Optimal stimulation parameters for Renzhong (DU 26) electro-acupuncture for improving motor function in a rat model of middle cerebral artery occlusion

The selection of electro-acupuncture parameters remains poorly unified between clinical studies. The present study observed the effects of electro-acupuncturing Renzhong （DU 26） with different stimulation parameters on motor function recovery following middle artery occlusion injury in rats. Results showed an optimal stimulation parameter for Renzhong electro-acupuncture that was low frequency and mild current （2 Hz, 1 mA） significantly improved cortical excitability and conductive function, and promoted recovery in a rat model of motor function in middle artery occlusion. Frequency had a greater impact than current or interaction, and played a critical role in electro-acupuncture therapy.

Motor outcomes of patients with a complete middle cerebral artery territory infarct

Detailed knowledge of motor outcomes enables to establish proper goals and rehabilitation strate-gies for stroke patients. Several previous studies have reported functional or motor outcomes in patients with a middle cerebral artery territory infarct. However, little is known about motor outcome in patients with a complete middle cerebral artery territory infarct. In this study, we investigated the motor outcomes in 23 patients with a complete middle cerebral artery territory infarct. All of these patients received comprehensive rehabilitative management, including movement therapy and neuromuscular electrical stimulation of the affected finger extensors and ankle dorsiflexors, for more than 3 months. Motor outcomes were measured at 6 months after stroke onset using the Medical Research Council, Motricity Index, the modified Brunnstrom Classification, and Functional Ambula-tion Category scores. The motor function of the lower extremities was found to be better than that of the upper extremities. After receiving rehabilitation treatments for 3–6 months, about 70% of these patients were able to walk independently （Functional Ambulation Category scores 3）, but no pa-tient achieved functional hand recovery.

In vivo tracking of human adipose-derived stem cells labeled with ferumoxytol in rats with middle cerebral artery occlusion by magnetic resonance imaging

Ferumoxytol, an iron replacement product, is a new type of superparamagnetic iron oxide ap- proved by the US Food and Drug Administration. Herein, we assessed the feasibility of tracking transplanted human adipose-derived stem cells labeled with ferumoxytol in middle cerebral artery occlusion-injured rats by 3.0 T MRI in vivo. 1 × 104 human adipose-derived stem cells labeled with ferumoxytol-heparin-protamine were transplanted into the brains of rats with middle cerebral artery occlusion. Neurologic impairment was scored at 1, 7, 14, and 28 days after transplantation. T2-weighted imaging and enhanced susceptibility-weighted angiography were used to observe transplanted cells. Results of imaging tests were compared with results of Prussian blue staining. The modified neurologic impairment scores were significantly lower in rats transplanted with cells at all time points except I day post-transplantation compared with rats without transplantation. Regions with hypointense signals on T2-weighted and enhanced susceptibility-weighted angiography images corresponded with areas stained by Prussian blue, suggesting the presence of superparamagnetic iron oxide particles within the engrafted cells. Enhanced susceptibility-weighted angiography image exhibited better sensitivity and contrast in tracing ferumoxytol-heparin-protamine-labeled human adipose-derived stem ceils compared with T2-weighted imaging in routine MRI.

Establishment of a rhesus monkey model of middle cerebral artery ischemia and reperfusion using a microcatheter embolization method

