Functional motor recovery is improved due to local placement of GDNF microspheres after delayed nerve repair

The majority of bioengineering strategies to promote peripheral nerve regeneration after injury have focused on therapies to bridge large nerve defects while fewer therapies are being developed to treat other nerve injuries, such as nerve transection. We constructed delivery systems using fibrin gels containing either free GDNF or polylactide–glycolic acid (PLGA) microspheres with GDNF to treat delayed nerve repair, where ELISA verified GDNF release. We determined the formulation of microspheres containing GDNF that optimized nerve regeneration and functional recovery in a rat model of delayed nerve repair. Experimental groups underwent delayed nerve repair and treatment with GDNF microspheres in fibrin glue at the repair site or control treatments (empty microspheres or free GDNF without microspheres). Contractile muscle force, muscle mass, and MUNE were measured 12 weeks following treatment, where GDNF microspheres (2 weeks formulation) were superior compared to either no GDNF or short‐term release of free GDNF to nerve. Nerve histology distal to the repair site demonstrated increased axon counts and fiber diameters due to GDNF microspheres (2 weeks formulation). GDNF microspheres partially reversed the deleterious effects of chronic nerve injury, and recovery was slightly favored with the 2 weeks formulation compared to the 4 weeks formulation. Biotechnol. Bioeng. 2013; 110: 1272–1281. © 2012 Wiley Periodicals, Inc.     

Biomimetic surface delivery of NGF and BDNF to enhance neurite outgrowth

Treatment for peripheral nerve injuries includes the use of autografts and nerve guide conduits (NGCs). However, outcomes are limited, and full recovery is rarely achieved. The use of nerve scaffolds as a platform to surface immobilize neurotrophic factors and deliver locally is a promising approach to support neurite and nerve outgrowth after injury. We report on a bioactive surface using functional amine groups, to which heparin binds electrostatically. X-ray photoelectron spectroscopy analysis was used to characterize the presence of nitrogen and sulfur. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were bound by electrostatic interaction to heparin, and the release profile evaluated by enzyme-linked immunosorbent assay, which showed that ca. 1% of NGF was released from each of the bioactive surface within 7 days. Furthermore, each surface showed a maximum release of 97% of BDNF. Neurotrophin release on neurite outgrowth was evaluated by primary dorsal root ganglion with a maximum neurite growth response in vitro of 1,075 µm detected for surfaces immobilized with NGF at 1 ng/ml. In summary, the study reports on the design and construction of a biomimetic platform to deliver NGF and BDNF using physiologically low concentrations of neurotrophin. The platform is directly applicable and scalable for improving the regenerative ability of existing NGCs and scaffolds.     

Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.     

人工神经导管原材料选择与功能设计的研究进展

人工神经导管（nerve guidance conduits,NGCs）作为一种合成的神经移植物,为神经再生提供结构与营养支持。理想的神经导管对生物相容性、机械强度、拓扑结构和导电性等均有较高要求,因此需对神经导管的设计不断改进并建立更完善的周围神经再生策略,以期满足临床需求。虽然NGCs在周围神经损伤的治疗中已经取得一定进展,但其对长距离神经离断伤的结构与功能修复仍不理想。本文分别从原材料选择、结构设计、治疗因子搭载及自供电元件集成4个方面对神经导管的设计进行综述,归纳总结NGCs在周围神经损伤治疗中的研究进展,以期推动NGCs的迭代更新与临床转化。     

Nanoparticle mediated controlled delivery of dual growth factors

Peripheral nerve functional recovery after nerve injury generally requires multiple growth factors by synergistic effect.However,the optical combination of multiple synergistic growth factors for axonal regeneration has been scarcely considered up to now.Meanwhile,the use of growth factors in promoting nerve regeneration was limited by its short biological half-life in vivo,its vulnerability to structure disruption or hydrolyzation,leading to loss of bioactivity.Herein,a novel polymeric nanoparticle delivery system composed of heparin andε-poly-L-lysine(PL)was prepared for control release of nerve growth factor(NGF)and basic fibroblast growth factor(bFGF).The nanoparticles were synthesized by polyelectrolyte complexation in aqueous solution at room temperature,followed by cross-linking with biological genipin.The obtained nanoparticles had a spherical shape,with a mean diameter of about 246 nm,and high growth factors encapsulation efficiency as well as good stability.NGF and bFGF were encapsulated in the nanoparticles and showed a continuous and slow release behavior in vitro.The bioactivities of the released growth factors were evaluated,and exhibited the synergistic effect.The controlled release of the dual synergistic growth factors would improve the treatment of peripheral nerve injury to mimic the natural cellular microenvironments.     

