HCN1/HCN2介导内向整合电流（Ih）调控脊髓损伤后运动系统跨突触变性的机制研究

Trans-synaptic degeneration is one of the major factors disturbing long-term recovery of motor function in the patients with spinal cord injury (SCI), and its mechanism remains unclear. In our previous studies, we demonstrated there is significant relationship between trans-synaptic degeneration and abnormal excitability of spinal motor neurons in the patients with SCI through the motor unit number estimation and the Hmax/Mmax of H-reflex. Furthermore, we studied the measurements of excitability of spinal motor neurons distal to the SCI and found obviously reduced inwardly rectifying current (Ih) in these spinal motor neurons. Previous studies demonstrated that Ih of spinal motoneurons are mainly mediated by hyperpolarization-activated cyclic-nucleotide gated (HCN) channel subtype HCN1/HCN2. Based on these findings, we hypothesize that SCI may cause the inhibition of HCN1/HCN2 in distal anterior horn motor neurons, which reduce the Ih of these motor neurons and eventually lead to the trans-synaptic degeneration. In this project, we try to clarify the role and its mechanism of HCN1/HCN2 mediating Ih in the regulation of trans-synaptic degeneration in motor system after SCI with immunofluorescent staining, threshold tracking, patch clamp, transmission electron microscope and flow cytometry, which may provide new ideas for the mechanism of trans-synaptic degeneration after SCI and good long-term rehabilitation of the patients with SCI.

跨突触变性是导致脊髓损伤患者远期运动功能恢复不佳的主要原因之一，但其机制尚未完全清楚。在前期研究中，我们采用运动单位数目估计及迟发反射Hmax/Mmax分析，证实跨突触变性与前角运动神经元的兴奋性相关；进一步采用阈值追踪技术分析脊髓损伤患者远端前角运动神经元兴奋性参数，发现脊髓损伤远端前角运动神经元存在明显内向整合电流（Ih）减少。既往文献证实：脊髓前角运动神经元Ih主要由超极化激活环核苷酸门控通道（HCN）亚型HCN1/HCN2介导。基于上述提示，我们推测脊髓损伤可能通过抑制远端前角运动神经元HCN1/HCN2，减少运动神经元Ih，进而造成跨突触变性。本申请项目拟采用免疫荧光、阈值追踪、膜片钳、透射电镜、流式细胞检测等方法初步阐明脊髓运动神经元HCN1/HCN2介导Ih在继发性运动系统跨突触变性中的调控作用及机制，为探索脊髓损伤后运动系统跨突触变性的机制、促进患者远期功能康复提供新思路。

相对原发性脊髓损伤，继发损伤破坏程度更大、范围更广，不仅局限于损伤局部，也发生于远隔部位，形成“跨突触变性”。探索脊髓损伤后跨突触变性的形成机制，对于促进远期康复具有重要意义。项目负责人在青年基金的研究中，揭示：1）脊髓损伤后远端未受累脊髓节段的HCN1分布及表达在脊髓损伤与假手术组之间无明显差异。同时，与前期研究中慢性脊髓压迫性损伤（例如，脊髓型颈椎病等疾病）患者内向整合电流（Ih）减少不同，我们发现急性创伤性脊髓损伤后患者或小鼠模型的远端运动轴突的HCN1介导的Ih在损伤后即刻出现了异常增加；同时，脊髓损伤小鼠鞘内注射HCN拮抗剂可有效缓解脊髓损伤远端神经系统的去极化表现，轻度改善运动单位的丢失，促进运动功能恢复。2）基于该致病机制的临床转化研究证实，脊髓损伤后的运动系统“跨突触变性”参与了颈椎病性肌萎缩症，颈脊髓中央损伤综合征、平山病等多种疾病的致病过程；同时，上述运动系统“跨突触变性”并非迟发性病变，其在脊髓损伤后即刻发生，通过早期干预及术中扩大减压范围等方法可以有效阻断这一病变过程，提高脊髓损伤预后。3）基于继发性脊髓损伤的进一步拓展性实验发现，CD73可通过促进Ado/cAMP/PKA/CREB途径抑制继发性神经元凋亡，而lncGm3749在脊髓损伤后的微环境调节中起重要作用，因此推测两者可能均与脊髓损伤后的继发性远隔部位的神经元变性存在一定相关性，可作为后续研究的靶点。本项目初步解析了脊髓损伤后运动系统“跨突触损伤”的潜在机制，并探讨了以“跨突触损伤”为目标的治疗措施的临床价值，为后续防治继发性脊髓损伤的课题提供了新的研究方向。