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.     

神经桥接与导管套接修复猫动眼神经形态学观察

目的探求导管修复颅内段动眼神经的可行性.方法 20只健康家猫随机分为2组.将右侧动眼神经于脑池段切断后,分组采用自体神经桥接和导管套接的方法修复.术后14周末光镜、电镜观察神经纤维的连续性、再生纤维数目和直径.结果神经修复14周后,神经桥接组67%、导管套接组75%的动物其动眼神经功能均有一定程度的恢复.形态学显示两组均取得较好的神经再生效果,两组间再生纤维直径差异无显著性(P>0.05),但导管套接组再生神经纤维数目较多,差异有显著性(P<0.05).结论导管套接法可作为颅内段动眼神经损伤后的一种修复方法,其疗效近似或稍优于神经桥接法.     

Controlled release of nerve growth factor from a polymeric implant

Recent studies suggest that neurotrophic factors applied directly to brain tissue may enhance regeneration in the central nervous system. Biocompatible polymeric implants providing a controlled release of nerve growth factor (NGF) for over one month were developed. The released nerve growth factor stimulated neurite sprouting in cultured PC12 cells. While a model polymer with demonstrated biocompatibility was used for the present study, the methods can be extended to other polymer systems. Controlled release implants may be useful in the treatment of Alzheimer's disease.     

Substrate-bound nerve growth factor promotes neurite growth in peripheral nerve

Nerve growth factor (NGF), in addition to its well-known effects as a soluble neurite growth-promoting factor, also appears to promote the elongation of neurites when it is adsorbed to tissue culture substrates. Peripheral nerve Schwann cells appear to possess a receptor for NGF on their surfaces which is induced substantially after axotomy. We have found that the adsorption of NGF onto cryostat sections of the distal stump of previously severed sciatic nerve enhances neurite growth over this tissue. This finding, coupled with the two previous observations, suggests that Schwann cell surface NGF receptors serve to bind to NGF-like growth factors so as to provide favorable surfaces for regenerating peripheral nerve axons.     

BD™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration

This study investigated the effects of a membrane conduit filled with a synthetic matrix BD™ PuraMatrix™ peptide (BD) hydrogel and cultured Schwann cells on regeneration after peripheral nerve injury in adult rats.After sciatic axotomy, a 10 mm gap between the nerve stumps was bridged using ultrafiltration membrane conduits filled with BD hydrogel or BD hydrogel containing Schwann cells. In control experiments, the nerve defect was bridged using either membrane conduits with alginate/fibronectin hydrogel or autologous nerve graft. Axonal regeneration within the conduit was assessed at 3 weeks and regeneration of spinal motoneurons and recovery of muscle weight evaluated at 16 weeks postoperatively.Schwann cells survived in the BD hydrogel both in culture and after transplantation into the nerve defect. Regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel when compared with the alginate/fibronectin hydrogel and alginate/fibronectin with Schwann cells. Addition of Schwann cells to the BD hydrogel considerably increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. The conduits with BD hydrogel showed a linear alignment of nerve fibers and Schwann cells.The number of regenerating motoneurons and recovery of the weight of the gastrocnemius muscle was inferior in BD hydrogel and alginate/fibronectin groups compared with nerve grafting. Addition of Schwann cells did not improve regeneration of motoneurons or muscle recovery.The present results suggest that BD hydrogel with Schwann cells could be used within biosynthetic conduits to increase the rate of axonal regeneration across a nerve defect.     

Application of weak electric field to the hindpaw enhances sciatic motor nerve regeneration in the adult rat

Direct current (DC) electrical stimulation of the hindpaw is shown to enhance sciatic motor nerve regeneration in the adult rat. Cathodal stimulation, using weak currents (10 microA/cm2; field strength approximately 100 mV/cm) increased the reinnervation of the hindpaw muscles as measured by evoked electromyograms. This enhanced regeneration only occurred after cut-and-suture lesions, but not after crushing injury of the sciatic nerve. This enhancement of motor nerve regeneration by weak DC fields had been previously described in amphibians but we are the first to describe this phenomenon in mammals.     

