Junichi Hamada

Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice.

We sought to clarify the involvement of caspase-12, a representative molecule related to endoplasmic reticulum (ER) stress-induced cell-death signaling pathways, in neuronal death resulting from ischemia/reperfusion in mice. Transient focal cerebral ischemia (1 h) was produced by intraluminal occlusion of the middle cerebral artery (MCA). We assessed the expression patterns of caspase-12, Bip/GRP78, an ER-resident molecular chaperone whose expression serves as a good marker of ER stress, and caspase-7 by Western blotting and/or immunohistochemistry. Double-fluorescent staining of caspase-12 immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-12 in cell death. We confirmed that ER stress was induced during reperfusion in our model, as witnessed by up-regulated Bip/GRP78 expression in the MCA territory. Western blot analysis revealed that caspase-12 activation occurred at 5-23 h of reperfusion, and immunoreactivity for caspase-12 was enhanced mainly in striatal neurons on the ischemic side at the same time points. We found the co-localization of caspase-12 immunoreactivity and DNA fragmentation detectable by the TUNEL method. We did not detect the presence of caspase-7 in the ER fraction at the period of caspase-12 cleavage. Our results imply that cerebral ischemia/reperfusion induces ER stress and that caspase-12 activation concurred with ER stress. Caspase-12 seems to be involved in neuronal death induced by ischemia/reperfusion. Caspase-7 is not likely to contribute to the cleavage of caspase-12 in our experimental model.

Upregulation of Akt phosphorylation at the early stage of middle cerebral artery occlusion in mice

Akt is a serine/threonine kinase that is believed to promote cell viability in many different cell types, including neurons. Here, we observed the state of Akt phosphorylation at several time points (1, 3, 6, 12, and 24 h) during permanent occlusion of the middle cerebral artery (MCA) in mice. We detected a transient upregulation of Akt phosphorylation at 1 h of MCA occlusion (MCAO) by Western blot analysis. Double immunostaining revealed that the enhanced phosphorylation of Akt occurred mainly in neurons located in the outer area of the MCA territory (ischemic penumbra). This phenomenon was accompanied by the nuclear translocation of Akt. We confirmed that Akt enzymatic activity is elevated in both the nuclear and cytosolic fractions of brain tissue subjected to 1 h of ischemia. cAMP-response-element-binding protein (CREB), an intranuclear target molecule of Akt, exhibited increased phosphorylation after 1 h of MCAO. In our ischemia model, caspase-3 was activated in the central part of the MCA territory as little as 1 h after MCAO. However, caspase-3 activation was not recognized at this time in the outer area of the MCA territory, where Akt activity was upregulated. These results suggest that prosurvival cell signaling is initiated in an active fashion before cell death pathways are activated in neurons situated in the ischemic penumbra at the early stage of ischemia.

Temporal Profiles of the Subcellular Localization of Bim, a BH3-Only Protein, During Middle Cerebral Artery Occlusion in Mice

BH3-only proteins are a subfamily of proapoptotic Bcl-2 proteins that act upstream of the mitochondrially mediated cell death pathway, and their association with the pathogenesis of brain ischemia remains largely unknown. The authors explored the temporal profiles of the expression levels and subcellular localization of BH3-only proteins in permanent middle cerebral artery occlusion (MCAO) by Western blot analysis. They observed an increased mitochondrial distribution of Bim at 3 to 6 hours of MCAO that appeared unrelated to transcriptional upregulation, as assessed by semiquantitative reverse transcription—polymerase chain reaction. At 3 to 6 hours of MCAO, Bim immunoreactivity was enhanced in neurons and oligodendrocytes in the ischemic regions. The increased mitochondrial localization of Bim coincided with a marked cytochrome c release and preceded the peak of caspase-9 activation. The authors observed an association of Bim with the dynein intermediate chain, a major component of the dynein motor complex, in the brain using a coimmunoprecipitation assay. Cerebral ischemia induced a time-dependent significant decrease in dynein expression, which started at 3 hours of MCAO. The authors deduced that the liberation of Bim from the dynein motor complex is a likely mechanism for the increased mitochondrial localization of Bim. During MCAO, Bad did not show any change in phosphorylation state or subcellular localization.

