Vessel Occlusion Model Research Articles

Correlation between extracellular glutamate release and neuronal cell death in an eleven vessel occlusion model in rat

The aim of this study was to define the effects of glutamate release on cell death in an eleven vessel rat occlusion model. Male Sprague–Dawley rats (250–350 g) were used for the 11 vessel occlusion ischemic model, which was induced by a 5- and 10-min transient occlusion. During the surgical procedure, the extracellular glutamate concentration was measured in real-time using a microdialysis amperometirc biosensor with cerebral blood flow. In order to confirm neuronal cell death, brains were removed 72 h after ischemia for the detection of the neuron-specific nuclear protein and cleaved caspase-3 levels, using double-immunofluorescence. A significant decrease in % cerebral blood flow was observed in both the 5- and 10-min 11 vessel occlusion models, while an increase in glutamate release was detected after the onset of ischemia that continued to rise during the ischemic period. However, a significantly higher level of glutamate release was observed in the 10-min ischemia group compared to the 5-min group. Unlike the small amount of brain damage in the 5-min group, the increased glutamate levels in the 10-min group resulted in ischemic cell death in the hippocampal region with the activation of cleaved caspase-3 and the inhibition of neuron-specific nuclear protein expression. This study suggests that the increased level of glutamate release induces apoptotic cell death in the 11 vessel occlusion ischemic model.

The Effect of Extracellular Glutamate Release on Repetitive Transient Ischemic Injury in Global Ischemia Model

During operations, neurosurgeons usually perform multiple temporary occlusions of parental artery, possibly resulting in the neuronal damage. It is generally thought that neuronal damage by cerebral ischemia is associated with extracellular concentrations of the excitatory amino acids. In this study, we measured the dynamics of extracellular glutamate release in 11 vessel occlusion (VO) model to compare between single occlusion and repeated transient occlusions within short interval. Changes in cerebral blood flow were monitored by laser-Doppler flowmetry simultaneously with cortical glutamate level measured by amperometric biosensor. From real time monitoring of glutamate release in 11 VO model, the change of extracellular glutamate level in repeated transient occlusion group was smaller than that of single occlusion group, and the onset time of glutamate release in the second ischemic episode of repeated occlusion group was delayed compared to the first ischemic episode which was similar to that of single 10 min ischemic episode. These results suggested that repeated transient occlusion induces less glutamate release from neuronal cell than single occlusion, and the delayed onset time of glutamate release is attributed to endogeneous protective mechanism of ischemic tolerance.

The effect of granulocyte-colony stimulating factor in global cerebral ischemia in rats

Granulocyte-colony stimulating factor (G-CSF) is an endogenous peptide hormone of the hematopoietic system that has entered Phase I/II clinical trials for treatment of ischemic stroke. Severe intraoperative hypotension can lead to global cerebral ischemia and apoptotic neuron loss within the hippocampus. We tested G-CSF in a rat model of global cerebral ischemia. Global cerebral ischemia was induced in male Sprague–Dawley rats (280–330 g) with the 2-vessel occlusion model (hemorrhagic hypotension to a mean arterial pressure of 30–35 mm Hg and bilateral common carotid artery occlusion for 8 min). Three groups of animals were used: global ischemia without treatment (GI, n = 49), global ischemia with G-CSF treatment (GI + G-CSF, n = 42), and sham surgery (Sham, n = 26). Rats in the treatment group received G-CSF (50 μg/kg, subcutaneously) 12 h before surgery, on the day of surgery, and on postoperative Day 1 and were euthanized on Days 2, 3, and 14. Mild hyperglycemia was observed in all groups. T-maze testing for spontaneous alternation demonstrated initial improvement in the G-CSF treatment group but no long-term benefit. Measurement of daily body weight demonstrated an initial trend toward improvement in the G-CSF group. Quantitative Nissl histology of the hippocampus demonstrated equivalent outcomes on Days 3 and 14, which was supported by quantitative TUNEL stain. Immunohistochemistry and Western blot demonstrated an initial increase in phosphorylated-AKT in the GI + G-CSF group on Day 2. We conclude that G-CSF treatment is associated with transient early improvement in neurobehavioral outcomes after global ischemia complicated by mild hyperglycemia, but no long-term protection.

Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus

Progesterone is neuroprotective in models of focal or global ischemia when treatment starts either before the insult or at the onset of reperfusion. In these cases the steroid may act during the occurrence of the early pathophysiological events triggered by ischemia or reperfusion. As opposed to this condition, the aim of the present study was to assess the effect of delayed, post-injury administration of progesterone on the preservation of pyramidal neurons of the hippocampus of rats 21 days after been exposed to global ischemia by the four vessel occlusion model. Progesterone (8 mg/kg, i.v.) or its vehicle, were administered at 20 min, 2, 6, and 24 h after the end of ischemia. At histological examination, brains of the ischemic vehicle-treated rats showed a severe reduction of the population of pyramidal neurons in the CA1 and CA2 subfields (12% and 29% remaining neurons, respectively), and a less severe neuronal loss in the CA3 and CA4 subfields of the hippocampus (68% and 63% remaining neurons, respectively), as compared to rats exposed to sham procedures. They also showed a two-fold enlargement of the lateral ventricles and 33% shrinkage of the cerebral cortex as compared to the sham group. Progesterone treatment resulted in a significant preservation of pyramidal neurons in CA1 and CA2 (40% and 62% remaining neurons), with no ventricular dilation and only a mild (12%) cortical shrinkage. Results suggest that progesterone is able to interfere with some late pathophysiological mechanisms leading both to selective neuronal damage in the hippocampal CA1 and CA2 subfields, and to shrinkage of the cerebral cortex.

Animal Model of Cerebral Ischemia

Cerebral ischemia is a complex and mutifactorial process. Animal models of cerebral ischemia have been widely used to make clear the pathophysiology of the ischemic brain injury and to test the therapies against cerebral ischemia. Many animal ischemia models have been developed in numerous species and in various forms of cerebral ischemia, namely global and focal, complete and incomplete, and permanent and transient. In global cerebral ischemia, selective cell vulnerability to ischemia is a most important factor. Neurons appear to be more susceptible to hyoxia and ischemia as compared with other components, such as glial cells and vessels. In global cerebral ischemia models, the global cerebro-circulatory arrest model by occluding the inominate, the left subclavian and both mammary arteries, the bilateral carotid occlusion with systemic hypotension model in rats, the 4 vessel occlusion model in rats, and the bilateral carotid occlusion model were decribed in here. On the other hand, the main issue in the focal cerebral ischemia is a concept of ischemic thresholds of cerebral blood flow. The so-called of ischemic penumbra is of particular interest as a zone of nonfunctioning but still viable tissue that may recover its function if blood flow can be restored. As the focal cerebral ischemia model, the middle cerebral artery occlusion model in cats and rats and the embolic stroke model were discussed in here.

Effect of late treatment with γ-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia in the rat

It has been previously described that γ-hydroxybutyrate (GHB) provides significant protection against transient global cerebral ischemia in the rat (four vessel occlusion model), when given 30 min before or 10 min after artery occlusion. Here, we show that in the same rat model, significant protection can also be obtained when treatment is started 2 h after the ischemic episode. In saline-treated animals, 30 min of global ischemia followed by reperfusion caused a massive loss of neurons in the hippocampal CA1 subfield (examined 63 days after the ischemic episode), and an impairment of sensory-motor performance (tested on the 51st and 63rd days after ischemia) and of spatial learning and memory (evaluated starting 46 days after the ischemic episode). Treatment with GHB—300 mg/kg intraperitoneally (i.p.) 2 h after the ischemia–reperfusion episode, followed by 100 mg/kg i.p. twice daily for the following 10 days—afforded a highly significant protection, against both histological damage and sensory-motor and learning-memory impairments. These data further suggest the possible therapeutic effectiveness of GHB in brain ischemia, and indicate that the underlying mechanism of action involves non-immediate steps of the ischemia-induced cascade of events.

Nitric oxide-mediated protection by s-nitroso-polyethylene glycol-conjugated hemoglobin of brain damage after transient cerebral ischemia/reperfusion in a rat 2-vessel occlusion model

Nitric oxide-mediated protection by s-nitroso-polyethylene glycol-conjugated hemoglobin of brain damage after transient cerebral ischemia/reperfusion in a rat 2-vessel occlusion model

Observation of arterial and venous thrombus formation by scanning and transmission electron microscopy

