Recombinant human fibroblast growth factor-2 promotes nerve regeneration and functional recovery after mental nerve crush injury

Several studies have shown that fibroblast growth factor-2 （FGF2） can directly affect axon regeneration after peripheral nerve damage. In this study, we performed sensory tests and histological analyses to study the effect of recombinant human FGF-2 （rhFGF2） treatment on damaged mental nerves. The mental nerves of 6-week-old male Sprague-Dawley rats were crush-injured for 1 minute and then treated with 10 or 50 μg/mL rhFGF2 or PBS in crush injury area with a mini Osmotic pump. Sensory test using von Frey filaments at 1 week revealed the presence of sensory degeneration based on decreased gap score and increased difference score. However, at 2 weeks, the gap score and difference score were significantly rebounded in the mental nerve crush group treated with 10 μg/mL rhFGF2. Interestingly, treatment with 10 μg/mL rhFGF had a more obviously positive effect on the gap score than treatment with 50 μg/mL rhFGF2. In addition, retrograde neuronal tracing with Dil revealed a significant increase in nerve regeneration in the trigeminal ganglion at 2 and 4 weeks in the rhFGF2 groups （10 μg/mL and 50 μg/mL） than in the PBS group. The 10 μg/mL rhFGF2 group also showed an obviously robust regeneration in axon density in the mental nerve at 4 weeks. Our results demonstrate that 10 μg/mL rhFGF induces mental nerve regeneration and sensory recovery after mental nerve crush injury.

Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion

Using electroacupuncture and moxibustion to treat peripheral nerve injury is highly efficient with low side effects. However, the electroacupuncture-and moxibustion-based mechanisms underlying nerve repair are still unclear. Here, in vivo and in vitro experiments uncovered one mechanism through which electroacupuncture and moxibustion affect regeneration after peripheral nerve injury. We first established rat models of sciatic nerve injury using neurotomy. Rats were treated with electroacupuncture or moxibustion at acupoints Huantiao （GB30） and Zusanli （ST36）. Each treatment lasted 15 minutes, and treatments were given six times a week for 4 consecutive weeks. Behavioral testing was used to determine the sciatic functional index. We used electrophysiological detection to measure sciatic nerve conduction velocity and performed hematoxylin-eosin staining to determine any changes in the gastrocnemius muscle. We used immunohistochemistry to observe changes in the expression of S100—a specific marker for Schwann cells—and an enzyme-linked immunosorbent assay to detect serum level of nerve growth factor. Results showed that compared with the model-only group, sciatic functional index, recovery rate of conduction velocity, diameter recovery of the gastrocnemius muscle fibers, number of S100-immunoreactive cells,and level of nerve growth factor were greater in the electroacupuncture and moxibustion groups. The efficacy did not differ between treatment groups. The serum from treated rats was collected and used to stimulate Schwann cells cultured in vitro. Results showed that the viability of Schwann cells was much higher in the treatment groups than in the model group at 3 and 5 days after treatment. These findings indicate that electroacupuncture and moxibustion promoted nerve regeneration and functional recovery; its mechanism might be associated with the enhancement of Schwann cell proliferation and upregulation of nerve growth factor.

Nerve growth factor and injured peripheral nerve regeneration

Nerve growth factor （NGF） exhibits many biological activities, such as supply of nutrients, neuroprotection, and the generation and rehabilitation of injured nerves. The neuroprotective and neurotrophic qualities of NGF are generally recognized. NGF may enhance axonal regeneration and myelination of peripheral nerves, as well as cooperatively promote functional recovery of injured nerves and limbs. The clinical efficacy of NGF and its therapeutic potentials are reviewed here. This paper also reviews the latest NGF research developments for repairing injured peripheral nerve, thereby providing scientific evidence for the appropriate clinical application of NGF.

