Middle Cerebral Artery Cortex Research Articles

CD44 expression in stem cells and niche microglia/macrophages following ischemic stroke.

CD44, an adhesion molecule in the hyaluronate receptor family, plays diverse and important roles in multiple cell types and organs. Increasing evidence is mounting for CD44 expression in various types of stem cells and niche cells surrounding stem cells. However, the precise phenotypes of CD44+ cells in the brain under pathologic conditions, such as after ischemic stroke, remain unclear. In the present study, using a mouse model for cerebral infarction by middle cerebral artery (MCA) occlusion, we examined the localization and traits of CD44+ cells. In sham-mice operations, CD44 was rarely observed in the cortex of MCA regions. Following ischemic stroke, CD44+ cells emerged in ischemic areas of the MCA cortex during the acute phase. Although CD44 at ischemic areas was, in part, expressed in stem cells, it was also expressed in hematopoietic lineages, including activated microglia/macrophages, surrounding the stem cells. CD44 expression in microglia/macrophages persisted through the chronic phase following ischemic stroke. These data demonstrate that CD44 is expressed in stem cells and cells in the niches surrounding them, including inflammatory cells, suggesting that CD44 may play an important role in reparative processes within ischemic areas under neuroinflammatory conditions; in particular, strokes.

Do All ASPECT Score Regions have the Same Predictive Power for Functional Outcomes?

Background and PurposeASPECTS (Alberta Stroke Program Early CT Score) is a 10-point topographic CT scan score that has been shown to be a strong prognostic factor in acute ischemic stroke. We investigated whether all ASPECTS regions have the same prognostic value. MethodsClinical characteristics, ASPECTS, and 3-month modified Rankin Scale (mRS) data were retrospectively collected in 350 patients who were diagnosed with middle cerebral artery (MCA) territory stroke. To describe the 3-month mRS data, an ordered categorical approach was applied using a proportional odds model. Furthermore, external validation was performed using additional data from 30 patients. ResultsAs expected, ASPECTS was an independently important predictor. However, when 10 regions were analyzed separately, the M1, M2, and M3 regions, related to MCA cortex, were not found to predict 3-month mRS scores in the final model. The odds ratios for ischemic change in other regions (except M1, M2, and M3) ranged from 2.6 to 3.8. Moreover, among clinical characteristics, only age was identified as a significant predictor. The sensitivity and specificity of the final model in the external validation were 91% and 88%, respectively. ConclusionsAll ASPECTS regions did not have the same predictive power for functional outcomes, defined as the 3-month mRS. The implementation of a proportional odds model allowed a proper description of the ordered categorical nature of the mRS and the identification of relevant predictors.

Effects of lipopolysaccharide priming on acute ischemic brain injury.

Infection has been implicated as a stroke risk factor. Activation and infiltration of polymorphonuclear neutrophils (PMNs) after cerebral ischemia may contribute to ischemic brain injury. This study was conducted to investigate how enhanced postischemic PMN infiltration by lipopolysaccharide (LPS) altered the acute ischemic outcomes. LPS (0.05 mg/kg SC) or vehicle was given to Long-Evans male rats 24 hours before ischemia. Focal cerebral ischemia was induced by temporary ligation of the right middle cerebral artery and both common carotid arteries for 45 minutes. Animals were killed 6 and 24 hours after reperfusion to determine the extent of PMN infiltration (myeloperoxidase assay), brain edema (wet-dry weight method), and vascular injury (fluorescein isothiocyanate-conjugated dextran extravasation). The infarct volumes were measured on the basis of TTC stain 24 hours after ischemia. LPS had little effect on body temperature or peripheral white count but substantially enhanced PMN infiltration into the ischemic right middle cerebral artery cortex on the basis of myeloperoxidase activity (6 hours: control, 0 U/g; LPS, 0.186+/-0. 025 U/g; 24 hours: control, 0.185+/-0.025 U/g; LPS, 0.290+/-0.040 U/g; P<0.001) and morphological studies. The extent of vascular injury defined by the extravasation of fluorescein isothiocyanate-conjugated dextran into the ischemic tissue (6 hours: control, 3.11+/-0.41 microliter/mg protein; LPS, 0.48+/-0.16 microliter/mg protein; 24 hours: control, 1.77+/-0.23 microliter/mg protein; LPS, 0. 90+/-0.19 microliter/mg protein; P<0.001) and brain edema determined by the brain water content (6 hours: control, 84.77+/-1.63%; LPS, 82. 09+/-1.25%; 24 hours: control, 89.40+/-0.43%; LPS, 87.88+/-0.58%; P<0.01) were paradoxically reduced by LPS priming. LPS-primed rats also had smaller infarct volumes (control, 135+/-5 mm(3); LPS, 108+/-12 mm(3); P<0.05). Enhanced postischemic PMN infiltration is anticipated to facilitate ischemic brain injury. Contrary to this expectation, results from the present study suggest that an increase in postischemic PMN infiltration after LPS priming was not detrimental. These findings challenge the notion that postischemic PMN infiltration is uniformly deleterious.

