Identification of oxidized galectin-1 as an initial repair regulatory factor after axotomy in peripheral nerves

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt the axons to send out processes in peripheral nerves after axotomy are not well understood. We have shown using two specific strategies that galectin-1 can play an important role in this initial stage. One used an in vitro nerve regeneration model that allowed us to monitor the initial axon and support cell outgrowth from the proximal nerve stump comparable to the initial stages of nerve repair. The other strategy was to clarify the axonal regeneration-promoting factor from kidney-derived cells. Using these strategies, we discovered that oxidized galectin-1 from the cell (COS1 cell) conditioned media acts as an axonal regeneration-promoting factor without the lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same activity at low concentrations (pg/ml range). A similarly low concentration also effectively promoted axonal regeneration in both transection and crush experiments in vivo. Moreover, the application of functional anti-galectin-1 antibody strongly inhibited the regeneration in vivo. Since galectin-1was shown to be secreted and localized in the regenerating sciatic nerve, this suggests that secreted galectin-1 may be oxidized and change its molecular structure to regulate initial repair after axotomy as a kind of cytokine.     

内源性神经营养因子-3对小鼠周围神经损伤后再生与髓化的影响

为了探讨内源性神经营养因子3(NT3)在小鼠周围神经损伤后再生与髓化中的作用,本研究建立小鼠坐骨神经压榨损伤模型,腹膜腔注射抗NT3血清为实验组,腹膜腔注射生理盐水(NS)和羊血清(NSS)为对照组。采用fastblue(FB)逆行束路追踪与电子显微镜相结合的方法观察周围神经损伤后再生与髓化的情况,并运用体视学方法测量单位面积髓化纤维的数密度、直径分布。结果显示(1)实验各组的脊髓前角运动神经元内均可观察到FB逆标细胞,但抗NT3组FB逆标细胞的数目明显少于NS组和NSS组(P<0.05);(2)半薄切片上观察到抗NT3组动物坐骨神经损伤远端,其再生纤维分布稀疏、直径小,但间质增多,有许多溃变细胞;NS组和NSS组损伤远端再生纤维数目多,分布均匀;(3)超薄切片上还可观察到抗NT3组损伤远端有髓纤维的数目明显减少,轴突内细胞器少,髓鞘薄;而NS组和NSS组损伤远端有髓纤维数目多,轴突内细胞器多,髓鞘板层结构清楚、呈致密有规律的排列。以上结果提示内源性NT3与损伤神经的再生有关,对不同直径神经纤维的髓化具有选择性作用,主要影响大直径神经纤维的髓化。     

刀豆球蛋白A处理异种移植神经对神经再生的影响

用刀豆球蛋白Ａ预先处理的异种移植神经，对神经再生的影响。含Ｃｏｎ Ａ５０ｍｇ的Ｒｉｎｇｅｒ液１０００ｍｌ（４℃）灌注日本大耳兔２ｈ后，取出兔的胫神经。麻醉Ｗｉｓｔａｒ大白鼠，切除其右坐骨神经５ｍｍ，移植入８ｍｍ长、经ＣｏｎＡ处理的兔胫神经。术后４、８、１２周，可见再生神经纤维通过近端吻合部，长入异种移植神经，继而越过远端吻合部，向远端坐骨神经生长。移植神经段和远端坐骨神经中再生神经纤维成一吵或散在     

大鼠坐骨神经移植的形态及电生理研究

目的：探讨异体坐骨神经移植后神经纤维再生。方法：大鼠３８只,在手术显微镜下将右侧坐骨神经切除１ｃｍ,然后将１ｃｍ异体或自体右侧坐骨神经移植于神经缺损处。 ２、４、８、１２周进行形态学观察及神经电生理学检查。结果：术后４周笛生神经纤维通过近铁合口进入移植神经,术后８周通过远侧吻合口进入受体坐骨神经。术后１２划体组与自体组再生神经纤维髓鞘的厚度、轴索的开矿及神经电生理功能无明显差异。结论：免疫排序反应     

Different multiple regeneration capacities of motor and sensory axons in peripheral nerve

