The Combination Of NDGA,Acellular Muscle Scaffold And AECs For The Treatment Of Rat Spinal Cord Injury

Spinal cord injury (SCI) is a serious debilitating illness resulting from traffic, sportsaccidents, and natural disasters. The pathological mechanisms in the SCI include theprimary injury and secondary injury. The primary injury causes tissue destruction,whereas secondary injury does damage to the rest neurons leading to continuedneuron loss, and inhibit the growth of nerve fibers. The secondary injury is the mainreason for the sensory-motor dysfunction after spinal cord injury. After SCI thereasons for the failure of axonal regeneration are mainly due to: (i) Spinal cordprimary damage and secondary inflammation lead to necrosis or apoptosis of thespinal cord neurons, resulting in persistent injury to the neurons; (ii) After spinal cordinjury, a large number of myelin-related inhibitory molecules for axon growth arereleased. The formation of glial scar also impedes axonal growth; (iii) Damage lead tothe local apoptosis of neurons, and reduce the secretion of the neurotrophic factor,undermining the microenvironment of neurons in the repair and axonal regeneration.Therefore, the inhibition of inflammation and change of spinal cord injurymicro-environment are conducive to the reconstruction of the nerve function.Currently, the treatment of SCI includes the following aspects: (i) Medication: thetreatment of a large doses of corticosteroids (methylprednisolone) effectively reducethe secondary inflammation of the spinal cord injury, and prevent further apoptosis ofthe remnants of damaged neurons. (ii) Cell transplantation: transplanted cells cansecrete various neurotrophins and chemokines, and can provide nutrition andprotection to the neurons. They can also induce the regeneration and remyelination ofinjured axons to restore spinal cord function. Transplanted cells in previous studies forSCI repair included embryonic stem cells (ESCs), Schwann cells (SCs), olfactory ensheathing cells. They all promoted functional recovery of spinal cord to a certainextent. (iii) Tissue engineering: the main purpose of tissue engineering is toreconstruct the anatomical structures of the spinal cord, to provide guidance in axonalregeneration, and to reduce the glial scar formation in the damage region. The basicpattern of tissue engineering in the treatment of SCI is "seeded cells + biomolecules +scaffolds". There are two kinds of scaffolds in tissue engineering for the spinal cordrepair: the synthetic scaffold, such as polylactic acid, polyglycolic acid and itscopolymers, etc.; autologous or allogeneic tissue transplantation, such as allogeneicembryonic spinal cord tissue,self-body muscle fiber, peripheral nerve. All haveachieved a certain therapeutic effects. (iv) Improvement of the spinal cord injurymicroenvironment: researchers remove the inhibitory molecules(myelin associatedglycoprotein, Nogo-A, oligodendrocyte myelin glycoprotein , etc.) throughtransfection of genes, RNAi technology to provide an appropriate environment foraxonal regeneration. (v)Combination therapy: due to the complicated mechanisms ofspinal cord injury, researchers use the combination of different approaches to promotenerve repair after SCI. The studies showed that the combination strategies for thetreatment of spinal cord injury were often more effective than single strategies.Previous studies have shown that NDGA is an extract from the tar bush, and haveanti-inflammatory activity. Amniotic epithelial cells have variety of advantages andmay be a good kind of seed cell for the treatment of spinal cord injury. Our previousstudies have shown that chemical extracted acellular muscle is a good tissueengineering scaffold for SCI.Therefore, this study tried to explore if the NDGA hasanti-inflammatory effects in spinal cord injury, and use the anti-inflammatorycharacteristics of NDGA to promote the survival of amniotic epithelial cellstransplanted into the injured spinal cord, and co-transplanted with chemical extractedacellular muscle to treat spinal cord injury. Details are as follows:I. Observe the effects of NDGA on SCIThe inflammatory response plays an important role in the secondary pathologicalresponse after SCI. We have tried to observe if the NDGA can inhibit the inflammatory response after spinal cord injury, reducing the secondary damage ofspinal cord. After the rat spinal cord hemisection injury models were made, 30mg/kgNDGA were given in the first 3 days after SCI. MPO chemical test kits were used todetect neutrophil infiltration in the damage area after spinal cord injury. In the otherexperiments, the rat spinal cord hemisected injury models were given 30mg/kg ofNDGA for 7 days. After that, the HE and cresyl violet staining were used to observethe structure and neuronal survival of injured spinal cord. The immunohistochemicalstaining was applied to observe ED1 positive macrophages / microglia infiltration andGFAP positive astrocytes in the injured spinal cord. HE staining and cresyl violetstaining results showed that the survival of neurons in NDGA group was significantlymore than in the control group. The results showed that the difference is statisticallysignificant, suggesting that NDGA promoted the survival of neurons after spinal cordinjury. MPO assay values in each group showed that compared with the MPO value ofinjury group, the MPO value of NDGA group is low, and the difference is statisticallysignificant. This indicated that the application of NDGA reduces the infiltration ofneutrophils in spinal cord injury. One week after operation, the ED1immunofluorescent staining results demonstrated that compared with the injury group,ED1 positive cells in the NDGA group was significantly reduced, suggesting that theapplication of NDGA reduces the number of macrophages /microglial cells in