Stimulation of Cervical Excitatory Interneurons to Restore Breathing After Chronic Cervical Spinal Cord Injury

呼吸功能障碍是颈椎脊髓损伤(CSCI)后发病率和死亡率的重要原因。有能力 恢复颈椎脊髓损伤(CSCI)后慢性期的呼吸是一个压倒性但重要的目标。这个 横隔肌是主要的吸气肌，由位于颈部的膈运动神经元(PMN)支配 脊髓(C3-C5)。颈髓间神经元与中性粒细胞突触并与膈神经同步放电 呼吸输出和调节呼吸。吻侧腹侧呼吸神经元群内的运动前神经元 脑干(RVRG)为脊髓呼吸回路提供主要的吸气驱动。颈髓 损伤(SCI)扰乱了rVRG和脊髓呼吸回路之间的通讯，导致明显的 呼吸系统受损。我们最近证实，对颈椎兴奋性的药物遗传刺激在- 在CSCI后，神经元(EIN)立即挽救小鼠的呼吸；然而，目前尚不清楚这一策略是否会 在CSCI慢性期有效改善呼吸。在慢性阶段，呼吸系统是 已知经历了重大的修改。此外，我们之前在非创伤性压迫损伤方面的工作 颈髓表明，颈髓在促进可塑性和主要... 尽管中性粒细胞大量丢失，但仍保持通风。鉴于颈椎病整合到呼吸网中-- 它们是自然呼吸恢复所必需的，它们成为关键的治疗焦油- GET用于创伤性CSCI后慢性期的呼吸恢复。我们假设提供SLEEC- 慢性脊髓损伤后期对存活的PMN的兴奋性输入将增加PMN的输出和呼吸 恢复。这项提案的第一个目标是对呼吸系统的状况有更重要的了解-- 慢性CSCI后的保守性神经网络。该提案的第二个目标将研究一种新的治疗方法。 选择性刺激颈内动脉促进慢性期呼吸恢复的策略 CSCI。 在目标1中，我们将使用单突触来评估C2半横断损伤(C2Hx)后中性粒细胞的存活。 逆行示踪剂霍乱毒素B亚单位，以特异性追踪中性粒细胞。在目标1的第二部分，我们将 同时绘制颈部呼吸要素、PMN和 隔前颈椎病、幼稚未受伤状态和CSCI后。在目标2中，我们将有选择地刺激 应用DREADD技术检测慢性颈脊髓损伤后膈核区的颈内病变。这项技术 由于其能够非侵入性地激活或沉默神经元群体，因此具有创新的应用。其效果 电刺激宫颈EINS对慢性CSCI后呼吸恢复的促进作用 肌电描记和全身体积描记。拟议实验的预期结果是不确定的-- 因为它将增加我们对慢性CSCI后脊髓呼吸系统网络的了解，并促进 CSCI慢性期恢复呼吸的新策略的开发。这些结果将有一个 对患有脊髓损伤的退伍军人的健康和生存产生了有意义的积极影响，这里研究的战略已经 慢性脊髓损伤后慢性期撤机的可能性 当促进复苏仍然是一项艰巨的任务时。此外，SCI代表了一个重要的重点领域 退伍军人，在美国脊髓损伤患者中，约7%的人在积极的同时保持了他们的脊髓损伤 上菜了。受伤通常是由于枪击或高威力爆炸装置等穿透伤造成的。 因此，患有脊髓损伤的军事人员甚至比患有脊髓损伤的平民面临更多的长期残疾。 SCI，而且这些问题在患有CSCI和呼吸功能障碍的退伍军人中更加明显。

Dysfunctional breathing is a significant cause of morbidity and mortality after cervical SCI (cSCI). The ability to restore breathing in the chronic phase after cervical SCI (cSCI) is an overwhelming yet important goal. The diaphragm, the major inspiratory muscle, is innervated by phrenic motoneurons (PMNs) located in the cervical spinal cord (C3-C5). Cervical interneurons synapse onto PMNs and discharge in synchrony with phrenic inspir- atory output and modulate breathing. Premotor neurons within the rostral ventral respiratory group of neurons (rVRG) in the brainstem provide the main inspiratory drive to the spinal respiratory circuitry. Cervical spinal cord injury (SCI) disrupts the communication between rVRG and the spinal respiratory circuitry resulting in significant respiratory compromise. We recently demonstrated that pharmacogenetic stimulation of cervical excitatory in- terneurons (eINs) immediately after cSCI rescues breathing in mice; however, it is not known if this strategy will be effective in improving breathing in the chronic phase of cSCI. At the chronic stage, the respiratory network is known to undergo significant modifications. Moreover, our previous work in non-traumatic compressive injury to the cervical spinal cord demonstrated that cervical eINs play an integral role in promoting plasticity and main- taining ventilation despite a significant loss of PMNs. Given that cervical eINs integrate into the respiratory net- work, and they are necessary for the spontaneous respiratory recovery, they emerge as critical therapeutic tar- gets for respiratory recovery in the chronic phase after traumatic cSCI. We hypothesize that providing selec- tive excitatory input to surviving PMNs at the late stage of cSCI will enhance PMNs output and respiratory recovery. The first objective of this proposal aims to gain more significant insights into the status of the respira- tory neural network after chronic cSCI. The second objective of this proposal will examine a novel treatment strategy involving selective stimulation of cervical eINs to promote respiratory recovery in the chronic phase after cSCI. In aim 1, we will assess the survival of PMNs following C2 hemisection injury (C2Hx) using the monosynaptic retrograde tracer Cholera Toxin Subunit B to specifically track the PMNs. In the second part of Aim 1, we will simultaneously map the input-output connectivity of the cervical respiratory elements, the PMNs and the prephrenic cervical eINs, in the naïve-uninjured state and after cSCI. In Aim 2, we will selectively stimulate the cervical eINs in the region of the phrenic nuclei after chronic cSCI using the DREADD technology. This technique has innovative applications due to its ability to activate or silence neuronal populations non-invasively. The effect of stimulating cervical eINs on promoting respiratory recovery after chronic cSCI will be assessed using electro- myography and whole-body plethysmography. The expected outcomes of the proposed experiments are inno- vative as it will increase our knowledge of the spinal respiratory network after chronic cSCI and facilitate the development of a novel strategy to restore breathing in the chronic phase of cSCI. These results will have a meaningful positive impact on the health and survival of veterans with SCI, and the strategy examined here has the potential to be implemented for weaning patients off mechanical ventilation in the chronic phase after cSCI when promoting recovery has remained a daunting task. Additionally, SCI represents a significant focus area of the veterans, about 7% of those living with SCI in the United States have sustained their SCI while actively serving. Injuries are often due to penetrating injuries such as from gunshots or high-powered explosive devices. As such, military personnel with SCI face even more long-term disabilities than those endured by civilians with SCI, and the issues more pronounced in veterans with cSCI and respiratory dysfunctions.