Usefulness of MRA-DWI mismatch in neuroendovascular therapy for acute cerebral infarction.

BACKGROUND: This study evaluated the usefulness of MR angiography (MRA)-diffusion-weighted imaging (DWI) mismatch in neuroendovascular therapy over 3 h after onset of acute cerebral infarction. METHODS: The subjects were 14 cases (age, 73 ± 8.4 years) who had an anterior circulation deficit on DWI/MRA on arrival and underwent neuroendovascular therapy over 3 h after onset. MRA-DWI mismatch (MDM) (+) was defined as 'major artery lesion (+) and diffusion-weighted image-Alberta Stroke Program Early CT Score (DWI-ASPECTS) ≥6'; MDM (-) was defined as 'major artery lesion (+) and DWI-ASPECTS <6'. RESULTS: Reperfusion was achieved in nine of 14 patients (64%) undergoing neuroendovascular therapy. Within the reperfusion group, in the five MDM (+) patients and the four MDM (-) patients, the outcome was a favorable clinical response in the MDM (+) group. The modified Rankin Scale (mRS) scores after 90 days were 0-2 in 3 (60%) and 3-6 in 2 (40%) of the MDM (+) group patients and 0-2 in 0 (0%) and 3-6 in 4 (100%) of the MDM (-) group patients. In the MDM (+) group, a good outcome was achieved. However, the number of cases was small, so this was not a significant difference. Within the non-reperfusion group, in the three MDM (+) patients and the two MDM (-) patients, the mRS scores after 90 days were 0-2 in 1 (33%) and 3-6 in 2 (67%) of the MDM (+) group patients and 0-2 in 0 (0%) and 3-6 in 2 (100%) of the MDM (-) group patients. In both groups, the outcome was poor. CONCLUSIONS: With neuroendovascular therapy, a good outcome with reperfusion was achieved in the MDM (+) group compared to the MDM (-) group. This suggests that the presence or absence of MDM may be useful in determining prognosis after reperfusion.

Up-regulation of the Ire1-mediated signaling molecule, Bip, in ischemic rat brain.

The endoplasmic reticulum (ER) is thought to play important roles in various neurological diseases via multifactorial and complex mechanisms. The Ire1-mediated signal is part of one ER signaling pathways; the signal induces the expression of an ER-resident protein, Bip/GRP78, and is thought to be involved in cell death under ER stress. In this study, we examined time-dependent Bip expression after transient middle cerebral artery occlusion and characterized the Bip-positive cells. Ire1- mediated molecules, Bip, were rapidly up-regulated in the ischemic area after 3.5 h recirculation. Their immunoreactivity continued to increase until 24-48 h. Immunofluorescence staining revealed Bip up-regulation in ischemic neurons, which were TUNEL positive. Our studies suggest that the Ire1-mediated signal might be associated with ischemic neuronal damage.

Phosphorylation of cyclic adenosine monophosphate response element binding protein in oligodendrocytes in the corpus callosum after focal cerebral ischemia in the rat.

Phosphorylation of cyclic adenosine monophosphate (AMP) response element binding protein (CREB) was examined immunohistochemically in the corpus callosum of the rat brain at various time points after 90-minute focal cerebral ischemia. Focal ischemia was induced by occlusion of the middle cerebral artery (MCA) using the intraluminal suture method. Sham animals showed that numerous oligodendrocytes (OLGs) constitutively express unphosphorylated CREB. Local cerebral blood flow (lCBF) measured by the 14C-iodoantipyrine method was reduced from 44.2 +/- 15.4 (mL 100 g(-1) min(-1)) to 18.4 +/- 3.8 and from 53.9 +/- 14.4 to 4.8 +/- 4.5 in the medial and the lateral regions of the corpus callosum, respectively, during MCA occlusion (MCAO). After release of the MCAO, lCBF recovered to the control level in each region. The medial region of the corpus callosum showed a marked increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, and it remained increased until 2 weeks of recirculation as it gradually declined. The activation of CREB phosphorylation in the OLGs was accompanied by expression of antiapoptotic protein bcl-2, normal staining with cresyl violet, and negative TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) staining. Myelination detected by immunostaining with anti-myelin basic protein (MBP) antibody and anti-myelin associated glycoprotein (MAG) antibody remained normal in the medial region of the corpus callosum. The lateral region of the corpus callosum showed a significant but only transient increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, which was followed by a rapid decrease during the subsequent recirculation period. Expression of bcl-2 was suppressed in this region, and demyelination became apparent. These findings suggest that signal transduction through CREB phosphorylation may be closely associated with survival of OLGs and maintenance of myelination in the corpus callosum after cerebral ischemia.

Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain.

BACKGROUND AND PURPOSE: Iba1 is a novel calcium-binding protein and is specifically expressed in microglia in the brain. It has been suggested that Iba1 plays an important role in regulation of the function of microglia. In the present study we examined time-dependent Iba1 expression after transient middle cerebral artery occlusion and characterized microglial activation in various brain regions. METHODS: Rat middle cerebral artery occlusion was induced by the intraluminal filament technique. After 1.5 hours of transient ischemia, Iba1 expression was examined by immunohistochemical and immunoblot analyses. The microglial activation in association with ischemic severity was characterized by double immunostaining with other specific markers. RESULTS: In the peri-ischemic area, heavily Iba1 immunoreactive cells rapidly appeared at 3.5 hours after reperfusion. Immunoreactivity was further increased and peaked at 7 days. In the ischemic core, round Iba1-positive cells, which may be blood-borne monocytes, appeared from 24 hours and reached a peak at 4 to 7 days. Double immunostaining revealed that activated microglia in the peri-ischemic area upregulated Iba1 expression but were negative for the macrophage marker ED1. ED1-positive cells were clearly restricted to the ischemic core. CONCLUSIONS: These findings suggest the following: (1) Iba1 expression may be associated with microglial activation in ischemic brain, and Iba1 immunostaining can be useful to evaluate the pathophysiological roles of activated microglia in ischemic injury. (2) Expression of ED1 antigen is strictly restricted to severe ischemic damage, whereas activated microglia in the peri-ischemic area showed Iba1 upregulation without ED1. Therefore, microglia may exhibit difference of antigenicity in the severity of ischemic brain injury.

Activation of NG2-positive oligodendrocyte progenitor cells during post-ischemic reperfusion in the rat brain.

This study examines the alteration of oligodendrocyte progenitor cells which express membrane NG2 chondroitin sulfate proteoglycan after focal ischemia in the rat brain. Adult male Sprague-Dawley rats were subjected to 90 min occlusion of the middle cerebral artery, followed by reperfusion time of up to 2 weeks. The distribution and morphological changes in NG2-positive oligodendrocyte progenitor cells were immunohistochemically examined. Stellate-shaped NG2-positive cells with multiple branched processes were detected in both the gray and white matter of normal brain. After 2 weeks of reperfusion, NG2-positive cells in the area surrounding the infarction site (peri-infarct area) clearly showed enlarged cell bodies with hypertrophied processes. These stained strongly for NG2. Although the number of NG2-positive cells was increased significantly in the peri-infarct area, it decreased markedly in the infarct core compared to controls. Double immunostaining revealed that these NG2-positive cells were neither astrocytes nor microglia, but NG2-positive oligodendrocyte progenitor cells. These progenitor cells are known to differentiate into oligodendrocytes. As such, this upregulation of NG2 expression may be an adaptive mechanism attempting to remyelinate rat brain tissue after ischemic insult. Only further study will elucidate this hypothesis.

Phosphorylation of signal transducer and activator of transcription-3 (Stat3) after focal cerebral ischemia in rats.