Evaluation of artificial nerve conduit and autografts in peripheral nerve repair in the rat model of sciatic nerve injury

Objective: To investigate the therapeutic effect of artificial nerve conduit in the sciatic nerve injury and repair in the rat model.

Methods: A total of 60 adult male Sprague Dawley rats were evenly randomized into five groups to build the model of sciatic nerve injury and perform the injury repair experiment. The five groups were: group A which was treated with artificial nerve conduit, group B which was treated with common carotid artery (CCA) autograft, group C which was treated with sciatic nerve autograft, group D which was treated with sham operation, and group E as the normal control. The injury was repaired by direct coaptation of the nerve ends. Postoperatively, the rats’ behavior, motor nerve conduction velocity (MNCV), incubation period, amplitude, remaining rate of wet weight of the gastrocnemius muscle, the diameter and section area of the gastrocnemius cell, and the histological changes were assessed. The results were analyzed by one-way ANOVA and two-way ANOVA.

Results: Twelve days postoperatively, 36 rats in groups A, B, and C presented with denervated adermotrophia on the injured ankle. The electrophysiological indicators in groups D and E were constant and similar. The values of MNCV and amplitude were group C > group A > group B, with an increasing tendency. The values of the incubation period were group C < group A < group B with statistical difference (p < 0.05) and showed a decreasing tendency. The wet gastrocnemius muscle in groups D and E showed plump morphology with luster and elasticity. Groups A and C had similar atrophic gastrocnemius muscles and reduced flexibility while the phenomena were more severe in group B. Progressive decrease of the cell diameter and sectional area was observed in groups A, B, and C. The adhesion between the sciatic nerve and the surrounding area in groups A, B, and C had statistical significance (P < 0.05), with group B the most serious.

Conclusions: The results suggest that artificial nerve conduit facilitated functional and morphological regeneration of the nerve. It seemed more effective than CCA but inferior to sciatic nerve autograft in repairing sciatic nerve injury in the rat model.     

Specific and nonspecific regeneration of motor axons after sciatic nerve injury and repair in the rat

The pattern of motor axon regeneration following unilateral sciatic nerve lesions (freezing or transection) was studied in adult rats. Transected nerves were repaired with epineurial or fascicular sutures. Four months after the lesion, the motor neuron cell body localization in the spinal cord of plantar or common peroneal nerve axons were examined bilaterally with retrograde transport of horseradish peroxidase. Motor neuron cell body localization was similar bilaterally after freezing, indicating that regenerating axons had reached their original peripheral innervation territory. However, after nerve transection, irrespective of whether epineurial or fascicular sutures were used, motor neuron cell body distribution on the operated side was abnormal with numerous labeled cell bodies located outside the area of the normal motor neuron pool. This finding indicates that after nerve transection the normal pattern of motor axon innervation is not restored even after fascicular nerve repair.     

维拉帕米与神经生长因子促进大鼠坐骨神经再生的协同作用

背景：实验证明周围神经损伤时,轴突的变性与神经元凋亡都与Ca2+的超载有着极其密切的关系。 目的：利用大鼠坐骨神经损伤模型观察L型钙离子通道阻滞剂维拉帕米联合神经生长因子促进周围神经再生的协同作用。 设计、时间及地点：随机对照动物实验,于2007-04/2008-11在辽宁医学院手外科实验室完成。 材料：同系健康雄性SD大鼠32只,体质量220~260 g;维拉帕米为辽宁卫星制药厂产品,国药准字H21022847;神经生长因子为sigma公司产品。 方法：同系SD大鼠32只随机分为4组,每组8只,分别在右侧梨状肌下缘5 mm切断坐骨神经后立即原位缝合造成坐骨神经损伤模型。①维拉帕米+神经生长因子组：腹腔注射维拉帕米4 mg/(kg•d),术侧腓肠肌肉注射神经生长因子0.6 μg/d。②维拉帕米组：腹腔注射维拉帕米4 mg/(kg•d),术侧腓肠肌注射等量生理盐水。③神经生长因子组：术侧腓肠肌注神经生长因子0.6 μg/d,并腹腔注射等量生理盐水。④空白对照组：分别腹腔,肌注等量生理盐水。以左侧坐骨神经为正常对照。 主要观察指标：术后12周对各组再生神经进行大体观察,神经电生理测定,组织学观察及有髓神经纤维计数。 结果：术后12周,维拉帕米+神经生长因子组足部溃疡的出现与愈合以及展抓反射出现的时间均早于其他各组。神经传导速度恢复率和有髓神经纤维计数恢复率分析表明：维拉帕米+神经生长因子组>维拉帕米组>神经生长因子组>空白对照组。光镜和电镜下可见：维拉帕米+神经生长因子组再生的神经纤维最多,轴突较为粗大。有髓神经纤维多,髓鞘完整,优于其他3组。神经纤维直径恢复率分析表明：维拉帕米+神经生长因子组>神经生长因子组>维拉帕米组>空白对照组。 结论：维拉帕米与神经生长因子对促进周围神经形态结构和功能的恢复均具有明显的协同作用。     

