The influence of bacterial collagenase on regeneration of severed rat sciatic nerves

Summary Regeneration of peripheral nerve fibers is impeded by the formation of scar tissue at the site of injury. The possible beneficial effect of collagenase on nerve regeneration was studied using clinical, neurophysiological (evoked potentials) and histological (nerve fiber counts) methods.The sciatic nerves of rats were transected and the severed ends abutted and sewn together. In one series, the area about the lesion was covered with fibrin adhesive and infused with either isotonic saline (controls) or collagenase (treatment group). In the other series, the severed ends of the nerve were inserted into a silicone tube and separated by a collagen plug, which was infused with either saline or collagenase. Compared to the controls, the treated animals showed a significant improvement of clinical and neurophysiological parameters. After 3 months of observation, the collagen content of the transection site was reduced, and in the silicone series, the total number of myelinated axons 5mm distal to the site of transection was increased, while the fiber diameter distribution was unchanged.Supported by a grant from the Deutsche Forschungsgemeinschaft (We 1248).     

Electrophysiological effect of HeNe laser on normal and injured sciatic nerve in the rat

Summary The effect of low energy CW HeNe laser irradiation on normal and dissected nerves in the rat was examined. The methods are described. Results are compared to the laser effect on other living tissues. HeNe irradiation was found to increase significantly the action potentials of the nerves. It was found to be a long-lasting effect, keeping an increase in the nerves action potential for more than eight months after irradition has been stopped. A possible explanation for the way the irradiation acts on the nerve is suggested.This work was supported by a joint grant from the Ministry of Industry and Commerce and Tel-Aviv University, Israel.     

Assessment of effects of pinealectomy and exogenous melatonin administration on rat sciatic nerve suture repair: an electrophysiological,electron microscopic,and immunohistochemical study

Summary Background. Collagen scar formation at the cut end of a nerve, an important problem in clinical practice for neurosurgeons in peripheral nerve surgery, obstructs sprouting of axons into appropriate distal fascicles, and thereby limits nerve regeneration. Researchers attempt to control collagen accumulation in the formation of neuroma by various physical and chemical methods, but these have yielded only limited functional success. This is the first experimental study investigating the effects of melatonin (MLT) on nerve repair and neuronal regeneration in rat sciatic nerve suture repair.Methods. The hypothesis that exogenous MLT administration may inhibit the formation of neuroma in peripheral nerve surgery was investigated in rat sciatic nerve model. In this study, a total of 80 rats were used for control groups (Groups Ia, Ib, IIa, and IId), MLT group (Group Ic), surgical pinealectomy (Px) groups (Groups IIb and IIc), and group of MLT treatment following Px procedure (Group IIe). All animals underwent a surgical intervention consisting of bilateral sciatic nerve section and primary suture repair. At 8 weeks after repair, the animals were killed following completion of recording of nerve action potentials (NAPs). Then, unilateral sciatic nerve specimens including the suture repair region were carefully removed and the excised segments were processed for electron microscopy examination. Afterwards, contralateral sciatic nerve specimens from two animals from each group were removed and stained for immunohistochemical analysis.Results. Results of morphometric analysis revealed that Px procedure caused an elevation of collagen content of the sciatic nerve and macroscopic neuroma formation, and that there was a statistically significant reduction in collagen content of the same region in pinealectomized animals treated with MLT (p<0.001). Accordingly, electrophysiological findings demonstrated that the stimulus intensities required to excite a NAP response were increased in surgical Px group, but the presence of a reduced threshold response was found in the group treated with MLT following Px procedure (p<0.01). Immunohistochemical staining for Type I collagen and Type III collagen was markedly more intense in the epineurium of animals after Px. Virtually no or only weak staining was observed in animals in control groups and the MLT treatment group. Results of immunohistochemical analysis revealed that surgical Px procedure caused a strong immunoreactivity for Type I collagen and Type III collagen in all connective tissue planes of the nerve, especially in the epineurium, and there was a statistically significant reduction in immunoreactivity of the repair region in animals receiving MLT treatment after Px procedure (p<0.001).Conclusion. This study demonstrates that exogenous MLT administration significantly inhibits collagen accumulation in the formation of neuroma in the suture repair site and thereby improves nerve regeneration. From a clinical standpoint, the positive effect of MLT administration on neuroma formation and nerve regeneration seems a particularly attractive treatment option. Therefore, we believe that nerve repair with addition of MLT may be a worthwhile option in addition to other treatment modalities in case of MLT deficiency, such as aging. However, further experimental and clinical studies using functional analysis warranted to confirm this result in future.     