JAK-STAT is the major downstream signal pathway of interleukin-6 (IL-6) cytokine family and is regulated by Tyr705 phosphorylation of Stat3. The present study examined the extent and the localization of phosphorylated Stat3 protein in brain tissue after focal ischemia in rats. The localizations of unphosphorylated and phosphorylated Stat3 were immunohistochemically examined in rats after 0.5 to 168 h of reperfusion following 1.5 h of middle cerebral artery occlusion (MCAO), induced by the intraluminal suture method. Absolute phosphorylated Stat3 immunoreactive cell counts were made in the cerebral cortex (ischemic core, peri-ischemia region, and contralareral cortex) and lateral striatal regions on both the ischemic and the contralateral sides. Stat3 protein was localized diffusely in cortical and striatal neurons in the sham-operated animals. Although weak Stat3 staining was detected in damaged neurons in the ischemic region, activated microglia, astrocytes, and endothelial cells clearly expressed Stat3 in this region. On the other hand, the sham group showed no phosphorylated Stat3 immunoreactivity. Phosphorylated Stat3 immunoreactivity was first detected in neurons after 3.5 h of reperfusion in each cortical and striatal region. Thereafter, Stat3 phosphorylation was marked in neurons in the peri-infarct region, peaked at 24 h, and then gradually declined throughout the reperfusion period. Endothelial cells expressed phosphorylated Stat3 in the ischemic core at 48 h of reperfusion. To identify the cellular source of phosphorylated Stat3, lectin histochemical study and immunohistochemical study with anti-microtubule-associated proten-2 and anti-glial fibrillary acidic protein antibodies were carried out. Double-staining immunohistochemistry with these cellular makers revealed phosphorylated Stat3 to be present in neurons, but in neither astrocytes nor microglia/macrophages. These results were also confirmed be western blot analysis. The present results indicate that Stat3 activation occurs in neurons and endothelial cells only during post-ischemic reperfusion despite widespread expression of IL-6 cytokines.

[Targets of treatment in the acute phase of cerebral infarction].

In the acute phase of cerebral infarction, many experimental data suggest that free radicals including superoxide, hydroxy radical and nitric oxide are one of the most important factors to cause brain damage. We have clearly detected nitrotyrosine (a marker of endogenous production of peroxynitrite, which is readily produced from superoxide and nitric oxide) in neurons and intraparenchymal vascular walls during post-ischemic reperfusion. Free radical scavengers thus seem to be very promising tools of treatment, and one of them (edaravone) has recently been approved for clinical use in Japan. CREB (cyclic AMP response element binding protein) is a DNA-binding transcription factor, and its function is activated by phosphorylation of Ser133 residue. CREB plays important roles in neuronal development, synaptic plasticity and regeneration. We have found that phosphorylation of CREB is significantly and persistently increased in surviving neurons and oligodendrocytes in post-ischemic brain, while this phosphorylation is only transiently increased in neurons and oligodendrocytes which eventually die. These data suggest that CREB phosphorylation plays an important role in protection of ischemic brain tissue. Oligodendrocyte progenitor cells (OPC) remain abundant throughout the adult brain, and retain their ability to become not only mature oligodendrocytes, but also neurons. We have recently found that OPC are significantly activated and proliferate in the peri-infarct area at 1-2 weeks after ischemia, suggesting that OPC may be involved in the repair mechanisms of ischemic brain. Future targets of stroke treatment should include enhancement of intrinsic protection mechanisms such as CREB phosphorylation and activation of progenitors cells.

Activated phosphorylation of cyclic AMP response element binding protein is associated with preservation of striatal neurons after focal cerebral ischemia in the rat.