Baseline effects of lysophosphatidylcholine and nerve growth factor in a rat model of sciatic nerve regeneration after crush injury

Schwann cells play a major role in helping heal injured nerves. They help clear debris, produce neurotrophins, upregulate neurotrophin receptors, and form bands of Büngner to guide the healing nerve. But nerves do not always produce enough neurotrophins and neurotrophin receptors to repair themselves. Nerve growth factor（NGF） is an important neurotrophin for promoting nerve healing and lysophosphatidylcholine（LPC） has been shown to stimulate NGF receptors（NGFR）. This study tested the administration of a single intraneural injection of LPC（1 mg/mL for single LPC injection and 10 mg/mL for multiple LPC injections） at day 0 and one（day 7）, two（days 5 and 7）, or three（days 5, 7, and 9） injections of NGF（160 ng/mL for single injections and 80 ng/mL for multiple injections） to determine baseline effects on crush ed sciatic nerves in rats. The rats were randomly divided into four groups： control, crush, crush-NGF, and crush-LPC-NGF. The healing of the nerves was measured weekly by monitoring gait; electrophysiological parameters： compound muscle action potential（CMAP） amplitudes; and morphological parameters： total fascicle areas, myelinated fiber counts, fiber densities, fiber packing, and mean g-ratio values at weeks 3 and 6. The crush, crush-NGF, and crush-LPC-NGF groups statistically differed from the control group for all six weeks for the electrophysiological parameters but only differed from the control group at week 3 for the morphological parameters. The crush, crush-NGF, and crush-LPC-NGF groups did not differ from each other over the course of the study. Single injections of LPC and NGF one week apart or multiple treatments of NGF at 5, 7 and 9 days post-injury did not alter the healing rate of the sciatic nerves during weeks 1-6 of the study. These findings are important to define the baseline effects of NGF and LPC injections, as part of a larger effort to determine the minimal dose regimen of NGF to regenerate peripheral nerves.

Biological characteristics of dynamic expression of nerve regeneration related growth factors in dorsal root ganglia after peripheral nerve injury

The regenerative capacity of peripheral nerves is limited after nerve injury.A number of growth factors modulate many cellular behaviors,such as proliferation and migration,and may contribute to nerve repair and regeneration.Our previous study observed the dynamic changes of genes in L4–6 dorsal root ganglion after rat sciatic nerve crush using transcriptome sequencing.Our current study focused on upstream growth factors and found that a total of 19 upstream growth factors were dysregulated in dorsal root ganglions at 3,9 hours,1,4,or 7 days after nerve crush,compared with the 0 hour control.Thirty-six rat models of sciatic nerve crush injury were prepared as described previously.Then,they were divided into six groups to measure the expression changes of representative genes at 0,3,9 hours,1,4 or 7 days post crush.Our current study measured the expression levels of representative upstream growth factors,including nerve growth factor,brain-derived neurotrophic factor,fibroblast growth factor 2 and amphiregulin genes,and explored critical signaling pathways and biological process through bioinformatic analysis.Our data revealed that many of these dysregulated upstream growth factors,including nerve growth factor,brain-derived neurotrophic factor,fibroblast growth factor 2 and amphiregulin,participated in tissue remodeling and axon growth-related biological processes Therefore,the experiment described the expression pattern of upstream growth factors in the dorsal root ganglia after peripheral nerve injury.Bioinformatic analysis revealed growth factors that may promote repair and regeneration of damaged peripheral nerves.All animal surgery procedures were performed in accordance with Institutional Animal Care Guidelines of Nantong University and ethically approved by the Administration Committee of Experimental Animals,China(approval No.20170302-017)on March 2,2017.

