骨髓基质干细胞在IKVAV多肽纳米纤维凝胶表面增殖、黏附及向神经细胞诱导分化的研究

目的研究骨髓基质干细胞（BMSCs）与IKVAV多肽纳米纤维凝胶复合的细胞假体应用于脊髓损伤治疗的可行性。方法合成IKVAV多肽两亲性分子，进行自组装，用透射电镜检测。将IKVAV多肽纳米纤维凝胶与BMSCs复合培养，采用倒置显微镜下观察、Calcein—AM／PI染色、CCK-8法、免疫荧光双标法，检测IKVAV多肽对BMSCs增殖、黏附及向神经细胞方向诱导分化的影响。结果IKVAV多肽可成功自组装成纳米纤维凝胶，其与BMSCs复合培养细胞生长良好，活细胞数达90％以上，IKVAV多肽对BMSCs增殖没有影响，可促进BMSCs的黏附，并在诱导BMSCs向神经细胞分化过程中提高神经元分化比例。结论BMSCs在IKVAV多肽纳米纤维凝胶材料表面可良好的增殖及黏附，并可提高其向神经细胞分化过程中神经元的分化比例。     

自组装IKVAV多肽纳米支架及其对背根神经节神经元细胞的作用

目的:应用自组装IKVAV多肽纳米支架与鼠背根神经节神经元细胞(DRGc)联合培养,观察其对DRGc的作用。方法:将多肽溶于0.1MNaOH溶液中,调整pH值为8.5,多肽浓度为0.01mg/μl,与等体积DMEM/F12混合触发多肽自组装为凝胶支架,透射电镜检测。采用原代分离培养方法获得DRGc单细胞悬液后分为实验组与对照组,实验组中DRGc接种于凝胶支架表面,对照组接种于多聚赖氨酸表面,倒置相差显微镜观察神经元生长情况,采用细胞计数结合免疫细胞化学染色方法,观察DRGc的存活和轴突生长情况,并行统计学分析。结果:透射电镜下显示自组装凝胶支架为编织状纳米纤维。实验组和对照组中DRGc培养1d时平均轴突长度分别为43.8±10.4μm、33.4±5.75μm;培养14d时实验组和对照组中神经元数目分别为36.50±1.78个/视野、19.70±3.71个/视野,神经元所占比例分别为(43.60±4.83)%、(26.97±4.90)%,两组间比较有显著性差异(P<0.05)。结论:自组装IKVAV多肽纳米支架能降低神经元的死亡率,并诱导轴突的发生和生长,具有支架及生物活性双重作用,可作为神经组织工程支架材料。     

自组袭多肽纳米支架与骨髓间充质干细胞的生物相容性研究

[目的]合成神经活性多肽IKVAV(isoleucine-lysine-valine-alanine-valine,异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸)两亲性分子,在体外进行自组装纳米纤维支架,并检测其与骨髓间充质干细胞(mesenchymal stemcells,MSCs)的生物相容性。[方法]IKVAV多肽两亲性分子委托上海波泰生物科技公司合成,并用高效液相色谱仪和质谱仪进行纯化和分析。将IKVAV多肽两亲性分子溶于0.1 M NaOH溶液中,加入稀盐酸降低pH值引发自组装,并利用透射电镜进行观察。分离兔骨髓间充质干细胞,流式分析其表面抗原标志。将MSCs与自组装支架体外共培养12 d,测定细胞粘附率,MTT方法检测IKVAV自组装材料对细胞生长增殖的影响,观察其生物相容性。[结果]IKVAV-PA可在一定条件下自组装形成凝胶,透射电镜可见其显示为纳米纤维,其直径为7.0～8.0 nm。分离的MSCs细胞表面标志CD105、CD44、CD34和CD45的阳性率分别为99.3%、99.5%、0.1%、0.4%。共培养过程后,随着时间延长,MSCs在多肽凝胶支架上黏附率上升,吸光度增加,细胞活性增强。[...     