Nonhuman primates are closest to humans in terms of lineage, and middle cerebral artery ischemia/reperfusion responses of nonhuman primates are most similar to ischemic stroke in humans. Therefore, nonhuman primates could be utilized to simulate the process of ischemic stroke in the human. Few studies, however, have reported the use of endovascular technology to establish a rhesus monkey stroke model. In the present study, seven adult, male, rhesus monkeys were selected and, following anesthesia, a microcatheter was inserted into one side of the middle cerebral artery via the femoral artery to block blood flow, thereby resulting in middle cerebral artery occlusion. After 2 hours, the microcatheter was withdrawn to restore the middle cerebral artery blood flow and to establish ischemia/reperfusion. Results from angiography and magnetic resonance angiography confirmed occlusion and reopening of the middle cerebral artery. Magnetic resonance imaging revealed the existence of ischemic brain lesions, and neurological examination showed sustained functional deficits following surgery. The rhesus monkey middle cerebral artery ischemia/reperfusion models established by microcatheter embolization had the advantage of non-craniotomy invasion and reproducibility. The scope and degree of ischemic damage using this model was controllable. Therefore, this nonhuman primate model is an ideal model for cerebral ischemia and reperfusion.

Apparent diffusion coefficient evaluation for secondary changes in the cerebellum of rats after middle cerebral artery occlusion

Supratentorial cerebral infarction can cause functional inhibition of remote regions such as the cerebellum, which may be relevant to diaschisis. This phenomenon is often analyzed using positron emission tomography and single photon emission CT. However, these methods are expensive and radioactive. Thus, the present study quantified the changes of infarction core and remote regions after unilateral middle cerebral artery occlusion using apparent diffusion coefficient values. Diffu- sion-weighted imaging showed that the area of infarction core gradually increased to involve the cerebral cortex with increasing infarction time. Diffusion weighted imaging signals were initially in- creased and then stabilized by 24 hours. With increasing infarction time, the apparent diffusion co- efficient value in the infarction core and remote bilateral cerebellum both gradually decreased, and then slightly increased 3-24 hours after infarction. Apparent diffusion coefficient values at remote regions （cerebellum） varied along with the change of supratentorial infarction core, suggesting that the phenomenon of diaschisis existed at the remote regions. Thus, apparent diffusion coefficient values and diffusion weighted imaging can be used to detect early diaschisis.

Intraoperative hemodynamic parameters of middle cerebral artery and other artery aneurysms utilizing transcranial Doppler ultrasonography

BACKGROUND： Hemodynamic changes accompany the initiation, development and rupture of middle cerebral artery （MCA） aneurysms. The complexity of the intraaneurysmal hemodynamic factors has not been completely clarified by the indirect measures and methods used in previous studies. OBJECTIVE： To evaluate correlations of intraoperative hemodynamic factors to initiation and rupture of MCA aneurysms. DESIGN, TIME AND SETTING： A case-control study was performed at the Department of Neurosurgery, Tiantan Hospital Affiliated to Capital Medical University, China between March and October 2008. PARTICIPANTS： A total of 12 consecutive patients diagnosed with MCA aneurysms （MCA aneurysms group） and five patients without middle cerebral artery aneurysms （with aneurysms located at other arteries, control group） were enrolled at the Department of Neurosurgery, Tiantan Hospital Affiliated to Capital Medical University, China. METHODS： The proximal and distal arteries of MCA aneurysms were exposed visibly in the MCA aneurysm group. The M1 segment of MCA without the aneurysm and the aneurysm on other arteries were also exposed visibly in the control group. Hemodynamic indices were then measured using an intraoperative 16 MHz probe installed in a Multi-Dop TCD8X4 device. MAIN OUTCOME MEASURES： Mean （time-averaged velocity） difference, maximum mean, pulsatility index difference, maximum pulsatility index, resistance index difference, maximum resistance index; correlation of development and rupture of MCA aneurysms to intraoperative hemodynamic factors of the parent artery. RESULTS： A total of 12 patients underwent microsurgery for treatment to occlude 15 MCA aneurysms. Of the 15 MCA aneurysms, 12 were located at the bifurcation, two at the M1 segment and one at the M3 segment; eight of the aneurysms were unruptured and seven were ruptured. The whole indices with combination mean difference, maximum mean, and maximum pulsatility index of the aneurysms were important factors influencing the rupture of MCA aneurysms （t = 2.92, P = 0.03, constant）. A higher velocity intraaneurismal flow at the bifurcation was identified （t = 3.48, P = 0.01, constant）. After the aneurysm was completely occluded, global high-velocity flow could not be detected in the parent arteries （t = 2.57, P=0.03, constant）. CONCLUSION： When short-term high-velocity blood flow is present, aneurysms can be easily initiated and ruptured at the bifurcation of MCA.