Phosphorylation of the DNA-binding transcription factor, cyclic AMP response element binding protein, has recently been suggested to provide neuroprotective signals in times of cellular stress. Medium-sized striatal neurons are among the cells that are most vulnerable to ischemic stress in the brain. In the present study, phosphorylation of cyclic AMP response element binding protein was immunohistochemically evaluated in rat striatum in order to examine the ischemic vulnerability of each striatal region from the standpoint of cyclic AMP response element binding protein. Rats were subjected to 90-min focal cerebral ischemia followed by various periods of recirculation. Focal ischemia was induced by occlusion of the middle cerebral artery by the intraluminal suture method. Local cerebral blood flow measured by the 14C-iodoantipyrine method in the lateral and the medial striatal regions during occlusion was 5.0+/-7. 1 and 42.5+/-8.1ml/100g/min, respectively. Cerebral blood flow in each region was restored to the control level during the recirculation period. The lateral and the medial regions of the striatum in the sham animals showed hardly any immunoreactivity with the specific antibody against phosphorylated cyclic AMP response element binding protein. By contrast, at 3.5h of recirculation, a number of phosphorylated cyclic AMP response element binding protein-positive neurons were detected in the medial striatal region on the occluded side, and the increase in the number of immunopositive cells continued until two weeks of recirculation with gradual decline. The lateral striatal region on the ischemic side showed only a mild increase in phosphorylated cyclic AMP response element binding protein-positive cells at 3.5h of recirculation, and the immunoreactivity rapidly disappeared during the subsequent recirculation period. Appreciable increase in immunoreactive cells was also noted in the contralateral striatum during the early phase of recirculation, and this increase seemed to be associated with spontaneous circling movements of the animals. Cresyl Violet staining revealed that striatal neurons in the medial region remained intact until two weeks of recirculation, whereas neurons in the lateral striatal region soon showed ischemic damage, followed by complete neuronal loss, and evolution of a frank infarct. Immunoreactivity for bcl-2, apoptosis-suppressive protein, was clearly detected in many neurons in the medial striatal region, but no such immunoreactivity was detected in the lateral striatal region. These findings suggest that persistently activated phosphorylation of cyclic AMP response element binding protein in the striatum during post-ischemic recirculation may be closely associated with protection of striatal neurons on the ischemic side, while it may be associated with spontaneous circling movements on the contralateral side.

Effects of blockade of voltage-sensitive Ca(2+)/Na(+) channels by a novel phenylpyrimidine derivative, NS-7, on CREB phosphorylation in focal cerebral ischemia in the rat.

NS-7 is a novel blocker of voltage-sensitive Ca(2+) and Na(+) channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca(2+) concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5-48-h recirculation after 1. 5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca(2+) and Na(+) channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.

Expression of interleukin-6 is suppressed by inhibition of voltage-sensitive Na+/Ca2+ channels after cerebral ischemia.

Expression of interleukin-6 (IL-6), a neurotrophic cytokine, is up-regulated after cerebral ischemia, but the underlying mechanism of the up-regulation remains unclear. NS-7 is a novel blocker of voltage-sensitive Ca2+ and Na+ channels and is known to reduce cerebral damage by ischemia. The present study was undertaken to examine the association between increases in intracellular Ca2+ concentration induced by membrane depolarization and IL-6 induction. IL-6 expression in rat brain was investigated by immunohistochemistry and Western blot analysis following 3.5-48 h of reperfusion after 1.5 h of occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was injected i.v. 5 min after the start of reperfusion. The saline group showed clear IL-6 expression in various cortical regions, which peaked at 24 h of reperfusion. By contrast, IL-6 expression was significantly suppressed in the NS-7 group throughout the reperfusion period. Microglia activation was also reduced in the NS-7 group. These findings suggest that IL-6 expression may be up-regulated by the increased intracellular Ca2+ concentration triggered by membrane depolarization after cerebral ischemia.

Immunohistochemical detection of leukemia inhibitory factor after focal cerebral ischemia in rats.

The cytokine leukemia inhibitory factor (LIF) modulates neuronal function during development and promotes neuronal survival after peripheral nerve injury, but little is known about LIF expression after cerebral ischemia. In the present study, the localization of LIF protein was immunohistochemically examined in rats after 3.5, 12, 24, 48, and 96 hours of reperfusion following 1.5 hours of middle cerebral artery occlusion (MCAO) induced by the intraluminal suture method. Double-staining immunohistochemistry with microtubule-associated protein-2 (MAP2), glial fibrillary acidic protein (GFAP), lectin histochemistry, and interleukin (IL) 6 was also performed. The sham group and immunosorption test did not show any clear LIF immunoreactivity. Definite LIF immunoreactivity was first detected after 12 hours of reperfusion in each of the brain regions examined: ischemic core, periinfarct region, and contralateral cortex. However, expression of LIF was most prominent in the periinfarct region at each time point, peaked at 24 hours, and then gradually declined until 96 hours of reperfusion. Some LIF-positive neurons in the periinfarct region expressed IL-6. At 96 hours of reperfusion, GFAP-labeled astrocytes around the infarct core also expressed LIF protein. Induction of LIF mRNA and protein was also confirmed by reverse transcription polymerase chain reaction and western blot analysis, respectively. These findings suggest that LIF expression in ischemically threatened neurons may reflect a repair or defense mechanism against the ischemic insult.