Induction of cyclooxygenase-2 messenger RNA after transient and permanent middle cerebral artery occlusion in rats: comparison with c-fos messenger RNA by using in situ hybridization.

Recently, two different cyclooxygenase (COX) genes, COX-1 and -2, were identified. In this study, topographic and chronological profiles of COX-2 messenger (m)RNA and c-fos mRNA expression were investigated using in situ hybridization after focal cerebral ischemia. Rats undergoing permanent ischemia were decapitated at 30 and 90 minutes and at 2, 4, 8, and 24 hours after middle cerebral artery (MCA) occlusion, and rats undergoing transient ischemia were decapitated at 4, 8, and 24 hours after MCA occlusion that lasted for either 30 or 90 minutes. After brief transient MCA occlusion, c-fos mRNA was induced in the whole MCA territory, adjacent cortex (cingulate cortex), and distant brain regions such as the hippocampus and substantia nigra. In contrast, COX-2 mRNA was not induced in the ischemic core (lateral striatum) but only in the penumbral area (MCA cortex). Long transient and permanent MCA occlusion did not induce c-fos and COX-2 mRNAs in the ischemic core but strongly induced both mRNAs in the penumbral area (medial striatum and periphery of MCA cortex) and adjacent cortex (cingulate cortex). In brain regions distant from the ischemic territory, although c-fos mRNA was induced in the thalamus, substantia nigra, and hippocampus after extended transient and permanent occlusion, COX-2 mRNA was only induced in the bilateral hippocampi. The induction of COX-2 mRNA persisted in all locations even at 24 hours after MCA occlusion. The distribution of COX-2 mRNA induction was apparently different from that of c-fos mRNA after MCA occlusion. These results pertaining to COX-2 mRNA agree well with the previous observations of changes in prostaglandin metabolism induced by focal cerebral ischemia. However, whether this induction of the COX-2 gene contributes to the histopathological outcome of cerebral ischemia remains to be elucidated.

Poly-phosphoinositide-mediated messengers in focal cerebral ischemia and reperfusion

The receptor-mediated poly-phosphoinositide (PI) signalling pathway is known to play an important role in maintaining intracellular calcium homeostasis, which in turn, is critical for mediating neuronal function. In this study, we examined the effects of focal cerebral ischemia induced in rats by temporary occlusion of the middle cerebral artery (MCA) and both common carotid arteries (CCAs) on this signal transduction pathway. Results indicate that several parts of the pathway are altered, both during the early phase of focal cerebral ischemic insult and after recirculation. Cerebral ischemia induced a decrease in levels of phosphatidylinositol 4,5-biphosphate (PIP 2) in the ischemic MCA cortex, due partly to stimulated poly-PI hydrolysis and partly to the depletion of ATP required for resynthesis of this substrate. ATP depletion during ischemia was also attributed to a sustained decrease in inositol 1,4,5-triphosphate (IP 3) levels. On the other hand, the decline in IP 3 3-kinase activity after 30 min of ischemic insult was not related to ATP depletion. During reperfusion upon prolonged ischemic insult, neither IP 3 level nor IP 3 3-kinase activity were able to show recovery after reperfusion, despite that ATP levels recovered by 80%. In situ hybridization studies indicated a decrease in mRNA expression of IP 3 receptor but not IP 3 3-kinase during the initial 4 h of reperfusion after a 45 min ischemic insult. Under this same condition, insulted cortical neurons started to show morphological changes between 4 and 8 h after reperfusion and extensive cell death could be observed by 16 h. Taken together, these results demonstrated early and delayed changes in the poly-PI signalling pathway due to focal cerebral ischemia. These effects are likely to cause impairment in neuronal function and underline the process of cerebral ischemic damage.

Expression of Glucose Transporters in Rat Brain Following Transient Focal Ischemic Injury

We have investigated the serial changes in the transcription and translation of the rat glucose transporter (GLUT) 1 and 3 genes after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. Northern blot analysis and in situ hybridization study were performed to determine the chronological change and regional expression. In the ipsilateral anterior cerebral artery (ACA) cortex, GLUT1 mRNA expression was increased at 12 h (11.6-fold) of reperfusion, and its expression was detected not only in vascular endothelial cells but also in neurons. At 48 h of reperfusion, GLUT3 mRNA expression was increased in the ipsilateral ACA (8.6-fold) and in the contralateral MCA cortex (9.1-fold). Immunohistochemical study failed to show GLUT1 protein synthesis in neurons in the ipsilateral ACA cortex. The immunoreactivity of GLUT3 protein was increased in neurons in ipsilateral ACA cortex and contralateral MCA cortex. Our results suggest that the expression of GLUT1 and GLUT3 is controlled differently after transient focal ischemic conditions. Furthermore, the postischemic localizations of both GLUT1 and GLUT3 expressions may be altered from the normal physiological expression pattern, which may be of importance in investigating postischemic cell function.