The impact of motor and sensory nerve architecture on nerve regeneration

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve's Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p < 0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p < 0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.     

Valproic acid enhances axonal regeneration and recovery of motor function after sciatic nerve axotomy in adult rats

It has recently been demonstrated that valproic acid (VPA) robustly promotes neurite outgrowth, activates the extracellular signal regulated kinase pathway, and increases growth cone-associated protein 43 and bcl-2 levels in cultured human neuroblastoma SH-SY5Y cells. We hypothesized that VPA could also enhance peripheral nerve regeneration in adult animals. To test this hypothesis, we examined the effects of VPA (300 mg/kg daily for 16 weeks) on sciatic axonal regeneration following single or conditional axotomies in rats. The results showed that in VPA-treated rats there was a significant increase in the total numbers of regenerated myelinated nerve fibers and reinnervated muscle fibers in comparison with those rats not treated with VPA. As measured by sciatic function index and toe spread index, the motor function of the reinnervated hind limbs of rats receiving single axotomy without VPA treatment significantly improved at week 8 and reached plateau levels at about week 11, whereas the motor function of the reinnervated hind limbs of rats receiving single axotomy plus VPA and rats receiving conditional axotomy with or without VPA treatment significantly improved at week 4 and reached plateau levels at about week 8; there was no significant difference of the motor function among the three later groups. The results demonstrated that VPA is able to enhance sciatic nerve regeneration and recovery of motor function in adult rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.     

神经生长因子治疗周围神经损伤的疗效观察

目的 观察神经生长因子治疗周围神经损伤的临床疗效.方法 纳入60例周围神经损伤患者,采用随机数字表法分为2组.试验组30例采用鼠NGF肌内注射治疗,对照组30例采用维生素B12治疗,治疗4周后观察疗效.观察指标包括疼痛(VAS)、麻木等临床症状和体征,同时观察单神经的神经电生理情况.结果 疼痛改善:试验组总有效率93.33％,对照组为53.33％;麻木改善:试验组总有效率86.67％,对照组为66.67％;2组比较差异均有统计学意义(P＜0.05).试验组恢复神经的感觉及运动电位的潜伏期时间均明显短于对照组,差异有统计学意义(P＜0.05);而波幅则均显著高于对照组,差异有显著性意义(P＜0.05).结论 神经生长因子能有效改善患者的疼痛、麻木症状,而且能对神经纤维的修复、电生理功能有促进作用.     

Does insulin-like growth factor I (IGF-1) trigger the cell body reaction in the rat sciatic nerve?

Regeneration was measured after the infliction of a crush lesion on rat sciatic nerves which 4 days earlier had been subjected to a distal conditioning transection. Such nerves exhibited an increased outgrowth of nerve fibers as compared to nerves subjected to a single crush lesion. This increased outgrowth could be prevented, if the nerve was locally perfused around the site of the transection during the 4 days conditioning interval, with cycloheximide, actinomycin D and vinblastine, inhibitors of protein-, RNA-synthesis and retrograde axonal transport, respectively. The inhibitory effect of cycloheximide could be overcome by simultaneous perfusion with insulin-like growth factor I (IGF-1). The results suggest that proteins including IGF-1 which are synthesised locally around a nerve lesion and then transported retrogradely could trigger regenerative events in the neuronal cell body.     

