Yunxia Duan

Safety and Efficacy of Remote Ischemic Preconditioning in Patients with Severe Carotid Artery Stenosis Prior to Carotid Artery Stenting: A Proof-of-Concept, Randomized Controlled Trial

Background: Remote ischemic preconditioning (RIPC) can inhibit recurrent ischemic events effectively in patients with acute or chronic cerebral ischemia. However, it is still unclear whether RIPC can impede ischemic injury after carotid artery stenting (CAS) in patients with severe carotid artery stenosis. Methods: Subjects with severe carotid artery stenosis were recruited in this randomized controlled study, and assigned to RIPC, sham, and no intervention (control) groups. All subjects received standard medical therapy. Subjects in the RIPC and sham groups underwent RIPC and sham RIPC twice daily, respectively, for 2 weeks before CAS. Plasma neuron-specific enolase and S-100B were used to evaluate safety, hypersensitive C-reactive protein, and new ischemic diffusion-weighted imaging lesions were used to determine treatment efficacy. The primary outcomes were the presence of ≥1 newly ischemic brain lesions on diffusion-weighted imaging within 48 hours after stenting and clinical events within 6 months after stenting. Results: We randomly assigned 189 subjects in this study (63 subjects in each group). Both RIPC and sham RIPC procedures were well tolerated and completed with high compliance (98.41% and 95.24%, respectively). Neither plasma neuron-specific enolase levels nor S-100B levels changed significantly before and after treatment. No severe adverse event was attributed to RIPC and sham RIPC procedures. The incidence of new diffusion-weighted imaging lesions in the RIPC group (15.87%) was significantly lower than in the sham group (36.51%; relative risk, 0.44; 96% confidence interval, 0.20–0.91; P<0.01) and the control group (41.27%; relative risk, 0.39; 96% confidence interval, 0.21–0.82; P<0.01). The volumes of lesions were smaller in the RIPC group than in the control and sham groups (P<0.01 each). Ischemic events that occurred after CAS were 1 transient ischemic attack in the RIPC group, 2 strokes in the control group, and 2 strokes and 1 transient ischemic attack in the sham group, but these results were not significantly different among the 3 groups (P=0.597). Conclusions: RIPC is safe in patients undergoing CAS, which may be able to decrease ischemic brain injury secondary to CAS. However, the mechanisms and effects of RIPC on clinical outcomes in this cohort of patients need further investigation. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01654666

Effects of Erythropoietin on Gliogenesis during Cerebral Ischemic/Reperfusion Recovery in Adult Mice

Erythropoietin (EPO) promotes oligodendrogenesis and attenuates white matter injury in neonatal rats. However, it is unknown whether this effect extends to adult mice and whether EPO regulate microglia polarization after ischemic stroke. Male adult C57BL/6 mice (25–30g) were subjected to 45 min of middle cerebral artery occlusion (MCAO). EPO (5000 IU/kg) or saline was injected intraperitoneally every other day after reperfusion. Neurological function was evaluated using the rotarod test at 1, 3, 7 and 14 days after MCAO. Brain tissue loss volume was determined by hematoxylin-eosin staining. Immunofluorescence staining and Western blot were also used to assess the severity of white matter injury and phenotypic changes in microglia/macrophages. Bromodeoxyuridine (BrdU) was injected intraperitoneally daily for 1 week to analyze the number of newly proliferating glia cells (oligodendrocytes, microglia, and astrocytes). We found that EPO significantly reduced Brain tissue loss volume, ameliorated white matter injury, and improved neurobehavioral outcomes at 14 days after MCAO (P<0.05). In addition, EPO also increased the number of newly generated oligodendrocytes and attenuated the rapid hypertrophy and hyperplasia of microglia and astrocytes after ischemic stroke (P<0.05). Furthermore, EPO reduced M1 microglia and increased M2 microglia (P<0.05). Taken together, our results suggest that EPO treatment improves white matter integrity after cerebral ischemia, which could be attributed to EPO attenuating gliosis and facilitating the microglial polarization toward the beneficial M2 phenotype to promote oligodendrogenesis.

A new idea about reducing reperfusion injury in ischemic stroke: Gradual reperfusion.