In order to examine the process of thrombosis formation in artery and vein, the reactions of the arterial and venous endothelial surfaces were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in the photothrombosis model. Thirty-nine rats were divided into the following 4 groups: 1) Sham group (n = 5) without illumination, 2) group A (n = 10) irradiated for 1 min, 3) group B (n = 10) irradiated for 5 min, 4) group C (n = 14) irradiated for 10 min at the level of the left common carotid artery and internal jugular vein. SEM provided no evidence of damage or adhesion of blood platelets to the endothelium of either the artery or vein in shams or group A animals. In group B, evidence of damage to endothelial cell membrane (e.g., plasmalemmal pits, crater-like structures associated with tears between endothelial cells, and decreased number of microvilli) was obtained in the arterial wall but not in the vein. In group C, there was extensive or widespread adhesion of blood platelets and other cells, tears between arterial endothelial cells, and a decrease in the number of microvilli in the artery but not in the vein (p < 0.05). Cell membrane injuries, tears between the endothelial cells, and endothelial detachment occur before adhesion of blood platelets and thrombus formation in the blood vessel occlusion model by photochemical reaction. These changes occur significantly earlier in the artery than in the vein.

Platelet adhesion and arteriolar dilation in the photothrombosis: observation with the rat closed cranial and spinal windows

The mechanism of cerebral infarction, in which thrombus formation and platelet–endothelium interaction play an important part, have not yet been clearly elucidated in vivo . The aim of this study was to observe rolling and adherent platelets and to analyze adherent leukocytes and vessel diameter change in vivo using a photothrombotic vessel occlusion model. A photothrombosis, which is mediated by free radicals, was induced in male Wistar rats in the presence of a photosensitizing dye (Photofrin II) and exposure to a filtered light. Rhodamine 6G-labeled platelets and leukocytes were visualized with intravital fluorescence videomicroscopy through a closed cranial or spinal window. The vessel diameter, photothrombosis and leukocyte adhesion were analyzed. Rolling and adherent platelets were observed during irradiation through the cerebral and spinal window. Before the platelets were recognized, the irradiated arteriole dilated significantly. After the photochemical occlusion of an arteriole, other arterioles also dilated and the adherent leukocytes increased in the venules. The photothrombi were almost completely composed of platelets according to electron microscopic analysis. The arteriolar dilation rate and the number of adherent leukocytes in the cerebrum were greater than those in the spinal cord. By combining the photochemical thrombus formation and the fluorescence microscope techniques, we were able for the first time to observe rolling and adherent platelets and microvascular responses during photothrombosis in the cerebral and spinal microvasculature. It is suggested that free radicals, which can lead to platelet aggregation, play an important role as a cerebral vasodilator. This model is useful for cerebral and spinal microcirculatory analysis to investigate the platelet–endothelium interaction, the platelet aggregation and the effect of free radicals on cerebral and spinal microcirculation.

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex.

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.

Effect of hypotension severity on hippocampal CA1 neurons in a rat global ischemia model

Neuronal death in the hippocampal CA1 subregion has been shown to occur in a delayed manner after transient global ischemia. The 2-vessel occlusion model is one of the most frequently used global ischemia paradigms in rodents. Although researchers often fail to induce bilateral delayed CA1 neuronal death, the importance of hypotension severity has not been fully discussed. We induced 10 min of global ischemia with 2-vessel occlusion and various severities of hypotension in rats, and the subsequent neuronal damage and neurogenesis in the hippocampal CA1 pyramidal cell layer were immunohistochemically studied. Neuronal apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL). The mean arterial blood pressure of 31-35 mmHg was the most appropriate range of hypotension in this model because of low mortality and consistent bilateral CA1 injury. Most of the neurons in the CA1 pyramidal cell layer lost neuron specific nuclear protein and became TUNEL-positive 3 days after ischemia. There was no evidence of apoptosis or neurogenesis at 7-28 days. There were ischemia-tolerant neurons in the CA1 pyramidal cell layer that survived delayed neurodegeneration, however, further studies are necessary to characterize the property of these neurons.

The effect of topical insulin on the release of excitotoxic and other amino acids from the rat cerebral cortex during streptozotocin-induced hyperglycemic ischemia

