Shigeru Nogawa

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.

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.

Upregulation of oligodendrocyte progenitor cells associated with restoration of mature oligodendrocytes and myelination in peri-infarct area in the rat brain

This study examines the alteration of oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes (OLGs) and myelination 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 infarct core showed a rapid and progressive decrease in the number of OPCs, OLGs, as well as the myelin density after 48 h of recirculation. The peri-infarct area exhibited a moderate reduction in the number of OLGs and the myelin density with a slight increase in the number of OPCs at 48 h of recirculation. Subsequently, a steady increase in the number of OPCs and a gradual recovery of the number of OLGs were noted in the peri-infarct area, which were accompanied by a gradual restoration of the myelin density, resulting in almost complete recovery of myelin density at 2 weeks of recirculation. OPCs in the peri-infarct area showed characteristic morphological changes such as mitotic figures, monopolar or bipolar shapes, and hypertrophied cell bodies and processes, all indicating active cell proliferation and migration. These findings suggest that the upregulation of OPCs may contribute to replenishment of OLGs and resultant remyelination in the peri-infarct area after ischemic insult.

Inhibition of nitric oxide synthesis impairs autoregulation of local cerebral blood flow in the rat.

The effect of intravenous administration of NG-monomethyl-L-arginine (L-NMMA, 30 mg kg-1), a specific inhibitor of nitric oxide (NO) synthesis, on the autoregulation of local cerebral blood flow (LCBF) was examined in the rat using the [14C]iodoantipyrine autoradiographic method. LCBF was significantly lower in various superficial regions such as the cerebral cortices and cerebellar cortex and in several deep brain regions in animals with haemorrhagic hypotension induced after L-NMMA infusion (the L-NMMA + HEM group) compared with animals without haemorrhagic hypotension after L-NMMA infusion (the L-NMMA group). The present findings suggest that NO synthesis may play a crucial role in the autoregulation of LCBF in response to a reduction in blood pressure in the cerebral cortices, cerebellar cortex and several deep brain regions.

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.

Familial idiopathic brain calcification with autosomal dominant inheritance

Article abstract-We report a family with brain calcification, predominantly in the basal ganglia, and no evident cause such as abnormal calcium or phosphorus metabolism. The proband, a 48-year-old man, had intellectual decline, parkinsonism, and mild cerebellar ataxia. He had bilateral and symmetric calcification of the basal ganglia, thalamus, dentate nucleus, cerebral cortex, subcortical white matter, and hippocampus on CT. Calcified areas showed low- or high-intensity signals on MRI T sub 1-weighted images, and low-intensity signals on MRI T2-weighted images. Two sons and both parents, all asymptomatic, also showed calcification of the basal ganglia, suggesting an autosomal dominant inheritance. Familial idiopathic brain calcification is a rare disorder with less than 20 previously reported families. Twelve families with autosomal dominant inheritance showed a relatively homogeneous clinical picture, which may represent a distinct clinical entity. Mental deterioration, parkinsonism, and cerebellar ataxia appear in adult life and progress gradually. CT imaging, rather than MRI, is a simple and useful means to screen family members for this condition. NEUROLOGY 1997;48: 645-649

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.

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.