Peripheral nerve autografts to the rat spinal cord: Study on the origin and course of regenerating fibres

Summary Autologous sciatic nerve grafts were implanted to the lower thoracic spinal cord (SC) of adult rats. The grafts were longitudinally placed on both sides of the SC midline over the dura mater and their cut ends were inserted into the dorsal white matter of the SC. Eight to 60 weeks later the grafts were exposed. In a first experimental group (A) either horseradish peroxidase (HRP) or lectin conjugated horseradish peroxidase (WGA-HRP) was injected into the grafts in order to investigate the origin and the course of regenerated fibres entering the grafts. In a second experimental group (B) the SC was acutely transected between the upper and lower graft insertions and either HRP or WGA-HRP was injected into the caudal stump of the SC in order to investigate the ability of regenerating axons once entered the grafts to re-enter the SC. In group A retrogradely labelled cells were found in the SC scattered in proximity of both the caudal and rostral graft insertions and in the ventral horns as far as 30 mm rostrally to the grafts. Labelled cells were also located in the spinal ganglia, at the level of the grafts and up to 6 segments caudal to them. In group B retrogradely labelled cells were present in the SC rostrally to the transection, both in proximity of the upper graft insertions and in the ventral horns as far as 30 mm rostrally to the grafts. These findings demonstrate that PN grafts implanted to the SC of adults rats can be innervated by regenerated axons arising from both intraspinal neurons and dorsal root ganglion cells (group A); furthermore axons from intraspinal neurons entered and elongated in the grafts can reenter the SC (group B).     

肌肉源22、35kD神经诱向因子对游离周围神经修复脊髓损伤的作用

将从３周龄大白鼠腓骨肌中提取的２２、３５ｋＤ的肌肉源神经诱向因子（ＭＮＴＦ），用１×１ｍｍＰｈａｓｔ凝胶块运载后放入游离周围神经移植与脊髓连接处。结果表明：术后４周，实验组游离神经与脊髓连接处无明显空洞反应，大量神经纤维长入神经内，再生的有髓纤维为对照组的１０．５倍（Ｐ＜０．０１）。ＨＲＰ示踪发现实验组有１４８０．３±４２３．５个阳性神经元，而对照组只有２０．３±６．５个神经元（Ｐ＜０．０１）。显然，ＭＮＴＦ具有增强脊髓及神经根背节神经元，尤其是脊髓运动神经元再生的能力，并可能有营养移植神经中雪旺氏细胞的作用。这对脊髓损伤修复具有一定的意义。     

周围神经移植修复脊髓损伤的实验研究

目的：探讨自体坐骨神经移植修复脊髓损伤的可行性。方法：将58只雌性Wistar大鼠分为二组，实验组：采用显微外科技术，将50只大鼠于T13水平切除左半侧脊髓10mm，再取右侧坐骨神经10mm移植到脊髓缺损处，近端接脊髓，远端接马尾，分别于术后2、4、6、8、12、22周在光镜和电镜下观察移植处坐骨神经、吻合口远端马尾神经、左后肢坐骨神经再生情况，并用摄像机记录患肢功能恢复情况。对照组：8只大鼠，于13水平切除左半侧脊髓10mm，不移植坐骨神经，观察脊髓缺损远端马尾神经和左右肢坐骨神经再生情况。结果：对照组坐骨神经的轴突及髓鞘部分崩解，密度降低，无再生轴突形成。实验组术后4周电镜下偶见移植处坐骨神经髓鞘及轴突形成，术后8周光镜及电镜下可见较多细的有髓神经纤维，22周时接近正常；同时观察到左后肢坐骨神经再生；大鼠后肢功能部分恢复，肌力达3级。结论：大鼠脊髓损伤后有再生能力，周围神经移植修复脊髓损伤是可行的。     

碎化的自体坐骨神经脊髓内移植对损伤脊髓再生的影响

将碎化的自体坐骨神经植入大鼠的半横断损伤脊髓内，用辣根过氧化物酶逆行示踪技术及组织学方法，检查了移植术后脊髓的再生能力。结果表明，自体坐骨神经脊髓内移植能明显增加损伤脊髓的再生，而且移植的神经组织与脊髓相互融合，并形成新的纤维性连接。     

