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We previously reported the effect of a selective inducer of BiP (a BiP inducer X; BIX) after permanent middle cerebral artery occlusion (MCAO) in mice. However, in acute stroke, almost all drugs have been used clinically after the onset of events. We evaluated the effect of post-treatment of BIX after permanent MCAO in mice, and examined its neuroprotective properties in in vivo mechanism. BIX (intracerebroventricular injection at 20μg) administered either at 5min or 3h after occlusion reduced both infarct volume and brain swelling, but at 6h after occlusion there was no reduction. BIX protected against the decrease in a dose-dependent manner. Furthermore, BIX reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells induced by the ischemia in ischemic penumbra. These findings indicate that post-treatment with BIX after ischemia has neuroprotective effects against acute ischemic neuronal damage in mice even when given up to 3h after MCAO. BIX may therefore be a potential drug for stroke.

Endoplasmic reticulum (ER) stress, which is caused by an accumulation of unfolded proteins in the ER lumen, is associated with stroke and with neurodegenerative diseases such as Parkinson’s and Alzheimer diseases. We assessed the expression patterns of immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein (GRP) 78 (an ER-resident molecular chaperone whose expression serves as a good marker of ER-stress), activating transcription factor (ATF)-4, and C/EBP homology protein (CHOP) by immunohistochemistry and/or Western blotting after transient forebrain ischemia in gerbils. Double-fluorescent staining involving CHOP immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of CHOP in cell death. Immunohistochemical and Western blot analyses of the hippocampal Cornet d’Ammon (CA)1 subfield showed that BiP expression was increased at 12 h, peaked at 3 days, then decreased (versus the control group). A transient increase was detected in CA3 at 1 day after ischemia, but BiP expression was unchanged in dentate gyrus and cortex. Signals for ATF-4 and CHOP were increased at 1 day and 3 days in CA1, and at 12 h in CA3. Co-localization of CHOP immunoreactivity and DNA fragmentation was detected by the TUNEL method at 3 days after ischemia in CA1, but not at 12 h in CA3. These findings are consistent with ER stress playing a pivotal role in post-ischemic neuronal death in the gerbil hippocampal CA1 subfield.

Rifampicin, an antibacterial drug, is highly effective in the treatment of tuberculosis and leprosy. Recently, it has been reported to have neuroprotective effects in in vitro and in vivo models. This study was designed to elucidate its neuroprotective effects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity (known as an in vivo mouse model of Parkinson's disease). Mice were injected intraperitoneally (i.p.) with MPTP (10 mg/kg) four times at 1- h intervals, and brains were analyzed 3 or 7 days later. Rifampicin at 20 mg/kg (i.p., twice) had protective effects against MPTP-induced neuronal damage (immunohistochemical changes in tyrosine hydroxylase) in both the substantia nigra and striatum. Rifampicin also protected against the MPTP-induced depletions of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum. The maximal concentrations of rifampicin between 30 and 240 min after a single rifampicin injection (20 mg/kg, i.p.) were 2.6 μM (at 30 min) in plasma and 0.77 μM (at 60 min) in striatum. Next, the effects of rifampicin on oxidative stress [lipid peroxidation in mouse brain homogenates and free radical-scavenging activity against diphenyl- p -picrylhydrazyl (DPPH)] were evaluated to clarify the underlying mechanism. At 1 μM or more, rifampicin significantly inhibited both lipid peroxidation in the striatum and free radical production. These findings suggest that in mice, rifampicin can reach brain tissues at concentrations sufficient to attenuate MPTP-induced neurodegeneration in the nigrostriatal dopaminergic neuronal pathway, and that an inhibitory effect against oxidative stress may be partly responsible for its observed neuroprotective effects.

