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Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has been demonstrated to protect against ischemic cerebral damage in vitro and in vivo. In this study, we aimed to further define the preclinical characteristics of Rd. We show that Rd passes the intact blood-brain barrier and exerts protection in both transient and permanent middle cerebral artery occlusion (MCAO) in rats. In the dose-response study, Rd (10–50 mg/Kg) significantly reduced the infarct volume on postoperative days (PODs) 1, 3, and 7. This protection was associated with an improved neurological outcome for as many as 6 weeks after transient MCAO, as assessed by modified neurological severity score, modified sticky-tape test, and corner test. For comparison, Rd was significantly more effective than edaravone and slightly more effective than N-tert-butyl-alpha-phenylnitrone (PBN). In the therapeutic window study, Rd exhibited remarkable neuroprotection, even when administered for as many as 4 h after the recirculation of transient MCAO or after the onset of permanent MCAO. Furthermore, in female rats or 16-month-old male rats, the salutary effects of Rd were also observed. These findings suggest Rd is a promising neuroprotectant and provide support for future clinical studies to confirm whether Rd is beneficial in ischemic stroke.

Background Signal transducer and activator of transcription 3 (STAT3) activation in ischemic brain has been verified. However, the mechanism and the role of STAT3 activation after cerebral ischemia-reperfusion are poorly elucidated. In the present study, we sought to test the hypothesis that STAT3 activation after cerebral ischemia-reperfusion was related to reactive oxygen species (ROS) production. Methods Adult male Sprague-Dawley rats were subjected to focal cerebral ischemia induced by middle cerebral artery occlusion. STAT3 activation was evaluated by immunohistochemistry and Western blotting. Rats were subjected to permanent ischemia or ischemia-reperfusion to clarify the temporal profile of STAT3 activation. The role of ROS in inducing STAT3 activation was assessed by administration of the ROS scavenger dimethylthiourea (DMTU). The effects of DMTU and the STAT3 activation inhibitor AG490 administration on brain ischemic injuries were evaluated by neurologic behavior scores and brain infarct volumes. Results The activation of STAT3 after middle cerebral artery occlusion was significantly increased within peri-ischemia neurons and astrocytes. STAT3 activation mainly occurred in the reperfusion phase rather than in the ischemia phase. In addition, DMTU suppressed STAT3 activation in a dose-dependent manner, indicating that STAT3 activation may be a subsequent event after ROS production. DMTU and AG490 significantly reduced infarct sizes and improved neurologic outcomes. Conclusion In comparison with ischemia, reperfusion is a more powerful stimulus for STAT3 activation. ROS scavenging is closely correlated with an inhibition of STAT3 activation. Neuroprotective effects are achieved through ROS scavenging and down-regulation of STAT3 activation.

Age is the single most important risk factor for cerebral stroke. Unfortunately, the effect of age on ischemic brain damage is less clear. In this study, we sought to examine the potential influence of aging on the histologic and functional outcomes after ischemia. Juvenile (4 weeks of age), young adult (4 months of age), mid-aged (11-12 months of age), and aged (18-19 months of age) mice were subjected to transient middle cerebral artery occlusion. There was no remarkable difference of infarct volume on postoperative days 1 and 3. However, on postoperative day 7, aged mice exhibited significantly worsened infarct volume compared with juvenile and young mice. Intriguingly, the increase of infarct volume was most prominent in the striatal area rather than in cortex. Accordingly, aged mice displayed a slower and incomplete functional recovery after stroke. We further evaluated the effects of aging on the oxidative damage and mitochondrial dysfunction following ischemia. Brain tissues were assayed for lipid, DNA, and protein peroxidation products, mitochondrial enzyme activities, mitochondrial membrane potential, production of reactive oxygen species, and antioxidant activities. Aging was associated with declined mitochondrial function and antioxidant detoxification following ischemia, thereby inducing a deteriorated oxidative damage. Regional subanalyses demonstrated that, in accordance with infarct area, the pro-oxidant/antioxidant imbalance occurred more prominently in subcortical areas. Collectively, these findings suggest mitochondria-mediated oxidative damage may be involved in the age-related aggravated injury in subcortical areas. Mitochondrial protection could be a promising target for neuroprotective therapy, especially in the aged population.