Angiotensin-converting enzyme gene polymorphism and middle cerebral artery stenosis in a Chinese Han population

The angiotensin-converting enzyme gene is a candidate gene of stroke. The present study involved 62 healthy volunteers and 148 patients with middle cerebral artery stenosis as confirmed by brain color ultrasound from a Han population in North China, and determined the peripheral blood angiotensin-converting enzyme genotype using PCR-restriction fragment length polymorphism analysis. The results showed that the frequencies of the DD genotype and D allele were increased in patients with middle cerebral artery stenosis, but the difference was not statistically significant compared with healthy controls. The findings of this study on the relationship between stroke genes and middle cerebral artery stenosis indicate no significant correlation between the frequencies of the DO genotype and D allele of angiotensin-converting enzyme and middle cerebral artery stenosis in this Han population from North China. In the future, studies will be carried out to investigate correlations between multiple stroke candidate gene synergy and middle cerebral artery stenosis to provide a foundation for the development of gene therapy.

An experimental study of the biomechanics of the middle cerebral artery in acute cerebral infarction

Local cerebrovascular paralysis was found in most cases of cerebral infaction. The extent and severity of vascular paralysis and its effect on contraction-relaxation function of vessels were dependent on the developmental rapidity and duration of vascular

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

A molecular probe carrying anti-tropomyosin 4 for early diagnosis of cerebral ischemia/reperfusion injury

In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.

不同针灸介入时机对大脑中动脉供血区急性脑梗死神经功能预后的影响

目的观察不同针灸介入时机对大脑中动脉供血区急性脑梗死神经功能预后的影响。方法回顾性选取2020年1月—2022年8月收治的116例大脑中动脉供血区急性脑梗死患者的临床资料进行分析,根据针灸介入时机分为两组。两组均进行静脉溶栓及常规药物治疗,观察组61例患者于发病72 h内给予针灸治疗,对照组55例患者于发病2周时给予针灸治疗。检测两组不同时间点侧支循环代偿情况、脑损伤标志物的水平,评估两组不同时间点简易精神状态检查(Mini-mental state examination,MMSE)评分、神经功能评分、Barthel指数(Barthel index,BI)评分、肢体运动功能评分、中医症状评分的差异,统计两组疗效。结果治疗前,两组侧支循环代偿情况比较,差异无统计学意义(P>0.05)。治疗4周和随访时,两组患侧大脑前动脉平均血流速度与对侧大脑中动脉平均血流速度的比值(Ratio of the average flow ve⁃locity of the affected anterior cerebral artery to the average flow velocity of the contralateral middle cerebral artery,iVACA/cVM⁃CA)较治疗前升高,观察组同时间点较对照组更高(P<0.05);两组患侧大脑后动脉平均血流速度与对侧大脑中动脉平均血流速度的比值(Ratio of the average flow velocity of the affected posterior cerebral artery to the average flow velocity of the contralateral middle cerebral artery,iVPCA/cVMCA)与治疗前比较,差异无统计学意义(P>0.05)。治疗前,两组脑损伤标志物比较,差异无统计学意义(P>0.05)。治疗4周和随访时,两组脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)较治疗前升高,观察组同时间点较对照组更高(P<0.05);两组钙结合蛋白β(Calcium binding proteinβ,S100β)、神经胶质纤维酸性蛋白(Glial fibrillary acid protein,GFAP)较治疗前下降,观察组同时间点较对照组更低(P<0.05)。治疗前,两组Fugl-Meyer评分、中医症状评分等相关评分比较,差异无统计学意义(P>0.05)。治疗4周和随访时,两组MMSE评分、BI评分及上肢和下肢Fugl-Meyer评分较治疗前升高,观察组同时间点较对照组更高(P<0.05);两组美国国立卫生院神经功能缺损(National institutes of health stroke scale,NIHSS)评分、中医症状评分较治疗前下降,观察组同时间点较对照组更低(P<0.05)。观察组总有效率为88.52%(54/61)高于对照组的72.73%(40/55),差异有统计学意义(P<0.05)。结论发病72h内采用针灸治疗可改善大脑中动脉供血区急性脑梗死脑损伤标志物的表达,改善脑血流,促进神经功能的恢复,有利于疾病的康复。