SUN N8075 is a novel antioxidant with neuroprotective properties. This study was designed to elucidate its neuroprotective effects against 6-hydroxy dopamine (6-OHDA)-induced cell death and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity (known as in vitro and in vivo models of Parkinson's disease, respectively). In the in vitro study, on human neuroblastoma SH-SY5Y cells, SUN N8075 decreased the hydrogen peroxide (H2O2)-induced production of reactive oxygen species and protected against 6-OHDA-induced cell death. In the in vivo study, SUN N8075, when injected intraperitoneally (i.p.) twice with a 5-h interval, inhibited lipid peroxidation (viz. the production of thiobarbituric acid reactive substance) in the mouse forebrain at 1 h after the second injection. Mice were injected i.p. with MPTP (10 mg/kg) four times at 1-h intervals, and brains were analyzed 7 days later. SUN N8075 at 30 mg/kg (i.p., twice) exhibited a protective effect against the MPTP-induced decrease in tyrosine hydroxylase (TH)-positive fibers in the striatum. Moreover, SUN N8075 at 10 and 30 mg/kg (i.p., twice) had a similar protective effect against the MPTP-induced decrease in TH-positive cells in the substantia nigra. Further, SUN N8075 30 mg/kg (i.p. twice) markedly suppressed the MPTP-induced accumulation of 8-hydroxy-deoxyguanosine (8-OHdG) in the striatum. These findings indicate that SUN N8075 exerts protective effects, at least in part via an anti-oxidation mechanism, in these in vitro and in vivo models of Parkinson's disease.

Endoplasmic reticulum (ER) stress, which is caused by the accumulation of unfolded proteins in the ER lumen, is associated with stroke and neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. We evaluated the effect of a selective inducer of immunoglobulin heavy chain binding protein (BiP) (BiP inducer X; BIX) against both tunicamycin-induced cell death (in SH-SY5Y cells) and the effects of global transient forebrain ischemia (in gerbils). BIX significantly induced BiP expression both in vitro and in vivo. Pretreatment with BIX at 2 or 5 microM reduced the cell death induced by tunicamycin in SH-SY5Y cells. In gerbils subjected to forebrain ischemia, prior treatment with BIX (intracerebroventricular injection at 10 or 40 microg) protected against cell death and decreased TUNEL-positive cells in the hippocampal CA1 subfield. These findings indicate that this selective inducer of BiP could be used to prevent the neuronal damage both in vitro and in vivo.

(2S)-1-(4-Amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl) phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate (SUN N8075) is a novel antioxidant with neuroprotective properties. We examined whether SUN N8075 inhibited the neuronal damage resulting from permanent focal cerebral ischemia, and examined its neuroprotective properties in vivo and in vitro mechanism. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice, and the resulting infarction, brain swelling, and neurological deficits were evaluated after 24 h or 72 h. Brain damage was assessed histochemically using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and antibody recognizing 4-hydroxynonenal histidine adduct (4-HNE). In the in vitro study, we examined the effects of SUN N8075 on 1) lipid peroxidation in mouse brain homogenates and 2) cell viability and caspase-3 protease activity under a hypoxic insult or FeSO(4) in rat cultured cerebrocortical neurons. SUN N8075 administered either 10 min before or at 1 h after the occlusion reduced both infarction size and neurological deficits. SUN N8075 reduced brain swelling when administered 10 min before, 1 h, or 3 h after occlusion. Furthermore, only pretreatment (administered 10 min before) decreased infarct volume and brain swelling at 72 h after middle cerebral artery occlusion. SUN N8075 reduced the number of TUNEL-positive cells and decreased the level of oxidative damage, as assessed by immunopositive staining to 4-HNE. SUN N8075 inhibited lipid peroxidation, leakage of lactate dehydrogenase, caspase-3 activation induced by in vitro hypoxia, and the neuronal damage induced by in vitro FeSO(4) exposure. These findings indicate that SUN N8075 has neuroprotective effects against acute ischemic neuronal damage in mice and may prove promising as a therapeutic drug for stroke.

