Temporary Focal Ischemia Research Articles

The Low Molecular Weight Heparin Enoxaparin Reduces Infarct Size in a Rat Model of Temporary Focal Ischemia

Low molecular weight heparins (LMWHs) have significantly reduced infarct size in animal studies but they have not been effective in clinical trials, probably because they were administered after ischemic injury had become irreversible. The present study was designed to explore the temporal characteristics of the LMWH enoxaparin with the objective of determining the duration of the treatment window in a rat model of temporary focal ischemia. Focal cerebral ischemia was induced by the intraluminal suture, middle cerebral artery occlusion (MCAO) method. Enoxaparin (10 mg/kg) was administered subcutaneously twice; the first dose was administered to different groups of animals either 1 h before or 1.5, 3, or 5 h after MCAO, and the second 4, 6, 9, or 25 h after the first dose. At 48 h animals were tested for motor coordination and brains were removed for determination of infarct size. Results showed that infarct size and degree of motor impairment were a function of time of the first and the second treatments. If the first treatment was given 1 h prior to MCAO, significant reductions in infarct size were obtained when the second treatment was given up to 6 h after the first (5 h after MCAO), but not when the second dose was given at longer intervals. If the first treatment was given 1.5 h after stroke onset, reduced infarct size was observed only in the group treated for the second time 4 h after the first injection. If the first treatment was given 3 h after MCAO, infarct size was not reduced in any group. However, if enoxaparin was administered intravenously rather than subcutaneously, significant reductions in infarct size were obtained when the first dose was given as long as 5 h after MCAO. These findings indicate that enoxaparin can reduce infarct size in rats when administered prior to stroke onset and suggest that the drug might be considered for prophylactic treatment in a phase 3 clinical trial.

Differential cerebral protein synthesis and heat shock protein 70 expression in the core and penumbra of rat brain after transient focal ischemia.

The purpose of this study was to correlate the cerebral protein synthesis (CPS) reductions in the ischemic core and penumbra with the metabolic stress response indicated by heat shock protein 70 (HSP70) synthesis. Rats were subjected to 90 minutes of temporary focal cerebral ischemia produced by occlusion of the middle cerebral artery, using the endovascular suture model. Regional CPS was qualitatively evaluated, with [(35)S]methionine autoradiography, after reperfusion for 2 to 72 hours. The observed changes were correlated with HSP70 immunoreactivity, as assessed in the same brain sections. The ischemic core in the striatum was characterized by HSP70 expression only in endothelial and/or glial cells, with an absence of expression in neurons. The penumbra was delineated as the cortical middle cerebral artery territory region in which HSP70 was also expressed in metabolically stressed neurons. After 2 hours of reperfusion, CPS was reduced to 30 +/- 16% of the homologous contralateral hemisphere value in the core and to 75 +/- 22% in the penumbra (P < 0.05). This difference was still present at 72 hours, when CPS values were 62 +/- 21% and 98 +/- 29% of the nonischemic contralateral hemisphere values in the core and penumbra, respectively (P < 0.05). Persistent inhibition of CPS in regions in which neuronal HSP70 expression is absent may distinguish core areas of infarction from penumbral regions in which neuronal HSP70 is present, which eventually recover from sublethal metabolic stress during reperfusion after temporary focal ischemia.

Adeno-associated virus-mediated delivery of BCL-w gene improves outcome after transient focal cerebral ischemia.

A recombinant adeno-associated virus (rAAV) vector was used to overexpress the anti-apoptotic Bcl-2-family protein, BCL-w, in rat brain. Three weeks after injecting the vector into cerebral cortex and striatum on one side, temporary focal ischemia was induced by occlusion of the ipsilateral middle cerebral artery for 90 min, followed by reperfusion for 24 h. BCL-w expression was increased in cerebral cortex and striatum--and in neurons, astroglia and endothelial cells--in the brains of rats that received the rAAV-BCL-w vector, compared to rats given phosphate-buffered saline or a control vector containing the gene for green fluorescent protein. Recipients of the rAAV-BCL-w vector also showed a 30% reduction in infarct size and a 33-40% improvement in neurological function, compared to the control groups. These results provide evidence for a role of BCL-w in regulating histological and functional outcome after focal cerebral ischemia.