异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸多肽纳米纤维对神经干细胞生物学行为的影响

目的制备异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸（IKVAV）多肽材料并检测其对神经干细胞（NSC）的生物学行为的影响。方法合成IKVAV多肽两亲性分子，进行自组装，用透射电镜（TEM）检测。培养NSC，加入IKVAV，用CCK．8检测吸光度（A值）显示NSC活性。同时检测IKVAV对NSC黏附、分化和轴突生长的影响。结果成功合成IKVAV并自组装成为凝胶，TEM显示为纳米纤维，直径7～8nm，长度可达到数百纳米。当IKVAV浓度为20mg／g和60mg／L时，A值有明显增加，和对照组相比差异有统计学意义（P〈0．01）。加入IKVAV后，神经球黏附率由4％上升到96％，神经球分化率由6％上升到100％，向神经元分化的比率由4．5％提高到5．6％，同时促进神经轴突生长。结论IKVAV可形成纳米纤维，并且具有良好的神经生物活性，是一种优良的神经生物材料。     

神经干细胞与功能化自组装多肽水凝胶的细胞相容性

目的 制备功能化自组装多肽水凝胶并检测其与神经干细胞(NSCs)的生物相容性.方法 合成自组装多肽RADA16 与多肽RADA16-IKVAV,将两者混合制备功能化自组装多肽水凝胶,用原子力显微镜(AFM)观察其形态学特征.采用无血清培养法从新生大鼠皮层分离培养NSCs.将NSCs分别接种到RADA16自组装多肽水凝胶(对照组)与功能化自组装多肽水凝胶(实验组)表面.激光扫描共聚焦显微镜(LSCM)观察细胞迁移.用CCK-8法测定吸光度(A)检测细胞增殖能力.应用Nestin、MAP2、GFAP和CC-1免疫荧光染色,检测神经干细胞分化.结果 AFM显示功能化自组装多肽水凝胶由纳米纤维组成,其纤维直径为20～30 nm,长度可达数百纳米.在细胞接种6 d后,对照组和实验组细胞在水凝胶中的迁移距离分别为(27.5±3.7)μm和(53.2±5.4)μm,两组差异有统计学意义(P＜0.05).复合培养7 d后,实验组中细胞A值明显高于对照组(P＜0.05).免疫荧光结果显示13 d后对照组中MAP2阳性细胞百分率为(22.44±3.52)%,实验组为(40.35±4.84)%,两组差异有统计学意义(P＜0.05).结论 功能化自组装多肽水凝胶与NSCs相容性良好.     

人工合成IKVAV多肽对星形胶质细胞增殖和Neurocan基因表达的影响

【摘要】 目的 观察人工合成IKVAV多肽对星形胶质细胞增殖及硫酸软骨素蛋白多糖核心蛋白Neurocan基因表达的影响,并探讨IKVAV潜在的促进受损脊髓功能恢复的作用。 方法 设计IKVAV序列,人工合成IKVAV多肽,将星形胶质细胞接种于1% IKVAV包被的培养板上,分别于培养1 d、3 d、5 d、7 d、9 d后在激光共聚焦显微镜下观察IKVAV多肽和星形胶质细胞的组织相容性,用CCK8法检测星形胶质细胞增殖情况,用荧光定量PCR法检测星形胶质细胞硫酸软骨素蛋白多糖核心蛋白Neurocan的表达,并与对照组比较,进行统计学分析。 结果 与对照组相比,实验组培养板上星形胶质细胞总数明显减少,但细胞存活率＞95%。荧光定量PCR结果显示实验组星形胶质细胞Neurocan的表达量较对照组显著降低。 结论 人工合成IKVAV多肽可抑制星形胶质细胞增殖、下调硫酸硫酸软骨素蛋白多糖的表达,从而抑制受损脊髓胶质瘢痕的形成,促进脊髓功能恢复。     