Recovery of decreased local cerebral blood flow detected by the xenon/CT CBF method in a patient with eclampsia.

A 23-year-old woman presented in our hospital with toxemia, underwent cesarean section at about 36 weeks gestation, and became eclamptic in the immediate postpartum period. Following a complex partial seizure a few hours after the cesarean section, the patient experienced drowsiness, then cortical blindness. Cranial computed tomography (CT) performed at about 24 hours after the onset of the seizure showed low density areas in the bilateral occipital lobes. Intravenous magnesium sulfate was given, and the neurological symptoms disappeared within three weeks. Xenon/CT cerebral blood flow (CBF) was measured during the acute and chronic stages of the patient's eclampsia and compared with cranial magnetic resonance imaging (MRI) performed at about the same time. In the acute stage, MRI showed abnormal T2-hyperintensity signals in the head of the left caudate nucleus and in the bilateral occipital lobes, predominantly in the white matter. Xenon/CT CBF measurement showed decreased local cerebral blood flow (LCBF) in the area of the left anterior cerebral artery (ACA), the bilateral posterior cerebral arteries (PCAs), and the watershed areas of the left hemisphere. In the chronic stage, abnormal T2-hyperintensity signals remained in that part of the left occipital lobe where, in the acute stage, a marked decrease in LCBF had been detected. The main mechanism of eclampsia in this patient is thought to be a reactive vasoconstriction against hypertension rather than a vasodilatation.

Local cerebral hemodynamic changes through the angiographic stages of moyamoya disease.

In order to elucidate the cerebral hemodynamic changes that occur in Suzuki's six angiographic stages of moyamoya disease, local cerebral blood flow (LCBF) during the stable state and CO2 responsiveness of LCBF (L-CO2R: delta %LCBF/delta PaCO2) were measured by the Xenon CT-CBF method. Nineteen patients with moyamoya disease (mean age: 36.8 +/- 11.6 years) and 11 age-matched normal volunteers were studied. The LCBF during the steady state at all stages was not significantly different from that in normal volunteers. At stage 6, however, the LCBF was slightly decreased in the anterior part of the brain, resulting in loss of "hyperfrontality." On the other hand, the L-CO2R in the anterior part of the brain tended to diminish with progression through the stages. Especially in the frontal cortex, the L-CO2R at stage 5 was significantly less than that in normal volunteers (p < 0.01) or at stage 3 (p < 0.05). In conclusion, the cerebrovascular reserve in the anterior circulation became insufficient after stage 4, although the posterior circulation was well maintained. Revascularization surgery involving the anterior circulation may be crucial to prevent ischemic events.

Persistent CREB phosphorylation with protection of hippocampal CA1 pyramidal neurons following temporary occlusion of the middle cerebral artery in the rat.

Phosphorylation of the DNA-binding transcription factor, cyclic AMP response element binding protein (CREB), was immunohistochemically examined in rat brain hippocampal CA1 in order to examine the ischemic vulnerability of this region from the viewpoint of CREB activation. The rat brain had been subjected to 90-min focal ischemia followed by various periods of recirculation. Focal ischemia was induced by occlusion of the middle cerebral artery using the intraluminal suture method. CA1 pyramidal neurons in the sham animals showed definite immunoreactivity with anti-CREB antibody, which binds to both unphosphorylated and phosphorylated CREB, while reactivity with anti-phosphorylated CREB antibody was barely detectable in these neurons. In contrast, at 3.5 h of recirculation, a significant increase in the number of phosphorylated CREB-positive neurons was noted in the CA1 on both sides, and the increase continued until 48 h of recirculation with a tendency for gradual decline. At each period, the ischemic side showed a more marked increase in the number of immunoreactive cells as compared to the nonischemic side. Cresyl violet staining revealed CA1 pyramidal neurons to be maintained intact until 14 day of recirculation, at which time CREB phosphorylation has returned to the control level. Transient global ischemia is known to induce only mild CREB phosphorylation in the CA1 followed by a frank neuronal loss in this region. These data suggest that CREB phosphorylation can be persistently activated in CA1 neurons after focal ischemia and that this phenomenon may be closely associated with protection of these neurons.

