Application of custom anatomy-based nerve conduits on rabbit sciatic nerve defects: in vitro and in vivo evaluations

The intermingling of regenerated nerve fibers inside nerve grafts is the main reason for mismatched nerve fibers. This is one of the key factors affecting limb function recovery after nerve injury. Previous research has shown that the accuracy of axon regeneration can be improved by a bionic structural implant. To this aim, iodine and freeze-drying high-resolution micro-computed tomography was performed to visualize the 3D topography of the New Zealand rabbit sciatic nerve (25 mm). A series of 1-, 2-, 3-, and 4-custom anatomy-based nerve conduits (CANCs) were fabricated based on the anatomical structure of the nerve fascicle. The match index, luminal surface, and mechanical properties of CANCs were evaluated before implanting in a 10-mm gap of the sciatic nerve. Recovery was evaluated by histomorphometric analyses, electrophysiological study, gastrocnemius muscle weight recovery ratio, and behavioral assessments at 12 and 24 weeks postoperatively. The accuracy of nerve regeneration was determined by changes in fluorescence-labeled profile number during simultaneous retrograde tracing. Our results showed that the optimal preprocessing condition for high-resolution micro-computed tomography visualization was treatment of the sciatic nerve with 40% Lugol’s solution for 3 days followed by lyophilization for 2 days. In vitro experiments demonstrated that the match index was highest in the 3-CANC group, followed by the 2-, 1-, and 4-CANC groups. The luminal surface was lowest in the 1-CANC group. Mechanical properties (transverse compressive and bending properties) were higher in the 3- and 4-CANC groups than in the 1-CANC group. In vivo experiments demonstrated that the recovery (morphology of regenerated fibers, compound muscle action potential, gastrocnemius muscle weight recovery ratio, pain-related autotomy behaviors, and range of motion) in the 3-CANC group was superior to the other CANC groups, and achieved the same therapeutic effect as the autograft. The simultaneous retrograde tracing results showed that the percentages of double-labeled profiles of the 2-, 3-, and 4-CANC groups were comparatively lower than that of the 1-CANC group, which indicates that regenerated nerve fascicles were less intermingled in the 2-, 3-, and 4-CANC groups. These findings demonstrate that the visualization of the rabbit sciatic nerve can be achieved by iodine and freeze-drying high-resolution micro-computed tomography, and that this method can be used to design CANCs with different channels that are based on the anatomical structure of the nerve. Compared with the 1-CANC, 3-CANC had a higher match index and luminal surface, and improved the accuracy of nerve regeneration by limiting the intermingling of the regenerated fascicles. All procedures were approved by the Animal Care and Use Committee, Xinjiang Medical University, China on April 4, 2017 (ethics approval No. IACUC20170315-02).

Nerve growth factor-basic fibroblast growth factor poly-lactide co-glycolid sustained-release microspheres and the small gap sleeve bridging technique to repair peripheral nerve injury

We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique.Multiple growth factors play a synergistic role in promoting the repair of peripheral nerve injury;as a result,in this study,we added basic fibroblast growth factors to the microspheres to further promote nerve regeneration.First,in an in vitro biomimetic microenvironment,we developed and used a drug screening biomimetic microfluidic chip to screen the optimal combination of nerve growth factor/basic fibroblast growth factor to promote the regeneration of Schwann cells.We found that 22.56 ng/mL nerve growth factor combined with 4.29 ng/mL basic fibroblast growth factor exhibited optimal effects on the proliferation of primary rat Schwann cells.The successfully prepared nerve growth factor-basic fibroblast growth factor-poly-lactide-co-glycolid sustained-release microspheres were used to treat rat sciatic nerve transection injury using the small gap sleeve bridge technique.Compared with epithelium sutures and small gap sleeve bridging alone,the small gap sleeve bridging technique combined with drug-free sustained-release microspheres has a stronger effect on rat sciatic nerve transfection injury repair at the structural and functional level.

miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Differential expression of mi RNAs occurs in injured proximal nerve stumps and includes mi RNAs that are firstly down-regulated and then gradually up-regulated following nerve injury.These mi RNAs might be related to a Schwann cell phenotypic switch.mi R-30 c,as a member of this group,was further investigated in the current study.Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1,4,7,14,21,and 28 days post injury for analysis.Following sciatic nerve injury,mi R-30 c was down-regulated,reaching a minimum on day 4,and was then upregulated to normal levels.Schwann cells were isolated from neonatal rat sciatic nerve stumps,then transfected with mi R-30 c agomir and co-cultured in vitro with dorsal root ganglia.The enhanced expression of mi R-30 c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells.We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of mi R-30 c agomir on myelin sheath regeneration.Fourteen days after surgery,sciatic nerve stumps were harvested and subjected to immunohistochemistry,western blot analysis,and transmission electron microscopy.The direct injection of mi R-30 c stimulated the formation of myelin sheath,thus contributing to peripheral nerve regeneration.Overall,our findings indicate that mi R-30 c can promote Schwann cell myelination following peripheral nerve injury.The functional study of mi R-30 c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

Microelectrode array for bioelectrical signal stimulation and recording

A microelectrode array（MEA） is presented, which is composed of 60 independent electrodes with 59 working ones and one reference one, and they are divided into 30 pairs. Except for the reference electrode, each pair consists of one stimulating electrode and one recording electrode. Supported by the peripheral circuits, four electrode states to study the bioelectrical signal of biological tissue or slice cultured in-vitro on the surface of the electrodes can be realized through each pair of electrodes. The four electrode states are stimulation, recording, stimulation and recording simultaneously, and isolation. The state of each pair of working electrodes can be arbitrarily controlled according to actual needs. The MEAs are fabricated in printed circuit board （PCB） technology. The total area of the PCB-based MEA is 49 mm × 49 mm. The impedance measurement of MEA is carried out in 0.9% sodium chloride solution at room temperature by means of 2-point measurements with an Agilent LCR meter, and the test signal for the impedance measurement is sinusoidal （AC voltage 50 mV, sweeping frequency 20 Hz to 10 kHz）. The electrode impedance is between 200 and 3 kΩ while the frequency is between 500 and 1 000 Hz. The electrode impedance magnitude is inversely proportional to the frequency. Experiments of toad sciatic nerve in-vitro stimulation and recording and signal regeneration between isolated toad sciatic nerves are carried out on the PCB-based MEA. The results show that the MEA can be used for bioelectrical signal stimulation, recording, stimulation and recording simultaneously, and isolation of biological tissues or slices in-vitro.

附子汤对蟾蜍坐骨神经动作电位的影响

目的:观察附子汤对蟾蜍离体坐骨神经动作电位阈刺激、不应期及传导速度的影响,研究其对坐骨神经电生理特性的作用。方法:将制备的蟾蜍坐骨神经干分为阈刺激、不应期、传导速度3组,引导神经干动作电位,分别测其正常时与加药后的阈刺激、不应期、传导速度的变化。结果:附子汤能够提高蟾蜍离体坐骨神经阈刺激,延长不应期,与对照组比有显著性差异(P<0.05,P<0.01);可减慢传导速度,但无显著性差异。结论:附子汤对蟾蜍坐骨神经动作电位的抑制作用与提高阈刺激、延长不应期相关。

茶对蟾蜍坐骨神经电生理特性的影响

采用含不同浓度茶水的任氏液在不同时间段内浸泡蟾蜍坐骨神经干的方法,研究了茶水对蟾蜍坐骨神经干动作电位的宽度、幅度、引起动作电位的阈强度、不应期及传导速度等电生理指标的影响。结果表明,低浓度茶水处理后,动作电位的宽度变小、幅度变大、引起动作电位的阈强度降低、不应期缩短、传导速度变快,随着茶水浓度的升高和浸泡时间的延长,以上结果均成反向趋势。可以得出如下结论:低浓度茶水使神经的兴奋性升高,传导速度加快;高浓度茶水或浸泡时间过长,可降低神经的兴奋性,减慢传导速度。

