Incomplete infarct and delayed neuronal death after transient middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: The clinical syndrome of transient ischemic attacks is accompanied in a significant percentage of patients by brain lesions or neuroimaging abnormalities whose structural counterparts have not been defined. The objective of this study was to analyze, in an experimental model of short-term (< 25 minutes) focal ischemia and long-term (< or = 28 days) reperfusion, the extent and nature of the structural abnormalities affecting neurons and glia located within the territory of the transiently occluded artery. METHODS: Adult Wistar rats (n = 121) had the origin of one middle cerebral artery (MCA) occluded with a nylon monofilament for periods of 10 to 25 minutes. Experiments of transient MCA occlusion were terminated at variable periods ranging from 1 day to 4 weeks. Control experiments consisted of (1) MCA occlusion without reperfusion (n = 7) lasting 7 to 14 days and (2) sham operations (n = 2) followed by 1- to 4-day survival. After in situ fixation, brain specimens were serially sectioned and subjected to detailed morphometric evaluations utilizing light and electron microscopes. The statistical method used to evaluate the results was based on ANOVA followed by Bonferroni's corrected t test and Student's t test comparisons. RESULTS: Brain lesions were not detectable in the sham-operated controls. All brains with permanent MCA occlusion (7 to 14 days) had large infarctions with abundant macrophage infiltration and early cavitation. Forty-five (37%) of the experiments involving transient MCA occlusion had no detectable brain lesions after 4 weeks. Selective neuronal necrosis was found in 76 of 121 rats (63%) with transient MCA occlusion. Neuronal necrosis always involved the striatum, and in 29% of the brains with ischemic injury, necrosis also included a short segment of the cortex. In the striatum, the length of the arterial occlusion was the main determinant of the number of necrotic neurons (20 minutes [22.6 +/- 19] is worse than 10 minutes [4.9 +/- 7]) (P < .0001). In the cortex, the length of reperfusion determined the number of necrotic neurons appearing in layer 3. Experiments with reperfusion of 4 to 7 days' duration yielded more necrotic neurons per microscopic field (2.02 +/- 3) than those lasting fewer days (0.04 +/- 0.1) (P < .05). The histological features of these lesions underwent continuous change until the end of the fourth week, at which time necrotic neurons were still visible both in the striatum and in the cortex. CONCLUSIONS: Arterial occlusions of short duration (< 25 minutes) produced, in 76 of 121 experiments (63%), brain lesions characterized by selective neuronal necrosis and various glial responses (or incomplete infarction). This lesion is entirely different from the pannecrosis/cavitation typical of an infarction that appears 3 to 4 days after a prolonged arterial occlusion. Delayed neuronal necrosis, secondary to a transient arterial occlusion or increasing numbers of necrotic neurons in experiments with variable periods of reperfusion, was a response observed only at a predictable segment of the frontoparietal cortex.     

Collateral hemodynamics after middle cerebral artery occlusion in Wistar and Fischer-344 rats

We investigated whether the difference in infarction volume after occlusion of a long proximal segment of the middle cerebral artery between Wistar and Fischer-344 rats, is caused by differences in collateral blood flow rate through leptomeningeal anastomoses. In view of the retrograde direction of collateral blood flow into the middle cerebral artery territory, we developed parasagittal laser-Doppler flowmetry. Using this method two laser-Doppler probes are placed on the cerebral cortex: probe 1 is placed near the anastomoses between the middle- and anterior cerebral artery, probe 2 is placed 2 mm further away from these anastomoses than probe 1. We found in both rat strains a comparable relation between the areas under the curve of the signal measured by both laser-Doppler probes for 2 h after middle cerebral artery occlusion. This relation is considered to be a measurement of the collateral blood flow rate into the middle cerebral artery territory through leptomeningeal anastomoses after middle cerebral artery occlusion. We conclude that collateral blood flow for the two strains were essentially similar for the initial 2 h after MCA occlusion. Although these collateral blood flows could have been different at a later time, it is unlikely that the interstrain difference in cerebral infarction volume between Wistar and Fischer-344 rats after proximal middle cerebral artery occlusion is caused by an apparent interstrain difference in the magnitude of collateral blood flow rate through leptomeningeal anastomoses. The parasagittal laser-Doppler flowmetry technique we developed for these experiments is currently successfully used in our laboratory to evaluate the efficacy of hemodynamically active pharmacotherapeutical agents in raising the collateral blood flow rate into the middle cerebral artery territory after middle cerebral artery occlusion.     