胚胎脊髓组织移植联合应用神经生长因子对损伤脊髓再生修复的形态学影响

选择胎龄为１４～１５天的大鼠胚胎脊髓植入成年大鼠半横断的脊髓损伤腔，同时局部应用神经生长因子。术后２～８个月，用组织学、免疫细胞化学、辣根过氧化酶示踪及超微结构的检查方法证实，移植组织在宿主脊髓内的存活率为９０％，并且分化成熟，具有正常的神经组织结构特征，大多数移植物充满损伤腔，与宿主脊髓形成良好的融合，移植组织与宿主组织出现新的纤维联系，使脊髓损伤的两端恢复了解剖的连续性。     

大鼠脊髓慢性压迫性损伤动物模型的建立

目的 建立一种大鼠脊髓慢性压迫性损伤模型。方法 应用平头不锈钢螺钉从大鼠C4椎体前侧钻入压迫脊髓腹侧，术后保留螺钉30d，并用联合行为评分(CBS)、神经电生理、光镜、电镜检查进行综合评判。结果 模型动物脊髓慢性压迫随着螺钉轻、中、重程度的不同，其术后行为学、运动诱发电位(MEP)及组织学观察符合脊髓慢性压迫症病理改变的特点。结论 建立了大鼠脊髓慢性压迫性损伤模型，此模型制作简便，可重复性强，脊髓损伤能表现出不同的压迫深度，为进一步研究脊髓慢性压迫损伤病理机制奠定了基础。     

The efficacy of erythropoietin on acute spinal cord injury. An experimental study on a rat model

Introduction  The accumulated knowledge of erythropoietin (EPO) interaction in neural injury has led to potentially novel therapeutic strategies. Previous experimental studies of recombinant human EPO (rhEPO) administration have shown favorable results after central and peripheral neural injury. In the present study we used the aneurysmal clip model to evaluate the efficacy of two different regimes of rhEPO administration on the functional outcome after severe acute spinal cord injury (ASCI). Materials and methods  Thirty rats were operated on with posterior laminectomy at thoracic 10th vertebra. Spinal cord trauma produced by extradural placement of the aneurysm clip, for 1 min. Animals were divided into three groups; the first group received a low total EPO dose (EPO-L), (2 doses of 1,000 IU each s.c.). The second group was administered the high total EPO dose (EPO-H), (14 doses of 1,000 IU each s.c.), and the third was the Control group, which received normal saline in the same time fashion with EPO-H group. Follow-up was for 6 weeks. Estimation of the functional progress of each rat was calculated using the locomotor rating scale of Basso et al, with a range from 0 to 21. Results  After surgery the animals suffered paraplegia with urinary disturbances. Rats that received EPO demonstrated statistically significant functional improvement compared to the Control group, throughout study interval. On the last follow-up at 6 weeks the EPO-L rats achieved a mean score 17.3 ± 1.15, the EPO-H 14.7 ± 1.82, and the control group 8.2 ± 0.78. Comparison between the two EPO groups reveals superior final outcome of the group treated with lower total dose. Conclusion  Our study supports current knowledge, that EPO administration has a positive effect on functional recovery after experimental ASCI. These data reflect the positive impact of EPO on the pathophysiologic cascade of secondary neural damage. However, we observed a dose-related effect on functional recovery. Interestingly, large doses do not seem to favor the neurological recovery as lower doses do.     

脊髓损伤两种修复方法的比较研究

目的：比较脊髓损伤后两种修复方法的效果。方法：损伤成鼠胸髓左后术,移植孕１４ｄ３胚胎脊髓（Ｆ组）,胚胎脊髓加自体带蒂正中神经（Ｖ＋Ｆ组）及不移植（对照组）,观察胚胎脊髓的生长、分化情况及对宿主的修复能力。结果：Ｖ＋Ｆ组胚胎脊髓体积增长速度、神经纤维数量和神经元密度显著高于Ｆ组（Ｐ〈０．０１）,细胞分化较好,突触较成熟,界面区无明显的胶质增生。Ｖ＋Ｆ组ＳＥＰ的Ｐ１、Ｎ１波潜伏期显著缩短（Ｐ〈０．０１     

Experimental bypass surgery between the spinal cord and caudal nerve roots for spinal cord injuries