Dextrorphan reduces infarct volume, vascular injury, and brain edema after ischemic brain injury.

Focal cerebral ischemia confined to the cerebral cortex in the right middle cerebral artery (MCA) territory was induced by temporary ligation of the MCA and both common carotid arteries (CCAs). Reperfusion was initiated by releasing all three arterial occlusions after 90 min of ischemia. Infarct volume was morphometrically measured after triphenyltetrazolium chloride staining 24 h postischemia. Blood-brain barrier breakdown was assessed 4 h postischemia by measuring vascular permeability to fluorescein isothiocyanate-conjugated dextran (FITC-D), a macromolecule tracer. Ischemic brain edema was measured based on percent water content, 24 h postischemia. Dextrorphan (DX) 20-10 mg/kg given ip 15 min before ischemia reduced infarct volume in a dose-dependent manner with an apparent U-shaped dose-response curve; best protection was observed at 30 mg/kg. Posttreatment at 30 min, but not 60 min, was still effective. DX (30 mg/kg, given 15 min before ischemia) also reduced the postischemic increase in vascular permeability and brain edema in the right MCA cortex. Results from this study support the idea that NMDA receptor activation contributes to blood-brain barrier breakdown and brain edema after ischemic insults

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion.

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [14C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4-16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3-4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A2 activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.

Inositol Trisphosphate, Polyphosphoinositide Turnover, and High-Energy Metabolites in Focal Cerebral Ischemia and Reperfusion

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. 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. 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. 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.

In situ hybridization of mRNA expression for IP3 receptor and IP3-3-kinase in rat brain after transient focal cerebral ischemia.

Loss of intracellular calcium homeostasis has been regarded an important factor underlying neuron cell death after cerebral ischemic insult. In the brain, a major mechanism for regulation of intracellular calcium is through the signal transduction pathway involving hydrolysis of poly-phosphoinositides and release of the second messenger, inositol 1,4,5-trisphosphate (IP3). IP3 mobilizes calcium by interacting with an intracellular receptor. Upon its release after agonist stimulation, this second messenger is catabolized by a 3-kinase and a 5-phosphatase. In this study, in situ hybridization was carried out to examine the mRNA expression of IP3, receptor (IP3R) and IP3 3-kinase (IP3K) in rat brain cortex after transient focal cerebral ischemia induced by temporary occlusion of the middle cerebral artery (MCA) and the common carotid arteries (CCAs). Results indicate a large decrease (52%) in IP3R mRNA levels in the ischemic cortex as compared to that in the contralateral side at 4 h after a 45 min ischemic insult. By 16 h, practically no IP3R mRNA could be detected in the ischemic cortex. On the other hand, IP3K mRNA levels remained unaltered until 16 h after reperfusion, during which time, expression in the infarct core decreased but that surrounding the core area increased instead. Hybridization of adjacent brain sections with probes for neuron specific enolase (NSE) and beta-actin indicated also a time-dependent decrease in mRNA levels after ischemia, but these changes were less dramatic as compared to IP3R. At 16 and 24 h after reperfusion, there was an increase in beta-actin mRNA in cortical areas outside the MCA cortex, suggesting of reactive gliosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15-60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of ischemic tolerance following brief focal ischemia in rat brain.

The objective of this study was to determine whether brief focal ischemia induces ischemic tolerance in rat brain. Focal ischemia was produced in Wistar rats by occluding the middle cerebral artery (MCA) for 20 min at a distal site. Following recovery for 24 h, the animals were subjected to a 10-min episode of forebrain ischemia using a combination of bilateral carotid artery occlusion and systemic hypotension. Histologic injury, assessed after a survival period of 3-4 days, consisted of selective neuronal necrosis bilaterally in cerebral cortex, striatum, hippocampus, and thalamus superimposed upon a small cortical infarct adjacent to the site of MCA occlusion. However, the intensity of neuronal necrosis in the MCA territory of the neocortex ipsilateral to MCA occlusion was markedly less than that in the contralateral MCA cortex. In contrast, the extent of neuronal necrosis in subcortical structures was similar in both hemispheres. Unexpectedly, animals in which the MCA was manipulated, but not occluded, also exhibited a marked reduction of neuronal necrosis in the ipsilateral MCA neocortex following forebrain ischemia. However, in animals with craniotomy alone, forebrain ischemia caused a similar extent of neuronal necrosis in the MCA neocortex of both hemispheres. Transient occlusion of the MCA induced the focal expression of the 72-kDa heat-shock protein (hsp72) in the MCA territory of the neocortex. Limited expression of hsp72 was also detected following sham occlusion, but not after craniotomy alone. These results demonstrate focal induction of ischemic tolerance in rat neocortex that may be related to expression of heat-shock proteins.