Thermosensitive heparin‐poloxamer hydrogel encapsulated bFGF and NGF to treat spinal cord injury

The application of growth factors (GFs) for treating chronic spinal cord injury (SCI) has been shown to promote axonal regeneration and functional recovery. However, direct administration of GFs is limited by their rapid degradation and dilution at the injured sites. Moreover, SCI recovery is a multifactorial process that requires multiple GFs to participate in tissue regeneration. Based on these facts, controlled delivery of multiple growth factors (GFs) to lesion areas is becoming an attractive strategy for repairing SCI. Presently, we developed a GFs‐based delivery system (called GFs‐HP) that consisted of basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and heparin‐poloxamer (HP) hydrogel through self‐assembly mode. This GFs‐HP was a kind of thermosensitive hydrogel that was suitable for orthotopic administration in vivo. Meanwhile, a 3D porous structure of this hydrogel is commonly used to load large amounts of GFs. After single injection of GFs‐HP into the lesioned spinal cord, the sustained release of NGF and bFGF from HP could significantly improve neuronal survival, axon regeneration, reactive astrogliosis suppression and locomotor recovery, when compared with the treatment of free GFs or HP. Moreover, we also revealed that these neuroprotective and neuroregenerative effects of GFs‐HP were likely through activating the phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signalling pathways. Overall, our work will provide an effective therapeutic strategy for SCI repair.     

Therapeutic Effect of Vinorine on Sciatic Nerve Injured Rat

Vinorine is a monoterpenoid indole alkaloid, a type of natural alkaloids. Growing reports exhibited the numerous pharmacology activities of vinorine such as anti-inflammation, anti-bacterial and anti-tumor. In this study, the effect of vinorine injection (7.5, 15 and 30 mg/kg) on motor function, sensation and nerve regeneration in sciatic nerve crush injury rat was investigated. The results of behavioral analysis, electrophysiological analysis and muscle histological analysis suggested that vinorine promoted the motor function recovery after sciatic nerve injury. The results of mechanical withdrawal thresholds assay and hot plate test demonstrated that vinorine improved the sensation recovery after sciatic nerve injury. The results of Fluoro-gold retrograde labeling, transmission electron microscope assay, toluidine blue and HE staining showed that vinorine attenuated the nerve damage caused by sciatic nerve injury and promoted the nerve regeneration. Furthermore, nerve growth factor (NGF) and its downstream extracellular signal-regulated kinase (ERK) signaling pathway participated in the neuro-recovery effect of vinorine after crush. In conclusion, vinorine treatment accelerated the sciatic nerve regeneration, motor function recovery and sensation recovery after crush injury via regulation of NGF and ERK activity. These results suggested that vinorine is a promising agent for never injury therapy.     

Drug-delivering nerve conduit improves regeneration in a critical-sized gap

Autologous nerve grafts are the current “gold standard” for repairing large nerve gaps. However, they cause morbidity at the donor nerve site and only a limited amount of nerve can be harvested. Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of GDNF can enhance axon growth and motor recovery. FK506, an FDA approved small molecule, has also been shown to enhance peripheral nerve regeneration. This paper describes the design of a novel hole-based drug delivery apparatus integrated with a polytetrafluoroethylene (PTFE) nerve conduit for controlled local delivery of a protein such as GDNF or a small molecule such as FK506. The PTFE devices were tested in a diffusion chamber, and the bioactivity of the released media was evaluated by measuring neurite growth of dorsal root ganglions (DRGs) exposed to the released drugs. The drug delivering nerve guide was able to release bioactive concentrations of FK506 or GDNF. Following these tests, optimized drug releasing nerve conduits were implanted across 10 mm sciatic nerve gaps in a BL6 yellow fluorescent protein (YFP) mouse model, where they demonstrated significant improvement in muscle mass, compound muscle action potential, and axon myelination in vivo as compared with nerve conduits without the drug. The drug delivery nerve guide could release drug for extended periods of time and enhance axon growth in vitro and in vivo.     