FK506对大鼠坐骨神经再生作用的实验研究

押目的对FK506促神经再生的作用及药物应用方法进行初步探讨。方法雄性SD大鼠60只,随机分成三组,切断双侧坐骨神经制成坐骨神经再生室模型。A组(对照组)再生室内注入生理盐水;B组(全身用药组)再生室内注入盐水,颈后皮下注射FK5061mg/kg,连续14天;C组(局部用药组)再生室内注入1μg/mlFK506。于术后一定时间内观察神经损伤局部的免疫反应熏检测腓肠肌湿重、组织形态学及图像分析、电生理学。结果B、C组神经损伤局部淋巴细胞浸润程度较A组轻微。6周时B组各测定结果明显优于A组;C组各指标好于A组,但不具有统计学意义。结论穴1雪全身应用FK506(1mg/kg)具有神经保护和神经营养作用,可加快神经功能的恢复。(2)局部应用FK506(1μg/ml)对早期损伤神经有一定的保护作用,但对神经再生的促进作用不确切。     

优化法去细胞大鼠神经同种异体移植修复坐骨神经缺损

[目的]以优化法去细胞大鼠神经移植,修复同种异体坐骨神经缺损,观察术后动物的免疫排斥、早期功能恢复及神经再生情况。[方法]以优化去细胞方法处理新鲜取材的成年SD大鼠坐骨神经,移植修复同种异体1.0cm坐骨神经缺损,以自体神经和新鲜异体神经移植为对照,术后1个月行坐骨神经功能指数评价、神经电生理和组织学检查,观察动物在功能恢复、免疫排斥及神经再生方面的情况。[结果]自体神经和去细胞异体神经移植组动物的坐骨神经功能指数无显著差异(P>0.05),大体观察均可见神经连续性良好。电生理检测表明2组动物移植神经均已恢复电传导能力,在传导速度(CV)上无显著差异(P>0.05),但均未达到正常神经水平(P<0.05)。组织学观察则显示2组再生神经纤维均已长入移植段远端。S-100免疫组化显示两者在雪旺氏细胞数、形态和排列等方面无明显差异。2组在CD8 T细胞和巨噬细胞免疫组化染色阳性面积百分比上差异无统计学意义(P>0.05)。计算移植神经中段轴突密度后表明两者无显著差异(P>0.05),但都比正常神经小(P<0.05),比新鲜异体神经移植组大(P<0.05)。[结论]优化去细胞神经移植组与自体神经移植组在免疫排斥、功能恢复及神经再生方面无显著差异。优化法去细胞神经在移植修复同种异体神经缺损时,可以达到免疫耐受,其早期功能恢复和神经再生情况良好,在修复周围神经缺损时可以作为自体神经移植的一种替代疗法。     

Comparative study of microepineurial anastomoses with the use of CO2 laser and suture techniques in rat sciatic nerves: Part 1. Surgical technique, nerve action potentials, and morphological studies

The possibility of utilizing the CO2 laser for neural anastomoses was investigated in a rat sciatic nerve model. One nerve in each animal was acutely divided and anastomosed using 10-0 nylon epineurial sutures, while the opposite side was joined by "welding" the opposed nerve ends together with CO2 laser pulses. The surgical incisions were reexplored 60 days postoperatively, action potentials were recorded across the anastomoses, and the nerves were removed for light and electron microscopy. The operative patency rate in the suture group was 100%, compared to 87% in the laser group. Among those animals with bilaterally intact nerves, action potentials could be recorded across the anastomotic site in 78% of the sutured preparations and in 85% of nerves spot-welded with the CO2 laser. Morphological studies showed a greater degree of scar tissue formation and constriction in the anastomotic zone of the nerves joined by sutures than was present in laser-treated animals. We believe these preliminary observations suggest that the CO2 laser may have a role to play in peripheral nerve surgery. Further study of this technique is warranted.     

