Chronic Neuromodulation of Phrenic Sympathetics to Rescue Diaphragm Function Following Cervical Spinal Cord Injury

摘要/项目摘要(30行文本) 呼吸机依赖型颈髓损伤(CSCI)患者的预期寿命显著缩短 呼吸道并发症。尽管最近在肺部医学方面取得了进展，包括膈神经刺激 对于CSCI诱导的呼吸机依赖，没有长期的解决方案。这个项目 通过测试和开发放大横隔膜的翻译原理来解决这一深刻的治疗差距 通过选择性神经调节膈交感神经减少或消除收缩强度 呼吸机依赖。虽然交感神经纤维广泛地与血管运动控制有关，但最近的研究 提供令人信服的证据表明骨骼肌的交感神经支配提供必要的营养支持 用于维持健康的神经肌肉连接(NMJ)和促进神经传递。在人类和在 对于动物来说，膈神经是支配横隔膜的交感纤维的管道。膈交感神经 (PS)起源于脊髓外的节后神经元，对脊髓没有直接损伤 电源线。值得注意的是，与所研究的所有肌肉相比，横隔肌的交感神经供应最丰富。 轴突终末与NMJ共同定位。这项研究将开发PS神经调节的翻译原则， 一种基于生物但未经测试的治疗策略，用于增强或恢复下列隔膜功能 颈髓损伤。在目标1中，慢性选择性PS刺激对长期隔膜的影响 将在C4-5光遗传CSCI小鼠模型中研究功能和NMJ健康状况 可光激发的PS纤维。将用蓝光对隔膜PS纤维进行慢性体内光刺激 通过植入的微型光电子设备。将进行终端实验以评估隔膜 收缩特性和横隔膜组织学评估慢性PS募集的程度 可以减轻观察到的CSCI引起的生理和组织学变化。AIM 2将开发出一种重组 基于发光素技术的腺相关病毒基因治疗方法 化学发生对PS神经元活动的控制。发光素是一种具有光敏功能的融合蛋白 视紫红质离子通道和发光荧光素酶赋予神经元两种控制模式； 1)视蛋白的光激活和2)荧光素酶的分子激活产生视蛋白激活的生物发光 灯。发光素方法允许对神经元活动进行光激活控制，但不需要 内部光源。将进行生存手术，以将工程rAAV通过 经腹入路。转导后2周的终末实验将比较 光和分子发光素激活对横隔膜的收缩力和招募PS活性。组织学 分析将基于发光素和PS标记物的共同标记来评估转导效率。由此产生的 发光素基因治疗方法将促进未来的大型动物研究和临床翻译 转基因和光遗传方法是不可用的，成本过高，或不适用。

Abstract/Project Summary (30 lines of text) Life expectancy of ventilator-dependent cervical spinal cord injury (cSCI) patients is significantly reduced by respiratory complications. Despite recent advances in pulmonary medicine, including phrenic nerve stimulation and diaphragm pacing, there are no long-term solutions for cSCI-induced ventilator dependency. This project addresses this profound therapeutic gap by testing and developing translational principles to amplify diaphragm contraction strength via selective neuromodulation of phrenic sympathetic innervation to reduce or eliminate ventilator dependency. While sympathetic fibers are widely associated with vasomotor control, recent studies provide compelling evidence that sympathetic innervations of skeletal muscles provide essential trophic support for maintaining healthy neuromuscular junctions (NMJs) and facilitates neurotransmission. In humans and in animals, the phrenic nerve is a conduit for sympathetic fibers innervating the diaphragm. Phrenic sympathetics (PS) originate from extra-spinal post-ganglionic neurons and are undamaged with direct injuries to the spinal cord. Notably, the diaphragm compared to all muscles studied, has among the richest supply of sympathetic axonal terminals colocalizing with NMJs. This study will develop principles to translation for PS neuromodulation, a biologically-based but untested therapeutic strategy, for enhancing or restoring diaphragm function following cervical spinal cord injury. In Aim 1, the effects of chronic selective PS stimulation on long-term diaphragm function and NMJ health will be studied in an optogenetic C4-5 hemocontusion cSCI mouse model with photoexcitable PS fibers. Chronic in-vivo photostimulation of diaphragm PS fibers with blue-light will be delivered by an implanted miniature optoelectronic device. Terminal experiments will be performed to assess diaphragm contractile properties and phrenic-diaphragm histology to evaluate the extent to which chronic PS recruitment can mitigate observed cSCI-induced physiological and histological changes. Aim 2 will develop a recombinant adeno-associated virus (rAAV) gene-therapy approach using luminopsin technology to impart selective opto- chemogenetic control over PS neuronal activity. Luminopsins are fusion proteins with a light-sensing channelrhodopsin ionotropic channel and a light-emitting luciferase imparting two modes of neuronal control; 1) photoactivation of opsin and 2) molecular-activation of luciferase generating opsin-activating bioluminescence light. The luminopsin approach permits photoactivation control over neuronal activity but obviates the need for internal light source. Survival surgeries will be performed to deliver the engineered rAAV to the diaphragm via a transabdominal approach. Terminal experiments, 2 weeks following transduction, will compare the effects of photo- and molecular luminopsin activation on diaphragm contraction force and recruited PS activity. Histological analysis will evaluate transduction efficacy based on co-labeling of luminopsin and a PS marker. The resultant luminopsin gene-therapy approach will facilitate future large animal studies and clinical translation where transgenic and optogenetic approach are unavailable, cost prohibitive, or inapplicable.