Spinal cord injuries often cause permanent neurological deficits and are still considered as inaccessible to efficient therapy. Injured spinal cord axons are unable to spontaneously regenerate in adult mammalians. Re-establishing functional activity especially in the lower limbs by reinnervating the caudal infra-lesional territories could represent an attractive therapeutic strategy. For several years, we have studied and developed surgical bypasses using peripheral nerve grafts bridging the supra-lesional rostral spinal cord to the caudal infra-lesional lumbar roots. Main objectives were: 1- to overcome the spinal cord lesion and the consecutive glial barrier blocking the axonal regeneration; 2- to find and bring an alternative source of regenerating axons; 3- to guide those axons toward precisely definite targets (for example, lower limb muscles). We report here the results of our experimental research, which led us from animal experimental models (rodents, primates) to the first human experimentation. Limitations of the method (especially technical pitfalls) are numerous. However, we have obtained encouraging results in our attempts to "repair" the motor pathway. Functional recovery with strong evidence of centrifugal axonal regeneration from the spinal cord to the periphery has been observed. Regarding the sensory pathway, we have found evidence of centripetal axonal regeneration from the periphery toward the spinal cord. Further studies are obviously advocated, but our experimental model of spinal cord - nerve roots bypasses may be integrated in future "repair" strategies of both motor and sensory pathways following spinal cord injury.     

Bladder inhibition or voiding induced by pudendal nerve stimulation in chronic spinal cord injured cats

AIMS: To investigate pudendal-to-bladder spinal reflexes in chronic spinal cord injured (SCI) cats induced by electrical stimulation of the pudendal nerve. METHODS: Bladder inhibition or voiding induced by pudendal nerve stimulation at different frequencies (3 or 20 Hz) was studied in three female, chronic SCI cats under alpha-chloralose anesthesia. RESULTS: Voiding induced by a slow infusion (2-4 ml/min) of saline into the bladder was very inefficient (voiding efficiency=7.3%+/-0.9%). Pudendal nerve stimulation at 3 Hz applied during the slow infusion inhibited reflex bladder activity, and significantly increased bladder capacity to 147.2+/-6.1% of its control capacity. When the 3-Hz stimulation was terminated, voiding rapidly occurred and the voiding efficiency was increased to 25.4+/-6.1%, but residual bladder volume was not reduced. Pudendal nerve stimulation at 20 Hz induced large bladder contractions, but failed to induce voiding during the stimulation due to the direct activation of the motor pathway to the external urethral sphincter. However, intermittent pudendal nerve stimulation at 20 Hz induced post-stimulus voiding with 78.3+/-12.1% voiding efficiency. The voiding pressures (39.3+/-6.2 cmH2O) induced by the intermittent pudendal nerve stimulation were higher than the voiding pressures (23.1+/-1.7 cmH2O) induced by bladder distension. The flow rate during post-stimulus voiding induced by the intermittent pudendal nerve stimulation was significantly higher (0.93+/-0.04 ml/sec) than during voiding induced by bladder distension (0.23+/-0.07 ml/sec). CONCLUSIONS: This study indicates that a neural prosthetic device based on pudendal nerve stimulation might be developed to restore micturition function for people with SCI.     

Ephrin-B1在大鼠脊髓损伤的表达

[目的]探讨红细胞生成素诱导肝癌细胞株配体-B1 (Ephrin-B1)在大鼠脊髓损伤的表达变化.[方法]将60只成年Wistar大鼠分正常组、假手术组、伤后3、7、14 d及28 d组.假手术组大鼠不损伤脊髓,损伤组大鼠采用Allen's氏打击法损伤T10脊髓.分别在各个时间点取材,免疫荧光和Western Blot检测Ephrin-B1蛋白表达情况.[结果]免疫荧光结果显示Ephrin-B1在正常、假手术和损伤脊髓的灰质和白质中均有表达,但在损伤脊髓组织中表达较高,脊髓组织中的星形胶质细胞表达Ephrin-B1.Western Blot显示损伤组比对照组Ephrin-B1蛋白含量显著增加(P＜0.01),从伤后3d开始持续到伤后28 d;伤后14 d蛋白含量达到高峰,明显高于对照组及伤后其他组(P＜0.01);伤后28 d蛋白含量较14 d减少(P＜0.01),与伤后7d相比无显著性差异(P＞0.05).[结论]Ephrin-B1在大鼠脊髓损伤后表达升高,伤后14d表达升高显著,表达于脊髓的星形胶质细胞.     