优化法去细胞兔神经移植修复大鼠坐骨神经缺损

目的 以优化法去细胞兔臂丛神经，移植修复大鼠坐骨神经缺损，观察免疫排斥情况、早期功能恢复及神经再生情况。方法 以优化法处理新鲜取材的新西兰大白兔臂丛神经分支，移植修复成年sD大鼠坐骨神经1．0cm缺损，分别于术后1个月及3个月行功能评价、电生理和组织学检查，观察免疫排斥、功能恢复及神经再生情况。结果 实验组与自体神经移植组在免疫排斥、功能恢复及神经再生方面无显著差异，却明显优于新鲜兔神经移植组。3个月取材时的神经再生及功能恢复情况优于1个月取材时。结论 优化法去细胞神经在移植修复异种神经缺损时，可以达到免疫耐受，其早期功能恢复和神经再生情况良好，在修复周围神经缺损时有望作为自体神经移植的一种替代疗法。     

应用Laminin促进周围神经再生的研究

目的探讨局部应用Laminin（LN）对周围神经再生的影响。方法将34只SD大鼠分为4组,1～3组每组10只,手术切除双侧10mm坐骨神经并用硅胶管套接,左侧套管内注入LN0．6μg,右侧注入生理盐水作对照。术后1、3、4个月分别进行电生理和组织学检查。另4只鼠于术后3个月行辣根过氧化物酶逆行示踪检查。结果LN治疗组术后1、3、4个月时的神经肌肉动作电位、运动神经传导速度、再生有髓神经纤维数量及髓鞘厚度均明显优于对照组。相应节段脊髓前角和背根神经节标记神经元明显多于对照组。结论LN可促进周围神经损伤后的再生。     

Carbon dioxide laser-assisted thyroarytenoid myomectomy

CO2 laser-assisted myomectomy of the thyroarytenoid muscle (TA) was carried out through a thyroplasty approach in ten laryngeal specimens and the TA was examined histologically. Using the CO2 laser at super-pulsed mode, the TA can be cut or resected using 3-5 W of laser power. There is good control of the depth and extent of TA resection. Histologically, there is little evidence of collateral injury, with sparing of vital surrounding vocal structures. The use of CO2 laser for TA resection offers better access and surgical hemostasis in an otherwise difficult surgical field. Laser-assisted myomectomy may be a feasible alternative to current methods to treat spasmodic dysphonia.     

Ultrastructural study of rabbit sciatic nerve regeneration following experimental excisional transection and autograft reconstruction

Summary This experimental study presents the ultrastructure of regenerating sciatic nerve of the rabbit, after transection and immediate end to end anastomosis, using perineural fascicular nerve autograft, in a sterile environment.Twenty-four hours, 1, 2, and 6 weeks after the anastomosis, the treated sciatic nerves were exposed and three segments were excised and studied. The first at the region of the graft and the others from the proximal and distal stump of the nerve, in the vicinity of the graft suture. The sections taken from the proximal part showed that the nerve structure was identical with the control. Degeneration and regeneration of nerve fibres were observed on the sections taken from the region of the grafts and from the distal parts. Macrophagic activity appeared mainly one week after the operation. Fibroblastic invasion started 24 hours after operation. A moderate amount of collagen fibres was gradually formed. The fibres were disposed in parallel with the neuraxon. Schwann cells were slightly affected initially but consequently they fully recovered and showed signs of extra-activity of the cytoplasm organelles,e.g., enlargement of the granular endoplasmic reticulum cisternae.The present study showed that the bridging of experimental gaps of rabbit's sciatic nerve, by means of autograft and by use of perineural suturing, was sucessful. The regenerating nerve fibres were growing through the graft towards the distal part of the nerve. In this process Schwann cells and fibroblastic activity play a key role, which is most favourably influenced by using the technique described in this paper.     