Long-term functional outcome following transient middle cerebral artery occlusion in the rat: Correlation between brain damage and behavioral impairment.

The assessment of both histological and functional long-term outcomes after cerebral ischemia is increasingly recommended for preclinical studies. Whereas correlations between behavioral impairments and primary ischemic lesion are documented, little is known about their relationships with remote nonischemic regions that undergo secondary degeneration, such as the thalamus. Anesthetized rats were subjected to mild (30 min) or severe (60 min) occlusion of the middle cerebral artery. Two months after ischemia, sensorimotor behavior was assessed according to the neurological score, limb-placing, adhesive-removal, and staircase tests; the final histological lesion was measured after this assessment. Cortical damage was correlated to all transient and long-lasting sensorimotor deficits, whereas striatal lesion was more consistently reflected by the forelimb-placing reflexes and adhesive-removal motor deficits. By contrast, the thalamic atrophy was not correlated to early neurological impairment, but rather to the late sensory deficit at the adhesive-removal test and to the skilled forepaw reaching alteration at the staircase test. This suggests that thalamus contributes, albeit moderately, to the ischemia-induced long-lasting sensorimotor deficits, some of which represent relevant targets for therapeutic interventions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Assessment of sensorimotor neglect after occlusion of the middle cerebral artery in the rat

Evaluating the efficacy of neuroprotective drugs in rat models of focal cerebral ischemia has involved histological and behavioral batteries to examine treatment outcome. However, the behavioral tests used to date provide little insight into the nature of the neurological impairments. To provide an analysis of a possible "neglect" syndrome after occlusion of the middle cerebral artery, M. I. Posner's (1980) visual attentional paradigm was adapted for use in the rat. A paw-reaching task and a test of somatosensory "neglect" also were used to assess forelimb sensorimotor function. The lesion group displayed unilateral deficits; however, there was no evidence of attentional dysfunction. Results are consistent with the conclusion that the behavioral deficits identified arise from a somatosensory deficit rather than hemineglect due to dysfunctional spatial attention.     

Monofilament intraluminal middle cerebral artery occlusion in the mouse

The rat middle cerebral artery (MCA) occlusion model with an intraluminal filament is well characterized with a two hour period of occlusion in widespread use. The recent availability of transgenic animals has led to an interest in adapting the MCA model in the mouse. To date the model has not been well characterized in the mouse. We performed the present study to compare different durations of MCA occlusion and to validate new functional assessments in this model. The MCA occlusion model (5-0 filament) was used. Swiss-Webster mice, 24-44 g, were randomly assigned to four groups: one hour of occlusion; two hours of occlusion; three hours of occlusion; or permanent occlusion. At 48 hours post-ischemia, the animals were rated on three neurologic function scales, and then the brains were removed for lesion size determination. Overall, there was a significant difference in lesion volume (p < 0.001) between the groups. In the permanent group of mice, the average lesion volume was 78.41 +/- 17.47 mm (n = 12); two and three hours of ischemia produced 51.29 +/- 29.82 mm3 (n = 11) and 54.85 mm3 (n = 13), respectively, significantly different than the one hour group 14.84 +/- 31.34 mm3 (n = 11). All three functional scoring systems found significant overall differences between the four groups with our detailed General and Focal scores producing more robust between group treatment differences and showing correlation coefficients of r = 0.766 and r = 0.788, respectively to infarct volume. The MCA filament occlusion model can be successfully adapted in the mouse with either two or three hour occlusions producing reliable infarcts. New functional scoring systems unique to the mouse appear to add additional information.     