The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. ER stress-related markers [immunoglobulin binding protein (BiP)/glucose-regulated protein (GRP) 78, activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP)] in the striatum and the cortex were investigated after permanent middle cerebral artery occlusion (MCAO) in mice. Using endoplasmic reticulum stress-activated indicator (ERAI) transgenic mice, which show splicing of X-box protein 1 (XBP-1) mRNA as green fluorescence, we monitored the regional changes in fluorescence after MCAO. BiP mRNA (by reverse-transcription polymerase chain reaction [RT-PCR] analysis) was increased in the cortex at 6 h. In immunohistochemical and/or Western blot analysis, the expressions of ER stress-related markers (BiP, ATF-4, and CHOP) were increased in the infarct region, more strongly in the cortex than in the striatum. ERAI fluorescence was observed in the ischemic area starting from 6 and 12 h, respectively, after MCAO, with the peaks at 1 day and the fluorescence co-localized with the 2,3,5-triphenyltetrazolium chloride (TTC)-visible extension of brain infarction. These findings suggest that permanent MCAO induces expression of ER-stress related genes mainly in the periphery of the MCA territory.

To test our hypothesis that somatosensory inputs would influence postural modulation of soleus H-reflex, eleven subjects were investigated under the head-out water immersion (HOWI) conditions. Subjects were supine or standing on a tilting bed in each condition. They were instructed to maintain an upright posture with both legs. The water was filled to the subject's neck level in a test tank to reduce 95% of the gravitational effect by buoyancy. Surface electromyography of the soleus and tibialis anterior was measured. The soleus H-reflex was elicited at a stimulation intensity of 1.05 times the motor threshold. The recruitment profile of the motor response was unchanged between the conditions. The background activities of the soleus and tibialis anterior were not detected in any condition. The peak-to-peak amplitude of the H-reflex was significantly different between the conditions while the stimulation intensity (small M size) was not different. The soleus H-reflex during standing was significantly decreased compared with being supine in the control condition, whereas it did not in the HOWI condition. It was concluded that somatosensory inputs due to gravity exert an influence on postural modulation of the soleus H-reflex to maintain static posture in humans.

To examine the association of level of functional fitness to demographic, health, and life behavioral or social factors, cross sectional data were obtained for 737 persons aged 60 years or older, and who were independently living in the community. Functional fitness was measured with a functional fitness test containing 4 task items: standing, walking, hand performance, and self-care performance. Among the demographic factors, statistically significant associations with functional fitness were found for age in both male and female and for the presence of spouse in male. Health status, previous or present history of circulatory diseases and musculo-skeletal diseases were significantly associated with lower levels of functional fitness in male, and previous or present history of musculo-skeletal diseases and presence of higher obesity associated with lower fitness level in female. With life behaviors, men who had habitual exercise activities and women who had no habitual nap but habitual exercise activities and frequent out-of-home activities showed significantly higher fitness level than their counterparts. These results suggest that level of functional fitness in independently living aged people in the community was significantly associated with the presence of spouse, history of circulatory and musculo-skeletal diseases, and habitual exercise activities in males; and with the history of musculo-skeletal diseases, obesity, and habitual exercise activities, napping, and frequent out-of-home activities in females.

A test of functional fitness defined as physical capacity to independently perform daily functional activities was developed for aged persons. The functional fitness test was composed of four physical capacity evaluation tasks representing physical abilities necessary to perform main activities of daily living; viz. sitting and standing up test, zig-zag walking test, hand working test with pegboard for dexterity evaluation, and rope working test for self-care evaluation. The reliability and feasibility of the test were examined with 765 aged persons living in the community. The distribution of measurement values in each item showed neither extreme skewness nor kurtosis. Retest reliabilities for each task were 0.857 to 0.942, but the second trial showed significantly reduced (p0.001) values than the first trial in test-retest. Significant relationships (r = 0.503 to 0.627) between measurement values in each items and chronological age were found in both male and female. Functional fitness test and physical fitness test scores were correlated 0.740. These results showed that the functional fitness test developed in the present study has a high reliability and feasibility to evaluate functional capacity of daily living in aged persons.