外源性表皮生长因子促进鼠坐骨神经再生的实验研究

目的 评价外源性表皮生长因子（exogenous epidemal growth factor,EGF）对神经再生的影响。方法 雄性SD大鼠48只,建立成鼠坐骨神经挤压伤模型。按术后注射药物的不同成分2组,每组24只鼠。损伤对照组：在神经损伤处注射生理盐水5μl;EGF组：注射EGF／生理盐水液（10μg/5μl）。于术后2、4、6周3个时间点测定坐骨神经功能指数、CMAP的潜伏期、最大语诱发电位的恢复率、组织学检测、电镜超微结构观察。结果 坐骨神经功能指数恢复率在各时间点,EGF组无明显优于对照组（P＜0．01）。CMAP潜伏期的延迟率,EGF组明显小于对照组（P＜0．01）;诱发电位恢复率EGF组明显好于对照组（P＜0．01）。组织学检查：有髓神经纤维数在术后2、4周时EGF组明显多于对照组（P＜0．01）;各时间点有髓神经纤维直径及截面积,EGF组明显优于对照组（P＜0．01）。超微结构观察：EGF组再生神经的有髓纤维数,髓鞘厚度,髓鞘的成熟度明显好于对照组。结论 外源性EGF对神经的再生和功能恢复有一定的作用。     

促红细胞生成素促进大鼠坐骨神经再生作用的实验研究

目的 探讨促红细胞生成素(Erythoropoietin,EPO)对大鼠坐骨神经损伤修复后神经再生的影响,为周围神经损伤的临床治疗提供实验依据.方法 雄性SD大鼠40只,随机分为两组,即EPO组和神经生长因子(NGF)组,用硅胶管桥接10 mm的坐骨神经缺损,EPO组和NGF组分别注射EPO和NGF.术后4周和8周时每组分别提取10只大鼠,以坐骨神经功能指数(SFI)、运动神经传导速度(MNCV)、形态学观察和蛋白基因产物9.5(PGP 9.5)免疫组织化学染色,评估EPO对大鼠坐骨神经再生的影响.结果 术后4周SFI,EPO组为[(-78.85±3.87),x-±s,下同],NGF组为(-79.98±4.58),差异无统计学意义(P>0.05);术后8周SFI,EPO组为(-60.26±2.91),NGF组为(-64.65±4.11),差异有统计学意义(P<0.05).术后4周和8周时,EPO组MNCV、有髓神经纤维数目以及PGP9.5免疫阳性神经纤维的平均光密度和积分光密度均优于NGF组,差异有统计学意义(P<0.05).结论 EPO 能促进大鼠坐骨神经损伤后的修复与再生.     

直径相异的神经连接方法对神经再生的影响

目的探讨直径相异神经的连接方法与神经再生的关系。方法以大鼠臂丛根性撕脱伤为模型,将膈神经移位于外侧束(直径比为1:4),采用干干、干束、包埋和端侧缝合及无间隙和小间隙静脉套接六种连接方法,术后用电生理学、组织学和功能测定评估神经再生和功能恢复情况。结果术后4个月时,各端端吻合组的各项观察指标优于端侧组;各端端吻合组间,除潜伏期和最大收缩持续时间恢复率无明显差异外,静脉套接组的有髓神经纤维通过率优于干束组,其余各项指标显示静脉套接组、干干组、包埋组优于干束组。结论端端吻合优于端侧吻合;干干对合优于干束对合;套接缝合优于直接缝合;无间隙与小间隙静脉套接无明显差异。     