FGL多肽自组装纳米纤维与神经干细胞生物相容性研究

目的 观察组织工程支架材料FGL多肽组装纳米纤维和神经干细胞(neural stem cells,NSCs)的生物相容性. 方法同相法合成FGL多肽两亲性分子(FGL peptide-amphiphile,FGL-PA),采用高效液相色谱仪和质谱仪进行纯化和分析.加入0.1mol/LHCI于FGL-PA溶液,降低pH值引发其自组装,透射电镜观察自组装后的材料.取新生1 d大鼠大脑皮质,培养大鼠NSCs,分别加入最终浓度为0、50、100、200、400 mg/L FGL-PA,采用CCK-8试剂盒检测FGL-PA对细胞增殖的影响.将NSCs分别加入分化培养基(对照组:DMEM/F12、2?7和10?S)和含FGL-PA的分化培养基(实验组:DMEM/F12、2?7、10?S和100 mg/L FGL-PA)诱导分化,采用免疫荧光法检测FGL-PA对NSCs分化的影响. 结果 FGL-PA可自组装形成凝胶,透射电镜示其为纳米纤维,直径为10～20 nm,长度可达数百纳米.加入各浓度FGL-PA 48 h后,当FGL-PA浓度为50、100、200 mg/L时,吸光度(A)值显著增加,差异有统计学意义(P＜0.05).NSCs诱导分化培养14 d,免疫荧光结果 显示对照组NSCs分化为神经元比例为46.35%4±1.27%,实验组为72.85%±1.35%,两组比较差异有统计学意义(P＜0.05). 结论 FGL-PA能自组装形成纳米纤维凝胶,具有良好的生物相容性和生物活性.     

人工合成IKVAV多肽对星形胶质细胞增殖和Neurocan基因表达的影响

目的观察人工合成IKVAV多肽对星形胶质细胞增殖及硫酸软骨素蛋白多糖核心蛋白Neurocan基因表达的影响,并探讨IKVAV潜在的促进受损脊髓功能恢复的作用。方法设计IKVAV序列,人工合成IKVAV多肽,将星形胶质细胞接种于1%IKVAV包被的培养板上,分别于培养1 d、3 d、5 d、7 d、9 d后在激光共聚焦显微镜下观察IKVAV多肽和星形胶质细胞的组织相容性,用CCK-8法检测星形胶质细胞增殖情况,用荧光定量PCR法检测星形胶质细胞硫酸软骨素蛋白多糖核心蛋白Neurocan的表达,并与对照组比较,进行统计学分析。结果与对照组相比,实验组培养板上星形胶质细胞总数明显减少,但细胞存活率〉95%。荧光定量PCR结果显示实验组星形胶质细胞Neu-rocan的表达量较对照组显著降低。结论人工合成IKVAV多肽可抑制星形胶质细胞增殖、下调硫酸硫酸软骨素蛋白多糖的表达,从而抑制受损脊髓胶质瘢痕的形成,促进脊髓功能恢复。     

两亲性含IKVAV的肽体内诱导血管生成的研究

目的设计合成含IKVAV的两亲性肽，探讨其体外自组装成三维多孔凝胶及体内诱导血管生成的可行性。方法固相法合成肽．自组装成凝胶，TEM观察凝胶结构。实验组：配制1wt％的肽溶液，pH值为9．0；对照组：配制16．67％的明胶，DH值为7．4。各取1mL，分别注射于大鼠脊柱两侧皮下，术后1周观察全身反应及局部皮肤。2周后取材，固定，常规HE染色及VEGF免疫组织化学染色。结果高效液相频谱仪及质谱仪示肽纯度及分子量分别为95．22％与1438,TEM示凝胶为交织成网状的纳米纤维。术后1周动物存活良好，无全身不良反应，局部皮肤无红肿、无坏死。实验组：1wt％的肽体内自组装成凝胶，血管长入凝胶内部，管腔内可发现红细胞；术后2周，VEGF阳性。对照组：明胶内未发现血管，VEGF阴性。结论本实验合成含IKVAV两亲性肽，在PBS溶液中可自组装成凝胶且体内可诱导新生血管生成。     