A new method to improve in-bore middle cerebral artery occlusion in rats: demonstration with diffusion- and perfusion-weighted imaging

BACKGROUND AND PURPOSE: In-bore middle cerebral artery occlusion (MCAO) enables investigators to acquire preischemic MRI data and to image ischemic changes immediately after occlusion. We have developed a highly successful in-bore MCAO method. This study describes the methods and pertinent techniques. METHODS: Sixty-seven Sprague-Dawley rats were subjected to temporary (n=36) or permanent (n=31) MCAO. The occluding device consisted of a supporting tubing, a driving line, and a silicone-coated 4-0 nylon suture occluder. Outside the magnet, the occluder was positioned in the carotid canal. MCAO was achieved in the magnet bore by remotely advancing the driving line until resistance was felt. Diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI) were acquired before and immediately after occlusion and were used to document the presence of MCAO. RESULTS: Fifty-nine (88.1%) rats were successfully occluded, demonstrating hyperintensity on DWI, perfusion deficits on PWI, and no subarachnoid hemorrhage at postmortem examination. The average values of the apparent diffusion coefficient in both the frontoparietal cortex and the lateral caudoputamen significantly decreased as early as 3 minutes after the onset of ischemia. The failures included preocclusion damage (1/67), sliding out of the occluder during occlusion (1/67), no occlusion (2/67), and arterial perforation (4/67). CONCLUSIONS: Our in-bore MCAO method is easily performed and is as successful as MCAO induced outside the magnet.     

Delayed treatment with YM90K, an AMPA receptor antagonist, protects against ischaemic damage after middle cerebral artery occlusion in rats

The neuroprotective effect of an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride] has been examined in a rat middle cerebral artery occlusion model. Intravenous infusion of YM90K (2.5-20mgkg(-l)h(-l) for 4h) starting immediately after occlusion of the middle cerebral artery significantly reduced the cortical infarct volume 24h after occlusion compared with the control group. The protection at the highest dose was 39% (P < 0.05). Similar protective effects were observed when YM90K (20mgkg(-1)h(-1) for 4h) was delayed up to 2h after middle cerebral artery occlusion (45% reduction, P < 0.05). CNS1102 [N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine hydrochloride], a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, also reduced the cortical infarct volume when 1.13mgkg(-1) was administered by intravenous bolus injection immediately after middle cerebral artery occlusion, followed by intravenous infusion at 0.785mgkg(-l)h(-1) for 4h (35% reduction, P<0.05). This neuroprotective effect was not observed when administration was delayed lh after middle cerebral artery occlusion. These results suggest that AMPA receptors might play a more important role than NMDA receptors in the late development of neuronal cell damage after focal cerebral ischaemia and that AMPA receptor blockade would be one beneficial strategy in treating acute stroke.     

Photothrombotic middle cerebral artery occlusion in spontaneously hypertensive rats: influence of substrain, gender, and distal middle cerebral artery patterns on infarct size

BACKGROUND AND PURPOSE: To analyze the effects of substrain and gender differences in spontaneously hypertensive rats (SHR) and distal middle cerebral artery (MCA) branching patterns on infarct size, we compared infarct volumes produced by photothrombotic distal MCA occlusion using SHR/Kyushu and SHR/Izumo (Izm). METHODS: Twenty-four male and 8 female SHR/Kyushu, 15 male and 5 female SHR/Izm, and 6 male Wistar-Kyoto rats (WKY)/Izm (5 to 7 months old) were subjected to photothrombotic distal MCA occlusion, and infarct volumes were determined. RESULTS: Although blood pressure levels were essentially the same between the two substrains of hypertensive rats, infarct volumes were significantly larger in the SHR/Kyushu substrain than in SHR/Izm of either sex (P<0.001); infarct volumes in male and female SHR/Kyushu were 83.8+/-11.7 and 58.5+/-9.2 mm3, and those in male and female SHR/Izm were 61.5+/-10.7 and 34.8+/-7.9 mm3, respectively (values are mean+/-SD). Male SHR/Kyushu that had simple Y-shaped MCA showed smaller infarcts (75.8+/-14.6 mm3, n=11) than those with more branching (regular) MCA (93.2+/-19.1, n=13), the difference being significant (P=0.022). Male SHR/Izm with simple distal MCA also produced smaller infarctions than those with regular MCA (51.0+/-3.7 versus 68.9+/-8.7 mm3, P=0.0004). CONCLUSIONS: Photothrombotic occlusion of distal MCA in hypertensive rats provides a simple and reproducible model of focal ischemia. Most importantly, this study emphasizes the substantial variabilities in infarct sizes caused by the differences in substrains of SHR, gender, and distal MCA patterns.     

