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

摘要/项目摘要（30行文本） 呼吸机依赖性颈脊髓损伤（cSCI）患者的预期寿命显著降低， 呼吸系统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 和隔膜起搏，对于cSCI诱导的呼吸机依赖性没有长期的解决方案。这个项目 通过测试和开发放大横膈膜的转换原理， 通过膈交感神经支配的选择性神经调节来减少或消除收缩强度 呼吸机依赖虽然交感神经纤维与血管控制广泛相关，但最近的研究表明， 提供了令人信服的证据，即骨骼肌的交感神经支配提供了必要的营养支持 用于维持健康的神经肌肉接头（NMJ）并促进神经传递。对人类以及 在动物中，膈神经是支配隔膜的交感神经纤维的管道。膈交感神经 (PS)起源于脊髓外节后神经元，并且不受脊髓直接损伤的损伤 线.值得注意的是，与所有研究的肌肉相比，横膈膜具有最丰富的交感神经供应。 轴突终末与NMJ共定位。本研究将开发PS神经调节的翻译原则， 一种基于生物学但未经测试的治疗策略，用于增强或恢复膈肌功能， 颈脊髓损伤。在目的1中，慢性选择性PS刺激对长期膈肌的影响 功能和NMJ健康将在光遗传学C4-5血挫伤cSCI小鼠模型中进行研究， 光激发PS纤维。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装置。将进行终末实验以评估隔膜 收缩特性和膈-膈肌组织学，以评估慢性PS募集的程度 可以减轻观察到的cSCI诱导的生理和组织学变化。Aim 2将开发一种重组 腺相关病毒（rAAV）基因治疗方法，使用发光蛋白技术，赋予选择性的光- PS神经元活性的化学遗传控制。发光蛋白是一种融合蛋白， 通道视紫红质离子通道和发光荧光素酶赋予两种模式的神经元控制; 1)视蛋白的光活化和2）荧光素酶的分子活化，产生视蛋白活化的生物发光 光光视蛋白方法允许光活化控制神经元活性，但不需要光激活。 内部光源。将进行存活手术以通过血管内注射将工程化的rAAV递送至横膈膜。 经腹入路在转导后2周的终末实验将比较 光和分子发光蛋白激活对膈肌收缩力和募集PS活性的影响。组织学 分析将基于发光蛋白和PS标记物的共标记来评估转导功效。所得 发光蛋白基因治疗方法将促进未来的大型动物研究和临床转化， 转基因和光遗传学方法是不可用的、成本过高的或不适用的。

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.