Bone marrow cells are able to increase vessels number during repair of sciatic nerve lesion

The aim of this study was to compare the outcomes of nerve autografts (GRF) and venous grafts containing mononuclear bone marrow cells (BMCs) in sciatic nerve-lesioned rats. Control animals underwent sham operations (SHAM), received empty venous grafts (EPV), or received venous grafts containing BMC vehicle (AGR). Outcome was evaluated through sciatic functional index (SFI), morphometric and morphologic analyses of the nerve distal to the lesion, and the number of spinal cord motor neurons positive for the retrograde tracer, Fluoro-Gold. All groups exhibited poor results in SFI when compared to SHAM animals throughout the postoperative period. All groups also had a significantly greater fiber density, decreased fiber diameter, and decreased motor neuron number than the SHAM group. No significant difference between the GRF and BMC groups was observed in any of these parameters. On the other hand, vessel density was significantly higher in BMC than all other groups. BMC-containing venous grafts are superior to nerve autografts in increasing vessel density during sciatic nerve regeneration.     

The end-to-side peripheral nerve repair. Functional and morphometric study using the peroneal nerve of rats

Morphologic and functional recovery following an end-to-side repair was studied comparatively with conventional end-to-end repair in a model of peroneal nerve lesion in rats. Twenty-eight rats were used and divided into four groups according to the reparative procedure following nerve division: (1) nerve stumps buried into neighboring muscles (n = 8); (2) conventional end-to-end repair (n = 7); (3) end-to-side repair onto the tibial nerve (n = 8); (4) sham operation (n = 5). The sciatic functional index (SFI) was evaluated at weekly intervals for 8 weeks, the peroneal nerve being resected on the 56th day for histologic and morphometric studies. The SFI progressively improved in Groups 2 (-16.9) and 3 (-22.7), although it did not reach normal values (around -8). The average nerve fiber density increased to normal values in both Groups 2 and 3, although accompanied by a marked decrease of average minimal and maximal nerve fiber diameter, myelin sheath area and G quotient. The differences between Groups 2 and 3 or Groups 2 and 4 were not significant. We conclude that, although resulting in significant morphologic and functional recovery, end-to-side repair is not as efficient as the conventional end-to-end nerve repair. However, end-to-side repair has a potential for application in selected cases in humans.     

Effects of local release of hepatocyte growth factor on peripheral nerve regeneration in acellular nerve grafts

Options for reconstructing peripheral nerve gaps after trauma are limited. The acellular nerve is a new kind of biomaterial used to reconstruct the peripheral nerve defect, but its use could be improved upon. We aimed to investigate the effect of adenoviral transfection with hepatocyte growth factor (HGF) on the functional recovery of transected sciatic nerves repaired by acellular nerve grafting. 30 Rats were divided into three groups (10/group) for autografting and acellular grafting, as well as acellular grafting with adenovirus transfection of HGF (1 × 10⁸ pfu) injected in muscles around the proximal and distal allograft coapation. Sciatic functional index (SFI) was evaluated every 4 weeks to week 16 by measuring rat footprints on walking-track testing. The three groups presented initial complete functional loss, followed by slow but steady recovery, with final similar SFIs. Weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization in the nerve grafts were evaluated at week 16. Autografting gave the best functional recovery, but HGF-treated acellular grafting gave better recovery than acellular grafting alone. Neovascularization was greater with HGF-treated acellular grafting than with autografting and acellular grafting alone. Axonal regeneration distance of autografting on the 20th postoperative day was the longest in the three groups,while that of acellular grafting alone was the smallest. Acellular nerve grafting may be useful for functional peripheral nerve regeneration, and with human HGF gene transfection may improve on acellular grafting alone in functional recovery.     

Anatomical and physiological assessments of peripheral nerve grafts in the dorsal columns of the spinal cord

Portions of 1 cm length of the sensory radial nerve from the cat forelimb were used to replace an excised portion of the dorsal columns in the upper lumbar spinal cord. Observations were made on the clinical recovery of the animals, and cine recordings were made of their ability to traverse a horizontal ladder 5 months after the grafting procedure. Evoked sensory potential studies performed 6 months after grafting showed that an impulse arising from a stimulus applied to the sciatic nerve could be recorded in the spinal cord caudal to the graft, in the graft and in the spinal cord rostral to the graft in 5 out of 8 animals. Tracing of nerve connections with injection of horseradish peroxidase into the grafts resulted in labelling of nerve cell bodies in dorsal root ganglia and the grey matter of the lumbar spinal cord up to a distance of 10 mm away from the graft. These results confirm that peripheral nerve grafts can provide a satisfactory environment for the regrowth of ascending fibres in the dorsal columns of the spinal cord. However, there is as yet no evidence that the regenerated fibres succeed in forming useful synaptic connections with other nerve cell bodies.     