修饰有BMP-7功能片段的功能化自组装多肽纳米纤维水凝胶RADKPS制备及其生物相容性研究

目的制备并评价载有BMP-7功能片段的新型功能化自组装多肽纳米纤维水凝胶支架材料RADKPS的生物相容性,为其在退变髓核再生领域中的体内研究提供实验依据。方法以自组装多肽RADA16-Ⅰ为原料,通过化学键链接BMP-7功能片段构建功能化自组装多肽RADA-KPSS,再与RADA16-Ⅰ等比例混合制备新型功能化自组装多肽RADKPS。通过大体观察、原子力显微镜评估RADKPS支架材料的结构特点。分离培养3月龄新西兰大白兔BMSCs,取第3代BMSCs与RADKPS复合培养7 d,通过扫描电镜、细胞荧光素二乙酸盐/碘化丙啶活性染色、MTT法检测RADKPS的细胞相容性;于6月龄新西兰大白兔离体椎间盘内注射1%RADKPS后培养并观察其髓核内细胞活性。采用溶血实验检测RADKPS的血液相容性。将RADKPS植入6~8周龄昆明小鼠皮下28 d,行大体观察及HE染色观察RADKPS组织相容性。结果 RADKPS在L-DMEM中成胶后呈均匀透明水凝胶状;原子力学显微镜示纳米纤维相互连接呈三维网状结构,纤维直径(25.68±4.62)nm,纤维长度(512.42±32.22)nm。RADKPS支架复合BM SCs 7 d后,扫描电镜示细胞在支架上黏附良好,细胞活性维持在90%以上;MTT检测示0.1%、0.05%、0.025%RADKPS溶液均不同程度促进新西兰大白兔BMSCs增殖。溶血实验结果示,不同浓度RADKPS溶液相对溶血率均5%,符合医用生物材料的溶血实验要求。小鼠皮下注射实验结果示,28 d后注射局部皮肤无水疱、红斑及焦痂形成;HE染色示淋巴细胞等炎性细胞浸润,大量纤维组织取代水凝胶支架,材料组织相容性良好。结论新型功能化自组装多肽纳米纤维水凝胶支架材料RADKPS具有良好的生物相容性和安全性,适合于髓核的组织工程修复与再生研究。     

利多卡因注射大鼠背根神经节致神经炎症的研究

背景选择性脊神经阻滞导致的脊神经或背根神经节（DRG）损伤是至今尚未充分研究的一种潜在的严重并发症。本实验假设局部麻醉药注入脊神经、DRG内可能会导致炎性反应与痛觉过敏。方法大鼠部分椎板切除后,于L5脊神经或背根神经节内注射4μl利多卡因或盐水,评价其炎性反应及行为反应。行为学测试在手术前、手术后分别进行,通过观察并记录大鼠对足部伤害性机械刺激的反应,并对痛觉过敏进行评估。摘取背根神经节并染色,对神经元周围起免疫反应的神经胶质细胞环进行计数。结果与脊神经利多卡因注射组相比,DRG利多卡因注射组注射利多卡因4天后出现了同侧足部的痛觉过敏。神经胶质原纤维酸性蛋白免疫阳性的神经胶质细胞环,即激活的卫星细胞的数量,在DRGs内的表达,DRG和脊神经利多卡因注射组均明显增加。同盐水注射组相比,利多卡因注射组神经胶质原纤维酸性蛋白阳性细胞明显增多,并在DRGs内发现了散在的、能代表激活的小神经胶质细胞的OX-42免疫阳性细胞。通过检测能标记激活的T淋巴细胞的Pan-T,并未发现T淋巴细胞。结论DRG内注射利多卡因可导致痛觉过敏,可能是通过激活固有的卫星神经胶质细胞。临床上,选择性脊神经阻滞时,应避免DRG内直接注射局部麻醉药。     

Phrenic nerve transfer in the treatment of brachial plexus avulsion: an experimental study of nerve regeneration and muscle morphology in rats

The regeneration of motor and sensory neurons and the morphological changes of the target muscle after phrenic nerve transfer were investigated in adult rats. Six months following nerve transfer, 326.0 +/- 16.31 phrenic motoneurons regenerated into musculocutaneous nerve, which is not different from the normal number of phrenic motoneurons. The regenerated motoneurons exhibited a 14% nonsignificant hypertrophy. Of the dorsal root ganglia (DRG) neurons, 255.8 +/- 45.26 regenerated, which was significantly lower than the number of normal phrenic DRG neurons. The regenerated phrenic DRG neurons showed a 24% close-to-significant atrophy. The target muscle fiber morphology changed considerably after reinnervation. The present results suggest that the phrenic nerve has very good regenerative ability in terms of its motoneurons and a relatively insufficient sensory neuronal regeneration.     

