Brain damage in a mouse model of global cerebral ischemia. Effect of NMDA receptor blockade.

The importance of particular genes in neuronal death following global cerebral ischemia can readily be studied in genetically modified mice provided a reliable model of ischemia is available. For that purpose, we developed a mouse model of global cerebral ischemia that induces consistent damage to different regions of the brain and with a low mortality rate. Twelve minutes of ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion under halothane anesthesia and artificial ventilation. Body and brain temperature were monitored and cortical cerebral blood flow in each hemisphere was measured by laser Doppler flowmeter before, during, and for 5 min after ischemia. Extensive damage was found in the striatum and marked cell damage was observed in the CA1 and CA2 regions of hippocampus and in thalamus. Mild damage was seen in the CA3 region, dentate gyrus and cortex. Hippocampal damage in the CA1 region is delayed and developed over 48 h. Intraischemic hypothermia of 33 °C provided a robust neuroprotection. The non-competitive N-methyl-Image-aspartate receptor blocker, MK-801, did not provide protection in the hippocampus, cortex, striatum or thalamus when administered 30 min prior to ischemia or 2 h after the end of ischemia, but selectively mitigated damage in the hippocampus, when administered immediately following ischemia. This model of global cerebral ischemia may be useful in pharmacological and genomic studies of ischemic brain damage

Free radical production and ischemic brain damage: influence of postischemic oxygen tension

It is now becoming increasingly clear that free radicals contribute to brain damage in several conditions, such as hyperoxia and trauma. It has been more difficult to prove that free radical production mediates ischemic brain damage, but it has often been suggested that it may be a major contributor to reperfusion damage, observed following transient ischemia. Recent results demonstrate that cerebral ischemia of long duration, particularly when followed by reperfusion, leads to enhanced production of partially reduced oxygen species, notably hydrogen peroxide (H2O2). It has also been suggested that postischemic hyperoxia, e.g. an increased oxygen tension during the recirculation period, adversely affects recovery following transient ischemia. Other data support the notion that brain damage caused by permanent ischemia (stroke) is significantly influenced by production of free radicals. The present study, however, fails to show that recirculation following brief periods of ischemia (15 min) leads to an enhanced H2O2 production, and that hyperoxia aggravates the ischemic damage. This study was undertaken to reveal whether variations in oxygen supply in the postischemic period following forebrain ischemia in rats affect free radical production and the brain damage incurred. To that end, rats ventilated on N2O/O2 (70:30) were subjected to 15 min of transient ischemia. Normoxic animals were ventilated with the N2O/O2 mixture, hyperoxic animals with 100% O2, and hypoxic ones with about 10% O2 (balance either N2O/N2 or N2) during the recirculation. At the end of this period, the animals were decapitated for assessment of H2O2 production with the aminotriazole/catalase method. This method is based on the notion that aminotriazole interacts with H2O2 to inactivate catalase; thus, the rate of inactivation of catalase in aminotriazole treated animals reflects H2O2 production. In a parallel series, animals ventilated with one of the three gas mixtures in the early recirculation period, respectively, were allowed to recover for 7 days, with subsequent perfusion-fixation of brain tissues and light microscopical evaluation of the brain damage. Animals given aminotriazole, whether rendered ischemic or not, showed a reduced tissue catalase activity, reflecting H2O2 production in the brain. Hyperoxic animals failed to show increased tissue H2O2 production, while hypoxic ones showed a tendency towards decreased production. However, all three groups (hypo, normo- and hyperoxic) had similar density and distribution of neuronal damage. These results suggest that although postischemic oxygen tensions may determine the rates of H2O2 production, variations in oxygen tensions do not influence the final brain damage incurred. In conclusion, there was thus no indication that variations in the postischemic oxygen supply altered production of free radicals, or modulated the damage incurred as a result of the ischemia. We conclude that free radical production may not be an important factor in the pathogenesis of brain damage following brief periods of ischemia, but may represent an important modulator following longer periods of ischemia, when a vascular component becomes an important factor in the pathological events

The Aldose Reductase Inhibitor Fidarestat Suppresses Ischemia-Reperfusion-Induced Inflammatory Response in Rat Retina.