Abstract After peripheral nerve injury, axons often project sprouts from the node of Ranvier proximal to the damage site. It is well known that one parent axon can sprout and maintain several regenerating axons. If enough endoneurial tubes in the distal stump are present for the regenerating axons to grow along, then the number of mature myelinated nerve fibers in the distal stump will be greater than the number in the proximal stump. "Multiple regeneration" is used to describe this phenomenon in the peripheral nerve. According to previous studies, a prominent nerve containing many axons can be repaired by the multiple regenerating axons sprouting from another nerve that contains fewer axons. Most peripheral nerves contain a mixture of myelinated motor and sensory axons as well as unmyelinated sensory and autonomic axons. In this study, a multiple regeneration animal model was developed by bridging the proximal common peroneal nerve with the distal common peroneal nerve and the tibial nerve. Differences in the multiple regeneration ratio of motor and sensory nerves were evaluated using histomorphometry one month after ablating the dorsal root ganglion (DRGs) and ventral roots, respectively. The results suggest that the motor nerves have a significantly larger multiple regeneration ratio than the sensory nerves at two different time points.     

Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats

The peripheral-type benzodiazepine receptor (PBR) is a protein predominantly located in the mitochondrial outer membrane that plays an important role in the regulation of cell survival and proliferation. Previous studies have shown an enhanced expression of PBR in the regenerating sciatic nerve, suggesting that this protein may be involved in the regenerative response. The rat sciatic nerve suffers important structural alterations with aging, including alterations in the morphology of myelin sheaths and a decrease in the number of myelinated fibers. In this study, we have assessed the effect of two PBR ligands, Ro5-4864 and PK-11195, to determine whether PBR may influence aging-associated morphological changes in the sciatic nerve. The treatment of 23-month-old, Sprague-Dawley male rats for 1 month with Ro5-4864 significantly reduced the percentage of fibers with myelin decompaction and increased the total number of myelinated fibers. In contrast, PK-11195, a PBR ligand that binds to a different site than Ro5-4864 in the PBR molecule, did not significantly affect any of the parameters analyzed. These findings support the potential role of PBR ligands to prevent aging-associated peripheral nerve degeneration.     

Beneficial effects of treadmill training in experimental diabetic nerve regeneration

OBJECTIVES:

We investigated the effects of treadmill training (10 weeks) on hindlimb motor function and nerve morphometric parameters in diabetic rats submitted to sciatic nerve crush.

MATERIALS AND METHOD:

Wistar rats (n = 64) were divided into the following groups: non-diabetic; trained non-diabetic; non-diabetic with sciatic nerve crush; trained non-diabetic with sciatic nerve crush; diabetic; trained diabetic; diabetic with sciatic nerve crush or trained diabetic with sciatic nerve crush. Diabetes was induced by streptozotocin injection (50 mg/kg, iv). Hindlimb motor function was evaluated weekly by assessing sciatic functional indices, and the proximal and distal portions of the sciatic nerve were used for morphometric analysis.

RESULTS:

At 13 weeks post-injury, the distal nerve portion of all injured groups and the proximal nerve portion of the diabetic with sciatic nerve crush group presented altered morphometric parameters such as decreased myelinated fiber diameter (∼7.4±0.3µm vs ∼4.8±0.2µm), axonal diameter (∼5±0.2µm vs ∼3.5±0.1µm) and myelin sheath thickness (∼1.2±0.07µm vs ∼0.65±0.07µm) and an increase in the percentage of area occupied by endoneurium (∼28±3% vs ∼60±3%). In addition, in the non-diabetic with sciatic nerve crush group the proximal nerve portion showed a decreased myelinated fiber diameter (7.4±0.3µm vs 5.8±0.3µm) and myelin sheath thickness (1.29±0.08µm vs 0.92±0.08µm). The non-diabetic with sciatic nerve crush, trained non-diabetic with sciatic nerve crush, diabetic with sciatic nerve crush and trained diabetic with sciatic nerve crush groups showed normal sciatic functional index from the 4^th, 4^th, 9^th and 7^th week post-injury, respectively. Morphometric alterations in the proximal nerve portion of the diabetic with sciatic nerve crush and non-diabetic with sciatic nerve crush groups were either prevented or reverted to values similar to the non-diabetic group by treadmill training.