Reduced Susceptibility of Magnocellular Neuroendocrine Nuclei of the Rat Hypothalamus to Transient Focal Ischemia Produced by Middle Cerebral Artery Occlusion

Intraparenchymal injections of glutamate analogues into the diencephalon near the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus selectively spare magnocellular neuroendocrine cells. In this study we investigated for the first time the susceptibility of this neuronal population to ischemia. Temporary focal ischemia was produced using a three-vessel occlusion method involving unilateral middle cerebral artery and bilateral common carotid artery occlusion (MCAO/CCAO). Most of the 3-h ischemic period was maintained without anesthesia and reversed by microclip removal of the contralateral common carotid artery occlusion. In one subset of rats transcardial perfusion with India ink was used to estimate the degree of ischemia produced during MCAO/CCAO in the SON, lateral magnocellular nucleus of the PVN (PVL), caudoputamen (CP), and frontoparietal cortex (COR). Computer-assisted densitometry measurements of ink density indicated significant reductions in ink penetration in the territory of the occluded MCA within the SON (46%), PVL (45%), CP (53%), and COR (76%). In contrast, neither sham-operated rats nor rats subjected to occlusion of the MCA alone showed differences in ink optical densities between the sides ipsilateral and contralateral to MCAO. The other subset of rats were perfused 48–72 h after recovery and brain sections were examined for neurodegenerative changes. While the incidences of cerebral and caudoputamen infarction after MCAO/CCAO were 98.4 and 52%, respectively, the histological features of the SON or PVL in ischemic rats were similar to those of control rats. Reduced susceptibility of magnocellular neuroendocrine cells to ischemia may be due to a number of mechanisms including neuronal resilience, neuroprotection by glia and vascular/perivascular cells, and access to perivascular cerebrospinal fluid.

Effects of LF 16-0687 Ms, a bradykinin B 2 receptor antagonist, on brain edema formation and tissue damage in a rat model of temporary focal cerebral ischemia

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein–kinin system, promotes neuronal tissue damage as well as disturbances in blood–brain barrier function through activation of B 2 receptors. LF 16-0687 Ms, a non-peptide competitive bradykinin B 2 receptor antagonist, was recently found to decrease brain swelling in various models of traumatic brain injury. We have investigated the influence of LF 16-0687 Ms on the edema formation, neurological outcome, and infarct size in temporary focal cerebral ischemia in rats. Sprague–Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Local CBF was bilaterally recorded by laser Doppler flowmetry. Study I: animals were assigned to one of three treatment arms ( n=11 each): (a) vehicle, (b) LF 16-0687 Ms (12.0 mg/kg per day), or (c) LF 16-0687 Ms (36.0 mg/kg per day) given repetitively s.c. over 3 days. The neurological recovery was examined daily. The infarct volume was assessed histologically 7 days after ischemia. Study II: brain swelling and bilateral hemispheric water content were determined at 48 h post ischemia in eight rats, subjected to the low dose regimen as described above, and in eight vehicle-treated control animals. All treated animals showed tendency to exhibit improved neurological recovery throughout the observation period as compared to the vehicle-treated controls, while this improvement was only significant within the low dose group from postischemic days 3 to 4. Low dose LF 16-0687 Ms significantly attenuated the total and cortical infarct volume by 50 and 80%, respectively. Furthermore, postischemic swelling (−62%) and increase in water content of the infarcted brain hemisphere (−60.5%) was significantly inhibited. The present findings provide strong evidence for an involvement of bradykinin-mediated secondary brain damage following from focal cerebral ischemia. Accordingly, specific inhibition of bradykinin B 2 receptors by LF 16-0687 Ms attenuated postischemic brain swelling, improved the functional neurological recovery, and limited ischemic tissue damage, raising its potential for clinical evaluation in patients with acute stroke.

Correlation between changes in apparent diffusion coefficient and induction of heat shock protein, cell-specific injury marker expression, and protein synthesis reduction on diffusion-weighted magnetic resonance images after temporary focal cerebral ischemia in rats.