Insulin has been demonstrated to be neuroprotective in brain and spinal cord ischemia. The mechanism of neuroprotection may involve alterations in metabolism, protein synthesis or uptake of GABA by astrocytes. Conversely, hyperglycemia increases the extent of neurologic damage observed during ischemia/reperfusion. Diabetic patients are 2–4 times more likely to suffer a stroke as normoglycemic patients and they also have worsened neurologic outcome. Determining if insulin, which many diabetics already use as therapy, can be neuroprotective, would be a possible means of alleviating the detrimental outcome from diabetic stroke. This study looked at the relationship between topically administered insulin (1 mIU insulin/ml and 100 mIU insulin/ml) during a four vessel occlusion model of global ischemia and the release of amino acids, especially glutamate, from the cortex in streptozotocin (STZ)-treated rats. The rats were utilized either 5–7 days (ASTZ) or 4–6 weeks (CSTZ) after a single STZ injection. In the ASTZ animals both doses of insulin increased the amount of the excitotoxic amino acids, aspartate and glutamate, released during reperfusion and the higher dose also increased the levels of taurine and GABA during reperfusion. In the CSTZ animals, both doses of insulin increased the amount of excitotoxic amino acids during reperfusion and the lower dose increased GABA levels released during reperfusion. The differences between the ACTZ and CSTZ animals may be due to metabolic differences in the utilization of glucose. Insulin may act as a neuroprotectant by increasing extracellular GABA resulting in neuroinhibition.

Hydroxyl Radical Production in the Cortex and Striatum in a Rat Model of Focal Cerebral Ischemia

Increases in hydroxyl radical production have been used as evidence of oxidative stress in cerebral ischemia/ reperfusion. Ischemia can also induce increased dopamine release from the striatum that may contribute to hydroxyl radical formation. We have compared hydroxyl radical production in the cortex and striatum as an index of oxidative stress in a rat model of focal cerebral ischemia with cortical infarction. Using a three vessel occlusion model of focal cerebral ischemia combined with bilateral microdialysis, hydroxylation of 4-hydroxybenzoate (4HB) was continuously monitored in both hemispheres in either the lateral striatum or frontoparietal cortex. The ischemia protocol consisted of one hour equilibration, 30 min of three vessel occlusion, then release of the contralateral common carotid artery (CCA) for 2.5 h. Induction of ischemia resulted in a 30-fold increase in dopamine release in the lateral striatum. Compared to the nonischemic striatum, the ratio of the hydroxylation product 3,4-dihydroxybenzoate (34DHB) to 4HB (trapping agent) in the ipsilateral striatum increased significantly 30 min after ischemia induction. In contrast, during the 30 min of three vessel occlusion there was no increase in the ratio in the cortex. Following the release of the contralateral CCA, the ratio from the ischemic cortex increased significantly compared to sham-operated animals. However, under all circumstances, the 34DHB/4HB ratio was greater in the striatum than in the cortex. The increase in the 34DHB/4HB ratio in the lateral striatum coincides with the increased dopamine release suggesting a role for dopamine oxidation in the increased production of hydroxyl radicals. The significant increase in the ratio from the ischemic cortex compared to that from the sham-operated animals is consistent with increased oxidative stress induced by ischemia. However, the lower 34DHB/4HB ratio in the cortex which does not receive dopaminergic innervation compared to the striatum suggests a different mechanism for hydroxyl radical production. Such an alternate mechanism may represent a more toxic oxidative insult that contributes to infarction.

Neuroprotective effect of 8-OH-DPAT in global cerebral ischemia assessed by stereological cell counting

The neuroprotective effect of the 5-HT 1A receptor agonist 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) was tested in a 2-vessel occlusion model in rats. The post-ischemic core temperature was carefully monitored for 24 h. After 7 days of survival, the viable CA1 neurons were counted in an 8-OH-DPAT (125 μg/kg/h) and vehicle-treated group using the optical fractionator method. The vehicle-treated ischemic rats had a median number of dorsal CA1 neurons of 49,900 whereas the 8-OH-DPAT-treated ischemic rats had a significant lower median number of dorsal CA1 neurons 105,200 ( P=0.018). 8-OH-DPAT significantly lowered the core temperature compared to the vehicle-treated group during the 24-h post-ischemic period. Hypothermia is proposed as a possible explanation of the neuroprotective effect of 8-OH-DPAT.