Foxo3a和p27kip1在大鼠坐骨神经损伤后背根神经节中的表达

目的 探讨坐骨神经夹伤后Foxo3a和p27kip1在腰段背根神经节(DRG)中表达变化及其意义.方法 将成年SD大鼠随机分为正常对照组和实验组.对实验组实行坐骨神经夹伤术.运用蛋白质印迹、免疫荧光双标,研究大鼠坐骨神经夹伤后,腰段DRG中Foxo3a和p27kip1的表达、分布以及细胞增殖和轴突再生情况.结果 Foxo3a在坐骨神经夹伤后1 d表达值(7.0±3.5)开始明显下降,2 d表达值(6.0±3.8)达到最低值,随之逐渐升高,p27kip1在坐骨神经夹伤后2 d表达值(29.0±3.5)表达开始明显下降,7 d表达值(21.0±3.0)达到最低值,随之逐渐升高;Foxo3a和p27kip1分布于神经元和神经胶质细胞内且在坐骨神经夹伤后2d DRG中,Foxo3a和p27kip1在神经元细胞[(37.8±5.7)%、(43.3±4.3)%]和神经胶质细胞[(22.4±3.9)%、(13.8±3.2)%]中表达较在正常组神经元细胞[(73.6±2.5)%、(84.1±3.7)%]和神经胶质细胞[(61.3±4.4)%、(68.7±5.6)%]减少;细胞核增殖抗原(proliferating cell nuclear antigen,PCNA)和神经元生长相关蛋白(GAP-43)在坐骨神经夹伤后2 d DRG中表达值[12±2.6,15±1.9]均开始上调,PCNA在7 d表达值(25.0±3.2)达到最高点,GAP-43则保持较高水平;此外,PCNA与神经胶质细胞共定位明显,与神经元细胞几乎无共定位.结论 大鼠坐骨神经损伤后,Foxo3a和p27kip1在腰段DRG中的表达减少与神经胶质细胞增殖和轴突再生密切相关.     

Harvested human neurons engineered as live nervous tissue constructs: implications for transplantation. Laboratory investigation

OBJECT: Although neuron transplantation to repair the nervous system has shown promise in animal models, there are few practical sources of viable neurons for clinical application and insufficient approaches to bridge extensive nerve damage in patients. Therefore, the authors sought a clinically relevant source of neurons that could be engineered into transplantable nervous tissue constructs. The authors chose to evaluate human dorsal root ganglion (DRG) neurons due to their robustness in culture. METHODS: Cervical DRGs were harvested from 16 live patients following elective ganglionectomies, and thoracic DRGs were harvested from 4 organ donor patients. Following harvest, the DRGs were digested in a dispase-collagenase treatment to dissociate neurons for culture. In addition, dissociated human DRG neurons were placed in a specially designed axon expansion chamber that induces continuous mechanical tension on axon fascicles spanning 2 populations of neurons originally plated approximately 100 microm apart. RESULTS: The adult human DRG neurons, positively identified by neuronal markers, survived at least 3 months in culture while maintaining the ability to generate action potentials. Stretch-growth of axon fascicles in the expansion chamber occurred at the rate of 1 mm/day to a length of 1 cm, creating the first engineered living human nervous tissue constructs. CONCLUSIONS: These data demonstrate the promise of adult human DRG neurons as an alternative transplant material due to their availability, viability, and capacity to be engineered. Also, these data show the feasibility of harvesting DRGs from living patients as a source of neurons for autologous transplant as well as from organ donors to serve as an allograft source of neurons.     

Effects of topically administered nerve growth factor on axonal regeneration in peripheral nerve autografts implanted in the spinal cord of rats

The effect of exogenous nerve growth factor (NGF) on axonal regeneration into autologous peripheral nerve (PN) grafts implanted to the spinal cord (SC) of rats was assessed by retrograde labeling of the parent soma of the regenerating axons with horseradish peroxidase. NGF was delivered at the graft site over periods of 15 and 30 days by using indwelling osmotic minipumps. In control rats, the minipumps were filled with saline. At 15 days after grafting in the NGF-treated rats, the mean number of SC as well as dorsal root ganglion (DRG) neurons that regenerated their axons into the peripheral nerve grafts was increased 55.3 and 26.4 times, respectively, as compared to the control group values. At 30 days, SC and DRG neurons in the NGF-treated group were 10.9 and 3.1 times greater than in the control group. In the NGF-treated group, the regenerating SC neurons were located within a range of 7 to 13 mm from the graft site as compared to 1 to 7 mm in the control group. Finally, the analysis of the soma diameters of the regenerating neurons showed that NGF enhanced and maintained with time the regenerative response from small-sized DRG neurons. Therefore, NGF is thought to promote directly the regenerative potential of SC as well as DRG neurons and to exert an indirect glial cell-mediated effect at the SC-graft interface.     