Around the world, stroke is the second most common cause of death and a major cause of disability. The main direct cause of stroke is the occlusion of intracranial artery, which leads to cell death in the core suffered region, or cell functional impairment surrounding the dead core (termed ischemic penumbra). Opening the occluded artery to save the ischemic penumbra is the aim of thrombolysis therapy. But the reperfusion induced injury counteracts the potential profit by thrombolysis. Herein, we assume that gradual reperfusion can reduce the reperfusion injury by reducing the production of free radicals during reperfusion. The reason is: free radicals are critical in the reperfusion injury; free radicals come from the penumbra during reperfusion; the respiratory chain is the main source of free radical; the enzyme activity of the respiratory chain is upgraded during ischemia; once reperfused, the activity upgraded enzymes in the respiratory chain meet normal amount of oxygen and glucose, which produces exceeding intermediates (free radicals); while gradual reperfusion reduces the production of free radicals, because it can confine the amount of oxygen and glucose.

Chrysophanol inhibits endoplasmic reticulum stress in cerebral ischemia and reperfusion mice

Abstract Endoplasmic reticulum (ER) stress plays a critical role in mediating ischemia/reperfusion (I/R) damage in the brain. Our previous study showed that Chrysophanol (CHR) alleviated cerebral ischemic injury in mice and nuclear factor‐&kgr;B (NF‐&kgr;B) involved in its neuroprotective effect, but the precise mechanism remains not fully understood. The present study investigated the effect of CHR treatment on I/R‐induced ER stress. Mice were subjected to middle cerebral artery occlusion (MCAO) for 45 min and received either vehicle or CHR (0.1 mg/kg) for 14 days after reperfusion. Terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in penumbral tissue. The expression of ER stress‐related factors including glucose‐regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2&agr; (p‐eIF2&agr;), CCAAT‐enhancer‐binding protein homologous protein (CHOP), and caspase‐12 as well as inhibitory &kgr;B‐&agr; (I&kgr;B‐&agr;), the inhibitor of NF‐&kgr;B, was assessed. Our results demonstrated that CHR treatment reduced MCAO‐induced upregulation of GRP78, p‐eIF2&agr;, CHOP, and caspase‐12 in the ischemic brain. Moreover, the TUNEL‐positive neuronal cells, which were colocalized with CHOP and caspase‐12, decreased in response to CHR treatment, indicating that CHR protects against I/R injury by inhibiting ER stress‐associated neuronal apoptosis. In addition, CHR reversed the decrease in I&kgr;B‐&agr; level induced by MCAO, which was attributed at least in part to the attenuation of translational inhibition induced by eIF2&agr; phosphorylation, indicating that CHR exerts anti‐inflammatory effects following I/R by inhibiting ER stress response. These results suggest that attenuation of ER stress may be involved in the mechanisms of neuroprotective effects of CHR.

Erythropoietin attenuates axonal injury after middle cerebral artery occlusion in mice

Abstract Objective: Erythropoietin (EPO) confers potent neuroprotection against ischemic injury through a variety of mechanisms. However, the protective effect of EPO on axons after cerebral ischemia in adult mice is rarely covered. The purpose of this study was to investigate the potential neuroprotective effects of EPO on axons in mice after cerebral ischemia. Methods: A total of 30 adult male C57 BL/6 mice were treated with EPO (5000 IU/kg) or vehicle after transient middle cerebral artery occlusion (MCAO). The mortality rate of each experimental group was calculated. Neurological function was assessed by Rota-rod test. Frozen sections from each mouse brain at 14 days after reperfusion were used to evaluate the fluorescent intensity of myelin basic protein (MBP) and neurofilament 200 (NF-200). Immunofluorescence staining and Western blotting were used to assess the protein level of β-amyloid precursor protein (β-APP) and glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. The protein levels of the myelin-derived growth inhibitory proteins, neurite growth inhibitor-A (Nogo-A), myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMG) were also examined by Western blot after MCAO. Results: The survival rate of the vehicle group 14 days after cerebral ischemia-reperfusion was 50%, which increased to 80% after EPO treatment at the start of reperfusion. EPO improved neurobehavioral outcomes at days 3 and 7 after MCAO was compared with the vehicle group (P < 0.05). Furthermore, EPO ameliorated demyelination, demonstrated by upregulation of the MBP/NF-200 ratio. Meanwhile, increased levels of β-APP, GFAP, Nogo-A, and MAG after MCAO were reduced by EPO treatment (P < 0.05). Conclusion: EPO treatment attenuates axonal injury and improves neurological function after cerebral ischemia in adult mice.