BACKGROUND: It has been reported that sevoflurane preconditioning can induce neuroprotection, the mechanisms of which, however, are poorly elucidated. We designed the present study to examine the hypothesis that sevoflurane preconditioning could reduce cerebral ischemia– reperfusion injury through up-regulating antioxidant enzyme activities before ischemic injury by generating reactive oxygen species (ROS). METHODS: In preconditioning groups, adult male Sprague–Dawley rats were pretreated with 1 hour sevoflurane exposure at a dose of 1%, 2%, or 4% for 5 consecutive days. At 24 hours after the last exposure, all rats were subjected to focal brain ischemia induced by middle cerebral artery occlusion for 120 minutes followed by 72-hour reperfusion. The role of ROS in ischemic tolerance was assessed by administration of the free radical scavenger dimethylthiourea and antioxidant N-acetylcysteine before each preconditioning. Brain ischemic injury was evaluated by neurologic behavior scores and brain infarct volume calculation. Antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase [GSH-px]) of brain tissue and blood serum were tested at 24 hours after the last sevoflurane preconditioning. RESULTS: Sevoflurane preconditioning reduced infarct size and improved neurobehavioral outcome in a dose-dependent manner. The neuroprotective effects of sevoflurane preconditioning were abolished by dimethylthiourea and N-acetylcysteine. The activities of catalase and glutathione peroxidase (GSH-px) in the brain tissue were elevated by sevoflurane preconditioning before ischemic injury. The up-regulated activity of GSH-px in serum negatively correlated with brain infarct volume percentage. CONCLUSION: Sevoflurane preconditioning induces cerebral ischemic tolerance in a dose– response manner through ROS release and consequent up-regulation of antioxidant enzyme activity before ischemic injury in rats. Serum GSH-px activity could be developed as a marker to assess the effectiveness of sevoflurane preconditioning before ischemia.

Background and Purpose— Our previous study has demonstrated that the rapid tolerance to cerebral ischemia by electroacupuncture (EA) pretreatment was possibly mediated through an endocannabinoid system-related mechanism. The purpose of this study was to investigate whether activation of epsilon protein kinase C (εPKC) was involved in EA pretreatment-induced neuroprotection via cannabinoid receptor type 1 in a rat model of transient focal cerebral ischemia. Methods— The activation of εPKC in the ipsilateral brain tissues after EA pretreatment was investigated in the presence or absence of cannabinoid receptor antagonists. At 2 hours after the end of EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 minutes in rats. The neurobehavioral scores, infarction volumes, neuronal apoptosis, and the expression of Bcl-2 and Bax were evaluated after reperfusion in the presence or absence of εPKC-selective peptide inhibitor (TAT-εV1–2) or activator (TAT–ψεRACK). Results— EA pretreatment enhanced εPKC activation. Systemic delivery of TAT–ψεRACK conferred neuroprotection against a subsequent cerebral ischemic event when delivered 2 hours before ischemia. Pretreatment with EA reduced infarct volumes, improved neurological outcome, inhibited neuronal apoptosis, and increased the Bcl-2 -to- Bax ratio after reperfusion, and the beneficial effects were attenuated by TAT-εV1–2. In addition, the blockade of cannabinoid receptor type 1, but not cannabinoid receptor type 2 receptor, reversed the increase in εPKC activation and neuroprotection induced by EA pretreatment. Conclusion— EA pretreatment may activate endogenous εPKC-mediated anti-apoptosis to protect against ischemic damage after focal cerebral ischemia via cannabinoid receptor type 1, which represents a new mechanism of EA pretreatment-induced rapid tolerance to focal cerebral ischemia in rats.

We previously found that ginsenoside Rd (Rd), one of the major active ingredients in Panax ginseng, protects neuronal cells from hydrogen peroxide and oxygen-glucose deprivation, an in vitro model of cerebral ischemia. In this study, we examined the protective effects of Rd in an animal model of focal cerebral ischemia. Rats administered with Rd or vehicle were subjected to transient middle cerebral artery occlusion (MCAO). Rd (50 mg/kg) significantly reduced the infarct volume by 52.8%. This reduction of injury volume was associated with an improvement in neurological function and was sustained for at least 2 weeks after the induction of ischemia. To evaluate the underlying mechanisms of Rd against stroke, brain tissues were assayed for mitochondrial enzyme activities, mitochondrial membrane potential (MMP), production of reactive oxygen species (ROS), energy metabolites, and apoptosis. Rd markedly protected mitochondria as indicated by preserved respiratory chain complex activities and aconitase activity, lowered mitochondrial hydrogen peroxide production, and hyperpolarized MMP. Microdialysis results illustrated that Rd significantly decreased the accumulation of lactate, the end product of anaerobic glycolysis, and increased pyruvate, the end product of aerobic glycolysis, hence inducing a lower lactate/pyruvate ratio. Additionally, in vitro studies further exhibited that Rd protected isolated mitochondria from calcium-induced damage by attenuating mitochondrial swelling, preserving MMP and decreasing ROS production. Moreover, Rd treatment reduced mitochondrial release of cytochrome c (CytoC) and apoptosis-inducing factor (AIF), thereby minimizing mitochondria-mediated apoptosis following ischemia. In conclusion, these findings demonstrated that Rd exerts neuroprotective effects in transient focal ischemia, which may involve an integrated process of the mitochondrial protection, energy restoration and inhibition of apoptosis.