颅脑超声对宫内窘迫新生儿脑损伤的早期诊断价值

目的探讨颅脑超声对宫内窘迫新生儿脑损伤的诊断价值。方法对146例宫内窘迫新生儿行颅脑超声及常规MRI检查,并将超声检查结果与MRI结果进行对比分析。根据脑损伤情况分为脑损伤组(n=46)和无脑损伤组(n=100),分析比较2组新生儿大脑中动脉和前动脉血流动力学指标[收缩期血流速度(PSV)、舒张末期血流速度(EDV)及阻力指数(RI)],采用受试者工作特征(ROC)曲线分析其对脑损伤的诊断价值。结果146例宫内窘迫新生儿中46例存在影像学异常征象,阳性检出率31.74%(46/146),其中MRI的检出率为91.30%(42/46),明显高于颅脑超声的65.22%(30/46,χ^(2)=9.200,P=0.002)。缺氧缺血性脑病(12.33%比5.48%,χ^(2)=4.222,P=0.040)、硬膜或蛛网膜下腔出血(2.74%比0,χ^(2)=4.056,P=0.044)的MRI检出率明显高于颅脑超声(P<0.05),脑室周围-脑室内出血的颅脑超声检出率明显高于MRI(11.64%比4.79%,χ^(2)=4.540,P=0.033)。脑损伤组新生儿大脑中动脉和前动脉PSV、EDV小于无脑损伤组,RI大于无脑损伤组(P<0.05)。ROC曲线显示,大脑中动脉和前动脉PSV、EDV和RI诊断脑损伤的曲线下面积分别为0.710、0.786、0.625和0.740、0.819、0.613。结论颅脑超声可早期发现宫内窘迫新生儿脑组织结构改变及血流动力学变化,在脑室周围-脑室内出血方面的检出率优于MRI,但脑损伤诊断效果不如MRI,临床可根据患儿情况选择合适检查方案。