The authors investigated the relationship between the time course of apparent diffusion coefficient (ADC) changes and stress protein induction, ischemic neuroglial damage, and cerebral protein synthesis (CPS) after temporary focal cerebral ischemia in rats. In Group I, ADC changes were measured on magnetic resonance (MR) images obtained during the second half of a 1-hour middle cerebral artery (MCA) occlusion, during a 1-hour reperfusion, and after 23 hours of reperfusion in rats. Immunohistochemical studies for heat shock protein (hsp) 70, glial fibrillary acidic protein (GFAP), and neuronal nuclear (NeuN) protein were performed. In Group II, CPS was assessed using autoradiographic studies obtained after occlusion. At 36 minutes of occlusion, MR imaging demonstrated significantly less ADC reduction in the frontoparietal cortex (82 +/- 9% of the contralateral hemisphere) than in the striatum (64 +/- 11%; p < 0.05). After 1 hour of reperfusion, the lesion resolved and the difference between cortex and striatum was no longer evident. After 23 hours of reperfusion, the ADC lesion recurred in striatum (76 +/- 12%) compared with frontoparietal cortex (100 +/- 11%; p < 0.05). Immunohistochemical studies showed hsp 70 expression and an increased GFAP reactivity localized in the frontoparietal cortex of the ischemic hemisphere, along with a significant drop in striatal NeuN immunoreactivity. A trend toward greater reduction in striatal CPS (53 +/- 15%) than in frontoparietal cortex CPS (78 +/- 23%) was also observed. Sequential ADC maps correlate with the expression of neuroglial stress and injury markers after temporary focal ischemia in rats, distinguishing the striatum (infarct core) from the cortex (ischemic penumbra). A greater reduction in striatal CPS further supports the conclusion that the striatum is more susceptible to temporary MCA occlusion than the cortex.

High-Dose Methylprednisolone Treatment in Experimental Focal Cerebral Ischemia

Previous studies using steroids for experimental focal stroke have demonstrated conflicting results, possibly related to dose used or ischemic models employed. In this study we examined high-dose methylprednisolone treatment following permanent and temporary focal cerebral ischemia in the rat. Focal stroke was induced in spontaneously hypertensive rats by permanent right common carotid and either permanent or 3 h of temporary middle cerebral artery (MCA) occlusion. Methylprednisolone (105 mg/kg) was administered intra-arterially. Infarct volume was measured at 24 h after permanent and temporary MCA occlusion. Cerebral edema was determined by measuring right and left hemispheric volumes and water content 24 h after permanent MCA occlusion in one experiment. Methylprednisolone, whether administered in divided doses over 12 h (n = 15 in each group) or a single bolus (n = 9 per group), had no effect on infarct volume after permanent MCA occlusion. Methylprednisolone treatment also had no influence on cerebral edema (n = 9 per group). In two different experiments, methylprednisolone given in divided doses over 12 h (n = 11, n = 25) after temporary MCA occlusion decreased infarct volume (P < 0.05) by 20% compared with saline controls (n = 10, n = 25). High dose methylprednisolone decreased infarct volume following temporary, but not permanent, focal ischemia. The benefit suggests that high dose methylprednisolone may prove useful clinically if reperfusion can be established with thrombolytic agents. Furthermore, the differential treatment effect in the setting of comparable ischemic insults implies that different modifiable biochemical processes may be present during temporary but not permanent focal ischemia, thus providing indirect evidence for reperfusion injury.

Estrogen decreases infarct size after temporary focal ischemia in a genetic model of type 1 diabetes mellitus.