spinalcord injury. The GFAP immunofluorescence staining results proved that comparedwith the injury group, the GFAP-positive cells in NDGA group was significantlyreduced. These results suggest that NDGA reduces the secondary pathologicalchanges of spinal cord injury through the inhibition of inflammatory cells.II. Determine the therapeutic dose of NDGACell transplantation is an important means for the treatment of SCI, but oneproblem for this method is that the survival of transplanted cells decreaseddramatically due to the attack of the inflammatory response. We used different dosesof NDGA to promote the survival of transplanted amniotic epithelial cells after SCI.The chemically extracted acellular muscle tissue engineering scaffold was prepared with SDS and Triton X-100. The amniotic epithelial cells were cultured in vitro andseeded into the acellular muscle scaffold. After that, the amniotic epithelial cells andacellular muscle scaffolds were co-transplanted into rat spinal cord hemisectioninjury models. The injured rats were given intraperitoneal injections of saline,20mg/kg, 30mg/kg and 40mg/kg NDGA respectively one week postoperative. Thenthe samples were obtained at the end of 1, 2, 4, and 8 weeks after injury. The frozensections were observed under fluorescence microscope to observe the survivalamniotic epithelial cells in different groups. The application of immunofluorescencestaining of VEGF and NG2 were used to determine the effects of NDGA on thevasoformation and NG2-positive cells in injured spinal cord. The results showed thatthe 30mg/kg and 40mg/kg of NDGA were obviously conducive to the survival of theamniotic epithelial cells, and NG2-positive cells were not affected by NDGA. But theNDGA inhibited the formation of blood vessels. As a result, the dose of 30mg / kgNDGA was chosen for treating spinal cord injury in our next experiment.III. Observe the treatment effects of NDGA-AECs-AMS transplantation forspinal cord injuryHow to effectively promote the axonal regeneration in a controlled direction is animportant goal of treatment of SCI. Our preliminary work has confirmed that theacellular muscle scaffolds (AMS) could guide the direction of axonal regeneration.The AECs have a variety of advantages in the field of regenerative medicine. In thisstudy, 30mg/kg of NDGA was used to promote the survival of the AECs aftertransplantation, with the combination of AMS, for treatment of spinal cord injury.The chemically extracted acellular muscles seeded with the amniotic epithelial cellswere transplanted into rat spinal cord hemisected injury models. The treated rat spinalcord hemisected injury models were given 30mg/kg of NDGA for 7 dayspostoperative. 8 weeks after injury, routine HE staining was used to observe theintegration between acellular muscle and spinal cord. The immunohistochemicalstaining of NF, CGRP, 5-HT and MBP was used to observe the different kinds ofregenerated nerve fibers and myelinogenesis. The BBB score test was used to observe the functional recovery of rat spinal cord. HE staining showed that in thescaffold transplantation group, scaffold and cell transplantation group, and NDGA,scaffold and cell transplantation group, the scaffold integrate well with the host spinalcord and form a tissue bridge between the rostral and caudal stumps of the spinalcord. NF immunohistochemical staining results showed that in all treated groups, alarge number of nerve fibers grew into the scaffolds. In contrast with this, in theinjury group alone, nerve fibers terminate at the end of the spinal cord stumps. Thenerve fiber counting results showed that, compared with the injury group, thetreatment groups have more regenerated nerve fibers. But the difference of number ofnerve fibers between the treatment groups was not obvious. CGRPimmunohistochemical staining results showed that in all treatment groups,CGRPpositive sensory nerve fibers grew into the grafts. However, the difference of thenumber of CGRP nerve fibers among the treated groups is not obvious. 5-HTimmunohistochemical staining showed that in the scaffold transplantation group,5-HT nerve fibers did not grow into the scaffolds. However, in the scaffold and celltransplantation group and NDGA, scaffold and cell transplantationgroup,5-HT-positive nerve fibers grew into the scaffolds. But the difference of thenumber of 5-HT nerve fibers in the these two treatment groups are not obvious.GFAP immunohistochemical staining revealed that there are no significant differenceamong the injury group, the scaffold transplantation group, and scaffold and celltransplantation group. Compared with other groups, GFAP positive cells wassignificantly reduced in NDGA, scaffold and cell transplantation group. MBPimmunohistochemical staining results showed that only small amount ofMBP-positive myelinated nerve fibers regenerated in the scaffold transplantationgroup, while the regenerated MBP-positive myelinated nerve fibers increasedsignificantly in the scaffold and cell transplantation group, and NDGA, scaffold andcell transplantation group. The difference between the number of myelinated nervefibers in the scaffold and cell transplantation group, and NDGA, scaffold and celltransplantation group is not obvious. BBB score results showed that transplantation groups with AECs better promote rats,functional recovery than the scaffoldtransplantation group. But NDGA have no significant effects in promoting the rats,functional recovery.In summary, the results of this study showed that NDGA significantly inhibited theinfiltration of inflammatory cells and the proliferation of reactive astrocytes. NDGAcould also significantly increase the survival number of transplanted AECs in ratinjured spinal cord. Nevertheless, the application of NDGA did not significantlypromote nerve regeneration and motor function recovery after rat SCI.