Function of human insulin-like growth factor-1 ( hIGF-1 ) transgene towards the regeneration of peripheral nerves in vivo

The aim of this study was to investigate the function of human insulin-like growth factor-1 (hIGF-1) transgene in vivo towards the regeneration of peripheral nerves. Thirty (30) male Wistar rats were randomly grouped after the sciatic nerve regeneration chamber model was set up. The hIGF-1 Treatment Group (TG) was injected the mixed solution of pcDNAhIGF-1 and LipfectAmine (hIGF-1 DNA was 4 ug) immediately at the spot of crush injury. The Treatment Model Group (MG) was injected 10 ul of the mixed solution of pcDNA3.1, LipfectAmine and physiological saline, while the control group (CG) was not injected any solution. The functional indexes of sciatic nerve were tested after 8 weeks of the injection. At the same time, electrophysiology of the regenerated nerve fibers, histology, morphology and ultrastructural organization observation were carried out. Compared with MG and CG, TG showed significant improvements in the fields of nerve conductive velocity of the regenerated nerve fibers, the greatest amplitude and electric potential of complex musculation electric potential (P<0.01). The axon diameter, medulla sheath thickness and medullated nerve fibers counts of the regenerated nerve fibers of TG were also higher than those of MG and CG (P<0.01). The degree of maturity of the regenerated nerve fibers of TG was better in the ultrastructural organization observation. Furthermore, other functional indexes were improved. hIGF-1 transgene in vivo could promote the regeneration of the injured peripheral nerves.


INTRODUCTION
The axonotmesis caused by the injury of peripheral nerves (IPN) would subsequently induce distal Fahrenheit Degeneration and proximal Retrogressional Degeneration of axonal injury, which would further lead to the interruption of axoplasmic transportation.If this situation could not be repaired, the atrophy of skeletal muscle, especially the degeneration of motor end-plate and sensory end-organ, and the death of spinal neuron cells would unavoidably happen.While because of the *Corresponding author.anatomical and functional specialties of peripheral nerves, the recovery of the nerve function could not reach an ideal status.So, how to find a new solution for the improvement of the speed and quality of the regeneration of IPN is still a very urgent problem.A large number of studies have reported the effect of neurotrophic factor in this field, whereas the effect of insulin-like growth factor on peripheral nerves needs more attention.Therefore, this study was designed to investigate the regeneration effect of human insulin-like growth factor-1 on peripheral nerves with the method of transgenesis.1.Currently, Tissue Engineering and Gene Engineering techniques, etc, which greatly enriched and developed the research of IPN.Within 30 years, the basic research has made people realize many nutrilites related to the nerve regeneration, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and insulin-like growth factor (IGF), etc, among which IGF was discovered in the latest 10 years.IGF is a kind of multi active bio-polypeptide, showing neurotrophic and neuroregulating activities in the center and periphery (Nguyen et al., 2012;Zheng et al., 2000), while the researches of IGF are mainly in the center, the reports about IGF in periphery are relatively rare.In this study, human insulin-like growth factor-1 was transgenically used to the regeneration chamber mode after injury of peripheral nerves in rats.The effect of human insulin-like growth factor-1 in the promotion of nerve regeneration, protection of spinal motor neurons, inhibition of denervation muscle atrophy were observed through the experimental results.2. According to the theory of Neurotization Interstice, namely a suitable interspace, based on the trophicity and chemotaxis of the regeneration of nerves, could realize more accurate opposing connection (D'Ercole et al., 2002;Kou et al., 2011).In this study, the regeneration chamber mode of sciatic nerve crush injury was firstly set up, then sciatic nerve functional indexes (SFI), electrophysiology, axon diameter of medullated nerve fibers, thickness of myelin and transmission electron microscope observation were carried out for the investigation of the regeneration function and mechanism of hIGF-1 transgene in vivo to peripheral nerves in different spaces and periods.

Animals
Thirty (30) male white Wistar rats were provided by Animal Experiments Center of Jilin University, body weight 200~250g.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Clinic Medical Academy of Yangzhou University.

Animal models and grouping
The rats were randomly divided into 3 groups, namely hIGF-1 Treatment Group (TG, 10), Treatment Model Group (MG, 10) and Control Group (CG, 10).After the anesthesia with intraperitoneal injection of 5% Ketamine Hydrochloride (130 mg/kg), the right sciatic nerve was exposed under sterile situation.Five millimeter (5 mm) of lower margin of piriform muscle was then clipped with special forceps clip 3 times, 10 s each with extruding width at 3 mm, which would appear to be film status under the 6x Operation microscope.TG was then injected the mixed solution of pcDNAhIGF-1 and LipfectAmine 10 μl immediately at epineurium.MG was injected the mixed solution of pcDNA3.1,Lipfect Amine Gu et al. 445 and 10 μl of distilled water, while CG was injected nothing.Each rat was fed in a separate cage after the operation.