It is unclear how genetic type 1 diabetes mellitus (DM) influences infarct size when blood glucose is tightly controlled. The aim of this study was to determine the effect of genetic type 1 DM, as occurs in BB rats, on infarct size after transient unilateral middle cerebral artery occlusion (MCAO) in male and female rats. In addition, studies suggest that male type 1 DM rats have a higher incidence of end-organ complications than do females. A second aim of this study was to determine the effect of chronic 17beta-estradiol (E(2)) administration on infarct size in male BB rats. Diabetic male (MDiab, n=14) and female (FDiab, n=8) BB rats were studied and compared with background strain Wistar rats (MWist, n=16; FWist, n=14). Two additional male cohorts (MWist+E(2), n=15; MDiab+E(2), n=14) received subcutaneous 25 microg E(2) implants 7 to 10 days before MCAO. Rats underwent 1 hour of MCAO followed by 22 hours of reperfusion. Physiological variables were controlled among groups, and the intraischemic laser Doppler flow signal was reduced similarly in all animals. Infarction volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining and image analysis. Preischemic blood glucose was 94+/-5, 127+/-13, 90+/-15, 63+/-18, 122+/-8, and 81+/-14 mg/dL in MWist, FWist, MDiab, FDiab, MWist+E(2), and MDiab+E(2) rats, respectively (mean+/-SE). Intraischemic laser Doppler flow was reduced to 20% to 25% of baseline in all groups. Striatal infarct size (percentage of ipsilateral caudate putamen) was increased in male diabetic rats relative to nondiabetic MWist rats (41+/-3% versus 28+/-3%). Striatal injury was not increased in FDiab rats, and infarction volume was smaller than that in FWist rats (23+/-4% in FWist versus 13+/-3% in FDiab). Chronic estrogen treatment reduced cortical and striatal infarction in MDiab+E(2) rats compared with untreated MDiab rats. Type 1 DM is associated with increased infarct size after temporary MCAO, despite tight control of blood glucose. The deleterious effect of DM is evident only in males rats; female diabetic BB rats sustain small infarcts. Chronic E(2) treatment reduced injury in the male BB rat, providing neuroprotection even in the presence of DM. These data suggest that genetic diabetes even with mild glucose elevation plays a role in determining neuropathology in experimental stroke. However, factors such as reproductive steroids also determine outcome in DM stroke.

The N-methyl-D-aspartate antagonist CNS 1102 protects cerebral gray and white matter from ischemic injury following temporary focal ischemia in rats.

Cerebral white matter is as sensitive as gray matter to ischemic injury and is probably amenable to pharmacological intervention. In this study we investigated whether an N-methyl-D-aspartate (NMDA) antagonist, CNS 1102, protects not only cerebral gray matter but also white matter from ischemic injury. Ten rats underwent 15 minutes of temporary focal ischemia and were blindly assigned to CNS 1102 intravenous bolus injection (1. 13 mg/kg) followed by intravenous infusion (0.33 mg/kg per hour) for 3.75 hours or to vehicle (n=5 per group) immediately after reperfusion. Seventy-two hours after ischemia, the animals were perfusion fixed for histology. The severity of neuronal necrosis in the cortex and striatum was semiquantitatively analyzed. The Luxol fast blue-periodic acid Schiff stain and Bielschowsky's silver stain were used to measure optical densities (ODs) of myelin and axons, respectively, in the internal capsule of both hemispheres, and the OD ratio was calculated to reflect the severity of white matter damage. Neuronal damage in both the cortex and the striatum was significantly better in the drug-treated group than in the placebo group (P<0.05). The OD ratio of both the axons (0.93+/-0.08 versus 0.61+/-0.18; P<0.01) and the myelin sheath (0.95+/-0.07 versus 0.67+/-0.19; P=0.01) was significantly higher in the CNS 1102 group than in the placebo group. The neurological score was significantly improved in the drug-treated group (P<0.05). The NMDA receptor antagonist CNS 1102 protects not only cerebral gray matter but also white matter from ischemic injury, most probably by preventing degeneration of white matter structures such as myelin and axons.

Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor

Neuronal expression of brain-derived neurotrophic factor (BDNF) has been implicated in the mechanism of infarct tolerance (resistance to stroke) (H. Yanamoto et al., Infarct tolerance accompanied enhanced BDNF-like immunoreactivity in neuronal nuclei, submitted to Brain Res.), a process that takes more than 7 days following a preconditioning of repetitive cortical spreading depression (CSD). To investigate whether an elevated level of BDNF protein in the brain solely protects neurons against temporary focal ischemia, recombinant (r)BDNF was infused into the rat neocortex. Recombinant BDNF (or vehicle: saline) was administered into the left neocortex via an implanted osmotic minipump for 2.5, 7, 10 or 14 days pre-ischemia, during ischemia and for 2 days post-ischemia (8 μg in total) in male Sprague–Dawley rats ( n=6 each). Temporary focal ischemia was induced in the left middle cerebral artery (MCA) territory by three-vessel occlusion of bilateral common carotid arteries (CCAs) and MCA for 2 h, and the cerebral infarct volume was analyzed 2 days after ischemia using TTC staining. Regional cerebral blood flow (rCBF) of the left neocortex was monitored after 14 days of intracerebral administration of BDNF or vehicle ( n=10 each). The distribution of BDNF following different periods of rBDNF or vehicle-infusion was analyzed using immunohistochemical techniques ( n=5 each). In the groups treated with 8 μg of rhBDNF for 7, 10, or 14 days pre-ischemia, there were significant reductions of neocortical infarct volume compared to in the control or vehicle-treated groups ( p<0.05). In the rCBF study, there was no significant change after the infusion of 8 μg rhBDNF for 14 days. In the histological study, a wide distribution of BDNF-like immunoreactivity in the neuronal nuclei in the ipsilateral neocortex was demonstrated after the infusion of 8 μg rhBDNF for 14 days. The BDNF-like immunoreactivity in the neuronal nuclei was enhanced at the time that the resistance to stroke was achieved by direct intra-cerebral infusion of exogenous rBDNF. Elucidating the function of the BDNF-like protein located in the neuronal nuclei should reveal a new strategy for neuroprotection against ischemic brain attack in humans.