蟾酥对蟾蜍坐骨神经电生理特性的影响

目的观察蟾酥对蟾蜍坐骨神经电生理特性的影响。方法将剥制的蟾蜍坐骨神经干分为4组,分别在林格溶液和浓度为0.1%,0.2%,0.4%的蟾酥溶液中浸泡,测定各组不同浸泡时间坐骨神经干动作电位的幅度、宽度、传导速度、引起动作电位的阈强度及不应期等电生理指标。结果经不同浓度蟾酥溶液处理后,引起神经干动作电位的阈强度和动作电位的宽度均增大,不应期延长,传导速度减慢。结论蟾酥可降低神经的兴奋性,减慢传导速度。

3种麻醉剂对蟾蜍离体坐骨神经电生理特性影响的比较研究

使用BL-420生物机能实验系统,采用细胞外记录神经干动作电位的方法,观察了氨基甲酸乙酯、水合氯醛和戊巴比妥钠3种麻醉剂对蟾蜍离体坐骨神经电生理特性的影响.结果:3种麻醉剂均可使坐骨神经的兴奋性降低、动作电位时程延长;另外,氨基甲酸乙酯和水合氯醛对神经的传导速度和动作电位幅度抑制作用较强,戊巴比妥钠对神经传导速度抑制作用较弱,但中浓度的戊巴比妥钠有增加动作电位幅度的作用.

川芎嗪对蟾蜍离体坐骨神经干电生理特性的影响

目的:应用电生理方法观察盐酸川芎嗪注射液对蟾蜍离体坐骨神经干电生理特性的影响。方法:将制备好的神经干置于标准任氏液中浸泡20min(作为对照组)后;随机再分3个实验组,即川芎嗪5mg、10mg和15mg浸泡组,各组又分别浸泡5、10、15、20、25、30 min 6个时间点。用BL-420E+生物机能实验系统测定各组各浸泡时间点神经干动作电位的阈强度、最适强度和不应期等电生理指标。结果:经不同浓度盐酸川芎嗪液处理后,引起神经干动作电位的阈强度和最适强度增大,不应期延长,与浸泡前(对照组)相比,差异极其显著(P<0.01)。3组浓度间比较:随川芎嗪液浓度增高对神经干电生理指标影响越明显(P<0.05,P<0.01)。经任氏液重新浸泡,消失的动作电位仍可恢复。结论:盐酸川芎嗪注射液能降低蟾蜍坐骨神经干的兴奋性,可逆性阻滞动作电位的产生。

茶多酚对蟾蜍坐骨神经干兴奋传导及骨骼肌收缩的影响

采用不同浓度的茶多酚溶液作用于蟾蜍坐骨神经—腓肠肌标本,发现10μg/L茶多酚就可极显著地增强腓肠肌的收缩张力,且随着其浓度的加大而张力增加,直至茶多酚浓度超过160μg/L时,肌肉张力开始回落,间接证明了茶多酚能够促进肌细胞内质网钙库中Ca2+向细胞质的释放。实验还发现,茶多酚能够显著地缩短蟾蜍坐骨神经—腓肠肌标本单收缩的潜伏期,但不影响动作电位单独在神经和肌肉细胞上的传导速度。这是由于茶多酚能提高蟾蜍神经—肌肉接头处微终板电位(MEPP)的频率和振幅,从而提高终板膜的兴奋性,使动作电位在神经—肌肉接头处的传递速度加快,缩短了突触延搁的时间。由此推测,这种兴奋性的加强还是源于茶多酚可促进神经纤维轴突末梢Ca2+内流,使细胞质中Ca2+浓度增加,导致乙酰胆碱释放量增加所致。