Sciatic nerve regeneration using a nerve growth factor-containing fibrin glue membrane

Our previous findings confirmed that the nerve growth factor-containing fibrin glue membrane provides a good microenvironment for peripheral nerve regeneration; however, the precise mechanism remains unclear, p75 neurotrophin receptor （p75NTR） plays an important role in the regulation of peripheral nerve regeneration. We hypothesized that a nerve growth factor-containing fibrin glue membrane can promote neural regeneration by up-regulating p75NTR expression. In this study, we used a silicon nerve conduit to bridge a 15 mm-long sciatic nerve defect and injected a mixture of nerve growth factor and fibrin glue at the anastomotic site of the nerve conduit and the sciatic nerve. Through RT-PCR and western blot analysis, nerve growth factor-containing fibrin glue membrane significantly increased p75NTR mRNA and protein expression in the Schwann cells at the anastomotic site, in particular at 8 weeks after injection of the nerve growth factor/fibrin glue mixture. These results indicate that nerve growth factor-containing fibrin glue membrane can promote peripheral nerve regeneration by up-regulating p75NTR expression in Schwann cells.

Sustained delivery of vascular endothelial growth factor mediated by bioactive methacrylic anhydride hydrogel accelerates peripheral nerve regeneration after crush injury

Neurotrophic factors,currently administered orally or by intravenous drip or intramuscular injection,are the main method for the treatment of peripheral nerve crush injury.However,the low effective drug concentration arriving at the injury site results in unsatisfactory outcomes.Therefore,there is an urgent need for a treatment method that can increase the effective drug concentration in the injured area.In this study,we first fabricated a gelatin modified by methacrylic anhydride hydrogel and loaded it with vascular endothelial growth factor that allowed the controlled release of the neurotrophic factor.This modified gelatin exhibited good physical and chemical properties,biocompatibility and supported the adhesion and proliferation of RSC96 cells and human umbilical vein endothelial cells.When injected into the epineurium of crushed nerves,the composite hydrogel in the rat sciatic nerve crush injury model promoted nerve regeneration,functional recovery and vascularization.The results showed that the modified gelatin gave sustained delivery of vascular endothelial growth factors and accelerated the repair of crushed peripheral nerves.

Can amino-functionalized carbon nanotubes carry functional nerve growth factor?

Carbon nanotubes can carry protein into cells to induce biological effects. Amino-functionalized carbon nanotubes are soluble and biocompatible, have high reactivity and low toxicity, and can help promote nerve cell growth. In this study, amino-functionalized ethylenediamine-treated multi-walled carbon nanotubes were used to prepare carbon nanotubes-nerve growth factor complexes by non-covalent grafting. The physicochemical properties, cytotoxicity to PC12 and chick embryo dorsal root ganglion, and biological activity of the carbon nanotubes-nerve growth factor complexes were investigated. The results showed that amino functionalization improved carbon nanotubes-nerve growth factor complex dispersibility, reduced their toxicity to PC12 cells, and promoted PC 12 cell differentiation and chick embryo dorsal root ganglion.

Mechanical tension promotes skin nerve regeneration by upregulating nerve growth factor expression

This study aimed to explore the role of mechanical tension in hypertrophic scars and the change in nerve density using hematoxylin-eosin staining and S100 immunohistochemistry, and to observe the expression of nerve growth factor by western blot analysis. The results demonstrated that mechanical tension contributed to the formation of a hyperplastic scar in the back skin of rats, in conjunction with increases in both nerve density and nerve growth factor expression in the scar tissue. These experimental findings indicate that the cutaneous nervous system plays a role in hypertrophic scar formation caused by mechanical tension.

Preparation and Characterization of Poly Lactic Acid/Graphene Oxide/Nerve Growth Factor Scaffold with Electrical Stimulation for Peripheral Nerve Regeneration in vitro