Effects of hyperosmolarity and ion substitutions on amino acid efflux from the ischemic rat cerebral cortex

The contributions of sodium and chloride ions and of osmotic stresses to the ischemia-evoked efflux of excitotoxic and other amino acids were explored using a rat four vessel occlusion model. Replacement of Na + with choline or N-methyl- d-glucamine (NMDG) and of Cl − with sulfate or gluconate was used to evaluate the contribution that these ions make to amino acid efflux. The contribution of ischemia-evoked swelling to amino acid release was studied by applying mannitol or sucrose to minimize the cell volume increases and the compensatory regulatory volume decrease evoked efflux of amino acids. Aliquots of artificial cerebrospinal fluid (aCSF), appropriately adjusted for ion replacement or 150 mM mannitol or sucrose, were pipetted into cortical cups 35 min prior to ischemia and perfusate samples were obtained prior to, during and following ischemia (20 min) and reperfusion (40 min). Replacement of Na + by NMDG depressed basal (normoxic) efflux of most amino acids, with choline substitution having little effect. During ischemia NMDG substitution increased glutamate and GABA efflux and choline enhanced the release of aspartate, glutamate, GABA and taurine. A reduction in extracellular Na + would facilitate reversal of Na +-dependent transporters with extrusion of amino acids. Another possible explanation for the elevated release is that the absence of Na + would inhibit the Ca 2+/Na + counter transport system, with a deleterious accumulation of intracellular Ca 2+. Chloride replacement with sulfate or gluconate enhanced the efflux of aspartate, glutamate, GABA and taurine during ischemia. Removal of Cl − would depolarize cells, and block the Cl −-dependent action of inhibitory amino acid transmitters, with both actions enhancing the ischemic injury and, consequently, amino acid release. Exposure to hyperosmotic mannitol (150 mM) aCSF enhanced ischemia-evoked release of some amino acids (taurine, GABA) and decreased that of aspartate and phosphoethanolamine. Sucrose aCSF enhanced the ischemia-evoked release of most amino acids. A potential explanation for these observations is that both agents may be able to rapidly penetrate the plasma membranes of ischemic neurons, actually contributing to the release of other osmolytes. The unanticipated nature of many of the observations made during these manipulations of the aCSF serves to accentuate the complex nature of the mechanisms responsible for the ischemia-evoked amino acid efflux into the interstitial spaces.

Laser-Doppler scanning of local cerebral blood flow and reserve capacity and testing of motor and memory functions in a chronic 2-vessel occlusion model in rats.

An animal model of incomplete forebrain ischemia resembling human hemodynamic insufficiency was established. The model allows examination of acute and chronic changes of local cerebral blood flow (lCBF) and reserve capacity in correlation with behavioral parameters. Anesthetized male Wistar-Kyoto rats underwent bilateral carotid occlusion (BCO). Laser-Doppler scanning of lCBF at baseline conditions and after acetazolamide was done 30 minutes after BCO, motor and memory function tests were administered after 1 and 2 days, and both investigations were repeated after 1, 2, 4, and 6 weeks. A sham-operated and a control group without any vessel manipulation served as controls. lCBF dropped within 60 minutes after surgery by 62% (P<0.001) in 10 animals surviving BCO (BCOsurvival) and by 69% in 5 rats that died within 9 days (BCOlethal). Acetazolamide increased lCBF to 142.33% in controls, to 136.66% in sham-operated rats (both significant), and to 104.80% in BCOsurvival (not significant), and it decreased flow by 23.1% in BCOlethal rats (P<0.001). Baseline lCBF normalized within 4 weeks. Total motor function scores were significantly reduced from 9 points preoperatively to 5.80+/-0.65 in BCOlethal and 6.68+/-0.54 points in BCOsurvival rats 1 day after occlusion. Memory retention function remained impaired after BCO, as did the acetazolamide response, which correlated with motor score and was inversely related to maze exploration time. This model allows long-term follow-up of cerebral function, lCBF, and reserve capacity in a pathophysiological setting similar to hemodynamic insufficiency in humans.

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.

Inhibition of tyrosine phosphorylation attenuates amino acid neurotransmitter release from the ischemic/reperfused rat cerebral cortex

Protein tyrosine phosphorylation plays an important role in neuronal function. In this study we have examined the effects of inhibition of tyrosine phosphorylation on the extracellular levels of four neurotransmitter amino acids (aspartate, glutamate, γ-aminobutyric acid (GABA) and glycine) and of the non-transmitter amino acid phosphoethanolamine during cerebral ischemia and reperfusion in a rat four vessel occlusion model. In comparison with the control group, the tyrosine kinase inhibitor genistein significantly depressed ischemia/reperfusion-evoked efflux of these amino acids, with the exception of GABA, into cerebral cortical superfusates. GABA efflux was non-significantly reduced. These results suggest that tyrosine phosphorylation is involved in the ischemia-evoked efflux of amino acids into the extracellular milieu, likely as a consequence of the phosphorylation of microtubule-associated protein kinase (MAP kinase) and downstream activation of PLA 2 in the plasma membrane. Amino acid efflux would occur, in part, as a consequence of the ensuing disruption of plasma membrane integrity and leakage of cytoplasmic constituents along their concentration gradients.