CONCLUSION:

Diabetic condition promoted delay in sciatic nerve regeneration. Treadmill training is able to accelerate hindlimb motor function recovery in diabetic injured rats and prevent or revert morphometric alterations in proximal nerve portions in non-diabetic and diabetic injured rats.     

Effets d'une congélation localisée d'un nerf périphérique sur la conduction de l'influx nerveux,au cours de la dégénérescence et de la régénération

Summary Using the cold to cause a lesion in myelinated fibers of the sciatic nerve of the rat, one can observe, as soon as the freezing ends, a sudden and durable conduction block in the frozen area. Conduction disappears progressively in the peripheral part of the nerve, and no action potential can be recorded after 48 h. Conduction appears again towards the 10th day at the level of the previously frozen area, and the functional recovery is uniform 6 months later.

     

The Role of Defensin NP-1 in Restoring the Functions of an Injured Nerve Trunk

This report presents results from studies on the actions of neutrophil defensin NP-1 on the initial stage of regeneration of the lesioned sciatic nerve in rats. The effects of defensin on the growth rate and functional characteristics of regenerating nerve fibers were assessed by recording total action potentials 21 days after transection and microsurgical suturing of the nerve. These experiments showed that defensin increased the rate of growth of regenerating nerve fibers by 30%: the distance over which nerve spike conductivity was restored in the lesioned nerve increased from 7.2 ± 1.2 (control) to 10.5 ± 0.8 mm (experiment) from the suturing site (p < 0.05). In addition, an increase in the excitation conduction rate along the regenerating nerve fibers by 20% compared with control was observed. Overall, the results provide evidence for the positive effects of defensin on restoration of the functions of the lesioned nerve trunk. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 3, pp. 309–313, March, 2005.     

The dependence of nerve regeneration through muscle grafts in the rat on the availability and orientation of basement membrane

Summary Nerve regeneration through grafts of basement membrane matrix, prepared by freezing of autogenous muscle followed by thawing in distilled water, was investigated in Sprague-Dawley rats. Electrophysiological evidence of recovery in distal nerve was observed at 51 days after implantation of treated grafts whose basement membrane tubes were coaxial with the proximal and distal ends of the transected sciatic nerve. This correlated with histological findings of well-developed myelinated nerve fibres within both grafts and distal nerve. However, whereas normal axon numbers were achieved in the grafts by 3 months, the regenerating nerve in these muscle grafts took 6 months to 1 year to recover normal axon diameter and myelination. Recovery was delayed through grafts whose basement membrane tubes were at right angles to the nerve fibres and through grafts of untreated muscle coaxially aligned. It is concluded that successful repopulation of the distal stump and functional recovery can follow nerve regeneration through treated muscle autografts. The rate of regeneration is dependent on the availability of empty basement membrane tubes. If these are unavailable or inappropriately orientated, regeneration can still occur but is significantly delayed.     

神经生长颗粒对大鼠腓总神经横断损伤的修复作用

目的 研究神经生长颗粒(NGG)对大鼠腓总神经横断损伤的修复作用.方法 SD大鼠50只,随机分为NGG高、中、低剂量组(剂量分别为5.2g生药/kg、2.6g生药/kg、1.3g生药/kg)、弥可保组(剂最为625μg/kg)、空白对照组.行大鼠腓总神经横断缝合术,术后每日灌胃给药.于术后2周、3周、4周行足迹实验,测定展趾功能,术后4周行电生理检测,测定复合肌动作电位和神经干动作电位检测,组织形态学分析,测定再生有髓神经纤维数、髓鞘厚度和胫前肌肌纤维截面积,观察NGG对大鼠腓总神经损伤的修复作用.结果 与空白对照组相比,NGG组展趾功能、复合肌动作电位和神经干动作电位波幅及恢复率、再生有髓神经纤维计数、髓鞘厚度及胫前肌横截面积均显著增高,且呈剂量依赖的量效关系.结论 NGG有利于轴突生长和髓鞘形成,可以促进大鼠损伤神经修复和神经功能的恢复.     

