Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, 233 South 10th Street, BLSB 245, Philadelphia, PA 19107, United States.
Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, 233 South 10th Street, BLSB 245, Philadelphia, PA 19107, United States.
Exp Neurol. 2018 May;303:108-119. doi: 10.1016/j.expneurol.2018.02.007. Epub 2018 Feb 14.
Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality in individuals suffering from traumatic cervical spinal cord injury (SCI). Repair of CNS axons after SCI remains a therapeutic challenge, despite current efforts. SCI disrupts inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla from their phrenic motor neuron (PhMN) targets, resulting in loss of diaphragm function. Using a rat model of cervical hemisection SCI, we aimed to restore rVRG-PhMN-diaphragm circuitry by stimulating regeneration of injured rVRG axons via targeted induction of Rheb (ras homolog enriched in brain), a signaling molecule that regulates neuronal-intrinsic axon growth potential. Following C2 hemisection, we performed intra-rVRG injection of an adeno-associated virus serotype-2 (AAV2) vector that drives expression of a constitutively-active form of Rheb (cRheb). rVRG neuron-specific cRheb expression robustly increased mTOR pathway activity within the transduced rVRG neuron population ipsilateral to the hemisection, as assessed by levels of phosphorylated ribosomal S6 kinase. By co-injecting our novel AAV2-mCherry/WGA anterograde/trans-synaptic axonal tracer into rVRG, we found that cRheb expression promoted regeneration of injured rVRG axons into the lesion site, while we observed no rVRG axon regrowth with AAV2-GFP control. AAV2-cRheb also significantly reduced rVRG axonal dieback within the intact spinal cord rostral to the lesion. However, cRheb expression did not promote any recovery of ipsilateral hemi-diaphragm function, as assessed by inspiratory electromyography (EMG) burst amplitudes. This lack of functional recovery was likely because regrowing rVRG fibers did not extend back into the caudal spinal cord to synaptically reinnervate PhMNs that we retrogradely-labeled with cholera toxin B from the ipsilateral hemi-diaphragm. Our findings demonstrate that enhancing neuronal-intrinsic axon growth capacity can promote regeneration of injured bulbospinal respiratory axons after SCI, but this strategy may need to be combined with other manipulations to achieve reconnection of damaged neural circuitry and ultimately recovery of diaphragm function.
呼吸神经回路的损伤以及随之而来的膈神经功能丧失，是创伤性颈脊髓损伤（SCI）患者发病率和死亡率的主要原因。尽管目前正在努力，但 SCI 后中枢神经系统轴突的修复仍然是一个治疗挑战。SCI 破坏了起源于延髓头端腹侧呼吸组（rVRG）的吸气信号向膈神经运动神经元（PhMN）的投射，导致膈神经功能丧失。我们使用大鼠颈半切 SCI 模型，通过靶向诱导雷帕霉素靶蛋白（mTOR）信号通路的激活物 Ras 相关蛋白富亮氨酸胶质瘤失活 1（Rheb），来刺激损伤的 rVRG 轴突再生，从而恢复 rVRG-PhMN-膈神经回路。Rheb 是一种调节神经元内在轴突生长潜力的信号分子。在 C2 半切后，我们在 rVRG 内注射腺相关病毒血清型 2（AAV2）载体，该载体驱动组成型激活形式的 Rheb（cRheb）的表达。通过检测磷酸化核糖体 S6 激酶的水平，我们发现 rVRG 神经元特异性 cRheb 表达在半切对侧的转导 rVRG 神经元群体中强烈增加了 mTOR 通路的活性。通过将我们新的 AAV2-mCherry/WGA 顺行/跨突触轴突示踪剂共注射到 rVRG 中，我们发现 cRheb 表达促进了损伤的 rVRG 轴突再生到损伤部位，而用 AAV2-GFP 对照则没有观察到 rVRG 轴突再生。AAV2-cRheb 还显著减少了损伤部位上方完整脊髓内 rVRG 轴突的退化。然而，cRheb 表达并没有促进同侧半膈功能的任何恢复，这可以通过吸气肌电图（EMG）爆发幅度来评估。这种功能恢复的缺乏可能是因为再生的 rVRG 纤维没有延伸回脊髓尾部，以与我们从同侧半膈用霍乱毒素 B 逆行标记的 PhMN 进行突触再神经支配。我们的发现表明，增强神经元内在的轴突生长能力可以促进 SCI 后损伤的延髓呼吸轴突的再生，但这种策略可能需要与其他操作相结合，以实现受损神经回路的重新连接，并最终恢复膈神经功能。