草乌对离体蟾蜍坐骨神经动作电位的影响

目的研究草乌对坐骨神经动作电位传导的阻滞作用。方法观察草乌水煎液对蟾蜍离体坐骨神经复合动作电位的振幅和传导速度的影响。结果10%,20%,40%3种浓度的草乌水煎液均使坐骨神经复合动作电位的振幅变小(P<0.01),传导速度变慢(P<0.01)并最终使坐骨神经动作电位消失,且动作电位的振幅和传导速度均能恢复。结论草乌能可逆地阻滞神经动作电位的传导。

碱化布吡卡因溶液对蟾蜍坐骨神经阻滞效能的研究

目的：了解布吡卡因碱化溶液的药效。方法：①离体坐骨神经法：取蟾蜍６０ 只，制备坐骨神经标本，分两组，实验组（ 碱化布吡卡因组） 和对照组（ 盐酸布吡卡因组） 各３０ 只，测定不同浓度的两组布吡卡因对离体坐骨神经动作电位（ＡＰ） 幅度的影响；②在体坐骨神经丛法：取蟾蜍６０ 只，分组同上，破坏第三脊椎以上的脊髓后，将不同浓度的两种布吡卡因注入蟾蜍的腹腔内，测定其缩腿反射消失的时间。结果：实验一显示，实验组与对照组ＥＤ５０ 之比为１∶２ ．７ ；实验二显示，实验组缩腿反射消失的时间明显短于对照组（ Ｐ＜ ０ ．０１） 。结论：碱化布吡卡因对传导麻醉的作用强度显著大于盐酸布吡卡因。

辣椒碱对离体蟾蜍坐骨神经动作电位的影响

目的探讨辣椒碱对坐骨神经动作电位传导阻滞的作用。方法观察辣椒碱溶液对蟾蜍离体坐骨神经复合动作电位的振幅和传导速度的影响。结果 0.125%,0.20%,0.25%3种浓度的辣椒碱溶液均使坐骨神经复合动作电位的振幅变小(P<0.01),传导速度变慢(P<0.01),并最终使坐骨神经动作电位消失。结论辣椒碱能阻滞神经动作电位的传导。

柴胡桂枝汤对离体蟾蜍坐骨神经动作电位的影响

目的:研究柴胡桂枝汤对蟾蜍离体坐骨神经干动作电位传导阻滞作用。方法:观察柴胡桂枝汤对蟾蜍离体坐骨神经复合动作电位的振幅和传导速度的影响。结果:柴胡桂枝汤作用0min、2min、4min、6min、8min、10min后均使坐骨神经干复合动作电位的振幅变小(P<0.01),传导速度变慢(P<0.01)并最终使坐骨神经动作电位消失。结论:柴胡桂枝汤能阻滞神经动作电位的传导。

夹竹桃叶对蟾蜍离体坐骨神经干动作电位的影响

为了研究夹竹桃叶水提取物对蟾蜍离体坐骨神经动作电位的影响,将蟾蜍离体坐骨神经分为3组,分别用10%、20%和40%的夹竹桃水提取物对其进行浸泡,通过BL-420F生物机能实验系统测定各组离体坐骨神经干动作电位的振幅、传导速度、阈强度及不应期。结果显示经不同浓度夹竹桃水提取物处理后,都可引起神经干动作电位振幅减小(P<0.05),传导速度减慢(P<0.05),不应期延长(P<0.05),阈强度增大(P<0.05),并最终使坐骨神经动作电位消失,且动作电位均能恢复。试验结果表明夹竹桃叶水提取物能可逆地阻滞神经动作电位的传导。

