Project 2: Pro-inflammatory Cytokines Engaged in Muscle Afferent-Mediated Sympathetic Responsiveness with Femoral Artery Occlusion

项目摘要/摘要--项目2 收缩骨骼肌产生的运动加压反射是两种神经之一。 激发交感神经和心血管调节运动的机制。这其中的反常之处 在心血管疾病中，反射影响运动肌肉的血流输送和氧气供应。 外周动脉疾病(PAD)是常见的致命性动脉粥样硬化性血管疾病的表现。 这个项目的主要目标是更好地理解促炎细胞因子在控制中的作用。 诱发运动加压反射的细纤维(组III和组IV)肌肉传入神经元的兴奋性 垫子。对于薄纤维肌肉传入，我们建议结合体外全细胞贴片的力量- 钳夹技术，这将确定传入兴奋的机制，与体内提供的见解 这将决定这些机制如何转化为更强的兴奋性。特例 注意具有河豚毒素敏感和耐药钠通道的传入神经。对于体外实验， 通过标记背根神经节(DRG)逆行神经元来识别肌肉传入 荧光示踪剂DiI注射到正常大鼠和慢性阻塞性肺疾病大鼠的小腿三头肌中 股动脉闭塞。此外，分离的DRG细胞将通过它们的瞬时表达来识别 绿色荧光蛋白，其表达由近端神经元特异性启动子区域驱动 TTX敏感的钠通道(Nav1.7)和抗TTX的钠通道(NaV1.8)。对于活体内 实验中，由肌肉收缩引起的交感反应性将在对照组大鼠和 股动脉闭塞大鼠。在体外和体内实验中，将特别关注 PICS，即IL-6和肿瘤坏死因子-α对传入神经元膜和放电特性的影响。 此外，细胞因子受体和控制细纤维肌肉兴奋性的通道之间的相互作用 将对传入信息进行检查。此外，在兴奋过程中参与的细胞内信号通路 图片将会被检查。我们的一般假设是，在肌肉中诱导更高水平的PIC受体 大鼠后肢血流阻断后背根神经节神经元的变化。这进而改变了细胞内的 转导途径和钠通道的表达和功能，从而导致 运动升压反射。拟议中的实验预计将提供关于 PICS对运动加压反射传入臂兴奋性的影响机制 垫子。

PROJECT SUMMARY/ABSTRACT – PROJECT 2 The exercise pressor reflex, which arises from contracting skeletal muscles, is one of the two neural mechanisms that evoke the sympathetic and cardiovascular adjustments to exercise. The abnormalities in this reflex affect blood flow delivery and oxygen supply to exercising muscles in cardiovascular diseases. Peripheral arterial disease (PAD) is a manifestation of common and lethal atherosclerotic vascular disorders. The major goal of this project is to better understand the role of pro-inflammatory cytokines (PICs) in controlling excitability of the thin fiber (group III & IV) muscle afferent neurons that evoke the exercise pressor reflex in PAD. With respect to thin fiber muscle afferents, we propose to combine the power of in vitro whole-cell patch- clamp techniques, which will determine mechanisms of afferent excitability, with the insights provided by in vivo physiology, which will determine how these mechanisms translate into increased excitability. Particular attention will be paid to afferents that have TTX sensitive and resistant Na+ channels. For in vitro experiments, muscle afferents will be identified by labeling dorsal root ganglion (DRG) neurons with the retrograde fluorescent tracer DiI that has been injected into the triceps surae muscles of control rats and rats with chronic occlusion of the femoral artery. Moreover, the isolated DRG cells will be identified by their transient expression of green fluorescent protein, the expression of which is driven by the proximal neuron specific promoter region of the TTX sensitive Na+ channel (NaV1.7) and TTX-resistant Na+ channel (NaV1.8). For the in vivo experiments, sympathetic responsiveness evoked by muscle contraction will be examined in control rats and rats with femoral artery occlusion. In both in vitro and in vivo experiments, particular attention will be paid to the effects of PICs, namely IL-6 and TNF-α, on the membrane and discharge properties of the afferent neurons. Also, the interplay between cytokine receptors and channels that control excitability of the thin fiber muscle afferents will be examined. Additionally, intracellular signaling pathways that are engaged during excitation by PICs will be examined. Our general hypothesis is that the higher levels of PIC receptors are induced in muscle DRG neurons after hindlimb blood flow occlusion in the rat model of PAD. This in turn alters the intracellular transduction pathways and the expression and function of Na+ channels, thereby leading to the augmented exercise pressor reflex. The proposed experiments are anticipated to provide new information regarding the mechanisms by which the excitability of the afferent arm of the exercise pressor reflex is affected by PICs in PAD.