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Background and Purpose: Angiogenesis occurs after cerebral ischemia, and the extent of angiogenesis has been correlated with survival in stroke patients. However, postischemic angiogenesis is short-lived and may be completely terminated within a few weeks after ischemic insult. The molecular mechanism underlying the dissolution of postischemic angiogenic processes is poorly understood. Although the expression of angiogenic genes has been studied in ischemic stroke models, the activation of angiostatic genes after cerebral ischemia has not been investigated. Thrombospondin (TSP)-1 and TSP-2 are naturally occurring angiostatic factors, which inhibit angiogenesis in vivo. The aim of the present study was to explore the expression of TSP-1 and TSP-2 in relation to the evolution of angiogenic process in a focal ischemia model in rats., Methods: Rats underwent cortical ischemia in the middle cerebral artery territory for 60 minutes and reperfusion for up to 2 weeks. Northern and Western blot analysis were used to study the temporal profile of TSP-1 and TSP-2 expression at the mRNA and protein level, respectively. In situ hybridization and immunohistochemical studies were used to examine the spatial expression patterns. Double immunostaining was applied to define the cellular origins of TSP-1 and TSP-2., Results: A biphasic expression of TSP-1 was noted after ischemia, peaking at 1 and 72 hours. Endothelial cells in the leptomeninges were the only source of the first TSP-1 peak, whereas endothelial, glial, neuronal, and macrophage cells contributed to the second peak of TSP-1 expression. TSP-2 expression occurred much later and in a monophasic manner, peaking 2 weeks after ischemia. TSP-2 immunoreactivity was observed in endothelial, neuronal, and macrophage, but not glial, cells. TSP-1 was expressed before the peak of angiogenesis, whereas robust TSP-2 expression occurred at the peak of angiogenesis and continued into the period when angiogenesis had completely resolved., Conclusions: Robust expression of TSP-1 and TSP-2, 2 major angiostatic factors, was noted in the ischemic brain with different temporal expression profiles from different cellular origins. The expression of these angiostatic factors, especially TSP-2, likely contributes to the spontaneous resolution of postischemic angiogenesis. Further studies are needed to explore the molecular mechanisms that regulate the balance of angiogenic and angiostatic factors in the ischemic brain.

Background and Purpose: Angiogenesis occurs after cerebral ischemia, but the relationship between angiogenesis and cerebral hemodynamic change is unknown. The aim of the present study was to investigate the relationship between ischemia-induced angiogenesis and hemodynamics in a well-defined 3-vessel occlusion model of the rat by using diffusion- (DWI), perfusion-, and T2-weighted MRI (T2WI)., Methods: Rats were subjected to 60 minutes of transient middle cerebral artery occlusion or sham operation. DWI and T2WI were used to characterize the extent of the ischemic lesion from 4.5 hours to 14 days after reperfusion. A flow-sensitive alternating inversion recovery method and dynamic susceptibility contrast MRI were used to evaluate the temporal changes in relative cerebral blood flow (CBF) and cerebral blood volume (CBV), respectively. Rats were randomly selected and killed at each time point for investigation of vascular density and for hematoxylin-eosin staining., Results: Ischemic lesions developed in the ipsilateral cortex, as demonstrated by DWI and T2WI. CBF was significantly increased in the ipsilateral cortex, especially in the cortical outer layer from day 1 to day 14, and peaked on day 7 (P<0.05), while CBV was significantly increased on day 7 (P<0.01). The vascular density on the ipsilateral brain surface was gradually increased from day 1 to day 5, peaked on day 7, and then decreased on day 14. Histology study showed pannecrosis in the cortex from day 1 to day 5 and partial liquefaction of the necrotic tissues on days 7 and 14., Conclusions: A delayed increase in both CBF and CBV is documented in the ipsilateral cortex after transient focal brain ischemia, and such an increase may be associated with angiogenesis.

Background and Purpose: A rapid but transient expression of c-fos after cerebral ischemia has been extensively documented. However, the mechanism of this induction and whether induction of c-fos is neuroprotective or detrimental to the brain after ischemia is presently not clear. Fasting before transient cerebral ischemia has been shown to reduce delayed neuronal necrosis and infarct volume. The purpose of the present study was to examine the effect of preischemic fasting for 24 hours on the expression of c-fos after transient focal cerebral ischemia., Methods: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries for 60 minutes. Male Long-Evans rats weighting 250 to 300 g were randomly divided into two groups: fed (control group) and food deprived for 24 hours (fasted group) before ischemic surgery. Infarct volumes were measured on the basis of triphenyltetrazolium chloride-delineated infarct areas, and plasma glucose levels were determined by the glucose oxidase method. Temporal and spatial expression of c-fos was assessed by Northern blot analysis, in situ hybridization, and immunohistochemistry., Results: Fasting for 24 hours before 60 minutes of ischemia resulted in a 26.6% decrease in preischemic plasma glucose levels and a 74.5% reduction in infarct volumes in the fasted group compared with the control group. A rapid but transient induction of c-fos mRNA was observed in the ischemic cortex in control animals after 60 minutes of ischemia. Fasting not only prolonged but also enhanced the intensity of c-fos expression in the ischemic cortex. Regional c-fos expression was also different between these two groups., Conclusions: The results support the contention that c-fos expression may be compatible with its purported neuroprotective role in selected experimental paradigms. The signaling mechanisms underlying the effect of fasting and subsequent lowering of plasma glucose levels on postischemic c-fos expression remain to be explored.