A novel conductive drug-loading system was prepared by using an improved emulsion electrostatic spinning method which contained polylactic acid (PLA),graphene oxide (GO),and nerve growth factor (NGF) coated with bovine serum albumin (BSA) nanoparticles.Firstly,the structure,mechanical properties,morphology and electrical conductivity of PLA/GO electro spun fiber membranes with different GO ratios were characterized.PLA/GO scaffolds can exhibit superior porosity,hydrophilic and biomechanical properties when the GO incorporation rate is 0.5%.The addition of GO in the PLA/GO electro spun fiber membranes can also create appropriate pH environment for the repair of injured nerve when the GO incorporation rate is above 0.5%.Secondly,PLA/GO/BSA/Genipin/NGF particles (with a ratio of BSA/NGF=3:1) prepared by modified emulsion electro spinning method will release more NGF than PLA/GO/NGF particles.In addition,PLA/0.5%GO/NGF scaffold can maintain its structure stability for at least 8 weeks observed by scanning electron microscope (SEM).Moreover,the degradation of PLA/0.5%GO/NGF scaffold is consistent with its weight loss.Finally,in vitro assay confirmes that PLA/GO composite scaffold exhibits low cytotoxicity to RSC96 cells.Cellular results have demonstrated that PLA/0.5%GO/NGF sustained-release drug sustained-release system with appropriate electrical stimulation (ES) can promote PC12 cell proliferation,and it can maintain its differentiation capability for at least 3 weeks.In conclusion,PLA/0.5%GO/NGF sustained-release drug sustained-release system can maintain its biological activity for at least 3 weeks and promote cell proliferation with appropriate ES.

Modified insulin-like growth factor 1 containing collagen-binding domain for nerve regeneration

Insulin-like growth factor 1 （IGF-I） is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-（4,5-dimethyl-2-thiazolyl）-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.

Effect of salviae miltiorrhizae and ligustrazine hydrochloride injection on axonal regeneration and nerve growth factor expression in a rat model of sciatic nerve injury

BACKGROUND： Studies have shown that both salviae miltiorrhizae and ligustrazine can promote protein expression of nerve growth factor （NGF） and regeneration of peripheral nerve. OBJECTIVE： To verify the effect of salviae miltiorrhizae and ligustrazine hydrochloride injection on axonal regeneration and NGF protein expression in a rat model of sciatic nerve injury. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Traditional Chinese Medicine and the Institute of Bioengineering of Jinan University from July to December 2008. MATERIALS： Salviae miltiorrhizae and ligustrazine hydrochloride injection （containing 20 mg salviae miJtiorrhizae and 100 mg ligustrazine per 100 mL injection） was provided by Guizhou Baite Pharmaceutical, China; salviae miltiorrhizae and ligustrazine decoctions （containing 1 g raw drug per 1 mL decoction） were provided by Guangzhou Baiyunshan Factory for Traditional Chinese Medicine, China; rabbit-anti-rat NGF monoclonal antibody was provided by Beijing Biosynthesis Biotechnology, China. METHODS： A total of 80 healthy, male, Sprague Dawley rats were used to establish a sciatic nerve injury model via neurotomy, and were then randomly assigned to 4 groups： salviae miltiorrhizae and ligustrazine hydrochloride injection group （intraperitoneal injection of 35 mL/kg per day salviae miltiorrhizae and ligustrazine hydrochloride injection）, saIviae miltiorrhizae group （intragastric peffusion of 2 mL salviae miltiorrhizae）, ligustrazine group （intragastric peffusion of 2 mL ligustrazine）, and model group （intraperitoneal injection of 35 mL/kg per day saline）, with 20 rats in each group. Thereafter, rats in each group were then divided into 4 subgroups according to varying time points of 1, 2, 4, and 8 weeks post-surgery, with 5 rats in each subgroup. MAIN OUTCOME MEASURES： Axons were quantified using chromotrope 2R-brilliant green and silver staining combined with image analysis to calculate the axonal regeneration rate; NGF expression was detected using immunohistochemistry and Western blot analysis; toe interspace was measured by behavior at 4 and 8 weeks. RESULTS： With increasing time after sciatic nerve expression, and toe interspace gradually increased njury, the axonal regeneration rate, NGF protein At 4 and 8 weeks post-surgery, axonal regeneration rate and NGF protein expression were significantly increased in the injured tissue of the salviae miltiorrhizae and ligustrazine hydrochloride injection, salviae miltiorrhizae, and ligustrazine groups, compared with the model group （P 〈 0.05 or P 〈 0.01）, and toe interspace was remarkably enlarged （P 〈 0.05 or P 〈 0.01）, especially in the salviae miltiorrhizae and ligustrazine hydrochloride injection group. CONCLUSION： Salviae miltiorrhizae and ligustrazine hydrochloride injection promoted axonal regeneration and NGF protein expression in the injured sciatic nerve, and also enhanced neurofunctional recovery. Its effect was superior to salviae miltiorrhizae or ligustrazine alone.