脊髓诱发电位的实验研究

为探索脊髓诱发电位（Spinalcordevokednotentials，SCEP）在术中监测的安全范围，将刺激和记录线电极分别置于24只大耳白兔T7～T8、L4～L5间隙硬障外，记录正常电位后对脊髓施加纵向牵张力，至实验要求的波幅。波幅下降至正常的80％组脊髓组织结构轻微改变，脊髓功能正常；下降至50％组及＜50％组则出现不可逆损害。结果表明，SCEP波形稳定，其波幅下降至80％是脊髓损伤的危险信号，下降至50％或50％以下将产生不可逆的脊髓损伤。     

不同方法移植胚胎脊髓促进成鼠损伤脊髓功能恢复的研究

目的：研究提供血运的胚胎脊髓移植对成鼠损伤脊髓功能恢复的作用。方法：将成年Ｗｉｓｔａｒ大鼠作为脊髓半切洞损伤模型,应用单纯胚髓组织、胚胎脊髓＋大网组织、胚社以半切洞损伤的脊髓中,手术后１、２、４、１２周进行联合行为发,感觉诱闰（ＳＥＰ）和运动诱发电位（ＭＥＰ）检查,并与一言堂３组为单纯损伤线比较。结果：联合行为评分,单纯移植级和胚胎脊髓＋大网膜组织移植组优于单纯损伤组,ＳＥＰ和ＭＥＰ潜峰时的恢复,     

狗脊髓截除自体隐神经大网膜移植的初步观察

实验用6只狗于T13处切除脊髓8mm，局部用隐神经、大网膜移植。对照组6只狗单纯切除脊髓8mm。术后3个月、6个月实验组动物全部恢复到C／Ⅲ级功能，组织学切片见移植神经有不同程度存活，可记录到股四头肌、胫前肌电位。对照组动物仅达到0／0～A／I织功能，组织学切片见脊髓缺损处为结缔组织，诱电位波形消失。分析了影响脊髓神经功能恢复的原因。     

周围神经不同移植方式修复治疗损伤脊髓的实验研究

目的：探讨自体肋间神经不同移植术式对治疗损伤脊髓疗效的影响。方法：采用SD大鼠，经T8、T9平面切除4mm长的半侧脊髓制成脊髓半切伤模型，用6～8条纤细的肋间神经节段按近端白质-远端灰质和近端白质-远端白质两种桥接术式填充缺损处，对照组用明胶海绵填充脊髓半切残腔。饲养4～6周，观察其后肢活动功能、斜板试验及运动功能，光镜下观察植入区的组纵结构。结果：两种术式受试鼠的后肢功能部分恢复，镜下见移植组织存活、充填于缺损处，部分与脊髓组织融合。组织、功能的恢复均明显优于对照组，但两种术式间无明显差别。结论：自体肋间神经的植入对损伤脊髓有一定的疗效，但不同的桥接术式之间无明显差别。     

兔胚胎脊髓移植修复周围神经缺损的实验研究

目的：对用胚胎脊髓移植修复周围神经缺损的可行性进行研究。方法：将36只新西兰家兔制成兔双侧坐骨神经缺损模型。按脊髓桥接长度分为3组,每组两侧神经桥接长度不同。第1组桥接长度为坐骨神经直径的4倍,第2组为8倍,第3组为16倍;左侧选用胎兔脊髓桥接,右侧为自体神经桥接。于术后1、2、3个月任选2只动物行辣根过氧化物酶（HRP)逆行示踪,另外10只取移植段中、远段组织作透射电镜观察。结果：在兔脊髓前角3组均发现被标记的神经元细胞。扫描电镜下见到胚胎脊髓移植段及远段有大量的正常神经纤维,其轴突直径、髓鞘厚度与对照侧相比,差异均无显著性意义（P＞0．05）。结论：用兔胚胎脊髓移植修复周围神经缺损的方法是可行的。     