Inhibition of cyclic AMP-dependent protein kinase in the acute phase of focal cerebral ischemia in the rat.

Binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase is an essential step in cyclic AMP-mediated intracellular signal transduction. In the present study, the binding capacity of cyclic AMP-dependent protein kinase for cyclic AMP was examined by autoradiography with local cerebral blood flow in focal cerebral ischemia in the rat, which was induced by occlusion of the middle cerebral artery using the intraluminal suture method. The binding capacity of cyclic AMP-dependent protein kinase and local cerebral blood flow were assessed by the in vitro [3H]cyclic AMP binding and the [14C]iodoantipyrine methods, respectively. At 3 h of occlusion, a significant reduction in the binding of cyclic AMP-dependent protein kinase to cyclic AMP was already noted in the lateral region of the caudate-putamen and the parietal cortex. Between three and five hours of occlusion, the area with reduced cyclic AMP binding was significantly expanded to the peri-ischemic regions including the frontal cortex and the medial region of the caudate-putamen. The threshold in local cerebral blood flow for reduced cyclic AMP binding was clearly noted at 5 h of ischemia, and was 45 ml/100 g per min in the cerebral cortices, and 38 ml/100 g per min in the caudate-putamen, respectively. No threshold was noted at 3 h of ischemia, since cyclic AMP binding showed a large variation ranging from reduced to normal values even when local cerebral blood flow was below 20 ml/100 g per min. Recirculation for 3.5 h following 1.5 h of ischemia restored the normal cyclic AMP binding in the cerebral cortices, but failed to normalize cyclic AMP binding in the caudate-putamen despite good recovery of local cerebral blood flow. Western blot analysis suggested that this reduction in cyclic AMP binding was not due to loss or degradation of the subunit protein of cyclic AMP-dependent protein kinase, and may therefore have resulted from conformational changes in the protein. A significant increase in cyclic AMP binding was noted after recirculation in the non-ischemic regions such as the frontal and the cingulate cortices on the occluded side and in the contralateral cortices. These data indicate that cyclic AMP-mediated signal transduction in the brain tissue may be very susceptible to ischemic stress, and the region of disrupted signal transduction may expand progressively from the ischemic core to peri-ischemic regions in the acute phase of ischemia. Such impairment of signal transduction may not be restored in the caudate-putamen even when cerebral circulation is fully recovered after short-term ischemia, suggesting that a regional vulnerability to ischemic stress may also exist in cyclic AMP-mediated signal transduction. A significant increase in cyclic AMP binding after recirculation in regions outside of ischemic area may be closely related with the protective mechanisms of brain tissue, since cyclic AMP has been reported to exert various neuroprotective actions.

Uncoupling of cerebral blood flow and glucose utilization in the regenerating facial nucleus after axotomy.

Axotomy is known to activate various metabolic processes including protein synthesis and glucose utilization in the motor nucleus. Although it is generally assumed that the local cerebral blood flow (CBF) fluctuates in response to the axonal reaction, there has been no direct evidence for changes in CBF in the motor nucleus following axotomy. In this study, the CBF in the facial nuclei was measured after axotomy of the facial nerve employing the [14C]iodoantipyrine method to evaluate the relation between the CBF and axonal reaction. Following unilateral facial nerve axotomy in neonates, which induced neuronal degeneration in the facial nucleus, the CBF and glucose uptake was significantly decreased on the operated nucleus, suggesting that CBF and glucose metabolism are coupled in the degenerating nucleus. In contrast, after axotomy in adults, which induced regeneration of neurons and glial reactions, glucose uptake was increased on the operated nucleus, while the CBF did not differ significantly between the operated and unoperated nucleus. These findings imply that glucose metabolism and CBF are uncoupled in the regenerating nucleus, suggesting that the relation between CBF and metabolism in the regenerating nucleus following axotomy may clearly contradict the classical concept of a tight coupling between CBF and metabolism.