Audiogenic seizures following global ischemia induced by chest compression in Long-Evans rats.

Transient global ischemia was used to produce a rat model of generalized tonic-clonic epilepsy. Controlled chest compression in ketamine-anesthesized Long-Evans rats produced transient global ischemia by mechanically preventing the heart from pumping blood. Circulation was restored by standard cardiopulmonary resuscitation techniques. With a temporal muscle (skull) temperature of 35 +/- 0.4 degrees C, 75% (76/102) of the rats survived 7 min of chest compression. Generalized seizures could be evoked in 78% (59/76) of the surviving rats by a 60 s exposure to a loud sound (bell, 110 dB) beginning 24 h after the ischemic episode. The seizure patterns seen resembled those described by Maresceaux (1987) for genetically seizure-prone Wistar rats. Susceptibility to sound-induced seizures declined with time, with wide variations in recovery rate between individuals; one rat showed a daily sound-induced seizure for over 5 months. Seizures were attenuated or blocked by treatment with carbamazepine or sodium valproate. This model is similar to the great vessel occlusion model used by Kawai et al. (1995), but is less invasive. We believe it will be useful in the evaluation of therapies for acquired generalized (grand mal) seizures.

Neuroprotective efficacy of lifarizine (RS-87476) in a simplified rat survival model of 2 vessel occlusion.

1. A new, modified rat two vessel occlusion model (with hypotension) was established and the neuroprotective efficacy of the novel agent lifarizine (RS-87476) was examined. 2. The two vessel occlusion model used in the study was a modification of the model described in the literature, whereby we have obviated the need to use a muscle relaxant and intubate the trachea to provide ventilatory support by providing a tight fitting face mask attached to the ventilator. Furthermore, the need to combine exsanguination and additional pharmacological means of inducing the mandatory hypotension (50 mmHg), required to decrease brain blood perfusion pressure, has been removed by simply manipulating the concentration of the already present halothane anaesthetic. 3. The appropriate level of hypotension having been reached, microvascular clips were applied to bilaterally occlude the common carotid arteries for 12 min. This resulted in a loss of the cortical EEG activity. Local cerebral blood flow was measured 6 min into the occlusion period, using the fully quantitative [14C]-iodoantipyrine autoradiographic technique, in a separate group of rats (n = 5). This illustrated the lack of any blood flow, in the areas under study, during the period when there was an isoelectric cortical EEG pattern. 4. The high grade global ischaemic lesion which occurred gave quantifiable neuronal damage in several vulnerable regions of the brain, namely, the hippocampal CA1 sub-field, cortex, thalamus, striatum, and cerebellar brain stem (Purkinje cells). 5. Following the global ischaemic insult the rats were allowed to recover for 72 h before assessment of the damage, during which time one group of rats (n = 11) received 100 micrograms kg-1 lifarizine i.a. 5 min post-occlusion, 500 micrograms kg-1 lifarizine i.p. 15 min post-occlusion, and 500 micrograms kg-1 lifarizine i.p. twice daily for 72 h. A second group of rats (n = 12) was treated with appropriate volumes of vehicle (0.4 ml kg-1 i.a. and 2 ml kg-1 i.p.) at identical time points. 6. Histopathological damage was assessed, from cresyl violet and haematoxyline/eosin stained sections, using a scoring system of 0-6 (no damage-complete neuronal death). The dosing regimen of lifarizine gave reduced damage in the hippocampal CA1 sub-field (4.1 +/- 0.3 to 2.8 +/- 0.6) and striatum (1.7 +/- 0.3 to 1.2 +/- 0.3) and significant neuroprotection in the anterior cortex (2.0 +/- 0.2 to 1.2 +/- 0.2; p < 0.05), thalamus (1.5 +/- 0.2 to 0.8 +/- 0.2; p < 0.01), posterior cortex (1.5 +/- 0.2 to 1.0 +/- 0.2; p < 0.05) and cerebellar brain stem (0.9 +/- 0.2 to 0.4 +/- 0.1; p < 0.01). The overall mean brain score was significantly reduced (from 1.5 +/- 0.1 to 0.9 +/- 0.2). 7. These data show that the newly modified 2 vessel occlusion model produced a quantifiable level of ischaemic damage and that the novel agent lifarizine is neuroprotective in the model.