Characterization of the induced neuropeptide Y-like immunoreactivity in primary sensory neurons following complete median nerve transection

In this study, we examined characteristics of the neuropeptide Y-like immunoreactive (NPY-LI) dorsal root ganglion (DRG) neurons after complete median nerve transection (CMNT). With fluorogold (FG) injection into normal median nerves, numerous FG-labeled DRG neurons were localized predominantly in the C6 and C7 DRGs, where the focal regions were examined after CMNT. With NPY immunohistochemistry, a few NPY-LI neurons were detected in the ipsilateral but not contralateral DRGs after FG injection into the nerve. As early as 3 days after CMNT a few NPY-LI neurons could be detected, reaching a maximum in the DRGs at 4 weeks, subsiding thereafter over 20 weeks. The NPY-LI DRG neurons were primarily medium-sized and large neurons. With FG injection into the transected median nerve, we found that approximately 99% of NPY-LI neurons were labeled for FG, suggesting that they were derived from the injured but not intact DRG neurons. Using double fluorescent dyes tracing, we detected that some of the injured DRG neurons were NPY-LI neurons that projected to the cuneate nucleus (CN). Following dorsal rhizotomy, our data indicated that after CMNT the induced NPY-LI fibers in the ipsilateral CN originated exclusively from the injured DRG neurons. Taken together, these findings suggest that injury-induced NPY-LI fibers in the CN may originate from the injured DRG neurons via the median primary afferent fibers, affect the excitability of cuneothalamic projection neurons (CTNs), and involve neuropathic sensation following CMNT.     

FK506缓释膜片促进外周神经端-侧吻合后神经再生的研究

目的 探讨免疫抑制剂FK506缓释膜片局部应用对周围神经端-侧吻合后神经再生的影响。方法 选用SD大白鼠40只，随机平均分成实验组和对照组。实验组：切断腓总神经，在邻近的胫神经干外膜上开一1mm窗口，将腓总冲经远端与胫神经干做端-侧神经吻合后在吻合口周围放置含有FK506分子可降解缓释膜片。对照组：单纯行神经端-侧吻合：两组分别于术后2、4、8、12周取材，对腓总神经和胫神经干用快蓝（FB）和荧光金（FG）注射标记，取背根神经节（DRG）和脊髓在荧光显微镜下观察被标记细胞。结果 实验组背根冲经节和脊髓内FB标记细胞明显多于对照组。结论 免疫抑制剂FK506能促进外周神经端-侧吻合后神经再生的速度和质量。     

Primary sensory neurons with dichotomizing axons projecting to the facet joint and the sciatic nerve in rats.

STUDY DESIGN: Dorsal root ganglion (DRG) neurons that have dichotomizing axons to the lumbar facet joint and to the sciatic nerve were investigated in rats using a double fluorescent labeling technique. OBJECTIVES: To clarify the existence of DRG neurons with dichotomizing axons projecting to the lumbar facet joint and to the sciatic nerve in rats. SUMMARY OF BACKGROUND DATA: DRG neurons having dichotomizing axons have been reported in several species and are considered to be related to referred pain. However, such DRG neurons have not been investigated in the lumbar spine. Clinically, pain from the lumbar facet joint is sometimes referred to the lower extremities innervated by the sciatic nerve. METHODS: Two kinds of neurotracers (DiI and FG) were used in the present double-labeling study. DiI crystals were placed in the left L5-L6 facet joint, and FG was applied to the ipsilateral sciatic nerve or along the midline of the L5 dermatome. Bilateral DRGs T13-S1 were observed by fluorescence microscope. RESULTS: DRG neurons double labeled with DiI and FG were recognized only in the ipsilateral DRGs from L3 to L6 levels. Approximately 3% of DRG neurons innervating the L5-L6 facet joint had other axons to the sciatic nerve. By contrast, no double-labeled neurons were observed after FG was applied to the L5 dermatome. CONCLUSIONS: In rats approximately 3% of DRG neurons innervating the lumbar facet joints have dichotomized axons projecting to the sciatic nerve.