Recent studies suggest that increased aldose reductase (AR) activity plays an important role in ischemia-reperfusion injury in the retina. The mechanisms are not completely understood, but may be linked to inflammation. In the present study, we investigated whether the AR inhibitor fidarestat suppressed the retinal inflammatory response induced by ischemia-reperfusion in a rat model. The inflammatory response was manifested by increased gene expression of tumor necrosis factor-alpha and intercellular adhesion molecule-1 (ICAM-1) as well as elevated protein levels of soluble ICAM-1. This response was partially suppressed by the AR inhibitor fidarestat. The findings may reveal beneficial effects of AR inhibition on retinal inflammation associated with ischemia-reperfusion and are in agreement with recent developments in pharmacological research suggesting that pathological conditions other than diabetes may benefit from AR inhibitors

Lack of neuroprotection by heat shock protein 70 overexpression in a mouse model of global cerebral ischemia.

Heat shock protein 70 (Hsp70) is induced in cells by a variety of stress conditions, is known to be cytoprotective, and has been proposed to be neuroprotective during brain ischemia. A recently developed mouse model of 12-min global cerebral ischemia by bilateral common carotid artery occlusion with artificial ventilation and bilateral monitoring of regional cerebral blood flow by laser Doppler was applied. We examined the expression and possible neuroprotective role of the inducible form of Hsp70 in the mouse brain following global cerebral ischemia. Ischemia induced a marked expression of Hsp70 in the ischemia vulnerable CA1-CA3 region of the hippocampus. Intraischemic hypothermia (33degreesC) prevented cell damage without noticeable expression of Hsp70. A transgenic mouse overexpressing Hsp70 was subjected to 12 min of global cerebral ischemia, and the brain damage was evaluated after 4 days. No neuroprotection of ischemia-induced brain damage in hippocampus, striatum, cortex or thalamus was found in Hsp70 transgenic animals compared with wild-type littermate mice. We suggest that overexpression of Hsp70 following cerebral ischemia is an indicator of cell stress. Also, constitutively overexpression of Hsp70 is insufficient to effectively influence cell death after global cerebral ischemia in the mouse

Models for studying long‐term recovery following forebrain ischemia in the rat. 2. A 2‐vessel occlusion model

ABSTRACT— A model is described in which transient ischemia is induced in rats anaesthetized with N2O:O2 (70:30) by bilateral carotid artery clamping combined with a lowering of mean arterial blood pressure to 50 mm Hg, the latter being achieved by bleeding, or by bleeding supplemented with administration of trimetaphan or phentolamine. By the use of intubation, muscle paralysis with suxamethonium chloride, and insertion of tail arterial and venous catheters, it was possible to induce reversible ischemia for long‐term recovery studies. Autoradiographic measurements of local CBF showed that the procedure reduced CBF in neocortical areas, hippocampus, and caudoputamen to near‐zero values, flow rates in a number of subcortical areas being variable. Administration of trimethaphane or phentolamine did not affect ischemic and postischemic flow rates, nor did they alter recovery of EEG and sensory‐evoked responses, but trimetaphan blunted the changes in plasma concentrations of adrenaline and noradrenaline. Recovery experiments showed that 10 min of ischemia gave rise to clear signs of permanent brain damage, with a small number of animals developing postischemic seizures that led to the death of the animals in status epilepticus. After 15 min of ischemia, such alterations were more pronounced, and the majority of animals died. It is concluded that the short revival times noted are explained by the fact that the model induces near‐complete ischemia, and that recovery following forebrain ischemia is critically dependent on residual flow rates during the period of ischemia