超声检查不同病情HDCP患者胎儿、新生儿大脑中动脉血流动力学改变及对新生儿脑损伤的预测价值

目的探讨超声检查不同病情妊娠期高血压疾病(HDCP)患者胎儿、新生儿大脑中动脉血流动力学改变及意义。方法选取2020年9月至2022年9月在该院就诊的131例HDCP患者作为研究对象,根据疾病严重程度将其分为子痫组(39例)、子痫前期组(44例)、妊娠期高血压组(48例)。另选取同期50例健康孕妇作为对照组。比较各组胎儿期、新生儿期大脑中动脉舒张末期流速(EDV)、收缩期峰值流速(PSV)、收缩期峰值流速/舒张末期流速(S/D)、搏动指数(PI)、阻力指数(RI)。统计不同病情程度HDCP患者新生儿脑损伤发生情况,将有脑损伤的新生儿纳入有脑损伤组,无脑损伤的新生儿纳入无脑损伤组。比较有脑损伤组、无脑损伤组胎儿期超声检查结果。绘制受试者工作特征(ROC)曲线分析EDV、PSV、S/D、PI、RI单独及5项联合检测预测HDCP新生儿脑损伤价值。结果子痫组、子痫前期组、妊娠期高血压组胎儿EDV、PSV均低于对照组,差异均有统计学意义(P<0.05);子痫组、子痫前期组胎儿S/D、PI、RI均低于对照组,且子痫组PSV、S/D、PI、RI均低于子痫前期组,差异均有统计学意义(P<0.05)。4组新生儿EDV、PSV比较,均为子痫组<子痫前期组<妊娠期高血压组<对照组,差异均有统计学意义(P<0.05);4组新生儿S/D、PI、RI比较,均为子痫组>子痫前期组>妊娠期高血压组>对照组,差异均有统计学意义(P<0.05)。有脑损伤组纳入29例新生儿,无脑损伤组纳入102例新生儿。有脑损伤组EDV、PSV均低于无脑损伤组,S/D、PI、RI均高于无脑损伤组,差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,EDV、PSV、S/D、PI、RI单独预测HDCP患者新生儿脑损伤的曲线下面积(AUC)分别为0.784、0.710、0.755、0.784、0.770,均低于5项指标联合检测的AUC(0.944)。结论进入子痫期后大脑中动脉血流动力学改变明显,易发生新生儿脑损伤;不同病情程度HDCP均可影响新生儿早期大脑血液循环,超声检查结果可为预测HDCP患者新生儿脑损伤提供参考。

基于PiggyBac转座子优化稳定表达细胞系的新型瞬时受体电位M2通道抑制剂筛选

目的:使用PiggyBac(PB)转座系统建立稳定表达瞬时受体电位M2(TRPM2)通道的人胚胎肾细胞HEK293T,并进行TRPM2通道抑制剂筛选,为治疗脑缺血等疾病寻找潜在的药物。方法:根据PB转座原理,构建pPB-hTRPM2真核表达载体。将重组质粒和辅助质粒共同转染至HEK293T细胞中,利用系统携带的荧光与膜片钳鉴定TRPM2基因表达,通过Z'因子评估细胞模型是否适用于钙成像法的高通量筛选。利用钙成像法和膜片钳对11个先导化合物分子进行初步活性评价,测定化合物分子对TRPM2通道的抑制活性。利用氧糖剥夺/再灌注(OGD/R)损伤模型和细胞计数试剂盒8(CCK-8)方法验证筛选得到的化合物分子对损伤细胞的保护作用。利用流式细胞术检测细胞活性氧水平。利用小鼠短暂性大脑中动脉栓塞(tMCAO)模型评价化合物的神经保护作用。结果:成功构建pPB-hTRPM2真核表达载体,构建出可高效表达TRPM2基因的HEK293T细胞系,并同时表达增强型绿色荧光蛋白(EGFP)。在筛选获得的嘌呤霉素抗性细胞中,所有细胞荧光明亮可见,诱导后细胞表现出经典的TRPM2通道电流特征,TRPM2通道电流值与瞬时转染对照组差异无统计学意义(P>0.05)。该筛选系统进行钙成像实验的Z'因子为0.5416,表明其适用于高通量筛选。通过钙成像法结合膜片钳筛选出化合物6,其具有显著的TRPM2通道抑制作用,且1.0μmol/L的化合物6能够显著提高OGD/R后SH-SY5Y细胞的存活率(P<0.05),降低活性氧水平(P<0.05),0.3、1.0 mg/kg的化合物6能降低tMCAO小鼠脑梗死体积百分比(均P<0.05)。结论:利用PiggyBac基因编辑技术成功构建了TRPM2基因稳定表达细胞系,利用钙成像法和膜片钳在候选化合物中成功筛选出一个高亲和力的新的TRPM2通道抑制剂。该抑制剂可以通过降低细胞活性氧水平缓解OGD/R后细胞损伤,并对小鼠脑缺血再灌注损伤具有保护作用。