Intranasal nerve growth factor bypasses the blood-brain barrier and affects spinal cord neurons in spinal cord injury

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an in-creased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deifcits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.

Chitosan conduits combined with nerve growth factor microspheres repair facial nerve defects

Microspheres containing nerve growth factor for sustained release were prepared by a compound method, and implanted into chitosan conduits to repair 10-mm defects on the right buccal branches of the facial nerve in rabbits. In addition, chitosan conduits combined with nerve growth factor or normal saline, as well as autologous nerve, were used as controls. At 90 days post-surgery, the muscular atrophy on the right upper lip was more evident in the nerve growth factor and normal sa- line groups than in the nerve growth factor-microspheres and autologous nerve groups. Electro- physiological analysis revealed that the nerve conduction velocity and amplitude were significantly higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. Moreover, histological observation illustrated that the di- ameter, number, alignment and myelin sheath thickness of myelinated nerves derived from rabbits were higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. These findings indicate that chitosan nerve conduits com- bined with microspheres for sustained release of nerve growth factor can significantly improve facial nerve defect repair in rabbits.

Efficacy and safety of nerve growth factor for the treatment of neurological diseases:a meta-analysis of 64 randomized controlled trials involving 6,297 patients

OBJECTIVE： China is the only country where nerve growth factor is approved for large-scale use as a clinical medicine. More than 10 years ago, in 2003, nerve growth factor injection was listed as a national drug. The goal of this article is to evaluate comprehensively the efficacy and safety of nerve growth factor for the treatment of neurological diseases. DATA RETRIEVAL： A computer-based retrieval was performed from six databases, including the Cochrane Library, PubMed, EMBASE, Sino Med, CNKI, and the VIP database, searching from the clinical establishment of nerve growth factor for treatment until December 31, 2013. The key words for the searches were ＂nerve growth factor, randomized controlled trials＂ in Chinese and in English. DATA SELECTION： Inclusion criteria： any study published in English or Chinese referring to randomized controlled trials of nerve growth factor; patients with neurological diseases such as peripheral nerve injury, central nerve injury, cranial neuropathy, and nervous system infections; patients older than 7 years; similar research methods and outcomes assessing symptoms; and measurement of nerve conduction velocities. The meta-analysis was conducted using Review Manager 5.2.3 software. MAIN OUTCOME MEASURES： The total effective rate, the incidence of adverse effects, and the nerve conduction velocity were recorded for each study. RESULTS： Sixty-four studies involving 6,297 patients with neurological diseases were included. The total effective rate in the group treated with nerve growth factor was significantly higher than that in the control group （P 〈 0.0001, RR： 1.35, 95%CI： 1.30-1.40）. The average nerve conduction velocity in the nerve growth factor group was significantly higher than that in the control group （P 〈 0.00001, MD. 4.59 m/s, 95%CI： 4.12-5.06）. The incidence of pain or sclero- ma at the injection site in the nerve growth factor group was also higher than that in the control group （P 〈 0.00001, RR： 6.30, 95%CI： 3.53-11.27）, but such adverse effects were mild. CONCLUSION： Nerve growth factor can significantly improve nerve function in patients with nervous system disease and is safe and effective.