重组人促红细胞生成素(rH-EPO)对急性脊髓水肿抑制作用的实验研究

目的研究重组人促红细胞生成素对急性脊髓水肿的抑制作用及其机制探究其临床应用的可能性。方法实验动物随机分为3组,采用静力加载技术于Wistar大鼠T13椎体水平造成急性脊髓损伤。生理盐水对照组,伤后立即给予生理盐水尾静脉注射。rHu-EPO治疗组,动物伤后立即给予rHu-EPO 1000unit/kg of bw尾静脉注射。甲基强的松龙治疗组,伤后立即给予甲基强的松龙30mg/kg of bw尾静脉注射。观察治疗前后实验动物神经功能评分变化、以及脊髓组织含水量的改变,细胞内游离钙含量([Ca2 ]i)的变化。结果(1)较之对照组,rH-EPO治疗组伤后1h运动功能已明显恢复(P<0.01)Rh-EPO治疗组受伤节段含水量在伤后各阶段均明显低于对照组(P<0.01),Rh-EPO在伤后12h对细胞内[Ca2 ]i含量的上升表现出明显的抑制作用(P<0.05)(。2)较之对照组,MP治疗组伤后伤后24h开始出现明显的功能恢复(P<0.05),伤后24h、48h含水量少于对照组(P<0.01),MP在伤后24h对细胞内[Ca2 ]i含量的上升表现出明显的抑制作用(P<0.05)。结论重组人促红细胞生成素对急性脊髓水肿具有明显的抑制作用,并且起效时间早,有效促进急性脊髓水肿时的神经功能恢复。     

活性巨噬细胞与周围神经联合移植治疗

目的脊髓损伤后轴突再生是脊髓功能恢复的基础,但再生轴突受神经自身条件及损伤微环境的限制。研究表明脊髓损伤后损伤区早期、大量的巨噬细胞聚积可改善局部微环境,减轻脊髓继发性损伤并促进其再生;而周围神经移植可为随后发生的再生轴突提供通道和营养物质,两者联合应用则为脊髓损伤的治疗提供一条有效的途径。方法将72只S—D大鼠采用随机的方法分为4组,A组大鼠在T。脊髓半切加洞性损伤;B组在上述基础上行肋间神经移植;C组行巨噬细胞移植;D组行巨噬细胞和肋间神经联合移植。术后1、3天和1、2、4、8周收集脊髓标本并冰冻切片、免疫组化染色,显微镜下巨噬细胞及神经纤维计数。结果D组大鼠术后4周以上时BBB运动功能评分平均提高1分,镜下观察见C、D组大鼠在伤后4周内巨噬细胞及再生轴突数目均高于A、B组。结论该方法可减轻脊髓损伤并促进其再生,可能是治疗脊髓损伤的一条有效途径。     

Effects of laser irradiation on the spinal cord for the regeneration of crushed peripheral nerve in rats

BACKGROUND AND OBJECTIVE: The purpose of the present study was to examine the recovery of the crushed sciatic nerve of rats after low-power laser irradiation applied to the corresponding segments of the spinal cord. STUDY DESIGN/MATERIALS AND METHODS: After a crush injury to the sciatic nerve in rats, low-power laser irradiation was applied transcutaneously to corresponding segments of the spinal cord immediately after closing the wound by using 16 mW, 632 nm He-Ne laser. The laser treatment was repeated 30 minutes daily for 21 consecutive days. RESULTS: The electrophysiologic activity of the injured nerves (compound muscle action potentials--CMAPs) was found to be approximately 90% of the normal precrush value and remained so for up to a long period of time. In the control nonirradiated group, electrophysiologic activity dropped to 20% of the normal precrush value at day 21 and showed the first signs of slow recovery 30 days after surgery. The two groups were found to be significantly different during follow-up period (P < 0.001). CONCLUSION: This study suggests that low-power laser irradiation applied directly to the spinal cord can improve recovery of the corresponding insured peripheral nerve.     

周围神经端侧吻合后神经再生的研究

目的研究周围神经端侧吻合后远端神经的再生及其临床应用价值。方法选用３７只Ｗｉｓｔａｒ大鼠，分成Ａ、Ｂ两组。Ａ组：切断腓神经，在邻近的胫神经干外膜上开一１ｍｍ小窗，将腓神经远端吻合到胫神经干侧方开窗处。Ｂ组：切断腓神经后，在胫神经干和远侧腓神经干外膜上各开一小窗，取对侧相应的腓神经以端侧吻合的方式桥接于胫、腓神经干两窗之间。两组分别于术后４、８、１２周取材，作神经组织学、电镜及图像分析仪检测：（１）神经再生的数量和质量；（２）定性定量观察神经再生情况；（３）再生神经的数目、密度及截面积。结果直接端侧吻合或神经桥接移植的端侧吻合，均可使神经再生，且再生纤维的质量与端端吻合组（对照组）无显著性差异（Ｐ＞０．０５）；对被吻合神经－胫神经的功能亦无影响。结论神经端侧吻合可作为神经损伤后一种新的修复方法