In vivo Application of Electrical Fields Directs Retinal Ganglion Cell Axon Regeneration

项目摘要 据估计，全世界有1800万人因视神经病变如晚期青光眼而合法失明。 恢复视力需要再生视神经，视神经是视网膜神经节细胞（RGC）轴突的集合， 离开眼睛与大脑连接。虽然有希望，但仅基于细胞移植的策略不足以 再生视神经，部分原因是移植的RGCs无法将轴突延伸出眼睛。同样，神经- 再生方法受到不能引导长距离轴突生长的限制。在这个项目中，我提出了一个创新的 使用外加电场（EF）引导RGC轴突生长的方法。最近，我证明了研资局 当在体外暴露于EF时，轴突朝向阴极定向生长。EFs是否能引导RGC轴突 体内生长，是未知的，因为是通过细胞的感觉和响应EF的机制。初步数据 本研究表明：1）EF可以沿着大鼠视神经产生，2）在体内应用EF促进 挤压损伤后RGC轴突再生，和3）Rho GT3家族成员Rac 1的共活化， 在体外协同指导RGC轴突生长。该提案旨在证明体内EF的可行性 应用作为治疗方式来指导RGC轴突再生和测试EFs指导RGC轴突的假设 通过激活Rho-GT3信号级联来促进再生。K 08职业发展奖将为我提供 研究方法、应用电气工程和电生理学及技术的结构化教育 转移以及结构化的指导，以填补知识的教育和经验的差距，发展技能， 领导力，工作生活平衡，赠款和手稿写作，这将使我过渡到一个独立的，国家卫生研究院- 受资助的临床科学家，是视神经再生领域的世界专家。我战略性地组织了一个 导师团队由电气工程师，材料科学家，电生理学家，细胞生物学家，神经外科医生， 神经生物学家，统计学家和翻译科学家，以补充我作为神经眼科医生的背景， 发展神经生物学家，指导我的学习和职业轨迹。该项目的成功完成将 使我成为一名有竞争力的R 01申请人，我计划在那里测试EF申请是否与 分子线索，可用于指导移植RGCs的轴突生长，以再生视神经并恢复视觉 在不同的视神经病变动物模型中的功能。如果成功的话，这个项目有可能取得很大的进展， 在视神经再生领域，将电调制带到了最前沿。

Project Summary It is estimated that 18 million people worldwide are legally blind from optic neuropathies such as advanced glaucoma. Restoration of vision requires regenerating the optic nerve, a collection of retinal ganglion cell (RGC) axons that have exited the eye to connect with the brain. Although promising, cell transplantation-based strategies alone are inadequate to regenerate the optic nerve, in part, because transplanted RGCs fail to extend an axon out of the eye. Similarly, neuro- regenerative approaches are limited by failure to direct long distance axon growth. In this project, I propose an innovative approach that uses applied electrical fields (EFs) to guide RGC axon growth. Recently, I have demonstrated that RGC axons grow directionally, towards the cathode, when exposed to an EF, in vitro. Whether EFs can direct RGC axon growth in vivo, is unknown, as are the mechanisms through which cells sense and respond to EFs. Preliminary data presented here shows that 1) an EF can be generated along the rat optic nerve, 2) in vivo application of EFs promotes RGC axon regeneration after crush injury, and 3) co-activation of Rac1, a member of the Rho GTPase family, synergistically directs RGC axon growth in vitro. This proposal aims to demonstrate the feasibility of in vivo EF application as a therapeutic modality to guide RGC axon regeneration and test the hypothesis that EFs direct RGC axon regeneration by activating the Rho-GTPase signaling cascade. The K08 Career Development Award will provide me with structured education in research methodology, applied electrical engineering and electrophysiology, and technology transfer as well as structured mentorship to fill in educational and experiential gaps in knowledge, develop skills in leadership, work life balance, and grant and manuscript writing that will allow me to transition to an independent, NIH- funded clinician-scientist who is a world expert in the field of optic nerve regeneration. I have strategically assembled a mentorship team consisting of electrical engineers, material scientists, electrophysiologists, cell biologists, neurosurgeons, neurobiologists, statisticians, and translational scientists to complement my background as a neuro-ophthalmologist and developmental neurobiologist and direct my learning and career trajectory. Successful completion of this project will position me to become a competitive R01 applicant where I plan to test whether EF application, in conjunction with molecular cues, can be used to direct axon growth of transplanted RGCs to regenerate the optic nerve and restore visual function in different animal models of optic neuropathies. If successful, this project has the potential to make large strides in the field of optic nerve regeneration, bringing electrical modulation to the forefront.