Sciatic nerve functional index (SFI)
According to the referenced method (Silva-Couto et al, 2012), SFI was calculated by determining the walk print of the targets in different time spots.

Electrophysiology
Eight weeks after the operation, rats were anesthetized and fixed, then sciatic nerve and triceps surae muscle of the operational side were exposed to the stimulating electrode and recording electrode, respectively to record the conductive velocity of the regeneration nerve and the greatest amplitude of the complex musculation electric potential.

Histology and photo analyses
After the check of nerve electrophysiologic study, 5 injured nervous tissues of each group, 10 mm each, within the injured spots were made to paraffin section of cross section, with thickness of 4-6 μm, Marsland and LFB double staining was used after the tissues were fixed with toasting method.HPIAS-1000 High Resolution Color Pathological Photo Analysis System was then used to determine the axon diameter of medullated nerve fibers and thickness of myelin in 5 random fields of vision.At the same time, nerve fibers counts should also be recorded in each vision field.

Transmission electron microscope observation
After the check of nerve electrophysiologic study, 5 injured nervous tissues of each group, 10 mm each, within the injured spots were selected, then solidified with 2.5% glutaral for the former end and 1% osmic acid for the latter end, respectively; then dehydrated with ethanol and entrapped with Epon812 epoxide resin.LKB-III ultramicrocuts were then performed and double stained with uranyl acetic acid and lead citrate.The counts of the regeneration nerve, degree of maturity of myelin, integrality of perimysium and hyperplastic Schwann Cell were observed and photoed with JEM-1200EX Transmission Electron Microscope.

Statistic analysis
SPSS software (version 11.5) was used for the statistic analysis, the creditable range of multi group mean analysis of variance was set at 95%, and the data were shown in the form of S.

SFI
SFI values got better as time passed by, which were much more obvious after 24 days, and in every time spot, the SFI values of TG were better than the other two groups (Figure 1).

Electrophysiology
Fifty-six days after operation, the regenerating motor nerve conduction velocity, the maximum amplitude and latency of compound muscle action potential of the right sciatic nerve in hIGF-1 TG were significantly improved than those of MG and CG (P<0.01).There was no significant difference between MG and CG (P>0.05)(Table 1).
Compared with CG, electrophysiology results of MG showed no significant difference (P>0.05), while TG showed significant difference (P<0.01).Related data are shown in Table 1.

Histology and photo analyses
Regenerating sciatic nerve fibers was stained with Marslan and LFB.Axon was black and myelin was blue.The number of myelinated nerve fibers, axon diameter and the thickness of myelin regeneration were measured in each group with sciatic nerve injury regeneration by the Pathological Photo automated analysis system microscope (×600) (Table 2, Figure 2).Compared with MG and CG, IGF-1 TG showed a significant difference (P<0.01).There was no significant difference between MG and CG (P>0.05).Compared with CG, there were no significant difference in diameter of regenerating neurite, myelin thickness and nerve fibers counts in MG (P>0.05), while while TG showed significant difference in the above mentioned items (P<0.01).The photo analysis results are shown in Table 2 and Figure 2.

Transmission electron microscope observation
Intramedullary spinal cord neuropil and regenerating sciatic nerve fibers were observed with transmission electron microscope 56 days after operation.The structure of intramedullary spinal cord neuropil in IGF-1 TG was normal, regenerating sciatic nerve fibers were abundant and matured.Nerve fibers were thick and myelin were thick and evenly.In MG, the gap of neuropil protruding was large, mitochondria had a dense formation in axons, and myelin of myelinated nerve was loose.Regenerating sciatic nerve fibers were relatively thinner, less matured than those in TG, in addition that myelin thickness was less and more different than those in TG.In CG, there were vacuoles in the neuropil.Regenerating sciatic nerve fibers were small and immatured.The myelin thickness was thin and irregular.
There were more and more mature regenerating sciatic nerve fibers of TG: thicker nerve fibers, thicker myelin sheath.MG showed slimmer and less matured regenerating sciatic nerve fibers with less thick myelin sheath and bigger difference.CG showed small regenerating sciatic nerve fibers, immature, and the thickness of myelin sheath was slim and irregular (Figure 3).

