The Function Of NRG-1 In The Process Of Regeneration After Auto-nerve Grafting

Microsurgical techniques have made a significant progress and development during these years, but the pathophysiological mechanisms of peripheral nerve injury or regeneration have not been clarified. Peripheral nerve injury or regeneration is still a major problem to surgeon. Accurately masterring the anatomy, pathophysiology of nerve repair and surgical reconstruction technique is the prerequisite for nerve repair treatment. For the neurologic deficit larger than 5cm, because of excessive tension, direct neural anastomosis would lead to treatment failure. Auto-nerve grafting or casing technology can be applied to solve this problem. Surprising innovation and breakthrough has been achieved in biomedical engineering, however, as a result of immune tolerance; they can not be effectively used in clinical treatment. Auto-nerve grafting remains the gold standard of therapy for nerve damage repair. The major problem of auto-nerve grafting is the slow repair process and the high necrosis rate following injury. In recent years, many researchers found NRG-1 participed and played an important regulation role in the reparation of neural injury. Firstly, we established SD rat auto-nerve grafting model using microsurgical technique, then observed the change of NRG-1 in the model. Besides, ASON was applied to inhibit the expression of NRG-1 m RNA. Furthermore, SB203580 was used to inhibit the activation of P38α MAPK in the model and analysed the function of NRG-1/Erb B/P38α MAPK path in the process of regeneration after the auto-nerve grafting. Our experiment is divided into three parts.Experiment One: The change of NRG-1 expression in the process of regeneration after the auto-nerve graftingObjective The purpose of this study was to investigate the change of NRG-1 expression in the process of regeneration after the auto-nerve grafting.Methods Clean healthy male SD(Sprague-Dawley) rats, 40, 250-300 g were randomly divided into experimental group and control group, each group of 20. Observations were carried out at five time points: the 3rd, 7th, 14 th, 21 st, 28 th day after operation. In the experimental group, reversed sciatic nerve autograft were carried out while in the control group, the sciatic nerve was just exposed. The footprint were recorded and analyzed. The MNCV(motor nerve conduction velocity) was obtained through electrophysiological examination. The nerve fiber regeneration of myelin sheath were observed and analyzed by transmission electron microscope. The expressions of type II or type III NRG-1 were evaluated using Western blot or RT-PCR technic after nerve transplantation at different time points.Results We found that at each time point, the SFI(sciatic functional index) and the MNCV in the experimental group were lower than that in the control group, and the SFI of experimental group gradually increased and the difference was statistically significant(P<0.01); Compared with the control group, the medullated fiber area of the experimental group is statistically different at 7th, 14 th, 21 st, 28 th day(P<0.05). The number is changed significantly at the 3rd, 14 th, 21 st, 28 th day(P<0.05), and the diameter of axon at the 7th, 14 th, 21 st day significantly statistical difference(P<0.05). Expression of NRG-1 type II at the 3rd day after operation was significantly increased till the 28 th day, compared with the control group, the difference is significant(P<0.01); Both the m RNA and the protein expression of NRG-1 type II or type III are higher than normal group after nerve transplantation with significant statistical difference(P<0.01).Conclusion The NRG-1 participate the regulation of the myelin regeneration after auto-nervegrafting, which may have different function.Experiment Two: The function of NRG-1 type II in the process of regeneration after the auto-nerve graftingObjective The purpose of this study was to investigate the function of NRG-1 type II in the process of regeneration after the auto-nerve grafting.Methods Clean healthy male SD rats, 54, 250-300 g were randomly divided into Blank group, Model group and ASON group, each group 18, is sacrificed at the 3rd, 7th, 14 th, 21 st, 28 th and the 35 th day after the operation. Reversed sciatic nerve autograft was performed in the Model group and ASON group. Besides, PBS buffer or NRG-1 type II antisense oligonucleotide was locally injected in the Model group and ASON Group respectively instantly after operation and the third day after surgery, while in the blank group the sciatic nerve was just exposed. The changes of the footprint were analyzed. Nerve fiber regeneration of myelin sheath was observed by transmission electron microscope. The expression of NRG-1 type II as well as its m RNA was analyzed by Western blot technic or RT-PCR technology. And the MNCV was obtained by the electrophysiological examination after transplantation at different time points.Results We found the SFI of each time point of the ASON group is lower than the Model group except the 3rd day, the difference was statistically significant(P<0.01). After 14 to 35 days of the transplantation, inhibiting the expression of NRG-1 type II may slow conduction velocity of sciatic nerve, which indicated the absence of NRG-1 type II may have the negative effect on the nerve regeneration. The morphological analysis of myelin demonstrate the NRG-1 type II play a modulatory role to the disintegration of myelin and myelin sheath of Schwann cells in the process from the 3rd to 28 th day of regeneration after the auto-nerve grafting.The protein and m RNA expression of NRG-1 type II of the ASON group was lower comparing with the Model group, the difference is significant(P<0.01).ConclusionThe NRG-1 type II play a modulatory role to the disintegration of myelin and myelin sheath of Schwann cells in the process from the 3rd to the 28 th day of regeneration after the auto-nerve grafting. Lack of NRG-1 type II can slow down the auto-nerve function recovery.Experiment Three: Transduction of NRG-1/Erb B signal mediated by P38 MAPK in the process of regeneration after the auto-nerve graftingObjective The purpose of this study was to investigate transduction of NRG-1/Erb B signal mediated by P38 MAPK in the process of regeneration after the auto-nerve grafting.Methods Clean healthy male SD rats, 54, 250-300 g were randomly divided into Blank group, Model group and P38α MAPK inhibitor group, each group of 18, is divided into six points at the 3rd, 7th, 14 th, 21 st, 28 th, 35 th day after the operation. In the Model group and P38α MAPK inhibitor group reversed sciatic nerve autograft were performed. Model Group was locally injected with PBS buffer, P38α MAPK inhibitor group was injected with SB2035802 instantly after operation and the third day after the surgery, and in the Blank group the sciatic nerve was just exposed. The changes of the footprint, nerve fiber regeneration of myelin sheath were analyzed. And the expression of P-P38α MAPK was investigated by Western blot. The expression of MBP m RNA was evaluated by RT-PCR technology and the MNCV was obtained by the electrophysiological examination after transplantation at different time points.Results We found the SFI of the P38αMAPK inhibitor group is lower than the Model group from the 7th to the 35 th day and the difference was statistically significant(P<0.01). MNCV value indicated that inhibiting the activation of P38α MAPK can slow down the MNCV of the rat sciatic in the 14-35 th day of transplantation. Analysis of the area of myelinated fiber indicated that there was statistically significant difference between the P38α MAPK inhibitor group and the Model on the 28 th, 35 th day(P<0.01). The number of myelinated fiber in the unit area of the P38α MAPK inhibitor group was not significantly different with that in the Model group. Evaluation of the diameter of the axonindicated that there were statistical difference between the P38α MAPK inhibitor Group and the Model Group at the 28 th and 35 th day(P<0.01). As for G-ratio, The P38α MAPK inhibitor group was higher compared with the Model Group at the last two time points, and there is significant statistical difference(P<0.01). The protein of P-P38α MAPK and the m RNA expression of MBP of the P38α MAPK inhibitor group was lower compared with the Model group and the difference is significant at each time point(P<0.01).Conclusion Inhibitting P38α MAPK activation was an obstacle to the recovery of neural function, the area myelinated of nerve fibers, the diameter of the axon and the G-ratio, which demonstrate P38α MAPK may be a downstream signal to the NRG-1/Erb B transduction pathways and play a facilitating role to the recovery of neurological function and myelin regeneration after the auto-nerve grafting.