Combination of olfactory ensheathing cells and human umbilical cord mesenchymal stem cell-derived exosomes promotes sciatic nerve regeneration

Olfactory ensheathing cells(OECs)are promising seed cells for nerve regeneration.However,their application is limited by the hypoxic environment usually present at the site of injury.Exosomes derived from human umbilical cord mesenchymal stem cells have the potential to regulate the pathological processes that occur in response to hypoxia.The ability of OECs to migrate is unknown,especially in hypoxic conditions,and the effect of OECs combined with exosomes on peripheral nerve repair is not clear.Better understanding of these issues will enable the potential of OECs for the treatment of nerve injury to be addressed.In this study,OECs were acquired from the olfactory bulb of Sprague Dawley rats.Human umbilical cord mesenchymal stem cell-derived exosomes(0–400μg/mL)were cultured with OECs for 12–48 hours.After culture with 400μg/mL exosomes for 24 hours,the viability and proliferation of OECs were significantly increased.We observed changes to OECs subjected to hypoxia for 24 hours and treatment with exosomes.Exosomes significantly promoted the survival and migration of OECs in hypoxic conditions,and effectively increased brain-derived neurotrophic factor gene expression,protein levels and secretion.Finally,using a 12 mm left sciatic nerve defect rat model,we confirmed that OECs and exosomes can synergistically promote motor and sensory function of the injured sciatic nerve.These findings show that application of OECs and exosomes can promote nerve regeneration and functional recovery.This study was approved by the Institutional Ethical Committee of the Air Force Medical University,China(approval No.IACUC-20181004)on October 7,2018;and collection and use of human umbilical cord specimens was approved by the Ethics Committee of the Linyi People’s Hospital,China(approval No.30054)on May 20,2019.

Neurotrophic factors and corneal nerve regeneration

The cornea has unique features that make it a useful model for regenerative medicine studies. It is an avascular, transparent, densely innervated tissue and any pathological changes can be easily detected by slit lamp examination. Corneal sensitivity is provided by the ophthalmic branch of the trigeminal nerve that elicits protective reflexes such as blinking and tearing and exerts trophic support by releasing neuromediators and growth factors. Corneal nerves are easily evaluated for both function and morphology using standard instruments such as corneal esthesiometer and in vivo confocal microscope. All local and systemic conditions that are associated with damage of the trigeminal nerve cause the development of neurotrophic keratitis, a rare degenerative disease. Neurotrophic keratitis is characterized by impairment of corneal sensitivity associated with development of persistent epithelial defects that may progress to corneal ulcer, melting and perforation. Current neurotrophic keratitis treatments aim at supporting corneal healing and preventing progression of corneal damage. Novel compounds able to stimulate corneal nerve recovery are in advanced development stage. Among them, nerve growth factor eye drops showed to be safe and effective in stimulating corneal healing and improving corneal sensitivity in patients with neurotrophic keratitis. Neurotrophic keratitis represents an useful model to evaluate in clinical practice novel neuro-regenerative drugs.

Local administration of icariin contributes to peripheral nerve regeneration and functional recovery

Our previous study showed that systemic administration of the traditional Chinese medicine Epimedium extract promotes peripheral nerve regeneration. Here, we sought to explore the ther- apeutic effects of local administration of icariin, a major component of Epimedium extract, on peripheral nerve regeneration. A poly（lactic-co-glycolic acid） biological conduit sleeve was used to bridge a 5 mm right sciatic nerve defect in rats, and physiological saline, nerve growth factor, icariin suspension, or nerve growth factor-releasing microsphere suspension was injected into the defect. Twelve weeks later, sciatic nerve conduction velocity and the number of myelinated fibers were notably greater in the rats treated with icariin suspension or nerve growth factor-releasing microspheres than those that had received nerve growth factor or physiological saline. The effects of icariin suspension were similar to those of nerve growth factor-releasing microspheres. These data suggest that icariin acts as a nerve growth factor-releasing agent, and indicate that local ap- plication of icariin after spinal injury can promote peripheral nerve regeneration.