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

功能性呼吸障碍是颈脊髓损伤（cSCI）后发病和死亡的重要原因。的能力 在颈脊髓损伤（cSCI）后的慢性期恢复呼吸是压倒性的但重要的目标。的 膈是主要的吸气肌，由位于颈部的膈运动神经元（PMNs）支配 脊髓（C3-C5）。颈中间神经元与中性粒细胞突触并与膈神经元同步放电。 自动输出和调节呼吸。前运动神经元在吻腹呼吸神经元群 脑干中的rVRG（rVRG）为脊髓呼吸回路提供主要的吸气驱动。颈髓 损伤（SCI）破坏rVRG和脊髓呼吸回路之间的通信，导致显著的 呼吸系统受损我们最近证明，药物遗传学刺激颈部兴奋性神经元- cSCI后立即使用terneurons（eIN）挽救小鼠的呼吸;然而，尚不清楚这种策略是否会 有效改善cSCI慢性期的呼吸。在慢性阶段，呼吸网络 已知会发生重大变化。此外，我们以前在非创伤性压迫性损伤方面的工作， 颈髓证实，颈段eINs在促进可塑性和主要神经元功能方面起着不可或缺的作用。 尽管中性粒细胞大量丢失，但仍保持通气。考虑到宫颈上皮内瘤细胞与呼吸系统结合- 工作，他们是必要的自主呼吸恢复，他们出现作为关键的治疗焦油， 用于创伤性脊髓损伤后慢性期的呼吸恢复。我们假设，提供选择- 在cSCI晚期，对存活的PMNs的积极兴奋性输入将增强PMNs的输出和呼吸。 复苏本提案的第一个目标旨在更深入地了解呼吸系统的状况， 慢性脊髓损伤后的神经网络。本提案的第二个目标是研究一种新的治疗方法， 选择性刺激宫颈上皮内神经促进慢性期呼吸恢复的策略 CSCI。 在目的1中，我们将使用单突触标记评估C2半切损伤（C2 Hx）后中性粒细胞的存活情况。 逆行示踪剂霍乱毒素亚单位B，以特异性地追踪中性粒细胞。在目标1的第二部分，我们将 同时映射颈部呼吸元件、PMNs和 膈前颈部eIN，在未受损伤状态和cSCI后。在目标2中，我们将选择性地刺激 使用DREADD技术观察慢性cSCI后膈核区域的颈部eINs。这种技术 由于其非侵入性地激活或沉默神经元群体的能力而具有创新应用。效果 刺激颈部eINs促进慢性cSCI后呼吸恢复的作用将使用电- 肌描记术和全身体积描记术。所提出的实验的预期结果是INNO- 因为它将增加我们对慢性cSCI后脊髓呼吸网络的了解， 开发一种新的策略来恢复cSCI慢性期的呼吸。这些结果将有一个 对SCI退伍军人的健康和生存产生了有意义的积极影响，这里研究的策略 cSCI后慢性期患者脱离机械通气的可能性 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， 在美国，大约7%的SCI患者在积极治疗SCI的同时， 服务.受伤通常是由于穿透伤，如枪击或高威力爆炸装置。 因此，SCI的军事人员面临的长期残疾甚至比那些患有SCI的平民所承受的更长。 这些问题在患有cSCI和呼吸功能障碍的退伍军人中更为明显。

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.