Regional cerebral blood flow during and after 2 hours of middle cerebral artery occlusion in the rat

In this study we explored if the secondary bioenergetic failure, which occurs a few hours after recirculation, following transient middle cerebral artery occlusion (MCAO) in rats, is caused by a compromised reflow. We induced 2 hours of MCAO and measured CBF at the end of the ischemia, as well as 15 minutes, 1, 2, and 4 hours after the start of recirculation, using autoradiographic or tissue sampling 14C-iodoantipyrine techniques. After 2 hours of MCAO, the autoradiographically measured CBF in the ischemic core areas was reduced to 3 to 5% of contralateral values. The reduction in CBF was less in neighboring, penumbral areas. After recirculation, flow already normalized in core tissues after 15 minutes, and remained close to normal for the 4 hours recirculation period studied. However, in penumbral tissues, recovery CBF values were usually below normal. The results show that tissues that are heavily compromised by the 2-hour period of ischemia and are destined to incur infarction, show a "relative hyperemia" during recirculation. In fact, some areas of the previously densely ischemic tissue showed overt hyperperfusion. This finding raises the question whether the relative or absolute hyperemia reflects events that are pathogenetically important. Because drugs that clearly ameliorate the final damage incurred fail to alter the relative hyperperfusion of previously ischemic tissues, it is concluded that vascular events in the reperfusion period do not play a major role in causing the final damage.     

Cathepsin B and middle cerebral artery occlusion in the rat

Lysosomal proteases, although tightly regulated under physiological conditions, are known to contribute to cell injury after various forms of tissue ischemia have occurred. Because cathepsin B is a prominent lysosomal protease found in brain parenchyma, the authors hypothesized that it may contribute to neuronal cell death after focal cerebral ischemia. The authors measured the expression and spatial distribution of cathepsin B within the ischemic brain in 43 animals by means of immunohistochemical analysis in a rat model of transient middle cerebral artery (MCA) occlusion. Cathepsin B activity was also measured within specific ischemic brain regions by using an in vitro assay (22 animals). In addition, the authors tested the therapeutic effect of preischemic intraventricular administration of stefin A, a cysteine protease inhibitor, on the volume of cerebral infarction after transient MCA occlusion (15 animals). Increased cathepsin B immunoreactivity was detected exclusively within the ischemic neurons after 2 hours of reperfusion following a 2-hour MCA occlusion. Cathepsin B immunolocalization in the ischemic region decreased by 24 hours of reperfusion, but then increased by 48 hours of reperfusion because the infarct was infiltrated by inflammatory cells. Increased immunolocalization of cathepsin B in the inflammatory cells located in the necrotic infarct core continued through 7 days of reperfusion. Cathepsin B enzymatic activity was significantly increased in the ischemic tissue at 2, 8, and 48 hours, but not at 24 hours of reperfusion after 2 hours of MCA occlusion. Continuous intraventricular infusion of stefin A, before 2 hours of MCA occlusion (15 animals), significantly reduced infarct volume compared with control animals (12 animals): the percentage of hemispheric infarct volume was 20+/-3.9 compared with 33+/-3.5 (standard error of the mean; p = 0.025). These data indicate that neuronal cathepsin B undergoes increased expression and activation within 2 hours of reperfusion after a 2-hour MCA occlusion and may be a mechanism contributing to neuronal cell death. Intraventricular infusion of stefin A, an inhibitor of cathepsin B, significantly reduces cerebral infarct volume in rats.     

