediated by acidosis and tissue bicarbonate depletion, with consequent-iron mobilization and oxygen radical formation during reperfusion. We have previously shown that reducing intracellular pH to below 5.7 and bicarbonate ion to below 1 to 2 mmol/L during hyperglycemic ischemia produces a profound secondary deterioration of brain ATP and cerebral blood flow during reperfusion. This study tested the hypothesis that pretreatment with free deferoxamine ameliorates metabolic decay and delayed hypoperfusion after global hyperglycemic ischemia. In addition, deferoxamine conjugated to a high-molecular-weight starch was administered to determine the importance of an intravascular site of action. Iron-loaded deferoxamine was used to determine whether the iron chelation properties of deferoxamine are important to postischemic viability as distinguished from the agent's significant radical scavenging potential. Cerebral ATP, phosphocreatine, and pH were measured by 31P magnetic resonance spectroscopy in anesthetized dogs. Tissue bicarbonate concentration was calculated from the Henderson-Hasselbalch equation. Incomplete cerebral ischemia was produced by intracranial pressure elevation for 30 minutes with plasma glucose at 540 +/- 15 mg/dL. Free deferoxamine, saline vehicle, hydroxyethyl starch-conjugated deferoxamine, hydroxyethyl starch vehicle, and deferoxamine loaded with equimolar ferric chloride were administered intravenously in five groups of dogs. The dose of deferoxamine was 50 mg/kg before ischemia, 50 mg/kg at the onset of reperfusion, and 50 mg/kg over the 180-minute reperfusion period. Ischemic hemispheric blood flow (mean, 6 to 8 mL/min per 100 g), intracellular pH (5.7 to 6.0), and bicarbonate levels (1 to 2 mmol/L) were similar in all groups. During reperfusion, cerebral pH and bicarbonate recovered only in the free-deferoxamine group. Both ATP and phosphocreatine initially increased in all groups, but recovery was sustained only in the free-deferoxamine group. Secondary losses of energy phosphates and cerebral oxygen consumption were observed in all other groups, accompanied by progressive reduction of perfusion. These data support the hypothesis that iron catalyzed oxygen radical production plays an important role in acidosis-mediated mechanisms of ischemic brain injury. The results with free and iron-loaded deferoxamine suggest that iron scavenging is an important, but not necessarily the principal, component of this mechanism. The poor recovery seen with conjugated deferoxamine indicates that the beneficial action of deferoxamine is not localized within the intravascular compartment.