DISCUSSION
Whether the peripheral nerve could be regenerated successfully after injuries mainly depends on the activities of central neuron and suitable regenerating microenvironment.Nerve Growth Factor (NGF) is one of the most important factors in regenerating microenvironment.Many researchers applied exogenous stimulating NGFs for the regeneration of IPN, while the half lives of these factors are short, which need repeated injection of these giant molecule substance if a traditional injection method is used, and still hard to reach the specified place and maintain effective concentration.With the development of Molecular biology, applying genetic engineering could solve the problem.It theoretically needs occassional, even once, application to reach the therapeutic purpose if a certain protein gene with therapeutic activity is transferred into specified tissue.Furthermore, gene therapy could limit the adverse effects, and show unexampled advantage.
IGF-1, a single-stranded polypeptide with 70 amino acids composition, belongs to the family of Insulin-Like Growth Factor and shows endocrine, autocrine and paracrine activities.Its molecular weight is about 7649 Kb.Since the first report from Salmon and Daughaday,    this factor, related closely to growth hormone (GF), has attracted researches in many fields, and the appearance of bio products of recombination of IGF-1 further has opened the research visions in the fields of disease diagnosis and therapy (Gehrig et al., 2012;Mu et al., 2012;Yashida et al., 2011).During this experiment, a 3 mm neurotization chamber was successfully set up with sciatic nerve clip.This suitable interstice could solve the problem of nerve dislocation growth through the neurotrophy and chemotaxis and realize much more accurate opposing connection.Many research have reported that 2~3 mm is best for regeneration (Buti et al., 1996;Zhang et al., 2009), and some researchers also reported that the crevice should not be over 10 mm.
Compared with viral vectors, plasmid vectors could hold Klenow fragment DNA.Furthermore, when the plasmids are transferred into the target cell, they would appear to be circular DNA, which could not be integrated and copied, so that they could not generate replicationcompetent viruses, and show no potential possibilities of viral infection and cancerigenic ability to the host.At the same time, plasmid vectors with suitable purities could avoid the target cell death caused by the immunoreactions to exogenous genes.In this study, the axon diameter of the regenerating neurite, diameter of myein, nerve fibers counts and degree of maturity of the regenerating nerve under the degree of maturity of TG were all significantly better than those of CG, and the conduction velocity of regenerating nerve and amplitude of target compound muscle action potential were also obviously higher than CG, which revealed that IGF-1 could promote the regeneration of peripheral nerves.
Previous researches working on IGF-1 concerned more about diabetic perineuropathy (Godinez-Gomez et al., 2006;Zeng et al., 2005), while few about the treatment of IPN.In recent years, some researchers reported the IGF-1 experiments of IPN in animals (Emel et al., 2011;Hu et al., 2004;Kiryakova et al., 2010), such as facial nerve and sciatic nerve.Some scientists also applied IGF-1 in the clinical treatment of Human Hypogonadism (Hayes et al., 2001).To solve the problem of short half life of IGF-1, the experiment applied the method of gene engineering, which came up with significant results.The data of the present study showed that the histology and ultrastructural organization of the regeneration nerve treated with hIGF-1 were better than the other groups after 8 weeks of operation, and the expression of hIGF-1 in dynamoneure cell in TG was obviously higher than those in MG and CG after 4 weeks of operation.
As for the transferring mechanism, protection and trophism activities of hIGF-1 towards the nerve tissue, it was still unclear.In central nervous system, ectogenic IGF-1 mainly acts through the action on IGFBP-2 target neuron (Govoni et al., 2011;Sizonenko et al., 2001), andNagano et al. (2003) considered that the phosphoinositide-3 kinase pathway might be the explanation, while Zhang et al. (2004) thought exogenous plasmid DNA was intaken for some external stimulation, such as wound, which would further improve the cell membrane permeability.The above theories still need further investigation.
The experiment proved that hIGF-1 did have some effects on the regeneration of nerve fibers after IPN, and just as the regeneration of nerve after IPN was connected with age, the results might be related with the following factors: 1. Rats at different ages might affect the results (Apel et al., 2010).2. The experimental results might change owing to the different counts and effects of hIGF applied in the experiment.3. Different administration methods: the half life of plasmid is short and would have been degraded soon in vivo, which therefore could not maintain the effective concentration.So, the effects of multi administration of hIGF-1 plasmid to a specific local with a certain time interval would be better than one administration.Moreover, the effects might be different if local administration methods were different, such as nerve pars affecta administration, target muscle injection, pars affecta pump administration, or the combination of two methods.
Transgene in vivo with human Insulin-like growth factor-1 can promote nerve regeneration after peripheral nerve injury.

Table 1 .
Electrophysiology results of right sciatic nerve of three groups rats after 56 days ( x  S).

Table 2 .
Pathological Photo Analysis of regenerating nerve fibers ( x  S).