Thread occlusion but not electrocoagulation of the middle cerebral artery causes hypothalamic damage with subsequent hyperthermia

Moderate changes in body temperature can influence the outcome of cerebral ischemic insults and the effect of drugs. Body temperature was measured continuously for 24 hours in rats subjected to permanent occlusion of the middle cerebral artery (MCA) by either coagulation or thread insertion, and the results correlated with the histology of the hypothalamus. The body temperature did not change after MCA occlusion by coagulation and the hypothalamus was intact in all rats. In contrast, the body temperature rapidly increased from about 38 degrees C to more than 39.5 degrees C after MCA occlusion using intraluminal thread, and hyperthermia continued for at least 6 hours in all rats. Histological evaluation revealed neuronal damage in the preoptic area of the hypothalamus in all rats undergoing thread occlusion. Long duration hyperthermia must be prevented after permanent MCA occlusion when the intraluminal thread occlusion model is used in chronic experiments.     

Testosterone increases and estradiol decreases middle cerebral artery occlusion lesion size in male rats

This study was undertaken to determine the effects of estrogen and testosterone on cerebral ischemic lesion size induced by middle cerebral artery (MCA) occlusion in male rats. Rats were gonadectomized and treated with testosterone, estrogen, or testosterone plus estrogen filled Silastic pellets. The animals were divided into 6 groups: intact, intact + estrogen (E2), castrate, castrate + testosterone (T), castrate + E2, and castrate + T + E2. One week after treatment, cerebral ischemia was induced by MCA occlusion for 40 min, followed by reperfusion. After 24 h, rats were sacrificed and slices were then stained to assess lesion size. The presence of testosterone increased and the removal of testosterone decreased lesion size. A strong positive correlation (r2 = 0.922) between plasma testosterone concentrations and ischemic lesion size was observed. Estradiol treatment reduced ischemic area. In summary, the present study provides evidence that testosterone exacerbates and estrogens ameliorate ischemic brain damage in an animal model of cerebral ischemia.     

An integrated analysis of the progression of cell responses induced by permanent focal middle cerebral artery occlusion in the rat

Defining the chronology and severity of cell damage in an evolving lesion after ischemia is important for understanding the underlying mechanisms in the development of therapeutic intervention. In the present study, we used a combination of histological and immunocytochemical methods to evaluate cell responses from 30 min to 48 h after permanent occlusion of the middle cerebral artery (MCAO) in the rat. Specific immunocytochemical markers clearly revealed acute early responses in neurons (neurofilament protein 200), astrocytes (glial fibrillary acidic protein), and microglia/macrophages (OX-42 and ED-1) such as enlarged, convoluted neuronal processes, and disintegration of glia. Progressive topographic changes in the developing lesion, pinpointed by immunolabeling, indicated the severity and extension of the cell damage. Proliferation and hypertrophy of astrocytes and microglia around the infarct, and contralaterally, occurred 24-48 h after MCAO and coincided with mass necrosis and infiltration of neutrophils and macrophages into the core. These observations corroborate the suggestion that the inflammatory process is involved in the progression of the infarct.     

Induction of spreading depression in the ischemic hemisphere following experimental middle cerebral artery occlusion: effect on infarct morphology

