In Vitro Model Construction And Mechanism Of Nerve Injury And Preliminary Study Of New Repair Unit

Traumatic brain injury caused by traffic accidents has been the case of common death and long-term disability in the world.According to its degree,the traumatic brain injury can lead to central nervous system diseases such as headache,memory loss and traumatic stroke.The pathological mechanism of the traumatic brain injury is very complicated,involving the changes of all levels from molecule,neuron to neural network.In particular,the pathological changes of the whole network of traumatic central nervous injury are still unclear,and the repair measures for traumatic neural network injury are relatively simple.On the one hand,this study starts from the construction of a traumatic central nervous system multi-point injury model in vitro to study the overall pathological changes of the neuronal network after injury;on the other hand,based on the design concept of integrating a variety of effective neural repair measures,a novel nanostructured neural implantable unit for nerve repair was prepared.The main research work and results are displayed as below:1.The construction of the traumatic nerve injury model in vitro and the changes of neuronal network morphology were studied.This thesis introduces a method based on excimer laser processing combined with injection molding technology to rapidly prepare graphically structured microfluidic array chip,and a microfluidic array chip with smooth underside was prepared.Biological neural networks with different densities were cultured on the prepared array chip to construct an in vitro model of traumatic nerve multi-point injury.Compared with biological neural networks cultivated in glass slices open space,the growth morphology of neural network was analyzed.The results showed that（i）both the neuronal network formed in the array chip（pathological model）and glass slices open space could grow normally;（ii）the density of neurons on the chip was lower than that in open space under the same culture days;and（iii）the average length of neurites in the chip is lower than that in open space under the same culture days.From the results,it can be deduced that the micro-pillars array promoted the growth of some neurites extending along the edges of the micro-pillars while restricting the growth of some neurites.This showed that abnormal growth of neuronal axons and neurites near the damaged area resulted in the changes in the morphology of the whole network,which further affected the signal transmission.2.The construction of in vitro model of traumatic nerve injury and the changes in functional connectivity of neural networks were studied.A microfluidic array chip was used to construct an in vitro model of traumatic nerve injury,and the electrophysiological information of the pathological model was collected with a microelectrode array（MEA）.The recorded electrophysiological data were analyzed by cross covariance analysis and graph theory analysis.The study found that（i）the discharge rate,cluster discharge rate,network cluster discharge rate and connection strength of the neural network cultured on the array chip（pathological model）and the open space increased when the number of days of cultivation extended;（ii）however,compared with the neural network cultured in open space,the density and small-world properties of the neural network cultured in the pathological model were improved,at the same culture time.The results showed that the functional connectivity of the neuron network has been changed under the pathological model,which corresponds to the morphological changes of the neural network.3.The preparation of multi-walled carbon nanotube/filament protein membrane blending method and the surface deposition method were studied.On this basis,the silk fibroin proteins around carbon nanotubes were carbonized by laser irradiation to form a network of carbon micron tubes（PCMTs）.Then,multi-walled carbon nanotubes/porous network carbon microtubes/silk fibroin protein implanted neural repair units were prepared.The composite materials were characterized and analyzed,by using scanning electron microscope,Raman spectrometer,etc..It is found that the nerve repair unit was a porous network structure with excellent porosity and mechanical performance,which can be used to improve the environment of nerve damage and slow release of drugs and nutritional factors.The cyclic voltammetry characteristics,electrochemical AC impedance,and maximum safe injected charge(Q_inj)of multi-walled carbon nanotubes/porous network carbon microtubes/silk fibroin protein electrodes were measured and analyzed by a three-electrode test system.The results showed that multi-wall carbon nanotubes/porous network carbon microtubes/silk fibroin protein electrodes possessed excellent electrochemical performance,and the Q_injcould reach 5.7mc/cm²,which could be safely used for electrical stimulation to repair nerve injury.It lays the foundation for the subsequent exploration on the growth and repair of neural networks.