Temporal profile and cellular localization of interleukin-6 protein after focal cerebral ischemia in rats.

Although interleukin-6 (IL-6) has various neuroprotective effects against cerebral ischemia, the topographic distribution and cellular source of IL-6 after cerebral ischemia remain unclear. In the current study, the localization of IL-6 protein was immunohistochemically examined in rats after 3.5, 12, 24, and 48 hours of reperfusion after 1.5 hours of middle cerebral artery occlusion. Middle cerebral artery occlusion was induced by the intraluminal suture method. The specificity of the anti-IL-6 antibody used in the current study was confirmed by Western blot analysis and an immunoabsorption test. To identify the cellular source, lectin histochemical study and immunohistochemical study with microtubule-associated protein-2, ED1, and glial fibrillary acidic protein also were carried out. The sham group did not show any clear IL-6 immunoreactivity. After 3.5 hours of reperfusion, IL-6 immunoreactivity was first detected on the reperfused side, and it was upregulated, especially in the periinfarct region, after 24 hours of reperfusion. Also, IL-6 was expressed after 3.5 hours of reperfusion in the contralateral cerebral cortex and bilateral hippocampi. Double staining showed that the cells containing IL-6 were neurons and round-type microglia, not astrocytes. The current findings suggest that IL-6 expression in ischemically threatened neurons and reactive microglia is closely associated with brain tissue neuroprotective mechanisms against cerebral ischemia.

Selective inhibition of inositol 1,4,5-triphosphate-induced Ca2+ release in the CA1 region of the hippocampus in the ischemic gerbil.

We examined the effect of ischemia on inositol 1,4,5-trisphosphate receptor-induced Ca2+ release by functional and morphological approaches, using the gerbil model after 6-h unilateral occlusion of the common carotid artery. Autoradiographic study revealed that the basal uptake of 45Ca2+ into the endoplasmic reticulum and caffeine-induced 45Ca2+ release from the endoplasmic reticulum were normal in the presence of ATP in each ischemic brain region, whereas inositol 1,4,5-trisphosphate receptor-induced 45Ca2+ release from the endoplasmic reticulum was inhibited only in the CA1 region of the hippocampus on the ischemic side. In moderately ischemic gerbils, electron microscopic study demonstrated aggregation of swollen endoplasmic reticulum in the CA1 region of the hippocampus, so that abundant endoplasmic reticulum assembled in close contact to form endoplasmic reticulum cisternal stacks. In severely ischemic gerbils, immunohistochemical analysis of the hippocampus showed loss of type 1 inositol 1,4,5-trisphosphate receptor protein with preservation of immunoreactivity for type 2 and 3 inositol 1,4,5-trisphosphate receptor proteins, which was confirmed by western blot analysis. Such selective inhibition of inositol 1,4,5-trisphosphate receptor-induced Ca2+ release and the loss of type 1 inositol 1,4,5-trisphosphate receptor in the CA1 region of the hippocampus in cerebral ischemia may be associated with its region-specific vulnerability to ischemia.

Temporal profile of CREB phosphorylation after focal ischemia in rat brain.

The phosphorylation of cAMP response element binding protein (CREB) in the rat brain was examined immunohistochemically at 3.5 h, 12 h, 24 h and 48 h of recirculation after focal ischemia induced by occlusion of the middle cerebral artery for 1.5 h. Brain sections were stained with affinity purified anti-phosphorylated CREB antibody. The ischemic core revealed a significant, but transient increase in number of phosphorylated CREB-positive cells at 3.5 h of recirculation, followed by a rapid decrease during the subsequent period. In the peri-ischemia area, the number of phosphorylated CREB-positive cells showed a more marked increase as compared to that in the ischemic core at 3.5 h of recirculation, and the increase continued until 48 h of recirculation with a tendency for gradual decline. Persistent enhancement of CREB phosphorylation may thus be closely related to the neuronal viability and neuroprotective mechanisms, whereas rapid disappearance of CREB phosphorylation may clearly precede neuronal death.