This study was undertaken to test whether transient depolarizations occurring in periinfarct regions are important in contributing to infarct spread and maturation. Following middle cerebral artery (MCA) occlusion we stimulated the ischemic penumbra with recurrent waves of spreading depression (SD) and correlated the histopathological changes with the electrophysiological recordings. Halothane-anesthetized, artificially ventilated Sprague-Dawley rats underwent repetitive stimulation of SD in intact brain (Group 1; n = 8) or photothrombotic MCA occlusion coupled with ipsilateral common carotid artery occlusion (Groups 2 and 3, n = 9 each). The electroencephalogram and direct current (DC) potential were recorded for 3 h in the parietal cortex, which represented the periinffarct border zone in ischemic rats. In Group 2, only spontaneously occurring negative DC shifts occurred; in Group 3, the (nonischemic) frontal pole of the ischemic hemisphere was electrically stimulated to increase the frequency of periinfarct DC shifts. Animals underwent perfusion-fixation 24 h later, and volumes of complete infarction and scattered neuronal injury ("incomplete infarction") were assessed on stained coronal sections by quantitative planimetry. Electrical induction of SD in Group 1 did not cause morphological injury. During the initial 3 h following MCA occlusion, the number of spontaneous periinfarct depolarization in Group 2 (7.0 +/- 1.5 DC shifts) was doubled in Group 3 by frontal current application (13.4 +/- 2.7 DC shifts; p < 0.001). The duration as well as the integrated negative amplitude of DC shifts over time were significantly greater in Group 3 than in Group 2 rats (duration, 5.7 +/- 3.8 vs. 4.1 +/- 2.5 min; p < 0.05). Histopathological examination disclosed well-defined areas of pannecrosis surrounded by a cortical rim exhibiting selectively damaged acidophilic neurons and astrocytic swelling in otherwise normal-appearing brain. Induction of SD in the ischemic hemisphere led to a significant increase in the volume of incomplete infarction (19.0 +/- 6.1 mm3 in Group 3 vs. 10.3 +/- 5.1 mm3 in Group 2; p < 0.01) and of total ischemic injury (100.7 +/- 41.0 mm3 in Group 3 vs. 66.5 +/- 24.7 mm3 in Group 2; p < 0.05). The integrated magnitude of DC negativity per experiment correlated significantly with the volume of total ischemic injury (r = 0.780, p < 0.0001). Thus, induction of SD in the ischemic hemisphere accentuated the development of scattered neuronal injury and increased the volume of total ischemic injury. This observation may be explained by the fact that with limited perfusion reserve, periinfarct depolarization are associated with episodic energy failure in the acute ischemic penumbra.     

"Exuberant" neuronal growth after brain damage in adult rats: the essential role of behavioral experience

Wilms tumor can be explained only partially by the "two hit" model that was originally developed for retinoblastoma. Heterogeneity of two kinds operates. The first is that four other primary tumors are regularly observed in children, and the second is that Wilms tumor itself appears to represent more than one genetic entity. All five of these primary renal tumors arise from primary or secondary mesenchyme, renal blastema, or renal epithelium. Mesoblastic nephroma, and possibly clear cell sarcoma, may have some genetic affinity with Wilms tumor, but rhabdoid tumor of the kidney and renal carcinoma do not. At least three different genes seem to be important in the origin of Wilms tumor. One, WT1, whose mutations may be associated with aniridia, may follow the "two hit" model in that there are cases in which both copies of the gene are defective or lost, as expected for a tumor suppressor gene. A second gene, which is associated with Beckwith-Wiedemann Syndrome (BWS) and which has not been cloned, appears to be imprinted in females, and may have an oncogene function. It is evidently activated by gain of a paternal allele or by loss of the inactive, but possibly trans-sensing, maternal allele. Activation of the insulin-like growth factor II gene may be a final common pathway for mutation in both WT1 and BWS. A third gene is unlinked to either of the other two, but its location and function are unknown. It shares with WT1 specificity for Wilms tumor, which is not true of the BWS gene.     

Increased endothelin-1 in the rabbit model of middle cerebral artery occlusion

Human neutrophils (5 x 10(4) incubated on fibronectin precoated wells released 2.83 +/- .25 nmoles of superoxide (0(2)-) (x +/- 1 SEM, n = 15) in response to 5.9 nM (100 ng/ml) Tumor Necrosis Factor Alpha (TNF). On the contrary, the 0(2)- production induced by interleukin-8 (IL-8) (doses ranging from 0.1 nM to 1 microM) was comparable to that of "resting" cells (< .6 nmoles/5 x 10(4) cells). IL-8 (100 nM) did not affect the TNF-dependent 0(2)- production when added with TNF at the beginning of the assay, but reduced it by approximately 80% when added with TNF on neutrophils previously incubated for 1 hour on fibronectin. As compared with IL-8, N-formyl-methionyl-leucyl-phenylalanine (FMLP, 100 nM) failed to suppress the TNF-triggering of the oxidative burst in neutrophils plated on fibronectin. The data suggest that the interaction of neutrophils with fibronectin uncovers the capacity of IL-8 to limit the cell response to TNF, without affecting the response to the combination of FMLP and TNF. Thus, although the chemotactic factors IL-8 and FMLP share the capacity of triggering the oxidative burst of neutrophils incubated in suspension, only IL-8 has the potential to down-regulate the responsiveness of fibronectin-adherent cells to TNF.     