Experimental surgery in spinal cord lesions by connecting upper motoneurons directly to peripheral targets

This research was aimed at assessing the possibility to connect central motoneurons with skeletal muscles through PNS segments bypassing a lesion of the spinal cord. The investigation was performed in 20 non-human primates (Macaca fascicularis). The surgical paradigm consisted of anastomosing the lateral bundle of the spinal cord directly with the sciatic nerve of the right hindlimb, using the peroneal nerve as a graft. The animals were followed-up clinically for 18 months; at the end of this observation period, they underwent electrophysiological examinations before being killed. Specimens were taken from the spinal cord, graft, sciatic nerve and potentially reinnervated muscles, and processed for routine light microscopy and immunohistochemistry. Postoperative mortality was fairly high (six monkeys), yet the overall outcome was regarded as very good because the animals were neither restrained nor intensively cared for. Five of the surviving monkeys showed clinical, electrophysiological and histological evidence of successful reinnervation. This research demonstrated that upper CNS motoneurons are potentially capable of elongating neuritic processes into the endoneural tubes of a connecting graft, up to reaching a peripheral nerve (sciatic), and restoring functional connections with the relevant skeletal muscles.     

Androgen induced acceleration of functional recovery after rat sciatic nerve injury

PURPOSE: Testosterone (T) treatment accelerates recovery from facial paralysis after facial nerve crush in hamsters. In this study, we extended those studies to another injury model and asked the following question: Will T treatment accelerate recovery from lower limb paralysis following sciatic nerve crush in the rat? METHODS: Castrated adult male rats received a right side sciatic nerve crush at the level of the sciatic notch, with the left side serving as control. Half the animals received a subcutaneous implant of a propionated form of T (TP), the others were sham-implanted. Weekly testing using the Sciatic Functional Index (SFI), a quantitative measure of locomotion, was done for 7 weeks postoperative (wpo). RESULTS: Between 3 and 5 weeks post-op, the average SFI score of the TP-treated group was higher than controls. This difference was significant at 4 wpo, indicating an accelerated degree of functional recovery. At these timepoints, the differences were attributable to the footprint or paw length and associated with calf muscle reinnervation rather than the toespreading component associated with intrinsic foot muscle rein-nervation. Beyond 5 wpo, there were no differences in the SFI scores. CONCLUSION: The results indicate that, as with facial nerve regeneration in the hamster, testosterone accelerates functional recovery from hind limb paralysis following sciatic nerve injury in the rat. While the responses of spinal motoneurons to injury can differ from those of cranial motoneurons, in this case it appears that they share a similar response to the trophic actions of androgen. This is important in the context of designing therapeutic strategies for dealing with direct trauma to motoneurons resulting from both peripheral and central nervous system trauma, such as spinal cord injury.     

Long term evaluation of experimental median nerve repair by frozen and fresh nerve autografts,allografts and allografts repopulated by autologous Schwann cells

The authors used different kinds of peripheral nerve grafts to reconstruct a terminal branch of the brachial plexus (the median nerve) gap of adult Sprague-Dawley rats, including fresh or frozen autografts and allografts from Norway rats. They also performed acellular allograft repopulation by autogenous Schwann cells, to improve the environment for nerve regeneration. Three, six, nine and twelve months after grafting, rats underwent histological assessment (muscle, nerve and spinal cord) and simple functional assessment by the grasping test. Initially, the functional recovery of frozen grafts was lower than fresh graft recovery, but twelve months after surgery it was similar for both types of graft.     

Directly applied low intensity direct electric current enhances peripheral nerve regeneration in rats

The influence of direct electric stimulation on nerve regeneration was studied in a model of crush injury of the sciatic nerve of rats. Forty-three rats were used and distributed in four groups according to the procedure: (1) intact nerve, inactive circuit; (2) crush injury, inactive circuit; (3) intact nerve, active circuit; (4) crush injury, active circuit. The low intensity continuous current circuit (1 microA) was implanted in the lumbar region, the anode being fixed to the muscles proximally and the cathode below the nerve distally to the lesion site. The Sciatic Functional Index (SFI) was evaluated at weekly intervals for 3 weeks, the sciatic nerve being resected on the 21st day for histologic and morphometric studies. The SFI progressively improved and the average fiber nerve density recovered to a nearly normal value in Group 2 and increased in Group 4 compared with the control groups (1 and 3), but this was accompanied by a decreased average fiber nerve diameter. Both number and diameter of inter and intra-fascicular blood vessels increased in the stimulated nerves. We conclude that low intensity direct electric stimulation enhances nerve regeneration following a controlled nerve crush injury and increases blood supply by increasing number and diameter of vasa nervorum.     

