Astrocytic Reprogramming In The Treatment Of Spinal Cord Injury

Background:Recovery from spinal cord injury(SCI)remains an unsolved problem.Injuries to the spinal cord may cause a large number of neuronal loss,forming a wide range of neuronal deficiency,leading to the interruption of the neuronal networks.The adult mammalian spinal cord lacks the ability to spontaneously produce new neurons,making it a key challenge to provide new neurons for spinal cord regeneration.On the other hand,astrocytes form a dense glial scar around the lesion core after SCI,which may prevent axon regeneration.However,the glial scar is actually playing a dual role in SCI,both protective and inhibitory,making it another challenge to manipulate it for therapeutic purposes.Cellular reprogramming strategy has been investigated in several central nervous system disorders,which can realize the reprogramming from glial cells into neurons in vivo.In the context of SCI,it may reprogram some of the scar-forming astrocytes into neurons,which may not only replenish the lost neurons but also reduce the density of the glial scar.However,astrocytic reprogramming has rarely been investigated in the field of SCI.Current studies in this field mainly focused on exploring the ways and mechanisms of reprogramming,but rarely concentrated on its role in the glial scar and SCI recovery.How does astrocytic reprogramming influence the glial scar?Does it propel axon regeneration?Does it improve functional recovery after SCI?Does it have any shortcomings?These questions may need further investigation.Objective:In this study,we aimed to explore the role of astrocytic reprogramming in treating SCI,especially focusing on the following questions:1.Whether adeno-associated virus(AAV)can induce astrocytic reprogramming after SCI?2.Whether it can replenish new neurons in the lesion area?3.Whether it can reduce the density of the glial scar?4.Whether it can propel axon regeneration?5.Whether it can improve functional recovery?6.Does it have any shortcomings?How can these shortcomings be remedied to further improve functional recovery?Methods:1.We established a T10 spinal cord crush injury model in mice.2.We built an AAV-mediated gene delivery system carrying Sry-related HMG-box 2(Sox2)gene.The viral vectors were injected into the glial scar one week after SCI.3.The expression and specificity of the viral vectors was verified one and ten weeks after injection.4.The reprogrammed neurons were identified by immunohistochemical staining of neuronal markers.5.The density of the glial scar was evaluated by immunohistochemical staining of astrocytic marker GFAP.6.Anterograde tract-tracing of the corticospinal tract(CST)and propriospinal neurons was performed to investigate whether astrocytic reprogramming promotes axon regeneration.7.Behavioral tests were performed to evaluate the functional improvement after astrocytic reprogramming.8.The astrocytic reprogramming was combined with rehabilitation strategy to investigate whether this combination therapy can improve functional recovery.Results:1.The viral vectors exhibited high specificity,and mostly expressed in astrocytes but rarely in neurons.2.Some of the targeted astrocytes were reprogrammed into neurons,which replenished the neuronal loss in SCI.3.Compared with the radical ablation of the glial scar in some previous studies,astrocytic reprogramming moderately reduced the density of the glial scar without interrupting its integrity.4.Astrocytic reprogramming propelled CST and propriospinal axon regeneration by not only replenishing the lost neurons but also moderately reducing the density of the glial scar without interrupting its integrity.5.Astrocytic reprogramming alone only slightly improved functional recovery without statistic difference.6.Rehabilitation may be an ideal supplementary therapy for astrocytic reprogramming,which significantly delayed the muscle degeneration and provided use-dependent plasticity.Astrocytic reprogramming combined with rehabilitation strategy significantly improved functional recovery after SCI,which was also better than either of them alone.Conclusions:1.In the context of SCI,scar-forming astrocytes in the glial scar can be reprogrammed into neurons in vivo to replenish required interneurons by the AAVs carrying Sox2 gene.2.Astrocytic reprogramming can not only replenish new neurons but also moderately reduce the density of the glial scar without interrupting its integrity.Through these two effects,it can also propel axon regeneration.3.These results may provide us with a new idea about how to manipulate the glial scar:to reduce the density of the glial scar without interrupting its integrity at two weeks after SCI.4.Astrocytic reprogramming alone may not lead to satisfactory functional recovery.When combined with rehabilitation strategy,it can significantly improve functional recovery,which was also better than reprogramming or rehabilitation alone.