Society of Neurosurgical Anesthesia and Critical Care: Annual Meeting Report.

Society of Neurosurgical Anesthesia and Critical Care: Annual Meeting Report.

Exogenous spermine reduces ischemic damage in a model of focal cerebral ischemia in the rat.

Alterations in polyamine metabolism during and after global or focal cerebral ischemia can produce a multiplicity of effects on brain such as modification in mitochondria calcium buffering capacity, exacerbating glutamate-mediated neurotoxicity, and impairment of the blood-brain barrier. In this study, the endogenous polyamine spermine was administered intravenously 30 min prior to temporary focal cerebral ischemia in rats induced by clipping of the left middle cerebral and bilateral common carotid arteries for 3 h. Three days after removal of the microclips, intracardiac perfusion with 2% 2,3,5-triphenyl tetrazolium chloride was performed. Coronal slices were cut, photographed, and examined for cortical infarct volume. Spermine reduced infarct volume in a dose-dependent fashion. This study demonstrates that the use of polyamines may be considered as a powerful tool in prevention of ischemic tissue damage following focal cerebral ischemia.

Temporal evolution of ischemic injury evaluated with diffusion-, perfusion-, and T2-weighted MRI.

Ischemic lesions seen on diffusion-weighted imaging (DWI) are reversible if reperfusion is performed within minutes after the onset of ischemia. This study was designed to determine whether acute reversibility of DWI abnormalities is transient following brief temporary focal brain ischemia and to characterize the temporal evolution of in vivo ischemic lesions. Eight rats were subjected to 30 minutes of temporary middle cerebral artery occlusion and underwent diffusion-, perfusion-, and T2-weighted MRI during occlusion; immediately after reperfusion; 30, 60, and 90 minutes after reperfusion; and 12, 24, 48, and 72 hours after reperfusion. Average apparent diffusion coefficient (ADCav) values and the cerebral blood flow index (CBFi) ratio were calculated in both the lateral caudoputamen and overlying cortex at each time point. The size of the in vivo ischemic abnormalities was calculated from the ADCav and the T2 maps. Postmortem triphenyltetrazolium chloride (TTC) staining was used to verify ischemic injury. Both the CBFi ratio and ADCav values declined significantly in the two regions during occlusion. The CBFi ratio recovered immediately after reperfusion and remained unchanged over 72 hours. However, ADCav values returned to normal at 60 to 90 minutes and secondarily decreased at 12 hours after reperfusion as compared with those in the contralateral hemisphere. The extent of the in vivo ischemic lesions maximized at 48 hours and was highly correlated with TTC-derived lesion size. Acute recovery of initial ADCav-defined lesions after reperfusion is transient, and secondary ADCav-defined lesions develop in a slow and delayed fashion.

Hyperbaric oxygen decreases infarct size and behavioral deficit after transient focal cerebral ischemia in rats