通督调神针法对MCAO大鼠缺血半暗带超微结构及SNAP-25蛋白的影响

[目的]观察通督调神针法对大脑中动脉闭塞(MCAO)模型大鼠行为学、缺血半暗带超微结构和突触体相关蛋白25(SNAP-25)的影响,探析电针疗法的神经保护机制。[方法]将40只SD大鼠随机分为空白组、假手术组、模型组和电针组,每组10只。空白组不做干预,假手术组大鼠仅接受皮肤切开及血管剥离术,模型组及电针组大鼠采用线栓法进行脑缺血模型制备。造模成功后电针组予通督调神针法干预,1次/日,每次30 min,连续干预7 d。空白组、假手术组及模型组术后在同等条件下抓取,回笼饲养。记录各组大鼠神经功能评分,采用2,3,5氯化三苯基四氮唑(TTC)染色法检测大鼠脑梗死面积,透射电镜观察大鼠脑组织缺血半暗带处超微结构,Western blot检测各组大鼠海马区SNAP-25蛋白表达水平。[结果]模型组大鼠神经功能评分明显低于假手术组和空白组(P<0.05);与模型组比较,电针组大鼠神经功能评分显著增加,差异有统计学意义(P<0.05)。模型组和电针组大鼠存在明显脑梗死区域,电针组脑梗死面积小于模型组(P<0.01)。空白组和假手术组大鼠脑组织神经元细胞结构完整,细胞核膜完整,结构清晰,整体细胞器完整;模型组大鼠脑组织神经元细胞结构受破坏,包括细胞膜不完整、细胞核肿胀、内质网减少等;与模型组比较,电针组大鼠脑组织具有较为完整的细胞膜及细胞核结构,线粒体空泡现象较少。与空白组、假手术组比较,模型组大鼠缺血半暗带脑组织的SNAP-25蛋白表达水平明显增加(P<0.01);与模型组比较,电针组大鼠缺血半暗带脑组织的SNAP-25蛋白表达水平明显减少(P<0.01)。[结论]通督调神针法可通过下调SNAP-25蛋白表达水平,抑制神经突触小体增殖及修复神经细胞结构,从而发挥神经保护作用。

Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response

Puerarin, a traditional Chinese medicine, exerts a powerful neuroprotective effect in cerebral ischemia/reperfusion injury, but its mechanism is unknown. Here, we established rat models of middle cerebral artery ischemia/reperfusion injury using the suture method. Puerarin （100 mg/kg） was administered intraperitoneally 30 minutes before middle cerebral artery occlusion and 8 hours after reperfusion. Twenty-four hours after reperfusion, we found that puerarin significantly improved neurological deficit, reduced infarct size and brain water content, and notably diminished the expression of Toll-like receptor-4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in the ischemic region. These data indicate that puerarin exerts an anti-inflammatory protective effect on brain tissue with ischemia/reperfusion damage by downregulating the expression of multiple inflammatory factors.

Influence of Ren and Du meridian electro-acupuncture on neural stem cell proliferation and extracellular signal-regulated kinase pathway in a rat model of focal cerebral ischemia injury

BACKGROUND： Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE： To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS： Mouse anti-rat bromodeoxyuridine （BrdU） monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase （ERK） specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS： A total of 126 rats were randomly assigned to four groups： model （n = 36）, Du meridian （n = 36）, Ren/Du meridian （n = 36）, and Ren/Du meridian ＋ PD98059 （n = 18）. Rats in the Ren/Du meridian ＋ PD98059 group were observed on days 7 （n = 6） and 14 （n = 12） after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong （DU 26） and Baihui （DU 20） acupoints in the Du meridian group, as well as Chengjiang （RN 24）, Guanyuan （RN 4）, Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian ＋ PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian ＋ PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES： BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 （pERK1/2） expression in the subventricular zone. RESULTS： On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group （P 〈 0.05）. PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/：ten and Du meridians （P 〈 0.05）. On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group （P 〈 0.05）. The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction （P 〈 0.05）. However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression （P 〈 0.05）. CONCLUSION： Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.