神经溃变及再生的组织化学法评价

本文观察了鼠坐骨神经切断后溃变及再生过程中的乙酰胆碱酯酶(AChE)组织化学染色特征。结果表明:鼠坐骨神经切断后15天,远端酶组织化学染色阴性,而近端酶反应正常。再生神经纤维一开始便具有AChE酶活性反应。这种酶活性反应在溃变与再生神经纤维中的显著差别,为用该酶组织化学法评价神经再生提供了依据。     

靶肌肉注射促红细胞生成素对大鼠周围神经再生的作用

目的探讨靶肌肉注射人重组促红细胞生成素（recombinant human erythropoietin，rh-EP0）对大鼠坐骨神经损伤后神经再生的作用。方法选用健康雄性SD大鼠12只，制备大鼠右侧坐骨神经钳夹损伤模型。实验动物随机分为2组，每组6只，EPO组：靶肌肉注射rh-EPO2500U／kg；对照组：注射同体积的生理盐水。术后第7d、14d、21d观察坐骨神经功能指数（SFI），第21d组织学观察脊髓腰膨大（L4～L6）、夹伤远端坐骨神经、损伤侧腓肠肌组织并作图象分析测定脊髓前角运动神经元数、再生有髓神经纤维数、髓鞘厚度、轴突直径和腓肠肌肌细胞横截面积等指标。结果术后第7d两组SFI无显著性差异，术后第14d、21dEPO组SFI恢复程度明显大于对照组，差别有显著性意义（P〈0．05）；术后第21d损伤侧脊髓前角运动神经元数、再生有髓神经纤维数、髓鞘厚度、轴突直径和腓肠肌肌细胞横截面积等指标，EPO组均优于对照组（P〈0．01，P〈0．05）。结论靶肌肉注射rh-EPO能促进周围神经再生和功能恢复。     

Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve

The basal laminae of muscle fibers, when treated by denaturing methods including freeze thawing, have been used as conduits for regenerating nerves. In this study, we developed a new method for denaturing skeletal muscle fibers through treatment with a biological detergent, sodium dodecyl sulfate. Laminin and type IV collagen proteins of muscle fiber basal laminae were preserved after the detergent treatment. A segment of detergent-denatured muscle was grafted to a 1-cm defect of the rat sciatic nerve. One week after grafting, regenerating axons immunostained for neurofilaments were seen extending within laminin-positive muscle fiber basal lamina tubes. Four weeks after grafting, numerous myelinated axons at a much higher level than the control unoperated sciatic nerve, were found in the middle of the graft. They were smaller in diameter than those in the control nerve. Distal host nerves were well reinnervated 4 weeks after grafting. These findings suggest that the basal laminae of detergent-denatured muscle fibers provide effective conduits for regenerating axons.     

Morphologic and functional evaluation of peripheral nerve fibers regenerated through polyimide sieve electrodes over long-term implantation

We evaluated by morphologic and functional analysis the regeneration of peripheral nerve fibers through polyimide regenerative-type electrodes over long-term implantation. Polyimide sieve electrodes were placed in silicone chambers and implanted between the severed ends of the sciatic nerve in rats. The sieve part had 281 round via holes of 40 microm in diameter, with nine integrated recording-stimulating electrodes arranged around the via holes. The degree of axonal regeneration was examined at 2, 7, and 12 months postimplantation (mpi). Regeneration was successful in 12 of the 13 animals implanted. Reinnervation of distal muscle and nerves increased with time, reaching a plateau about 7 mpi. The number of myelinated fibers increased from 2 to 7 months, at which time it was similar to control values. With time the myelinated fibers matured, with significant increases in axon diameter and myelin thickness. Only 0.6% of the regenerated axons showed evidence of compression near the implanted electrode. The majority of the myelinated fibers that crossed the via holes and had been regenerated through the distal nerve had a normal appearance. Sieve electrodes were useful for nerve stimulation at postimplantation. Stimulation through different active electrodes excited nerve bundles, evoking compound muscle action potentials of varying shape and amplitude, indicative of selective axonal stimulation.     