Cortical neuronal function during ischemia. Effects of occlusion of one middle cerebral artery on single-unit activity in cats

To assess the effects of ischemia on neuronal function, the action potentials of 261 individual cortical neurons were recorded extracellulary and related to regional cerebral blood flow (CBF) measured by hydrogen clearance in 19 cats, seven of which had the left middle cerebral artery occluded during a recording. The onset of ischemia could be associated with transient increases of activity, including "seizure discharges," as well as cessation of activity. No activity was noted at CBF less than 0.18 ml/gm/min; at higher (but ischemic) values for CBF, abnormal patterns of activity frequently were recorded. One neuron recovered function after cessation in association with an increase of CBF, indicating a potential for the restoration of function of ischemic neurons by effective therapeutic measures.     

Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: Glial cell line-derived neurotrophic factor (GDNF) plays important roles in the survival and recovery of some mature neurons under pathological conditions. However, the effect of GDNF in ameliorating ischemic brain injury has not been well documented. Therefore, we investigated a possible effect of GDNF on the changes of infarct size, brain edema, DNA fragmentation, and immunoreactivities for caspases after permanent middle cerebral artery occlusion (MCAO) in rats. METHODS: For the estimation of ischemic brain injury, we calculated the infarct size of MCA region and also measured the brain water content as edema formation at 24 hours after the MCAO. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining was performed for the detection of DNA fragmentation. Immunoreactivities for caspase-1 (ICE), caspase-2 (Nedd-2), and caspase-3 (CPP32) were stained. RESULTS: Both infarct size and brain edema after permanent MCAO were significantly reduced by topical application of GDNF (48% and 30% decreases, P=0.01). TUNEL staining and immunoreactivities for caspases were markedly induced at 12 hours after permanent MCAO in the vehicle-treated animals. However, the spatial distribution of those immunohistochemically positive cells was dissociative in each caspase. Induction of TUNEL staining and immunoreactivities for caspases-1 and -3 was greatly reduced with GDNF treatment, whereas the reduction of caspase-2 staining was only minimum. CONCLUSIONS: These data suggest that the reduction of infarct size and brain edema by GDNF was greatly associated with the reduction of DNA fragmentation and apoptotic signals predominantly through caspases-1 and -3 cascades.     

Spatial stability of extracellular potassium ion and blood flow distribution in rat cerebral cortex after permanent middle cerebral artery occlusion

Extracellular potassium ion activity ([K+]o) increases precipitously during brain ischemia when blood flow falls below threshold values less than approximately 15 mL/100 g/min. This flow threshold for increase of [K+]o occurs also in focal ischemia producing gradient from ischemic core to adjacent normally perfused brain. In this study we investigated the spatial and temporal stability of extracellular potassium ion and blood flow gradients after permanent middle cerebral artery occlusion (MCAO) in rats. [K+]o and regional CBF were measured, respectively, with K+-sensitive and polarographic hydrogen-sensitive microelectrodes at different cortical locations in the middle cerebral artery distribution region. Spatial assessment of [K+]o and regional CBF was conducted at 30, 90, and 180 minutes after MCAO. [K+]o in the more lateral cortex (core) increased from near 3 mmol/L before MCAO to greater than 50 mmol/L and was associated with flow values less than 25% of pre-ischemic levels. Measurements medial to the core (penumbra) indicated progressively decreasing levels of [K+]o and improvement of CBF. There was a tendency for [K+]o in penumbral zones to decrease toward normal levels with time, but there was little dissipation of [K+]o in core regions. In contrast, the spatial CBF profile remained remarkably constant for the entire recording period. Thus, unlike infarction which has been reported to expand with time after focal ischemia, the spatial [K+]o disturbance tends to contract primarily due to decreasing [K+]o with time in the penumbra. Thus, steady state levels of [K+]o after focal ischemia may not be a valuable predictor of cell viability.