Preclinical Study Of Nimodipine And Human Embryonic Stem Cell-derived Spinal GABAergic Neurons In The Treatment Of Spinal Cord Injury

Preclinical Study of Nimodipine in the Treatment of Spinal Cord Injury in RatsObjective: Spinal cord injury(SCI)is a devastating condition that results in severe motor,sensory,and autonomic dysfunction.The initial traumatic impact results in a sustained cascade of secondary injury,including apoptosis,ischemia,vasospasm,disruption of ionic homeostasis and inflammation,which propagate the damage and worsen the function.Nimodipine(NMD)is an L-/T-type calcium channel blocker and has been approved for the treatment of subarachnoid hemorrhage by the U.S.Food and Drug Administration.The effects of NMD mainly include central nervous system-specific vasodilation and blockade of the flux of extracellular calcium through L-/T-type calcium channels.NMD has been tested in preclinical and clinical SCI research;however,its efficiency remains to be elucidated.Perhaps one of the reasons may be the short duration of the treatment.This study aims to evaluate the efficiency and possible mechanisms of long-term therapy with NMD on a preclinical SCI model.Methods: In this study,a 25-mm contusion SCI model was established in female rats.We chose female rats because manual expression of the bladder is more easily accomplished due to the shorter and wider urethra in females than in male rats.In addition,contusion SCI model is considered to be relevant to clinical pathophysiology.Then,SCI rats were randomly divided into the NMD group and the control group.Starting 1 h after injury,NMD group and control group were subcutaneously injected with NMD(10 mg/kg)or vehicle once a day for 6 weeks.We monitored the motor function,hind limb grip strength,pain-related behaviors,bladder function,spasticity-like symptoms and open-field spontaneous activity in this study to assess the efficacy of NMD in rats with SCI.Histologically,SCI lesion areas and perilesional neuronal numbers,gliosis and calcitonin gene-related peptide(CGRP+)fiber sprouting in the lumbar spinal cord and the expression of K+-Cl-cotransporter 2(KCC2)on lumbar motor neurons were also observed to further explore the possible protective mechanisms of NMD.Results: Rats treated with NMD showed improvements in locomotion,pain-related behaviors,and spasticity-like symptoms,but not in open-field spontaneous activity,hind limb grip strength or bladder function.Compared with the control group,NMD group showed greater tissue preservation with reduced lesion areas and increased perilesional neuronal sparing.NMD group also showed reduced gliosis and CGRP+ fiber sprouting in the lumbar spinal cord and higher KCC2 membrane expression on lumbar motor neurons.Conclusion: These results indicate that long-term treatment with NMD improves functional recovery after SCI.NMD may merit additional study as a possible therapeutic agent for the treatment of SCI.Preclinical Study of Human Embryonic Stem Cell-derived Spinal GABAergic Neurons in the Treatment of Neuropathic Pain after Spinal Cord Injury in RatsObjective: Neuropathic pain is one of the most debilitating sequelae for patients with chronic spinal cord injury(SCI).This severe sensory deficit prevails among 70-80% of SCI patients and interferes with these patients’ quality of life and their rehabilitation.Currently,neuropathic pain is often managed pharmacologically.However,these treatments are often accompanied by untoward side effects,and pain relief is often inadequate in many patients.Moreover,long-term treatment with conventional analgesics can elicit tolerance,sedation,and even addiction.Pre-clinical research has used foetal progenitor cells or human embryonic stem cells(h ESCs)-derived medial ganglionic eminence-like interneuron precursors to alleviate pain-related behaviors in SCI model.However,given their multipotent potential or foetal origin,they may be unsuitable for clinical application.The aim of this study was to evaluate the effect of h ESCs-derived spinal d I4 GABAergic neurons on neuropathic pain after SCI in rats.Methods: A 25-mm contusion SCI model was established in female rats.Giving that manual expression of the bladder in female rats are easier than male rats after SCI,only female rats were used in this study.SCI rats were randomly divided into two groups: cell transplantation group and vehicle transplantation group.h ESCs-derived spinal d I4 GABAergic progenitors or vehicle were transplanted 7 days post-injury.BBB score was used to evaluate the motor function of rats.Pain-related behaviors were assessed by Von Frey test and the Hargreaves test.In addition,with the DREADD-expressing cells as the donor,we assessed whether the function of the transplanted rats could be modulated by chemogenetic drugs clozapine-N-oxide(CNO)(cell excitation)or Salvinorin B(SALB)(cell silencing)at 12 and 24 weeks after transplantation.The rats were sacrificed 24 weeks after transplantation,and spinal cord sections were examined for cell survival,maturation,innervation,projection,the potential synaptogenesis and chemogenetics validity using immunofluorescence staining.Results: Transplanted rats showed improvement in locomotor function and mitigated pain-related behaviors.Exciting or silencing engrafted cells drives no obvious locomotor changes in transplanted rats.However,pain-related behaviors could be mitigated or enhanced by CNO-or SALB-based control of graft function,respectively.Transplanted cells survived in the injured spinal cord,expressed mature neuronal markers,received potential host input,projected to the lumbar spinal cord and showed potential synapse formation between grafted and host neurons.Moreover,by using c-Fos staining as a marker of neuronal activity,our results showed that DREADD-expressing cells were activated by CNO.Conclusion: This study suggests that transplantation with spinal GABAergic neural progenitors is a potentially effective therapy for SCI associated neuropathic pain.In the future,it may be necessary to combine multiple cell types to improve the therapeutic efficacy.