优化法去细胞神经同种异体及异种移植的比较

目的 分别以优化法去细胞(OA)大鼠坐骨神经及兔臂丛分支移植修复大鼠坐骨神经缺损,观察免疫排斥情况、早期功能恢复及神经再生情况,以比较此优化法处理的同种异体及异种神经移植物修复周围神经缺损的能力. 方法 以新鲜新西兰大白兔臂丛分支、自体坐骨神经、OA处理过的新鲜取材的SD大鼠坐骨神经及新西兰大白兔臂丛分支,移植修复成年SD大鼠坐骨神经1.0 cm缺损,即新鲜异种神经移植组、自体神经移植组、OA异种神经移植和OA异体神经移植组,分别于术后1个月及3个月行功能评价(SFI)、电生理(CV)和组织学检查,观察免疫排斥、功能恢复及神经再生情况. 结果 术后1个月,OA异体和OA异种神经移植组的SFI、CV、轴突密度分别为:61.38±5.59、(32.23±0.91)m/s、(22.26±1.74)m/s和(0.782±0.081)(个/100 μm2),3个月分别为59.00±5.40、(31.80±0.99)m/s、(23.35±2.40)m/s和(0.778±0.046)(个/100μm2);术后1个月,异体和异种神经移植CD8+T细胞和巨噬细胞染色阳性百分比分别为0.17385±0.01805、0.09299±0.01565和0.30223±0.09449、0.19537±0.02010.同种异体神经移植组与异种神经移植组在免疫排斥、功能恢复及神经再生方面差异无统计学意义(P＞0.05),且都明显优于未处理新鲜神经移植组(P＜0.05).3个月时的神经再生及功能恢复情况优于1个月组(P＜0.05). 结论 优化去细胞法处理的同种异体及异种神经移植物在修复周围神经缺损时,均可以达到免疫耐受,移植动物早期功能恢复和神经再生情况良好.     

Comparative study of rat sciatic nerve microepineurial anastomoses made with carbon dioxide laser and suture techniques: Part 2. A morphometric analysis of myelinated nerve fibers

Sciatic nerves of rats were severed with steel scalpel blades and subsequently anastomosed with epineurial sutures and laser-aided techniques. Morphometric analysis of myelinated nerve fibers proximal and distal to the anastomosed region revealed that the laser had no deleterious effects on the degree of retrograde axonal degeneration or regeneration potential as compared to the traditional suture technique. Although no significant difference in myelinated nerve fiber population was evident between the two methods, future experimentation with various laser parameters might lead to more effective use of lasers in nerve repair.     

Carbon dioxide laser-assisted nerve repair: effect of solder and suture material on nerve regeneration in rat sciatic nerve

In order to further improve and explore the role of lasers for nerve reconstruction, this study was designed to investigate regeneration of sharply transected peripheral nerves repaired with a CO(2) milliwatt laser in combination with three different suture materials and a bovine albumin protein solder as an adjunct to the welding process. Unilateral sciatic nerve repair was performed in 44 rats. In the laser group, nerves were gently apposed, and two stay sutures (10-0 nylon, 10-0 polyglycolic acid, or 25 microm stainless steel) were placed epi/perineurially. Thereafter, the repair site was fused at 100 mW with pulses of 1.0 s. In the subgroup of laser-assisted nerve repair (LANR), albumen was used as a soldering agent to further reinforce the repair site. The control group consisted of nerves repaired by conventional microsurgical suture repair (CMSR), using 4-6 10-0 nylon sutures. Evaluation was performed at 1 and 6 weeks after surgery, and included qualitative and semiquantitative light microscopy. LANR performed with a protein solder results in a good early peripheral nerve regeneration, with an optimal alignment of nerve fibers and minimal connective tissue proliferation at the repair site. All three suture materials produced a foreign body reaction; the least severe was with polyglycolic acid sutures. CMSR resulted in more pronounced foreign-body granulomas at the repair site, with more connective-tissue proliferation and axonal misalignment. Furthermore, axonal regeneration in the distal nerve segment was better in the laser groups. Based on these results, CO(2) laser-assisted nerve repair with soldering in combination with absorbable sutures has the potential of allowing healing to occur with the least foreign-body reaction at the repair site. Further experiments using this combination are in progress.