Yasuo Fukuuchi

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.

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.

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.

Immunohistochemical analysis of cyclic AMP response element binding protein phosphorylation in focal cerebral ischemia in rats

Phosphorylation of cyclic AMP response element binding protein (CREB) is one of the most important mechanisms controlling various gene transcriptions. In the present study, the phosphorylation of CREB was examined immunohistochemically at 24 h of recirculation following 1.5 h of middle cerebral artery occlusion (MCAO) in rats. MCAO was induced by the intraluminal suture method. The infarct core revealed a significant reduction in the number of immunoreactive cells with the anti-phosphorylated CREB and with the anti-CREB antibody, which binds to both unphosphorylated and phosphorylated CREB. In contrast, the peri-infarct area exhibited a marked increase in the number of immunopositive cells as well as in the intensity of nuclear staining with each antibody, so that almost all of the cells expressing CREB demonstrated phosphorylation of CREB. On the other hand, about half of the CREB immunopositive cells reacted weakly with the anti-phosphorylated CREB antibody in the sham group. These findings indicated that the expression as well as phosphorylation of CREB protein was significantly activated in the regions surrounding the infarct area. Since phosphorylation of CREB has recently been implicated in signal transductions that promote the survival and differentiation of neurons, the present data suggest that tissue repair mechanisms may be markedly activated in the peri-infarct area.

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.

Cerebral neurons express interleukin-6 after transient forebrain ischemia in gerbils

Markedly increased interleukin-6 (IL-6) mRNA levels occur in experimental cerebral ischemia, although the protein production and cellular sources of IL-6 remain unclear. We examined the cellular localization of IL-6 protein in gerbil brain following transient forebrain ischemia employing immunohistochemistry and Western blot analysis. The ischemia/recirculation groups revealed distinct IL-6 immunoreactivity predominantly in cortical and hippocampal neurons after 3 hours to 3 days recirculation. At 12 h recirculation, the IL-6 expression declined specifically in the hippocampus CA1. Microglia, but not activated astrocytes, also expressed IL-6 immunoreactivity. The sham group showed no apparent immunoreactivity. IL-6 protein may thus be expressed mainly in neurons following transient forebrain ischemia. Its transient decline in the CA1 at 12 h recirculation could reflect the specific vulnerability of this region.

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.

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.

Calcineurin inhibitor, FK506, prevents reduction in the binding capacity of cyclic AMP-dependent protein kinase in ischemic gerbil brain

We examined the effects of FK506, a specific inhibitor of calcineurin, on the binding capacity of cyclic AMP-dependent protein kinase (cAMP-DPK) in gerbils subjected to 2-h cerebral hemispheric ischemia. FK506 (0.1 mg/kg) was infused intravenously at 15 min prior to the induction of ischemia by common carotid artery occlusion. The binding capacity of cAMP-DPK was evaluated by autoradiographic analysis of the cAMP binding, and cerebral blood flow (CBF) was measured by the [14C] iodoantipyrine method. In the sham-operated gerbils, FK506 significantly increased mean arterial blood pressure and tended to decrease CBF, suggesting that FK506 may constrict systemic blood vessels as well as cerebral blood vessels. On the other hand, cAMP binding was not altered by FK506 in the sham-operated gerbils. In the ischemia group of gerbils, FK506 prevented any significant reduction of cAMP binding in the hippocampus CA1 and cerebral cortices on the ischemic side, whereas it exerted no significant influence on the cAMP binding of the nonischemic side. The values of CBF were comparable between the vehicle-treated gerbils and FK506-treated gerbils in the ischemic regions. Preservation of cAMP binding indicates that intracellular signal transduction via cAMP-DPK can be maintained by FK506 despite ischemia, suggesting that this agent may be beneficial for reducing ischemic tissue damage.