The effects of rapamycin in murine peripheral nerve isografts and allografts

The FKBP-12-binding ligand FK506 has been successfully used to stimulate nerve regeneration and prevent the rejection of peripheral nerve allografts. The immunosuppressant rapamycin, another FKBP-12-binding ligand, stimulates axonal regeneration in vitro, but its influence on nerve regeneration in peripheral nerve isografts or allografts has not been studied. Sixty female inbred BALB/cJ mice were randomized into six tibial nerve transplant groups, including three isograft and three allograft (C57BL/6J) groups. Grafts were left untreated (groups I and II), treated with FK506 (groups III and IV), or treated with rapamycin (groups V and VI). Nerve regeneration was quantified in terms of histomorphometry and functional recovery, and immunosuppression was confirmed with mixed lymphocyte reactivity assays. Animals treated with FK506 and rapamycin were immunosuppressed and demonstrated significantly less immune cell proliferation relative to untreated recipient animals. Although every animal demonstrated some functional recovery during the study, animals receiving an untreated peripheral nerve allograft were slowest to recover. Isografts treated with FK506 but not rapamycin demonstrated significantly increased nerve regeneration. Nerve allografts in animals treated with FK506, and to a lesser extent rapamycin, however, both demonstrated significantly more nerve regeneration and increased nerve fiber widths relative to untreated controls. The authors suggest that rapamycin can facilitate regeneration through peripheral nerve allografts, but it is not a neuroregenerative agent in this in vivo model. Nerve regeneration in FK506-treated peripheral nerve isografts and allografts was superior to that found in rapamycin-treated animals. Rapamycin may have a role in the treatment of peripheral nerve allografts when used in combination with other medications, or in the setting of renal failure that often precludes the use of calcineurin inhibitors such as FK506.     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

肌肉注射人神经生长因子基因逆轴突传递修复神经损伤的研究进展

神经生长因子(NGF)促进中枢及外周神经系统神经元细胞存活、分化、轴突再生等重要作用已得到临床的广泛证实。目前临床上主要以局部或肌肉注射NGF蛋白的方式对神经系统的损伤进行治疗。但NGF半衰期短、局部应用副作用大、费用昂贵、难以透过血脑屏障等缺点而限制临床应用。长期以来,科研工作者致力于寻求一种理想的途径或方法以克服这一缺陷。随着基因工程技术的飞速发展,研究人员发现通过骨骼肌肌肉注射途径,以非病毒载体介导外源的NGF基因体内表达并逆轴突传递到神经损伤部位,有望解决这一难题。本文将就NGF及受体的基本结构和特性、逆轴突传递的机制、非病毒载体结合骨骼肌肌肉注射的基因治疗等方面进行总结和阐述。     

Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury

Up-regulation of neurotrophin synthesis is an important mechanism of peripheral nerve regeneration after injury. Neurotrophin expression is regulated by a complex series of events including cell interactions and multiple molecular stimuli. We have studied neurotrophin synthesis at 2?weeks time-point in a transvertebral model of unilateral or bilateral transection of sciatic nerve in rats. We have found that unilateral sciatic nerve transection results in the elevation of nerve growth factor (NGF) and NT-3, but not glial cell-line derived neurotrophic factor or brain-derived neural factor, in the uninjured nerve on the contralateral side, commonly considered as a control. Bilateral transection further increased NGF but not other neurotrophins in the nerve segment distal to the transection site, as compared to the unilateral injury. To further investigate the distinct role of NGF in regeneration and its potential for peripheral nerve repair, we transduced isogeneic Schwann cells with NGF-encoding lentivirus and transplanted the over-expressing cells into the distal segment of a transected nerve. Axonal regeneration was studied at 2?weeks time-point using pan-neuronal marker NF-200 and found to directly correlate with NGF levels in the regenerating nerve.     