Background and Purpose: Although the signaling pathway involving polyphosphoinositide (poly-PI) hydrolysis and release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism for regulation of neuronal calcium homeostasis, the effect of cerebral ischemia-reperfusion on this calcium signaling pathway is not well understood. Because activity of this pathway is dependent on availability of ATP, this study is aimed at examining the poly-PI signaling pathway and high-energy metabolites in a rat stroke model., Methods: Focal cerebral ischemia in rats was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries. Levels of Ins(1,4,5)P3 were determined by use of the radioreceptor binding assay. Poly-PI turnover in rat cortex was assessed with an in vivo protocol involving intracerebral injection of [3H] inositol and systemic administration of lithium. High-energy metabolites (ATP, ADP, and AMP) were analyzed by high-performance liquid chromatography., Results: Ischemia induced an increase in poly-PI turnover in the right middle cerebral artery cortex, but reperfusion led to a decline in this signaling activity. However, Ins(1,4,5)P3 levels decreased during ischemia, and these levels were not restored if ischemic insults were longer than 30 minutes. ATP levels decreased to 26% of control during ischemia and recovered to 80% of control during the initial 4 hours of reperfusion; these changes were followed by a second phase of decline., Conclusions: Results show an important relationship between ischemia-induced depletion of high-energy metabolites and poly-PI signaling activity. However, the uncoupling between Ins(1,4,5)P3 and ATP during reperfusion after severe ischemia suggests that metabolism of Ins(1,4,5)P3 is more stringently regulated than ATP.

Background and Purpose: Increased release of excitatory neurotransmitters leading to activation of signal transduction has been noted after cerebral ischemia. One of the biochemical events in the signaling processes is an alteration of gene expression. A focal cerebral ischemia-reperfusion model in the rat was used to study the postischemic alteration of selected proto-oncogene and neurotrophin gene expression., Methods: Focal ischemia in the cerebral cortex irrigated by the right middle cerebral artery was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries for 30 or 90 minutes. Expression of selected proto-oncogenes and neurotrophin genes was studied with Northern blot analysis and in situ hybridization., Results: Northern blot analysis showed that an increase in the expression of selected proto-oncogenes including c-fos, c-jun, jun B, zif/268, and nur 77 were noted 30 to 90 minutes into postischemic reperfusion. In situ hybridization revealed different regional distribution of c-fos mRNA signal under different experimental conditions. Nuclear run-on experiments showed that the increase in c-fos and jun B mRNA signals was due to an increase in the transcription rate. An increase in AP-1 binding activity in the ischemic cortex was also noted. Increased expression of brain-derived neurotrophic factor and nerve growth factor mRNAs were noted subsequent to the expression of immediate early genes in the ischemic cortex and adjacent areas in a biphasic pattern., Conclusions: Results are in agreement with the contention that increased c-fos and jun B expression leads to increased AP-1 binding activity, which in turn has been linked to the enhanced expression of neurotrophin genes.

Background and Purpose: Infarct volume is one of the common indexes for assessing the extent of ischemic brain injury following focal cerebral ischemia. Accuracy in the measurement of infarct volume is compounded by postischemic brain edema that may increase brain volume in the infarcted region. We evaluated the effect of brain edema on infarct volume determined by triphenyltetrazolium chloride and hematoxylin and eosin stains in a focal cerebral ischemia model in rats., Methods: In a middle cerebral artery occlusion model in rats, infarction is confined to the cerebral cortex. The infarct was delineated by triphenyltetrazolium chloride stain and, in selected samples, by hematoxylin and eosin stain. We determined infarct size at different times after the ischemic insult (6 hours to 7 days) in relation to the evolution of brain edema by the direct measurement of infarct volume. Indirect measurement to reduce the effect of edema on infarct volume was also conducted in the same brain samples., Results: Direct measurement showed that infarct volume fluctuated with the evolution of brain edema (one-way analysis of variance, p < 0.0001). Infarct volume determined by indirect measurement was independent of the extent of brain edema and remained stable from 6 hours to 3 days after ischemia. There was a good correlation between triphenyltetrazolium chloride and hematoxylin and eosin stains in delineating infarct volume with both direct and indirect measurement., Conclusion: Traditional direct measurement of infarct volume is associated with an overestimation of infarct volume during the development of brain edema in the first 3 days after ischemia. This artifact can be reduced with indirect measurement, which is based on noninfarcted cortex volume.

Using a rat model of stroke, we examined the effects of focal cerebral ischemia on the metabolism of polyphosphoinositides by injecting 32Pi into both the left and right cortices. After equilibration of the label for 2-3 hours, ischemia induced a significant decrease (p less than 0.001) in the concentrations of labeled phosphatidyl 4,5-bisphosphates (66-78%) and phosphatidylinositol 4-phosphate (64-67%) in the right middle cerebral artery cortex of four rats. The phospholipid labeling pattern in the left middle cerebral artery cortex, which sustained only mild ischemia and no permanent tissue damage, was not different from that of two sham-operated controls. However, when 32Pi was injected 1 hour after the ischemic insult, there was a significant decrease (p less than 0.01) in the incorporation of label into the phospholipids in both cortices of four ischemic rats compared with four sham-operated controls. Furthermore, differences in the phospholipid labeling pattern were observed in the left cortex compared with the sham-operated controls. The change in labeling pattern was attributed to the partial reduction in blood flow following ligation of the common carotid arteries. We provide a sensitive procedure for probing the effects of focal cerebral ischemia on the polyphosphoinositide signaling pathway in the brain, which may play an important role in the pathogenesis of tissue injury.