The Promotion Of Peripheral Nerve Regeneration By Chitooligosaccharides And The Study Of Relative Mechanisms

Objective: The purposes of this study were to examine the possible benefits oftreatment with COSs on regeneration of rat crushed sciatic nerves, and to investigate therelative mechanisms in vitro.PartⅠ The promotion of peripheral nerve regeneration by chitooligosaccharidesin the rat nerve crush injury modelMethods:1. The animal models of rat crushed sciatic nerves were established: Forty adultSprague-Dawley (SD) rats of either gender, and were performed left sciatic nerve crushoperation. Following surgery, all rats were randomized into four groups of10rats each,and administered intraperitoneally daily with3or6mg/kg body weight of COSs (low orhigh dose groups),130μg/kg body weight of mecobalamin (positive control group), andnormal saline (negative control group), respectively. These agent treatments lasted for3weeks. The contralateral, nonoperated side of animals served as normal control.2. The withdrawal reflex latency (WRL) was measured at4,8,12,16dayspost-surgery.3. Walking track analysis was carried out at8,12,16,20days post-surgery. Thesciatic function index (SFI) was calculated for the assessment of motor nerve recovery.4. Electrophysiological tests were performed and compound muscle action potentials(CMAPs) were recorded at21days post-surgery.5. Electron microscopy was performed for regenerated sciatic nerves, the samples ofwhich were taken from three randomly selected rats for each group. The samples werefixed and embedded. The average thickness of regeneration myelin sheath was measured.6. The gastrocnemius muscles from both the operated and contralateral, nonoperatedlimbs of rats were removed, and weighed for determining the muscle wet weight. The fixedmuscle specimens were embedded and cut into5μm thick sections, to which Haematoxylin and Eosin (HE) staining was applied. The cross-sectional area (CSA) of the muscle fiberswas measured.7. Immunohistochemistry of neurofilament (NF)-200was applied to the regeneratedsciatic nerves for all groups. The average numbers of regenerated nerve fibers weremeasured.Results:1. WRL values were used to evaluate motor performance and nociceptive function ofperipheral nerves. The results showed that the WRL values for high dose COSs group weresignificantly lower than those for control group at days8,12post-surgery, respectively(P<0.05).2. The results of SFI scores indicated that at days16,20post-surgery, the SFI scoresfor low or high dose COSs groups were significantly increased as compared to those forcontrol group (P<0.05).3. CMAPs examinations offer an important index for the conduction function ofperipheral nerves. The results of electrophysiological tests showed that the recovery indexof CMAPs of high dose COSs was significantly greater than that of control group either indistal or proximal stimulating sites (P<0.05).4. The muscle wet weight ratio, defined as the ratio of muscle wet weight of theoperated side to the contralateral nonoperated side, is a more reliable measure than themuscle wet weight itself. The results of muscle wet weight ratio showed that the wetweight ratio of high dose COSs groups was significantly higher than that of control group(P<0.01).5. The results of CSA indicated that high COSs induced a significant increase in CSAof gastrocnemius muscle fibers at the injured side versus control group (P<0.05).6. The results of anti-NF immunohistochemistry showed that the average numbers ofregenerated nerve fibers in high dose COSs groups were significantly higher than that ofcontrol group (P<0.01).7. The results of electron microscopy showed that the average thickness ofregenerated myelin sheath in high dose COSs groups was significantly higher than that ofcontrol group (P<0.01).PartⅡ The study of relative mechanisms about promotion of peripheral nerveregeneration by chitooligosaccharides (1) Effect of chitooligosaccharides on the neurite outgrowth of rat dorsal rootganglionsMethods:1. DRG explant and dissociated DRG neuron were cultured, and NF-H fluorescentimmunocytochemistry was performed to detect the neurite growth of cultured DRG andDRG neuron treated with COSs (0.05,0.1and0.2mg/ml).2. Western blot was used to analyze the expression changes of NF-H and GAP43aftertreating with COSs in the dissociated DRG neuron.Results:1. The result of NF-H fluorescent immunocytochemistry showed that COSs (0.1mg/ml and0.2mg/ml) promoted the neurite outgrowth of cultured DRG (P<0.01), versuscontrol group.2. The effect of different concentrations of COSs on neurite outgrowth of DRGneuron was similar to the effect on DRG. Versus control group,0.2mg/ml COSs promotedthe neurite outgrowth of cultured DRG neuron (P<0.05).3. The results of Western blot showed that versus control group, the protein levels ofNF-H and GAP43were up-regulated in disscciated DRG neurons after treated with0.2mg/ml COSs for12h (P<0.05and P<0.01).