Nedd9 Protein, a Cas-L Homologue, Is Upregulated After Transient Global Ischemia in Rats Possible Involvement of Nedd9 in the Differentiation of Neurons After Ischemia

Background and Purpose— Some proteins involved in self-repair after stroke in the adult brain are primarily expressed during embryonic development and strongly down-regulated during the early postnatal phase. Neuronal precursor cell-expressed, developmentally down-regulated gene (Nedd) 9 was recognized to be identical to Crk-associated substrate lymphocyte type (Cas-L), a docking protein that associates with a variety of signaling molecules, such as focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), and Crk. We investigated the involvement of these proteins in the pathophysiology of global cerebral ischemia. Methods— The mouse Cas-L/Nedd9 cDNAs were cloned. The expression and function of Cas-L/Nedd9 protein in the pathogenesis of global ischemia in rats was investigated by RT-PCR, Western blot analysis, and immunohistochemistry. The neurite outgrowth of the transfectants of Nedd9 deletion mutants in PC-12 cells was also assessed to clarify the function of the Nedd9 protein. Results— Nedd9 was a splicing variant of Cas-L and was selectively induced in neurons of the cerebral cortex and hippocampus 1 to 14 days after the ischemia. Induced Nedd9 protein was tyrosine phosphorylated and was bound to FAK in dendrite and soma of neurons after the ischemia. Finally, it was demonstrated that Nedd9 promoted neurite outgrowth of PC-12 cells. Conclusions— Our study may support the potential of Nedd9 for participation in the differentiation of neurons after global ischemia in rats.

FK506 abrogates delayed neuronal death via suppression of nitric oxide production in rats

BACKGROUND AND PURPOSEnThe mechanism of the neuroprotective effect of FK506 in relation to nitric oxide (NO) production has not been clarified in vivo. We have investigated the effect of FK506 on ischemia-induced NO production in association with the pathogenesis of delayed neuronal death (DND) in rats.nnnMETHODSnIn vivo microdialysis was performed in the hippocampus of male Sprague-Dawley rats (250-350 g). Dialysate samples were collected every 3 min. In the ischemia group (n=16), global ischemia was induced for 21 min and reperfusion was achieved. In the FK506 treatment group (n=25), FK506 (1 mg/kg, i.v.) was administered 21 min prior to the onset of global ischemia. Sham operations were done (n=15). The levels of NO(2)(-) in the dialysate samples were determined by the Griess reaction. The animals were decapitated 7 days after ischemia. Coronal brain sections were stained with hematoxylin and eosin.nnnRESULTSnIn the ischemia group, the NO(2)(-) level significantly increased during ischemia. In the FK506 treatment group, there was no significant change in the NO(2)(-) level during ischemia. In histological examinations, FK506 treatment showed a neuroprotective effect against DND.nnnCONCLUSIONSnThe effect of FK506 inhibiting NO production contributes to the neuro-protective effect of FK506 on DND in the hippocampus.

Additive amelioration of oxidative stress and cardiac function by combined mineralocorticoid and angiotensin receptor blockers in postinfarct failing hearts.