Regeneration of nerve fibers by laser irradiation of spinal nerve projections

The effect of short infrared laser irradiation on regeneration of nerve fibers in rat sciatic nerve was studied. Starting from the first day after nerve crushing, the projection of proximal portion of the nerve of the test group rats was transcutaneously stimulated by laser irradiation with the wavelength of 890 nm and total dose of 42 mJ/cm². The test rats demonstrated higher rate of restoration of cutaneous sensitivity in comparison with control rats, which were operated but not irradiated. The local vestibular reaction occurred 2 days earlier in the test group than in the control. On day 30 after crushing there were no significant differences in the number of myelinated fibers and mass of regenerating soleus muscle in the test and control groups. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 3, pp. 348–350, March, 1998     

小鼠神经缺损管式修复后长时程功能恢复与再生评价

目的了解壳聚糖/聚乳酸-乙醇酸共聚体(PLGA)人工神经移植物修复小鼠神经缺损后神经功能长时程恢复水平与再生神经成熟度。方法采用人工神经移植物桥接修复小鼠坐骨神经缺损(n=6),以自体神经修复(n=6)和缺损组(n=6)为对照,术后1年采用热痛阈测定、电生理学、激光多普勒血流检测评定神经功能,采用靶肌湿重比、组织学和电子显微镜等技术综合评定神经重支配和再生神经成熟度。结果人工神经移植物组足底痛觉反应潜伏期、神经源性血管扩张程度、腓肠肌复合肌动作电位(CMAPs)波幅和潜伏期、靶肌湿重比、再生轴突数量等指标与自体神经修复组相近,但与健侧相比CMAPs潜伏期较长,髓鞘较薄,轴突直径分布滞后。结论人工神经移植物修复小鼠神经缺损术后1年感觉及自主神经功能、再生神经数量和靶肌重支配水平与自体神经修复相当,但再生神经纤维成熟度未达正常。     

Insulin-like growth factor I promotes nerve regeneration: an experimental study on rat sciatic nerve

Insulin-like growth factor I (IGF-I; somatomedin C) has previously been demonstrated, with immunohistochemical methods, to accumulate locally at the site of trauma of an injured peripheral nerve. In the experiments reported here a Y-shaped silicone-chamber system was used to test if local infusion of IGF-I had supportive effects on nerve regeneration. The proximal end of a cut sciatic nerve was inserted into one channel of the Y-shaped chamber and the length and growth direction of the regenerating myelinated axons were evaluated after 1 month. When IGF-I (250 micrograms/ml 0.5 microliters/h) was infused into one channel by an osmotic pump, the length of the regenerating axons increased significantly compared to the control groups with no IGF-I added. In some instances the regenerating axons grew towards the osmotic pump. It is concluded that local infusion of IGF-I at appropriate concentration promotes regeneration of a peripheral nerve. It exerts a neuronotrophic but not a clear chemotactic effect.     

Electronmicroscopical evaluation of short-term nerve regeneration through a thin-walled biodegradable poly(DLLA-epsilon-CL) nerve guide filled with modified denatured muscle tissue

The aim of this study was to evaluate short-term peripheral nerve regeneration across a 15-mm gap in the sciatic nerve of the rat, using a thin-walled biodegradable poly(DL-lactide-epsilon-caprolactone) nerve guide filled with modified denatured muscle tissue (MDMT). The evaluation was performed using transmission electron microscopy and morphometric analysis. Evaluation times ranged from 3 to 12 weeks after reconstruction. Already, 3 weeks after reconstruction, myelinated nerve fibers could be observed in the distal nerve stump. Twelve weeks after reconstruction, the number of (non)myelinated nerve fibers had significantly increased in the distal nerve stump. From this study, we can conclude that a thin-walled biodegradable poly(DL-lactide-epsilon-caprolactone) nerve guides filled with MDMT can be successfully applied in the reconstruction of severed nerves in the rat model. Furthermore, we showed fast nerve regeneration across the 15-mm nerve gap and found that the use of MDMT functioned as a mechanical support preventing a collapse of this thin-walled nerve guide.