葛根素对蟾蜍离体坐骨神经干电生理特性的影响

目的:应用电生理方法观察葛根素对蟾蜍离体坐骨神经干电生理特性的影响,为探讨葛根素对外周神经系统的作用及其机制提供理论依据。方法:将制备好的神经干置于标准任氏液中浸泡20min(作为对照组)后;随机再分3个实验组,分别为葛根素25、40、50mg浸泡组,各组又分别浸泡5、10、15、20、25min。用BL-420E+生物信号分析系统测定各组各浸泡时间点神经干动作电位的阈强度、最适强度和不应期等电生理指标。结果:经不同浓度葛根素处理后,引起神经干动作电位的阈强度除25mg组和40mg组各浸泡5min后无变化外,其余各实验组的阈强度、最适强度和不应期均随浸泡时间的延长而增大或延长,与浸泡前(对照组)相比,差异极其显著(P<0.05,P<0.01)。3组浓度间比较:葛根素50mg组对神经干动作电位电生理特性的影响明显大于葛根素25mg组和40mg组(P<0.05,P<0.01)。3组神经干动作电位消失后置新鲜任氏液浸泡均可再现。结论:葛根素能降低蟾蜍坐骨神经干的兴奋性,可逆性阻滞动作电位的产生。

乙醇与甲醇对中华大蟾蜍坐骨神经动作电位影响的比较研究

应用电生理方法观察了不同浓度的乙醇和甲醇对中华大蟾蜍离体坐骨神经干刺激阈值、最大刺激值、动作电位峰值和传导速度等四种生理指标的影响.结果表明:低浓度的乙醇(0.1%,0.3%,0.5%,1%,3%,5%)对神经干刺激阈值和最大刺激值的影响不明显,能引起动作电位峰值和传导速度先缓慢上升再缓慢下降;中、高浓度的乙醇(8%,10%,12%)对上述四种生理指标影响显著,随着浓度的递增,刺激阈值和最大刺激值逐渐增大,动作电位峰值逐渐下降,传导速度逐渐变慢.甲醇随着浓度的递增,对神经干上述四种生理指标的影响与乙醇基本一致,只是同浓度的甲醇较乙醇影响更为显著.这说明,甲醇对坐骨神经干动作电位的抑制作用要大于乙醇.

蛇床子提取液对离体蟾蜍坐骨神经动作电位的影响

目的:研究蛇床子提取液对蟾蜍离体坐骨神经动作电位传导阻滞作用。方法:观察3种浓度蛇床子提取液(1g·ml-1、0.5g·ml-1、0.2g·ml-1)对蟾蜍离体坐骨神经复合动作电位的振幅和传导速度的影响。结果:3种浓度的蛇床子提取液均可使坐骨神经复合动作电位的振幅变小(P<0.01),传导速度变慢(P<0.01)并最终使坐骨神经动作电位消失。结论:蛇床子提取液能阻滞神经动作电位的传导。

四数九里香挥发油对离体蟾蜍坐骨神经动作电位的影响

目的:研究四数九里香挥发油对离体蟾蜍坐骨神经动作电位的传导阻滞作用。方法:采用水蒸气蒸馏法提取四数九里香挥发油,观察四数九里香挥发油对离体蟾蜍坐骨神经复合动作电位的振幅和传导速度的影响。结果:四数九里香挥发油使坐骨神经复合动作电位的振幅变小(P<0.01),传导速度变慢(P<0.01),并最终使坐骨神经动作电位消失。结论:四数九里香挥发油能阻滞神经动作电位的传导。

不同因素对蟾蜍坐骨神经干动作电位的影响

实验采用电生理学方法,观察几种物质对蟾蜍坐骨神经干动作电位幅度及传导速度的影响.结果显示:1)与对照组相比,高渗KCl,NaCl,CaCl2,AlCl3溶液使动作电位幅度显著降低,传导速度显著减慢(P<0.05),呈时间依赖性;2)10%乙醇1、0%甲醇1、0%甲醛使动作电位幅度不同程度下降,传导速度显著减慢(P<0.05),呈时间依赖性;3)10%姜蒜汁处理神经干,动作电位幅度和传导速度也明显降低(P<0.05);4)20%葡萄糖使神经干动作电位幅度降低,传导速度显著减慢(P<0.05),呈时间依赖性;而10%葡萄糖使神经干动作电位幅度上升,传导速度加快(P<0.05).