PACAP facilitate the nerve regeneration factors in the facial nerve injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported as a strong neurotrophic factor in the various sites of nervous system. The facial nerve injury is one of the common problems in patients at the Otolaryngology since the nerve damage could occur easily due to the anatomical characteristics. Once it happens, the regeneration is little observed and functional recovery is poor. Thus, we investigated that PACAP might have some influence for regeneration after the facial nerve transaction in the guinea pig. PACAP treatment accelerated time for the appearance of compound muscle action potentials (CMAP) after the nerve transaction (first appeared at 1 versus 2 weeks in control) and shortened the latency at 4 weeks. The number of myelinated fibers increased at 4 weeks. Histochemical demonstration of GAP-43, a growth cone protein, was observed at the injury area at 2-4 days. PACAP increased the level of glial cell line-derived neurotrophic factor (GDNF), a neurotrophin, in facial target muscles at 1 day-4 weeks. These data indicated that PACAP promotes the regeneration factors and increases the possibility of functional recovery following the facial nerve injury.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.     

神经生长因子促进坐骨神经再生修复的酶组织化学研究

目的研究对兔右坐骨神经损伤后局部给予蛇毒神经生长因子(NGF),观察坐骨神经酶活性变化和超微结构的恢复情况,探讨NGF对神经再生的影响.方法乙酰胆碱酯酶(AChE)、酸性磷酸酶(ACPase)的酶组织化学技术和电镜技术.结果神经损伤后:AChE活性明显下降,NGF组的AChE活性恢复快于盐水对照组;ACPase活性逐渐增高,NGF组的ACPase活性恢复时间短于盐水对照组.坐骨神经的超微结构在神经损伤后也发生变化,NGF组的变化程度小于盐水对照组,恢复时间短于对照组.结论NGF可通过影响酶物质的代谢而起到加快受损神经恢复的作用.为临床上应用蛇毒NGF治疗周围神经损伤提供形态学依据.     

神经生长因子药效学研究

目的考察神经生长因子对视神经损害的疗效。方法腹腔注射二硫化碳致视神经损害的大鼠模型,给药组球后注射或肌内注射神经生长因子,每天一次,每周6d,共计3周,空白对照组球后注射等量生理盐水,于治疗前后测定大鼠模式翻转（FREP）和闪光视觉诱发电位。结果球后注射高剂量组大鼠在治疗10d和20d时各波潜伏时比对照组有显著缩短,而球后注射低剂量组和肌内注射高剂量组大鼠于治疗10d时FVEP各波潜伏时也显著缩短,肌内注射低剂量组大鼠于治疗20d时FEP的P1和P2波潜伏时也显著或非常显著缩短。结论神经生长因子对大鼠视神经损害有明显的治疗作用。     

神经生长因子对兔坐骨神经损伤后脊髓前角运动神经元乙酰胆碱酯酶的影响

钳夹损伤兔右坐骨神经,于损伤处注射蛇毒ＮＧＦ４００Ｂｕ／ｋｇ／日,损伤术后１,３,７天和２,３,４,６,８周动态观察脊髓腰段伤侧第Ⅸ板层外侧群的大型运动神经元的ＡＣｈＥ活性改变。结果表明术后１,３天实验组（指损伤给药组）和对照组（指损伤对照组）ＡＣｈＥ活性均下降（Ｐ＞００５）;术后１,２,３周对照组ＡＣｈＥ活性明显下降,而实验组ＡＣｈＥ活性逐渐趋于恢复（Ｐ＜００１）;术后６周实验组ＡＣｈＥ活性恢复至正常水平（Ｐ＜００１）。本研究显示蛇毒ＮＧＦ对坐骨神经损伤后脊髓前角运动神经元ＡＣｈＥ活性恢复有促进作用,从而对运动神经元可起一定的保护作用和促进恢复的作用     

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.