We previously found that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, attenuates neuronal oxidative damage in vitro induced by hydrogen peroxide and oxygen-glucose deprivation. In this study, we sought to investigate the potential protective effects and associated mechanisms of Rd in a rat model of focal cerebral ischemia. Rats administered with Rd (0.1-200mg/kg) or vehicle was subjected to transient middle cerebral artery occlusion. Rd at the dose of 10-50mg/kg significantly reduced the infarct volume and improved the long-term neurological outcome up to 6 weeks after ischemia. To evaluate the underlying mechanisms, in vivo free radical generation was monitored using microdialysis, oxidative DNA damage was identified by 8-hydroxy-deoxyguanosine immunostaining, oxidative protein damage was identified by the assessment of protein carbonyl and advanced glycosylation end products, and lipid peroxidation was estimated by determining the malondialdehyde and 4-hydroxynonenal formations. Microdialysis results displayed a prominent inhibitory effect of Rd on the hydroxy radical formation trapped as 2,3- and 2,5-DHBA. Early accumulations of DNA, protein and lipid peroxidation products were also suppressed by Rd treatment. Although Rd partly preserved endogenous antioxidant activities in the ischemic penumbra, in sham rats without stroke, endogenous antioxidant activities were not affected by Rd. Furthermore, we assayed sequential inflammatory response in a later phase after ischemia. Rd significantly eliminated inflammatory injury as indicated by the suppression of microglial activation, inducible nitric oxide synthase and cyclooxygenase-2 expression. Collectively, these findings demonstrated that Rd exerts neuroprotection in transient focal ischemia, which may involve early free radicals scavenging pathway and a late anti-inflammatory effect.

Currently available therapeutics has been less effective in promoting functional recovery from stroke or other injuries in the central nervous system (CNS). Axonal damage is a characteristic pathology seen in CNS injuries. Previously, it was reported that Nogo-A extracellular peptide residues 1-40 (NEP1-40), a competitive antagonist of Nogo-66 receptor (NgR1), has the ability to promote axonal regrowth and functional recovery after CNS injury. However, delivery of the therapeutic proteins into the brain parenchyma is limited due to its inability to cross the blood-brain barrier (BBB). We first generated a biologically active NEP1-40 fusion protein containing the protein transduction domain (PTD) of the transactivator of transcription (TAT), TAT-NEP1-40, which crosses the BBB in vivo after systemic delivery. The TAT-NEP1-40 can protect PC12 cells against oxygen and glucose deprivation (OGD) and promote neurite outgrowth when added exogenously to culture medium. The TAT-NEP1-40 protein transduced into the brain continued to sustain biological activities and protected the brain against ischemia/reperfusion injury through inhibition of neuronal apoptosis. Collectively, our data suggest that TAT-NEP1-40 may be a novel therapeutic candidate for axonal regeneration and functional recovery from CNS injuries such as cerebral hypoxia-ischemia, cerebral hemorrhage, brain trauma, and also for spinal cord injury.

Background We have previously reported that electroacupuncture (EA) pretreatment induced tolerance against cerebral ischemic injury, but the mechanisms underlying this effect of EA are unknown. In this study, we assessed the effect of EA pretreatment on the expression of α7 nicotinic acetylcholine receptors (α7nAChR), using the ischemia-reperfusion model of focal cerebral ischemia in rats. Further, we investigated the role of high mobility group box 1 (HMGB1) in neuroprotection mediated by the α7nAChR and EA. Methods Rats were treated with EA at the acupoint "Baihui (GV 20)" 24 h before focal cerebral ischemia which was induced for 120 min by middle cerebral artery occlusion. Neurobehavioral scores, infarction volumes, neuronal apoptosis, and HMGB1 levels were evaluated after reperfusion. The α7nAChR agonist PHA-543613 and the antagonist α-bungarotoxin (α-BGT) were used to investigate the role of the α7nAChR in mediating neuroprotective effects. The roles of the α7nAChR and HMGB1 release in neuroprotection were further tested in neuronal cultures exposed to oxygen and glucose deprivation (OGD). Results Our results showed that the expression of α7nAChR was significantly decreased after reperfusion. EA pretreatment prevented the reduction in neuronal expression of α7nAChR after reperfusion in the ischemic penumbra. Pretreatment with PHA-543613 afforded neuroprotective effects against ischemic damage. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and HMGB1 release following reperfusion, and the beneficial effects were attenuated by α-BGT. The HMGB1 levels in plasma and the penumbral brain tissue were correlated with the number of apoptotic neurons in the ischemic penumbra. Furthermore, OGD in cultured neurons triggered HMGB1 release into the culture medium, and this effect was efficiently suppressed by PHA-543,613. Pretreatment with α-BGT reversed the inhibitory effect of PHA-543,613 on HMGB1 release. Conclusion These data demonstrate that EA pretreatment strongly protects the brain against transient cerebral ischemic injury, and inhibits HMGB1 release through α7nAChR activation in rats. These findings suggest the novel potential for stroke interventions harnessing the anti-inflammatory effects of α7nAChR activation, through acupuncture or pharmacological strategies.