Self Assembling Peptide Nanofiber Scaffold Enhanced With RhoA Inhibitor CT04Improves Axonal Regeneration In The Transected Spinal Cord

This study was designed to explore a new therapeutic strategy--Self-assembling peptide nanofiber scaffold combined RhoA inhibitor to promote spinal cord regeneration.In present, there are many potential strategies to treat spinal cord injury in the animal models, such as Operative treatment, physicotherapeutics, adjuvant traditional Chinese medicine transportation treatment, to fill injury cavity with biomaterial and the elimination of inhibition factors. To some extent, these studies have contributed to the repair of spinal cord injury. However, transplantation has its source and ethical limitations, inhibitory factor is difficult to be eliminated completely, and single treatment strategy could not to response all the problems of spinal cord injury.Therefore, to seek new biomaterials and to explore new therapeutic strategies are the focus of the spinal cord injury study. In recent years, various new therapeutic strategies and new biomaterials continue to emerge.The recent studies show that RhoA in spinal cord injury plays an important role. RhoA belongs to a small molecule Rho family GTPase. It’s all eukaryotic cells actin cyto skeleton regulator. On the developmental stage of the nervous system, RhoA plays an important role on the neuronal migration and neurite growth; but the adult neurons do not express RhoA. When the neurons damaged, RhoA expression began to increase within1day and peak around1week later.3weeks after injury, the RhoA expression still can be detected and activated in the spinal cord. Re-expression and activation of RhoA in neuronal injury plays an important role to hinder the central nervous system regeneration.Most of the relevant factors that inhibit nerve regeneration are through the activation of its downstream RhoA signaling pathway to play a role. Increased RhoA expression and activation after spinal cord injury can result in axonal growth cone collapse and neurons damage, which are main reasons cause regeneration difficulty after spinal cord injury. Therefore, trying to reduce the expression of RhoA or inhibit signaling pathway of RhoA may be led a new treatment strategy to promote restoration of spinal cord injury.RhoA inhibitor can change the active GTP form by up-regulation of RhoA protein expression to inactive GDP form,then the up-regulated RhoA cannot educe the effection of the failure of axonal regeneration after SCI. The methods of RhoA inhibitor used in the central nervous system tissue are mainly two ways:the intravenous injection and local microinjection. However, intravenous injection needs large amount intravenous drug as is difficult to avoid systemic side effects, local microinjection can cause unnecessary secondary damage. It is necessary to explore a more practical therapeutic strategy.Recent years, Zhang S Research Group in the Massachusetts Institute of Technology, American, invented a new type of nano-materials, SAPNS. It has proven that it can play a good results in a variety of tissue repair. In tissue engineer field, it is considered the most promising tissue repair material. Compared to other biomaterials, SAPNS has the following advantages:no significant immune rejection and inflammatory responses, the nano-fiber diameter is very close to the extracellular matrix, can provide a three-dimensional environment for cell survival and migration, can minimize the risk of biological pathogens and contamination, with excellent biocompatibility, no cytotoxicity, can be utilized by the host.In precent study, based on spinal cord injury model, composite materials include RhoA-inhibitor (CT04) and SAPNS, was filled into the injury cavity,to meet with the complex factors in spinal cord injury. In order to repair the injury cavity and inhibit the activation of RhoA at the same time, explore a new method for structure and function recovery in spinal cord injury.This study consists of two parts. Part I:Construction of graft material and the model of spinal cord injury and transplantationUnder sterile condition, take a tube of RhoA inhibitors (CT04), centrifuge in the10000r/5mins speed,then add20μ150%glycerin, turn into the concentration of1μg/μl, using the method of percussion and shock dissolves, centrifuge with8000r/3mins speed, packing by2μl/pipe under sterile environment, stored in-20degrees. Every time take2μ1CT04and13μ1SAPNS, mixing quickly in clean petri dishes. Then hatch10mins in0.1M PBS solution at room temperature to make SAPNS fully solidification, the material of SAPNS group replaced by2μ1of50%glycerin, transplantation material were15μ1.As a kunming mice, anesthesia with1%sodium pentobarbital by intraperitoneal injection, dose for30mg/kg. After anesthesia, sham group open cone board in the T8-10position, only exposed spinal cord. The other in the same position need exposure spinal cord with microsurgery cutting further open endorhachis, and then cut off1mm length of a spinal cord in the vertical position T9, fish out the damage cavity completely. SAPNS+CT04group, SAPNS group and saline groups fill the the transplantation of materials above or amount of15μ1saline solution inside the cavity respectively, and then suture step by step.Two mice which transplant into the SAPNS+Dextran were sacrified at1w after surgery. The T6-11segments of spinal cord and cerebral cortical motor areas were dissected and cryosected. By fluorescent microscope, record the status of Dextran into the spinal cord neurons. The results showed that some of the fluorescrnce was observed to diffuse into the surrounding host cord from the SAPNS,while the implant still retained high fluorescent signal. The fluorescent dye used here confirmed that the chemicals released were from the SAPNS implants, which indicated that SAPNS can effectively serve as a platform for further controlled release of exogenous therapeutic moleculars of interest that have been introduced into the injured spinal cord, like its application is reported in the other tissues.Each of groups were sacrified at12w after surgery. A significant gap was developed in the lesion site in the saline group. In contrast, the initial lesion gap was bridged well by the grafted SAPNS implants with or without CT04incorporation. Of note the implants integrated smoothly with the surrounding host tissue without obvious cavities or gaps occurring between the implants and host. Part II:Evaluation of the injured spinal cord repair effect.The locomotoral functions of all the remaining mice were detected by climbing test and BMS score at lw,6w,12w after surgery.Fluorogold(FG) was injected into the caudal spinal cord of the transected site.7d after FG injected,the cortical motor evoked potential(CMEP) of the mice were detected,then sacrifice,cryosection and morphology serious studies were performed.The results showed that.1. The recovery effects to the SCI mice with grafts were enhanced.2. Compared to the SCI control group, there were some FG-labeled cells in the rostral spinal cord of transected site,the red neuclei,and the pyramidal layer of senserimotor cortex in the experimental group3. Compared to the SCI control group, the latency of CMEP decreased, and the amplitude increased in the grafted groups.4. Compared to the SCI control group, the glial scar cells, inflammatory cells, apoptotic cells obviously reduced near the damaged area;5. CT04and SAPNS can boycott the activity of RhoA, compared with pure SAPNS, the axon has high growth rate in the damage area, so CT04+SAPNS group has well function recovered from the rest group, the difference between groups with a statistical significance.