Neuroprotection by peripheral nerve electrical stimulation and remote postconditioning against acute experimental ischaemic stroke

Abstract Objective: Local electrical stimulation (ES) was reported to protect the brain during ischaemic injury, while the protective effect of limb remote ischaemic postconditioning (RIPostC) was confirmed. The aim of this study was to explore whether remote peripheral nerve ES exerted neuroprotection and whether this procedure shared the same neuroprotective mechanism underlying RIPostC. Methods: Stroke in Sprague-Dawley rats was induced by distal middle cerebral artery occlusion (dMCAO). Rats were divided into five groups: dMCAO, RIPostC, ES, nerve resection (NR) + ES and RIPostC+ES. Twenty-four hours after reperfusion, rats were examined for neurobehavioural function, including forelimb fault placing test, Ludmila Belayev 12 score test, and infarct volume. The expression of Bcl-2 and cleaved-caspase-3 in ischaemic cortex was assessed by Western blot. Results: In forelimb fault placing test, as compared to the highest score in the stroke-only group, RIPostC, ES and RIPostC+ES groups showed a significantly (P < 0.01) lower score. The results were similar for the Ludmila Belayev 12 score test. The infarct volume of the treatment groups also exhibited significant (P < 0.01) reduction as compared to the stroke-only group. The volume of infarct tissue in the combination of RIPostC+ES was significantly less than RIPostC and ES alone (P < 0.05). Furthermore, NR blocked the ESs protection (P < 0.05) as compared to the ES group by using above-mentioned methods. Bcl-2 was upregulated, while cleaved-caspase-3 was downregulated in the experimental groups as compared to the control group. No difference was found among the experimental groups. Discussion: Peripheral nerve ES appears to have a neuroprotective effect in a rat dMCAO model. This effect may indicate a neural protective mechanism underlying beneficial effect of RIPostC.

Dihydrocapsaicin (DHC) enhances the hypothermia-induced neuroprotection following ischemic stroke via PI3K/Akt regulation in rat

OBJECTIVE Hypothermia has demonstrated neuroprotection following ischemia in preclinical studies while its clinical application is still very limited. The aim of this study was to explore whether combining local hypothermia in ischemic territory achieved by intra-arterial cold infusions (IACIs) with pharmacologically induced hypothermia enhances therapeutic outcomes, as well as the underlying mechanism. METHODS Sprague-Dawley rats were subjected to right middle cerebral artery occlusion (MCAO) for 2h using intraluminal hollow filament. The ischemic rats were randomized to receive: 1) pharmacological hypothermia by intraperitoneal (i.p.) injection of dihydrocapsaicin (DHC); 2) physical hypothermia by IACIs for 10min; or 3) the combined treatments. Extent of brain injury was determined by neurological deficit, infarct volume, and apoptotic cell death at 24h and/or 7d following reperfusion. ATP and ROS levels were measured. Expression of p-Akt, cleaved Caspase-3, pro-apoptotic (AIF, Bax) and anti-apoptotic proteins (Bcl-2, Bcl-xL) was evaluated at 24h. Finally, PI3K inhibitor was used to determine the effect of p-Akt. RESULTS DHC or IACIs each exhibited hypothermic effect and neuroprotection in rat MCAO models. The combination of pharmacological and physical approaches led to a faster and sustained reduction in brain temperatures and improved ischemia-induced injury than either alone (P<0.01). Furthermore, the combination treatment favorably increased the expression of anti-apoptotic proteins and decreased pro-apoptotic protein levels (P<0.01 or 0.05). This neuroprotective effect was largely blocked by p-Akt inhibition, indicating a potential role of Akt pathway in this mechanism (P<0.01 or 0.05). CONCLUSIONS The combination approach is able to enhance the efficiency of hypothermia and efficacy of hypothermia-induced neuroprotection following ischemic stroke. The findings here move us a step closer towards translating this long recognized TH from bench to bedside.

Effects of hypoxic preconditioning on synaptic ultrastructure in mice.