(2) Effect of chitooligosaccharides on the viability and proliferation of rat SchwanncellsMethods:1. The primary rat Schwann cells were isolated and purified in vitro. The cell puritywas assessed by anti-S100immunohistochemistry.2. Methyl thiazolyl tetrazolium (MTT) assay was used to observe the effect of COSs(0.25,0.5and1.0mg/ml) on viability of rat primary Schwann cells.3. The Schwann cells were stained by Hoechst33342, and cell numbers werecalculated after treatment with COSs (0.25,0.5and1.0mg/ml) for24h and48h, and thencell growth curve was drawn.4. EdU(5-ethynyl-2'-deoxyuridine)staining and flow cytometry (FCM) analysistechnology were also used to detect the effect of COSs on proliferation of Schwann cells.5. Western blot was used to analyze the expression changes of Cyclin-D1, N-Cadherinand β-catenin protein after treating with COSs in the Schwann cells. 6. The RNA interference (RNAi) was used to further examine the relativemechanisms of COSs on the proliferation of Schwann cells.Results:1. The results of anti-S100immunohistochemistry indicated that the purity of culturedprimary Schwann cells was more than90%after purification.2. The results of MTT assay showed that versus control group, the viability ofSchwann cells treated with1.0mg/ml COSs for48h was significantly increased (P<0.05).3. The results of cell growth curve indicated that versus control group, the numbers ofSchwann cells treated with0.5mg/ml COSs for48h were significantly increased (P<0.05).However, the numbers of Schwann cells treated with1.0mg/ml COSs for24h and48hwere significantly higher than that of control group (p<0.05and P<0.01).4. The results of FCM analysis showed that versus control group, the proliferationindex of Schwann cells treated with1.0mg/ml COSs was significantly higher than that ofcontrol group (P<0.01).5. The results of EdU staining indicated that the EdU-positive cell numbers of1.0mg/ml COSs treatment group were significantly than that of control group (P<0.05).6. The results of Western Blot indicated that the expressions of Cyclin D1andβ-catenin in Schwann cells of the groups of0.5mg/ml and1.0mg/ml COSs were higherthan that of control group (P<0.05and P<0.01). The expression of N-Cadherin in Schwanncells treated with1.0mg/ml COSs was significantly higher than that of control group(P<0.05).7. The expression of N-Cadherin in Schwann cells transfected with N-CadherinsiRNA was significantly lower than that of control (P<0.05). EdU-positive cell numbers inN-Cadherin siRNA transfected group were lower than that in N-Cadherin siRNA negativetransfected group (P<0.05).(3) Effect of chitooligosaccharides on myelination of rat Schwann cellsMethods:1. MBP and MAG fluorescent immunocytochemistry was performed to detectmyelination of coculture of DRG and Schwann cells by COSs.2. Western blot was used to analyze the expression changes of MBP and MAG proteinafter treating with COSs in myelination of coculture of DRG and Schwann cells. Results:1. The result of MBP and MAG fluorescent immunocytochemistry showed that COSscould promote myelination of DRG. Versus control group, the MBP or MAG positivemyelinated segments in unit area were higher than that in control group (P<0.05andP<0.01).2. The results of Western blot indicated that versus control group, COSs promoted theexpression of MBP and MAG in cocultured of DRG and Schwann cells (P<0.05andP<0.01).Conclusion:1. COSs could promote peripheral nerve regeneration with the desired functionalrecovery in the sciatic crush injury model.2. COSs could promote the neurite outgrowth of cultured DRG and DRG neuron, andincrease the expression of NF-H and GAP43.3. COSs could promote the viability and the proliferation of primary rat Schwanncells, and increase the expression of Cyclin D1, N-Cadherin and β-catenin in Schwanncells. The promotion of the proliferation of Schwann cells may be mediated throughN-Cadherin.4. COSs could promote myelination of Schwann cells.