无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复坐骨神经缺损

背景：作者前期已经成功将无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经,并证明可以促进周围神经再生。 目的：构建组织工程人工神经,观察和验证桥接大鼠坐骨神经缺损后的神经功能恢复情况。 方法：成年雄性SD大鼠60只构建大鼠坐骨神经15 mm缺损模型。随机分成3组,每组20只。桥接大鼠坐骨神经缺损,实验组采用组织工程人工神经,空白对照组采用无细胞组织工程神经支架,自体神经对照组采用自体神经移植。桥接后12周通过大体观察、胫骨前肌湿质量、组织学等方法分析坐骨神经组织学及功能恢复情况。 结果与结论：桥接术后12周：实验组大鼠肢体可以支撑着地,钳夹大鼠手术侧足底皮肤出现逃避反射,足底皮肤s-100蛋白染色呈阳性反应。实验组与自体神经移植组胫骨前肌湿质量比差异无显著性意义(P > 0.05)。实验组辣根过氧化物酶逆行示踪实验显示脊髓、后根神经节均可见数量不等的辣根过氧化物酶标记阳性细胞。实验组移植物与自体神经移植组有髓神经纤维数、髓鞘厚度、神经组织面积比较差异无显著性意义。实验结果验证了无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损,可以促进神经组织学的修复重建和功能的恢复。     

Grafting of choroid plexus ependymal cells promotes the growth of regenerating axons in the dorsal funiculus of rat spinal cord: a preliminary report

Nerve regeneration in the central nervous system has been studied by grafting various tissues and cells. In the present study, we demonstrated that choroid plexus ependymal cells can promote nerve regeneration when grafted into spinal cord lesions. The choroid plexus was excised from the fourth ventricle of adult rats (Wistar), minced into small fragments, and grafted into the dorsal funiculus at the C2 level in adult rat spinal cord from the same strain. Electron microscopy and fluorescence histochemistry showed that ependymal cells of the grafted choroid plexus intimately interacted with growing axons, serving to support the massive growth of regenerating axons. CGRP-positive fibers closely interacted with grafted ependymal cells. HRP injection at the sciatic nerve showed that numerous HRP-labeled regenerating fibers from the fasciculus gracilis extended into the graft 7 days after grafting. This regenerating axons from the fasciculus gracilis was maintained for at least 10 months, with some axons elongating rostrally into the dorsal funiculus. Evoked potentials of long duration were recorded at a level ca. 5 mm rostral to the lesion in the rats 8 to 10 months after grafting. These findings indicate that choroid plexus ependymal cells have the ability to facilitate axonal growth in vivo, suggesting that they may be a promising candidate as graft for the promotion of nerve regeneration in the spinal cord.     

Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts

Experiments were carried out to determine if neurons damaged by injury to the spinal cord retain the ability to regenerate their axonal process for a prolonged period of time after the initial response to injury and if peripheral nerve (PN) grafts could support the regrowth of these processes. True blue (TB) was injected into one side of the adult rat lumbar spinal cord to label neurons with axons coursing through this region. Seven days later spinal cord tissue surrounding the injection sites was removed by aspiration to create a hemisection cavity 3-4 mm in length. Four weeks later scar tissue lining the lesion cavity was removed prior to grafting 1 cm segments of autologous tibial nerve to the rostral and the caudal surfaces of the cavity wall. The distal end of each graft was ligated and left unapposed to spinal cord tissue. Four weeks later the distal end of each PN graft was exposed to nuclear yellow (NY) to retrogradely label neurons that had grown an axon into the graft. Neurons containing both TB and NY were deemed capable of axonal regeneration while in a chronically injured state. Double-labeled (TB/NY) neurons were found in the ipsilateral spinal cord in laminae IV through X, excluding IX, and in Laminae VI and VII contralateral to the lesion. Most neurons were located within 10 mm of the lesion, with the majority caudal to the lesion. Nearly 50% (range 24-74%) of lumbar dorsal root ganglion neurons containing TB also were labeled with NY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spinal cord-skeletal muscle cografts: trophic and functional interactions