Abstract: Reactive oxygen species are exacerbating factors in failing hearts. We examined whether spironolactone, a mineralocorticoid receptor antagonist, provides additional effects to olmesartan, an angiotensin II receptor blocker, on oxidative stress in postinfarct failing hearts. Congestive heart failure due to myocardial infarction (MI) was induced by the coronary artery ligation in rats. Three weeks later, the rats were divided into 4 groups: an untreated MI group, spironolactone (100 mg·kg−1·d−1)-treated MI group, olmesartan (10 mg·kg−1·d−1)-treated MI group, and combination-treated (spironolactone and olmesartan) MI group. After 7 weeks of MI, monotherapy improved left ventricular dilatation and function, and suppressed myocardial lipid peroxidation, in association with an attenuation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–dependent and mitochondrial superoxide production. Moreover, combination therapy caused a synergistic improvement in these indices. In experiments using cultured myocytes, aldosterone (100 nmole/L) and angiotensin II (100 nmole/L) enhanced both sources of superoxide production, although these humoral factors affected NADPH oxidase subunits (p47phox and gp91phox) differently. In conclusion, aldosterone and angiotensin II increase NADPH oxidase–dependent and mitochondrial superoxide production in myocytes, and the combination of an angiotensin II receptor blocker and mineralocorticoid receptor antagonist has a synergistic attenuation of cardiac oxidative stress, leading to an improvement in cardiac function in postinfarct failing hearts.

Sweating Function and Retinal Vasomotor Reactivity in Migraine

Sweating function and retinal arterial reactivity in patients with migraine were examined during headache-free intervals. The sweat glands were stimulated by intradermal injection of pilocarpine hydrochloride, and molds of sweat droplets were obtained using Silastic. The number of droplets in classic migraine was significantly lower than that in the controls. The retinal arterial response to changes in perfusion pressure was measured by means of a fundus camera. The retinal vasomotor index (-change in retinal arterial diameter (%)/change in effective MABP (mmHg)) was significantly lower in classic and in common migraine than in controls. The above results suggest that sweating function and retinal arterial reactivity are impaired in patients with migraine and that the impairment may play an important role in the pathophysiology of migraine attacks.

Brainstem auditory evoked responses and CT findings in multiple system atrophy

Brainstem auditory evoked responses (BAERs) and CT findings were comparatively studied in 11 patients with Shy-Drager syndrome and 10 patients with olivopontocerebellar atrophy. The I-III interpeak latencies (IPLs) were prolonged in 6 patients with Shy-Drager syndrome and in 6 patients with olivopontocerebellar atrophy. Mean values of the I-III IPLs were 2.54 +/- 0.28 ms (Shy-Drager syndrome) and 2.62 +/- 0.15 ms (olivopontocerebellar atrophy). In each disease, the I-III IPLs correlated well with the degree of the pontine atrophy estimated from the CT scan. The patients with Shy-Drager syndrome could be clinically divided into two varieties. In addition to autonomic dysfunction, one variety (4 patients) was linked with parkinsonism, and the other (7 patients) with signs of multiple nervous systems involvement. Prolongation of I-III IPLs and pontine atrophy were noted in 6 out of the latters patients, whereas the former patients did not show such abnormalities. The combination of BAERs and CT scan provides useful clinical information on multiple system atrophy.

Delayed phosphorylation of p38 mitogen-activated protein kinase in the AT1a knock-out mouse striatal neurons during middle cerebral artery occlusion and reperfusion.

To investigate whether the phosphorylation of p38 in cerebral ischemia occurs via angiotensin II receptor type 1a (AT1a), we examined the time course of phosphorylation of p38 and proline-rich tyrosine kinase 2 in AT1a knock-out mouse striatal neurons during middle cerebral artery occlusion (MCAO) and reperfusion. Phosphorylated-p38 was observed after 2 h and 5 h of reperfusion after 1 h of MCAO in C57/B6 mice and AT1a knock out mice, respectively. We demonstrated a delay of phosphorylation of p38 in the reperfusion model of the AT1a knock-out mouse, and detected microglia in the striatum on the ischemic side that were phosphorylated-p38-positive after 71 h of reperfusion in both animals. However, there was no association between AT1a and delayed neuronal cell death, or between AT1a and activation of caspase-9 in cerebral ischemia/reperfusion.

Autonomic Nervous Action Potential of Cerebral Veins

1. Averaged action potential waves were recorded from cerebral arteries and from cerebral veins during cervical sympathetic stimulation. The averaged potential waves were suppressed after the administration of hexamet honium or tetrodotoxin.