Axonal regeneration in an articular branch following rat sciatic nerve lesions

This study examines the outcome of axonal regeneration in the posterior articular nerve of the adult rat knee joint (PAN), after sciatic nerve lesions. Some animals had previously been subjected to chemical sympathectomy with guanethidine. In crushed cases the number of myelinated PAN axons was 50% above control level. The occurrence of C-fibers was doubled, mainly due to an increased number of sympathetic efferents. In neurotomy/suture cases the number of myelinated fibers was clearly elevated, but the number of C-fibers was close to normal. Most C-fibers were sensory. Similar, but less marked, post-regeneration abnormalities were seen in the nerve to the lateral gastrocnemius muscle. The sural nerve exhibited moderately increased numbers of myelinated and unmyelinated fibers in crushed cases. In neurotomy cases, the myelinated axons had increased and the C-fibers had decreased in number. The size distribution of myelinated PAN axons was less abnormal in crushed cases than after neurotomy, like in the other nerves. These results show that the outcome of axon regeneration in an articular branch of the lesioned rat sciatic nerve differs from that in non-articular branches, and suggest that joints may become hyperinnervated by C-fibers after nerve crush lesions.     

神经生长因子促再生坐骨神经血管生成的作用

BACKGROUND： It remains to be determined whether nerve growth factor （NGF） can promote angiogenesis in regenerating peripheral nerves during repairing peripheral nerve injury.
OBJECTIVE： To evaluate the effects of NGF on angiogenesis, and to analyze the influencing mechanisms of NGF, according to the expression patterns of CD34, von Willebrand factor （vWF）, vascular endothelial cell growth factor （VEGF）, and the NGF receptor TrkA in proliferating vascular endothelial cells from a rat model of sciatic nerve injury.
DESIGN, TIME AND SETTING： Randomized, controlled study performed at the Research Institute of Field Surgery, Daping Hospital affiliated to the Third Military Medical University of Chinese PLA, between October 2003 and July 2005.
MATERIALS： Forty-five healthy, adult, Wistar rats underwent sciatic nerve injury. The rats were randomly divided into four groups： NGF ＋ chitosan （n = 15）, NGF ＋ chitosan ＋ anti-VEGF （n = 10）, chitosan （n = 10）, and physiological saline （n = 10）. METHODS： A 1 -cm defected sciatic nerve was bridged with a silica gel conduit. NGF ＋ chitosan group： 100 μ L chitosan and 5 μ L NGF （20 mg/L） were injected into the silica gel conduit; NGF ＋ chitosan ＋ anti-VEGF group： an additional 5μ L anti-VEGF monoclonal antibody （1 g/L） was injected into the silica gel conduit; chitosan group： 100μL chitosan and 5 μL physiological saline were injected into the silica gel conduit; physiological saline group： only 5μL physiological saline was injected into the silica gel conduit.
MAIN OUTCOME MEASURES： CD34 and vWf were used to label blood capillaries and large-diameter blood vessels in the regenerating peripheral nerves, respectively. At day 14 following surgery, immunohistochemistry was used to detect and semi-quantitatively analyze expressions of CD34, vWf, VEGF, and TrkA in proliferating vascular endothelial cells in the regenerating sciatic nerve. A confocal laser microscope was used to determine co-expression. RESULTS： Expressions of TrkA, CD34, vWf, and VEGF in the NGF ＋ chitosan group were significantly greater than the physiological saline and chitosan groups （P 〈 0.05-0.01）. Expressions of CD34 and VEGF in the NGF ＋ chitosan ＋ anti-VEGF group were completely inhibited, while expressions of vWf and TrkA gradually decreased, compared with the NGF ＋ chitosan group （P 〈 0.01）. Confocal microscopy revealed strong co-expression of VEGF and CD34 in the regenerating sciatic nerve, and CD34 expression positively correlated with VEGF expression. In addition, VEGF expression was greater than CD34 expression, and coexpression of VEGF and vWf was also strong.
CONCLUSION： VEGF was expressed in blood capillaries and large-diameter blood vessels, while exogenous NGF promoted VEGF expression in regenerating sciatic nerves, thereby increasing angiogenesis.     

Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Methods: Fifty Sprague‐Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit‐8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57 : 821–828, 2018     

组织工程周围神经修复坐骨神经缺损应用研究

目的应用组织工程方法构建周围神经以修复坐骨神经缺损.方法体外培养的雪旺细胞(SC)与牛去细胞基质(BAM)、胎牛血清和培养液按一定的比例混合注入聚乳酸聚羟基己酸共聚物(PLGA)导管中,构建成组织工程周围神经.30只SD大鼠随机分为3组,实验组:使用组织工程周围神经修复坐骨神经缺损;对照组:用不含雪旺细胞的导管修复;自体神经组:自体神经移植.16周后通过免疫组化、电生理、透射电镜、辣根过氧化物酶(HRP)逆行示踪及坐骨神经功能指数(SFI)等方法检测神经再生及坐骨神经功能恢复情况.结果 PLGA导管至16周已基本吸收,再生神经已通过缺损区长至远端,组织工程周围神经的修复效果接近自体神经组,优于空白组.结论体外构建的组织工程周围神经可以修复周围神经缺损.     

Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts

The aim of this study was to investigate the effects of vascular endothelial growth factor (VEGF) on regeneration of the rat sciatic nerve in vivo. To that end we used 10-mm long cell-free nerve grafts to bridge a gap in the sciatic nerve. The grafts were pretreated with either VEGF (50, 100 or 250 ng/ml), nerve growth factor (NGF, 100 ng/ml) or laminin (100 ng/ml) before implantation. Outgrowth of axons, Schwann cells, blood vessels and macrophages were studied 10 days post-implantation by the use of immunocytochemistry and histochemistry. Grafts pretreated with VEGF stimulated the outgrowth of Schwann cells and blood vessels but not axons. In such grafts, the Schwann cells also exhibited a dramatic change in morphology and became filled with large lipid-containing vacuoles. These cells also showed an intense immunoreactivity for the VEGF receptor flk-1. Neither pretreatment with laminin nor NGF affected the outgrowth of Schwann cells. However, NGF treatment increased the number of axons in the graft but was not able to counteract injury-induced downregulation of substance P in the dorsal root ganglia. The results show that local application of VEGF promotes at least two events, invasion of Schwann cells and neovascularization, which are important during nerve regeneration. The findings suggest that the effects of the pretreatment by the growth factors is local and limited to the graft, whereas central events like neuropeptide synthesis is not affected.     

Injection injury of the sciatic nerve (370 cases)

Injury to peripheral nerves due to injections of therapeutic and other agents is common. The postulated mechanisms of injury include direct needle trauma, secondary constriction by scar, and direct nerve fiber damage by neurotoxic chemicals in the injected agent. Neurological sequelae can range from minor transient sensory disturbance to severe sensory disturbance and paralysis with poor recovery. The recommended treatment has ranged from a conservative approach to immediate operative exposure and irrigation, and has also included early neurolysis of delayed exploration with neurolysis or resection and anastomosis. We present 370 cases of injection injury of the sciatic nerve in children treated during the last 20 years at the Neurosurgical Department of the Hospital La Paz in Madrid, Spain. Pathology, clinical course, treatment, and results are discussed.     

Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats

Adult rat sciatic nerve was transected and sutured with an entubulation technique. The nerve interstump gap was filled with either collagen gel (COL) or collagen gel mixed with glial cell line-derived neurotrophic factor (COL/GDNF). Four weeks after nerve transection, horseradish peroxidase (HRP)-labelled spinal cord motoneurons and the myelinated distal stump axons were quantified. Compared with the COL group, the percentages of labeled spinal somas and axon number were significantly increased after topically applied glial cell line-derived neurotrophic factor (GDNF). The functional recovery of the transected nerve was improved in COL/GDNF group. GAP-43 expression was also significantly higher in COL/GDNF group 1 and 2 weeks after sciatic nerve axotomy vs. COL group. These data provide strong evidence that GDNF could promote axonal regeneration in adult rats, suggesting the potential use of GDNF in therapeutic approaches to peripheral nerve injury and neuropathies.