Hypoxic preconditioning (HPC) elicits resistance to more drastic subsequent insults, which potentially provide neuroprotective therapeutic strategy, but the underlying mechanisms remain to be fully elucidated. Here, we examined the effects of HPC on synaptic ultrastructure in olfactory bulb of mice. Mice underwent up to five cycles of repeated HPC treatments, and hypoxic tolerance was assessed with a standard gasp reflex assay. As expected, HPC induced an increase in tolerance time. To assess synaptic responses, Western blots were used to quantify protein levels of representative markers for glia, neuron, and synapse, and transmission electron microscopy was used to examine synaptic ultrastructure and mitochondrial density. HPC did not significantly alter the protein levels of astroglial marker (GFAP), neuron‐specific markers (GAP43, Tuj‐1, and OMP), synaptic number markers (synaptophysin and SNAP25) or the percentage of excitatory synapses versus inhibitory synapses. However, HPC significantly affected synaptic curvature and the percentage of synapses with presynaptic mitochondria, which showed concomitant change pattern. These findings demonstrate that HPC is associated with changes in synaptic ultrastructure. Synapse 69:7–14, 2015.

Synergistically Induced Hypothermia and Enhanced Neuroprotection by Pharmacological and Physical Approaches in Stroke

Hypothermia is considered as a promising neuroprotective treatment for ischemic stroke but with many limitations. To expand its clinical relevance, this study evaluated the combination of physical (ice pad) and pharmacological [transient receptor potential vanilloid channel 1 (TRPV1) receptor agonist, dihydrocapsaicin (DHC)] approaches for faster cooling and stronger neuroprotection. A total of 144 male Sprague Dawley rats were randomized to 7 groups: sham (n=16), stroke only (n=24), stroke with physical hypothermia at 31ºC for 3 h after the onset of reperfusion (n=24), high-dose DHC (H-DHC)(1.5 mg/kg, n=24), low-dose DHC (L-DHC)(0.5 mg/kg, n=32) with (n=8) or without (n=24) external body temperature control at ~38 ºC (L-DHC, 38 ºC), and combination therapy (L-DHC+ ice pad, n=24). Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Infarct volume, neurological deficits and apoptotic cell death were determined at 24 h after reperfusion. Expression of pro- and anti-apoptotic proteins was evaluated by Western blot. ATP and reactive oxygen species (ROS) were detected by biochemical assays at 6 and 24 h after reperfusion. Combination therapy of L-DHC and ice pad significantly improved every measured outcome compared to monotherapies. Combination therapy achieved hypothermia faster by 28.6% than ice pad, 350% than L-DHC and 200% than H-DHC alone. Combination therapy reduced (p<0.05) neurological deficits by 63% vs. 26% with L-DHC. No effect was observed when using ice pad or H-DHC alone. L-DHC and ice pad combination improved brain oxidative metabolism by reducing (p<0.05) ROS at 6 and 24 h after reperfusion and increasing ATP levels by 42.9% compared to 25% elevation with L-DHC alone. Finally, combination therapy decreased apoptotic cell death by 48.5% vs. 24.9% with L-DHC, associated with increased anti-apoptotic protein and reduced pro-apoptotic protein levels (p<0.001). Our study has demonstrated that combining physical and pharmacological hypothermia is a promising therapeutic approach in ischemic stroke, and warrants further translational investigations.

Cerebral watershed infarcts may be induced by hemodynamic changes in blood flow

Abstract Objectives: A watershed infarct is defined as an ischemic lesion at the border zones between territories of two major arteries. The pathogenesis of watershed infarcts, specifically whether they are caused by hemodynamic or embolic mechanisms, has long been debated. In this study, we aimed to examine whether watershed infarcts can be induced by altering the hemodynamic conditions in rats. Materials and Methods: In phase one, to determine the proper clamping duration for a reproducible infarct, 30 rats were equally divided into 5 subgroups and underwent bilateral common carotid artery (CCA) clamping for different durations (0.5, 1.0, 1.5, 2.0, and 3.0 hours). In phase two, to analyze the types of infarcts induced by bilateral CCA clamping, 40 rats were subjected to bilateral CCA clamping for 2 hours. As a control, 8 rats underwent all the operation procedures except bilateral CCA clamping. We performed 7.0T magnetic resonance imaging on the surviving rats on the second day to evaluate the extent of the infarcts. We further identified and examined the infarcts with brain slices stained using 2, 3, 5-triphenyltetrazolium chloride (TTC) on the third day. Results: After 2 hours of bilateral CCA clamping, cerebral infarction occurred in 42% of surviving rats (13/31). The majority of the ischemic lesions were located in watershed regions of the brain, demonstrated by both MRI and TTC staining. Conclusion: Watershed infarcts were induced through changing hemodynamic conditions by bilateral CCA clamping in rats. This method may lead to the development of a reliable rodent model for watershed infarcts.