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

项目摘要 据估计，全世界有1800万人在法律上因晚期青光眼等视神经疾病而失明。 恢复视力需要再生视神经，这是一组视网膜神经节细胞(RGC)轴突，具有 离开眼睛，与大脑相连。尽管前景看好，但仅基于细胞移植的策略并不足以 再生视神经在一定程度上是因为移植的视网膜节细胞不能将轴突延伸出眼睛。同样，神经- 再生方法因未能引导长距离轴突生长而受到限制。在这个项目中，我提出了一种创新的 使用外加电场(EFS)引导RGC轴突生长的方法。最近，我已经证明了研资局 在体外，当轴突暴露在EF中时，轴突向阴极方向生长。EFS能否定向RGC轴突 在体内的生长，以及细胞感知和响应EFS的机制是未知的。初步数据 结果表明：1)大鼠视神经可产生EF；2)体内应用EFS可促进 挤压伤后RGC轴突再生，以及3)Rho GTP酶家族成员rac1的共激活， 协同作用指导RGC轴突的体外生长。这项建议旨在论证体内EF的可行性 应用EFS作为治疗手段引导RGC轴突再生并验证EFS引导RGC轴突的假说 通过激活Rho-GTPase信号级联来进行再生。K08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以及技术方面的结构化教育 转移以及结构化的指导，以填补教育和经验方面的知识空白，发展技能 领导力，工作和生活的平衡，以及拨款和手稿写作，这将使我过渡到一个独立的，NIH- 获得资助的临床医生和科学家，是视神经再生领域的世界专家。我策略性地组建了一个 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，并指导我的学习和职业轨迹。这个项目的成功完成将 让我成为一名有竞争力的R01申请者，我计划在那里测试EF申请是否与 分子信号，可以用来指导移植的视网膜节细胞的轴突生长，以再生视神经和恢复视力 在不同视神经病变动物模型中的作用。如果成功，这个项目有可能取得长足进步 在视神经再生领域，使电调制走在了前列。

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.