Transplanted Cerebrospinal Fluid-Contacting Neurons Alleviate Spasticity Following Spinal Cord Injury in Mice

Introduction/ Objective: Spasticity following spinal cord injury (SCI) remains a challenging condition with limited effective treatments. Transplantation of stem cells to replace the lost GABAergic neurons represents a promising therapeutic strategy. Our previous work has confirmed that cerebrospinal fluid-contacting neurons (CSF-cNs) exhibit characteristics of neural stem cells and can differentiate into GABAergic neurons. This study aims to investigate whether transplanted CSF-cNs can differentiate into GABAergic neurons and alleviate spasticity following SCI. METHODS: CSF-cNs were isolated and purified from adult mice in vitro. Following induction culture, differentiation was characterized by immunofluorescence. One week post-SCI, the CSF-cNs were transplanted into the injured area. The survival and differentiation of transplanted cells were assessed via immunofluorescence. Early progression of spasticity was assessed using the hindlimb grasp reflex. Late-stage spasticity relief was evaluated through electrophysiological and gait footprint analysis. RESULTS: Transplanted CSF-cNs survived within the SCI region, with 58% of GFP⁺ cells co-expressing NeuN. These GFP⁺ cells also expressed GABA, the GABA-synthesizing enzyme glutamate decarboxylase (GAD65/67), and the vesicular GABA transporter (VGAT). Within three weeks post-transplantation, the hindlimb grip reflex scores in the transplant group were significantly improved (P < 0.05). By 6 and 12 weeks, the H-reflex rate-dependent depression (RDD) in the transplant group was significantly superior to that of the control group (P < 0.05). Additionally, the stride length, step width, and hindlimb coordination were markedly improved in the transplant group compared to controls. DISCUSSION: This study provides the first experimental evidence that transplanted CSF-cNs survive and differentiate into GABAergic neurons within the SCI microenvironment. Simultaneously, CSFcN transplantation effectively alleviates spasticity following SCI. This outcome aligns with previous findings using stem cell-derived GABAergic progenitor cells for spasticity treatment. Therefore, we hypothesize that the alleviation of spasticity observed in this study may be associated with the differentiation of CSF-cNs into GABAergic neurons. Admittedly, this correlation requires further investigation. Additionally, although both behavioral and electrophysiological results demonstrated significant symptomatic relief, recovery remained incomplete. This indicates that even if CSF-cNs differentiate into GABAergic neurons and supplement the deficient inhibitory neurons in the damaged spinal cord circuits, this process is insufficient to completely reconstruct the complex spinal cord network. Future investigations will explore mechanisms beyond cell replacement, such as the potential role of transplanted cells in modulating synaptic efficacy (for instance, by restoring KCC2 transporter function). Overall, the evidence presented in this study suggests that CSF-cN transplantation represents a viable strategy for alleviating spasticity following SCI. CONCLUSION: Transplanted CSF-cNs can differentiate into GABAergic neurons and that their transplantation can alleviate spasticity in mice following SCI.