Skeletal muscle from embryonic day 20 (E20) was combined with E15 rat spinal cord in the anterior chamber of the eye of adult albino rats. The two grafts were either transplanted concomitantly or sequentially, in which case muscle tissue was added 4 months after the spinal cord. Control groups received a single graft of either spinal cord or skeletal muscle. Survival and intraocular growth were observed through the cornea. After maturation in oculo, the double grafts were examined immunohistologically utilizing antisera to neurofilament (NF) and acetylcholinesterase (AChE). The grafts were also evaluated using electrical stimulation to determine functional connectivity. The spinal cord and skeletal muscle grafts were found to exert reciprocal tropic effects on each other, evidenced as a larger muscle mass in skeletal muscle grafts allowed to develop in the presence of spinal cord tissue, and a larger volume of spinal cord grafts allowed to develop together with a skeletal muscle graft, respectively. Immunohistochemistry revealed NF-positive nerve fibers leaving the spinal cord graft and entering the muscle tissue. AChE-positive endplates developed in the muscle grafts. Electrical stimulation of the spinal cord part of double-graft combinations generally elicited contractile responses in specific areas of the muscle cograft. These results demonstrate both structural and functional connections between grafts of spinal cord and skeletal muscle tissue in vivo. The fact that such connections were also established between a mature (adult) spinal cord graft and fetal skeletal mu cle tissue suggests that some α-motoneurons are able to survive for many months in the intraocular grafts without an appropriate target, and that they are able to subsequently innervate skeletal muscle targets.     

Peripheral nerve grafts after cervical spinal cord injury in adult cats

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.     

Omental graft improves functional recovery of transected peripheral nerve

The omentum has several properties that are advantageous for neuronal sprouting and direction. We have therefore analyzed functional recovery following transection of rat sciatic nerve using omental graft to bridge the nerve defect. In group 1, a 25-30-mm nerve defect was produced and bridged with omental graft, whereas in group 2, an end-to-end repair was performed. The sciatic function index (SFI) was assessed at 2-week intervals until 8 weeks after surgery. Functional recovery was faster in group 1 than in group 2. After 8 weeks, SFI was improved significantly from -100% to -45% (+/- -4%) in group 1 (P < 0.001) compared to -72% +/- -2% in group 2 (n = 10). Histologically, the omental graft contained more newly developed nerve fibers and less scar tissue than the end-to-end repair. Thus, omental graft appears to improve directional growth of regenerating axon sprouts and may be a means of treating peripheral nerve injury.     

Reinnervation of denervated muscle by transplantation of fetal spinal cord to transected sciatic nerve in the rat

When motor neurons in the spinal cord are destroyed, regeneration of motor axons and muscle reinnervation cannot be expected. We attempted reinnervation of the denervated muscle, i.e. motor unit reconstruction, using transplantation of the fetal spinal cord to the peripheral nerve. The sciatic nerve of an adult rat was resected for 20 mm, and a cavity was prepared using an autologous femoral vein at the distal stump of the nerve. The fetal spinal cord was then transplanted into the venous cavity. After 3–6 months, no voluntary muscle contraction was observed due to the absence of communication with the central nervous system. However, reinnervation of the muscles via the sciatic nerve by the transplanted spinal neurons was demonstrated electrophysiologically and histochemically. This suggested that a motor unit can be reconstructed by fetal spinal cord transplantation even if the original motor neurons in the spinal cord are not available.     

Electron microscopic observation of cells ensheathing axons and their association with basal lamina in sciatic-nerve-grafted spinal cords

In transected and reanastomosed peripheral nerve, cells proliferate in the scar which unites the proximal and distal cut ends of the nerve. Those cells are considered to produce favorable effects on the advancement of naked axons, and ultrastructural studies have revealed that the naked axons are partially or completely ensheathed by cells in the union scar. On the other hand, in a spinal cord model, structure with a histological appearance similar to that of the union scar in the transected peripheral nerve has been produced experimentally by delayed autogenous sciatic nerve grafting into the transected spinal cord gap. In the present study, the same spinal cord model was used and an electron microscopic study of the junctional area between the spinal cord and grafted nerve was carried out in an attempt to answer the following questions: (1) Do cells in the spinal cord-nerve graft junction ensheath axons? And, if so, (2) is it possible to identify these cells? Five adult dogs were used for the experiment. One week after the first spinal cord transection, the wound was opened, necrotic materials in the gap were carefully removed microsurgically, and segments of autogenous sciatic nerve were placed in the gap of the spinal cord. The dogs were killed at 1 and 3 weeks, 3, 9 and 12 months after the delayed nerve grafting and electron microscopic observations were made. The cells which ensheathed axons at the graft junction were classified into five morphologically identifiable cell types: migratory Schwann cells, committed Schwann cells, astrocytes, oligodendrocytes and macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