Cerebral hypoxia is a major component of immediate and secondary cell damage caused by ischemia. Hyperbaric oxygen (HBO) is a potent means to increase the amount of oxygen dissolved in blood plasma. The effectiveness of HBO in clinical and experimental cerebral ischemia, however, is controversial. We sought to determine whether treatment with HBO initiated early after focal cerebral ischemia-onset protects the brain when experimental conditions such as brain temperature are controlled. Male Wistar rats ( n=57) underwent reversible filament occlusion of the right middle cerebral artery (MCA) for 75 min. Animals were awakened after filament introduction and assessed for presence of forelimb paresis. Rats then underwent a 60-min course of either 100% O 2 at 1.0 atmosphere absolute (ata; control group), HBO 1.5 ata, or HBO 2.5 ata in a customized HBO chamber allowing physiological monitoring and pericranial temperature control. The filament was then removed. Seven days after ischemia, rat behavior was scored from 3–18 (18=normal) and brains were removed for histological analysis of infarct volume. Rats treated with HBO 2.5 ata had better mean±standard deviation (S.D.) behavioral scores (14±2; p<0.05) than control (10±3) or HBO 1.5-ata-treated animals (11±3). Similarly, total infarct volumes (mean±S.D.) were smaller in animals receiving HBO at 2.5 ata (76±65 mm 3; p<0.05) compared to control (129±83 mm 3) and HBO 1.5-ata (119±68 mm 3)-treated groups. Cortical infarction occurred less frequently in HBO 2.5-ata-treated than in control animals (44% vs. 71%; p<0.05). We conclude that HBO can improve outcome after temporary focal ischemia when treatment is started early after ischemia-onset but HBO dose appears important. Potential mechanisms include enhanced oxygen supply to marginally perfused cells.

Increased expression of apoptosis-linked gene 2 (ALG2) in the rat brain after temporary focal cerebral ischemia

Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calcium-binding proteins have been implicated in calcium-regulated signal transduction. Apoptosis-linked gene 2 is a calcium-binding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosis-linked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosis-linked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosis-linked gene 2 protein was also up-regulated in the ischemic border-zone of parietal cortex 24 h after 20 min of focal ischemia, and was remarkably over-expressed in the caudate–putamen and parietal cortex, (where cells are destined to die) 24 h after 90 min of ischemia. The expression pattern of apoptosis-linked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay. Our results suggest that apoptosis-linked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia.

Combination of intraischemic and postischemic hypothermia provides potent and persistent neuroprotection against temporary focal ischemia in rats.

It is not known whether a combination of intraischemic and postischemic mild hypothermia provides extra neuroprotection and if so, whether the neuroprotection is persistent. Sixty-eight Sprague-Dawley rats were used. In group 1, ischemia and reperfusion were performed under normothermic (N) conditions (control, N-N). In group 2, ischemia was induced and maintained under hypothermic conditions (33 degrees C for 2 hours) and reperfusion was performed under normothermic conditions, H-N. In group 3, both ischemia and reperfusion were performed under hypothermic conditions for an additional 21 hours after the surgery, H-22H. In group 4, ischemia was induced and maintained under hypothermic conditions and reperfusion was performed under hypothermic conditions only for the initial 3 hours (H-3H). In group 5, ischemia was induced and maintained under normothermic conditions and reperfusion was performed under hypothermic conditions (33 degrees C) (N-22H). All rats were perfused 48 hours after the induction of ischemia. In addition, the normothermic or hypothermic therapy used for groups 1, 3, and 4 was performed again, and these rats were killed 30 days after the induction of ischemia. Furthermore, neurological deficits were monitored in groups N-N and H-22H for 4 weeks. In the H-3H and H-22H groups, the total infarct volume was significantly reduced by 41% or 66%, respectively, assessed 48 hours after ischemia. The significant reduction in group H-22H was again confirmed 30 days after ischemia, ie, 50% reduction was observed. In contrast, the reduction in group H-3H (31%) was not significant. The neurological deficits were significantly more severe in the N-N group than in the H-22H group during week 4. The neuroprotective effects against temporary focal ischemia evaluated by infarct volume and neurological functions by the combination therapy with intraischemic and prolonged postischemic mild hypothermia were persistent in rats. Appropriate design of mild hypothermia therapy extending into the late reperfusion period is important to maximize the neuroprotective effects of hypothermia.

Biphasic opening of the blood-brain barrier following transient focal ischemia: effects of hypothermia.