Regulation of brain-derived neurotrophic factor gene expression after transient middle cerebral artery occlusion with and without brain damage

Levels of mRNA for c-fos, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), TrkB, and TrkC were studied using in situ hybridization in the rat brain at different reperfusion times after unilateral middle cerebral artery occlusion (MCAO). Short-term (15 min) MCAO, which does not cause neuronal death, induced elevated BDNF mRNA expression confined to ipsilateral frontal and cingulate cortices outside the ischemic area. With a longer duration of MCAO (2 h), which leads to cortical infarction, the increase was more marked and elevated BDNF mRNA levels were also detected bilaterally in dentate granule cells and CA1 and CA3 pyramidal neurons. Maximum expression was found after 2 h of reperfusion. At 24 h BDNF mRNA expression had returned to control values. In the ischemic core of the parietal cortex only scattered neurons were expressing high levels of BDNF mRNA after 15 min and 2 h of MCAO. Analysis of different BDNF transcripts showed that MCAO induced a marked increase of exon III mRNA but only small increases of exon I and II mRNAs in cortex and hippocampus. In contrast to BDNF mRNA, elevated expression of c-fos mRNA was observed in the entire ipsilateral cerebral cortex, including the ischemic core, after both 15 min and 2 h of MCAO. Two hours of MCAO also induced transient, bilateral increases of NGF and TrkB mRNA levels and a decrease of NT-3 mRNA expression, confined to dentate granule cells. The upregulation of BDNF mRNA expression in cortical neurons after MCAO is probably triggered by glutamate through a spreading depression-like mechanism. The lack of response of the BDNF gene in the ischemic core may be due to suppression of signal transduction or transcription factor synthesis caused by the ischemia. The observed pattern of gene expression after MCAO agrees well with a neuroprotective role of BDNF in cortical neurons. However, elevated levels of NGF and BDNF protein could also increase synaptic efficacy in the postischemic phase, which may promote epileptogenesis

Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat

The immunosuppressant drug cyclosporin A (CsA) is considered to be inherently protective in conditions of ischemia, e.g. in hepatic and cardiac tissue. However, investigations of effects of CsA on neuronal tissue have been contradictory, probably because the blood-brain barrier (BBB) is virtually impermeable to CsA. In the present study, we exploited the finding that the insertion of a syringe needle into brain parenchyma obviously disrupts the BBB and allows influx of CsA, and explored whether CsA, given as intraperitoneal injections daily for 1 week before and 1 week after forebrain ischemia of 7 or 10 min duration, ameliorates the damage incurred to the hippocampal CA 1 sector. In other experiments, the needle insertion and the first i.p. injection of CsA were made 30 min after the start of recirculation, with continued daily administration of CsA during the postinsult week. In animals which were injected with CsA in daily doses of 10 mg kg-1, but in which no needle was inserted, the drug failed to ameliorate CA1 damage, whether the ischemia had a duration of 7 or 10 min. Likewise, needle insertion had no effect on CA1 damage if CsA was not administered. In contrast, when CsA was given to animals with a needle insertion, CA1 damage was dramatically ameliorated, whether treatment was initiated 1 week before ischemia, or 30 min after the start of recirculation. The effect of CsA seemed larger than that of any other drug proposed to have an anti-ischemic effect in forebrain/global ischemia. Injection of tritiated CsA in one animal with BBB disruption lead to detectable radioactivity throughout the ventricular system, suggesting a generalised increase of the entry of CsA across the BBB. The results demonstrate that immunosuppressants of the type represented by CsA markedly ameliorate delayed neuronal damage after transient forebrain ischemia, provided that they can pass the BBB. It is discussed whether the effect of the drug is one involving calcineurin, a protein phosphatase, or if CsA counteracts a permeability transition of the inner mitochondrial membrane, assumed to occur in response to adverse conditions, e.g. gradual accumulation of Ca2+ in the mitochondria in the postischemic period