HRP逆行示踪评价复合骨髓间充质干细胞的无细胞神经移植物

背景：作者前期将无细胞神经移植物与骨髓间充质干细胞复合培养,成功构建了组织工程人工神经。 目的：应用辣根过氧化物酶(HRP)神经逆行示踪技术对无细胞神经移植物复合骨髓间充质干细胞构建的神经移植复合体桥接大鼠坐骨神经缺损后运动神经元的保护作用进行评价。 方法：成年清洁级健康雄性SD大鼠,随机分成3组：①实验组：采用复合骨髓间充质干细胞的无细胞神经移植物桥接大鼠坐骨神经缺损。②空白对照组：采用无细胞神经移植物桥接大鼠坐骨神经缺损。③自体神经对照组：采用自体神经移植桥接大鼠坐骨神经缺损。术后12周应用辣根过氧化物酶神经逆行示踪技术对脊髓前角运动神经元的再生进行评价。 结果与结论：术后12周脊髓前角运动神经元再生评价结果显示：实验组优于无细胞神经移植物组,而与自体神经移植物组相比差异无显著性意义。证实无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损,对大鼠脊髓运动神经元具有保护作用,可能达到与自体神经移植相似的效果。 关键词：无细胞神经移植物;骨髓间充质干细胞;辣根过氧化物酶;神经移植;大鼠     

An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks

There is no widely accepted quantitative method for evaluating the functional effects of peripheral nerve damage in animals. In the present study, a method for evaluating sciatic nerve damage was developed from measurements of the prints of the hind feet of walking rats preserved on X-ray film. Four variables were measured from these tracks, and comparisons between the damaged (experimental) and intact (normal) side were converted to percent deficits and averaged to obtain a “sciatic functional index” (SFI). The SFI was then measured under normal conditions, after nerve transection, nerve crush, and sham procedures. Reliability and repeatability of the SFI were found to be excellent. The effects of sciatic nerve transection and nerve crush evaluated by this method agreed very well with other methods of evaluating nerve damage. We conclude that the SFI provides a simple, accurate, reliable, and repeatable method for evaluating the functional condition of sciatic nerve in rats.     

Inhibitor of DNA binding 2 accelerates nerve regeneration after sciatic nerve injury in mice

Inhibitor of DNA binding 2 (Id2) can promote axonal regeneration after injury of the central nervous system. However, whether Id2 can promote axonal regeneration and functional recovery after peripheral nerve injury is currently unknown. In this study, we established a mouse model of bilateral sciatic nerve crush injury. Two weeks before injury, AAV9-Id2-3×Flag-GFP was injected stereotaxically into the bilateral ventral horn of lumbar spinal cord. Our results showed that Id2 was successfully delivered into spinal cord motor neurons projecting to the sciatic nerve, and the number of regenerated motor axons in the sciatic nerve distal to the crush site was increased at 2 weeks after injury, arriving at the tibial nerve and reinnervating a few endplates in the gastrocnemius muscle. By 1 month after injury, extensive neuromuscular reinnervation occurred. In addition, the amplitude of compound muscle action potentials of the gastrocnemius muscle was markedly recovered, and their latency was shortened. These findings suggest that Id2 can accelerate axonal regeneration, promote neuromuscular reinnervation, and enhance functional improvement following sciatic nerve injury. Therefore, elevating the level of Id2 in adult neurons may present a promising strategy for peripheral nerve repair following injury. The study was approved by the Experimental Animal Ethics Committee of Jinan University (approval No. 20160302003) on March 2, 2016.

Chinese Library Classification No. R456; R745; R364.3+3     

无细胞神经支架复合骨髓间充质干细胞构建组织工程人工神经修复坐骨神经缺损*☆

背景：作者已经成功制备了无细胞神经移植物,并且复合骨髓间充质干细胞构建组织工程人工神经桥接大鼠坐骨神经缺损。 目的：无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损后运动功能的恢复。 方法：成年雄性SD大鼠构建大鼠坐骨神经15 mm缺损模型,分别应用组织工程人工神经、组织工程神经支架或自行神经桥接坐骨神经缺损。桥接后20周再生神经电生理学测定,手术侧胫骨前肌湿质量、腓肠肌组织学及透视电镜分析。 结果与结论：桥接20周后,组织工程人工神经与自体神经移植组胫骨前肌湿质量比较,差异无显著性意义(P > 0.05),神经干传导速度为(30.56±2.15)m/s。结果提示,无细胞神经移植物复合骨髓间充质干细胞构建的组织工程人工神经桥接大鼠坐骨神经缺损后,可以促进再生神经运动功能的恢复。