Tracer constants (Ki) for blood-to-brain diffusion of sucrose were measured in the rat to profile the time course of blood-brain barrier injury after temporary focal ischemia, and to determine the influence of post-ischemic hypothermia. Spontaneously hypertensive rats were subjected to transient (2 hours) clip occlusion of the right middle cerebral artery. Reperfusion times ranged from 1.5 min to 46 hours, and i.v. 3H-sucrose was circulated for 30 min prior to each time point (1 h, 4 h, 22 h, and 46 h; n = 5-7 per time point). Ki was calculated from the ratio of parenchymal tracer uptake and the time-integrated plasma concentration. Additional groups of rats (n = 7-8) were maintained either normothermic (37.5 degrees C) or hypothermic (32.5 degrees C or 28.5 degrees C) for the first 6 hours of reperfusion, and Ki was measured at 46 hours. Rats injected after 1.5-2 min exhibited a 10-fold increase in Ki for cortical regions supplied by the right middle cerebral artery (p < 0.01). This barrier opening had closed within 1 to 4 hours post-reperfusion. By 22 hours, the blood-brain barrier had re-opened, with further opening 22 and 46 hours (p < 0.01), resulting in edema. Whole body hypothermia (28 degrees C-29 degrees C) during the first six hours of reperfusion prevented opening, reducing Ki by over 50% (p < 0.05). Transient middle cerebral artery occlusion evokes a marked biphasic opening of the cortical blood-brain barrier, the second phase of which causes vasogenic edema. Hypothermic treatment reduced infarct volume and the late opening of the blood-brain barrier. This opening of the blood-brain barrier may enhance delivery of low permeability neuroprotective agents.

Reproducibility of cerebral cortical infarction in the wistar rat after middle cerebral artery occlusion

Although middle cerebral artery (MCA) occlusion in the rat is often used to study focal cerebral ischemia, the model of ischemia affects the size and reproducibility of infarction. The purpose of this experiment was to methodically examine different preparations to determine the optimum focal cerebral ischemia model to produce a reproducible severe ischemic injury. Eighty-two Wistar rats underwent either 1 hour, 3 hour, or permanent MCA occlusion combined with no, unilateral, or bilateral common carotid artery artery (CCA) occlusion. Three days after ischemia, the animals were prepared for tetrazolium chloride assessment of infarction size. One-hour MCA occlusion produced a coefficient of variation (CV) of 200% with an infarction volume of 20.3+/-10.5 mm(3). Adding unilateral or bilateral CCA occlusion resulted in a CV of 134% and 101%, respectively. Three-hour MCA occlusion combined with bilateral CCA occlusion decreased the CV to 58% with a cortical infarction volume of 82.6+/-12.1 mm(3), P<05, compared with 1-hour MCA occlusion with or without CCA occlusion. Permanent MCA occlusion combined with 3 hours of bilateral CCA occlusion resulted in a CV of 47% with a cortical infarction volume of 89.6+/-16.0 mm(3). These results indicate that 3-hour MCA occlusion combined with bilateral CCA occlusion provide consistently a large infarction volume after temporary focal cerebral ischemia.

808 Increased Infarct Size Occurs Following Temporary Focal Ischemia in a Genetic Model of Type 1 Diabetes Mellitus (DM)

Toung, Thomas K. M.D.; Sieber, Frederick E. M.D.; Hurn, Patricia D. Ph.D.; Traystman, Richard J. Ph.D. Author Information

Reversal of acute apparent diffusion coefficient abnormalities and delayed neuronal death following transient focal cerebral ischemia in rats.

Twenty-two rats were subjected to 8, 15, 30, or 60 minutes of temporary middle cerebral artery occlusion (n = 5 per group) or sham occlusion (n = 2) in the magnetic resonance imaging unit. Diffusion-, perfusion-, and T2-weighted imaging were acquired before and during occlusion, and after reperfusion. A coregistration method was used to correlate the acute changes of the average apparent diffusion coefficient (ADCav) with the histology after 72 hours at the same topographic sites. The initially reduced ADCav values recovered completely in both the lateral caudoputamen and upper frontoparietal cortex in the 8-, 15-, and 30-minute groups, partially in the cortex, and not at all in the caudoputamen in the 60-minute group. The histology showed that the caudoputamen was either normal or had mild neuronal injury in the 8-minute group and invariably had some degree of neuronal death in the 15-, 30-, and 60-minute groups, whereas the cortex was either normal or had varying degrees of neuronal injury in all groups. No histological abnormalities were seen in the sham-operated rats. Our data suggest that acute ADCav reversal does not always predict tissue recovery from ischemic injury and that temporary focal ischemia for even 8-minute duration can cause delayed neuronal death that is more severe in the caudoputamen where the initial ADCav decline was greater than in the cortex.