Comparison of the anti-apoptotic effects of 15-and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underly- ing mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion （usually from 30 minutes to 1 hour） is longer than traditional suspended moxibustion （usually 15 minutes）. However, the effects of 15- and 35-minute suspended moxibustion in rats with cerebra/ischemia/reperfusion injury are poorly understood. In this study, we performed 15- or 35-minute suspended moxibustion at acupoint Dazhui （GV14） in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxy- lin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Caspase-9 and caspase-3 expression at the in- jury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expres- sion was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.

Neuroprotective effect of penehyclidine hydrochloride on focal cerebral ischemia-reperfusion injury

Penehyclidine hydrochloride can promote microcirculation and reduce vascular permeability. However, the role of penehyclidine hydrochlodde in cerebral ischemia-reperfusion injury remains unclear. In this study, in vivo middle cerebral artery occlusion models were established in experimental rats, and penehyclidine hydrochloride pretreatment was given via intravenous injection prior to model establishment. Tetrazolium chloride, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and immunohistochemical staining showed that, penehyclidine hydrochloride pretreatment markedly attenuated neuronal histopathological changes in the cortex, hippocampus and striatum, reduced infarction size, increased the expression level of BcI-2, decreased the expression level of caspase-3, and inhibited neuronal apoptosis in rats with cerebral ischemia-reperfusion injury. Xanthine oxidase and thiobarbituric acid chromogenic results showed that penehyclidine hydrochloride upregulated the activity of superoxide dismutase and downregulated the concentration of malondialdehyde in the ischemic cerebral cortex and hippocampus, as well as reduced the concentration of extracellular excitatory amino acids in rats with cerebral ischemia-reperfusion injury. In addition, penehyclidine hydrochloride inhibited the expression level of the NR1 subunit in hippocampal nerve cells in vitro following oxygen-glucose deprivation, as detected by PCR. Experimental findings indicate that penehyclidine hydrochloride attenuates neuronal apoptosis and oxidative stress injury after focal cerebral ischemia-reperfusion, thus exerting a neuroprotective effect.

Combination of mild therapeutic hypothermia and adipose-derived stem cells for ischemic brain injury

Mild therapeutic hypothermia has been shown to mitigate cerebral ischemia, reduce cerebral edema, and improve the prognosis of patients with cerebral ischemia. Adipose-derived stem cell-based therapy can decrease neuronal death and infiltration of inflammatory cells, exerting a neuroprotective effect. We hypothesized that the combination of mild therapeutic hypothermia and adipose-derived stem cells would be neuroprotective for treatment of stroke. A rat model of transient middle cerebral artery occlusion was established using the nylon monofilament method. Mild therapeutic hypothermia（33°C） was induced after 2 hours of ischemia. Adipose-derived stem cells were administered through the femoral vein during reperfusion. The severity of neurological dysfunction was measured by a modified Neurological Severity Score Scaling System. The area of the infarct lesion was determined by 2,3,5-triphenyltetrazolium chloride staining. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling（TUNEL） staining. The regeneration of microvessels and changes in the glial scar were detected by immunofluorescence staining. The inflammatory responses after ischemic brain injury were evaluated by in situ staining using markers of inflammatory cells. The expression of inflammatory cytokines was measured by reverse transcription-polymerase chain reaction. Compared with mild therapeutic hypothermia or adipose-derived stem cell treatment alone, their combination substantially improved neurological deficits and decreased infarct size. They synergistically reduced the number of TUNEL-positive cells and glial fibrillary acidic protein expression, increased vascular endothelial growth factor levels, effectively reduced inflammatory cell infiltration and down-regulated the m RNA expression of the proinflammatory cytokines interleukin-1β, tumor necrosis factor-α and interleukin-6. Our findings indicate that combined treatment is a better approach for treating stroke compared with mild therapeutic hypothermia or adipose-derived stem cells alone.