Osteoarthritis Progression And Sensory Pathway Alterations

描述(由申请人提供)：骨关节炎(OA)疼痛是一个巨大的健康负担。患者在疾病过程中描述了不同类型的疼痛，包括具有机械特征的疼痛、神经病理因素和间歇性自发性疼痛。躯体感觉障碍的迹象，如机械性痛觉异常、本体感觉丧失和振动障碍已有报道。专门的感觉神经元调节不同的感觉，包括触摸、本体感觉、振动和不同类型的伤害性刺激。因此，我们认为，骨性关节炎的“痛”不仅仅是关节内伤害性神经元被激活的一种状态，而是涉及到伤害性神经元的特殊亚群和非伤害性A纤维中的感觉神经元的广泛变化。我们的总体目标是确定进行性骨性关节炎过程中神经元亚群的这些变化。我们将使用一个经过验证的小鼠模型，内侧半月板失稳(DMM)，它捕捉到了OA的长期进展。在这个模型中，疼痛依赖行为随着时间的推移而改变，早期出现膝关节痛觉过敏和机械性超敏，而持续疼痛的症状直到DMM后8-16周才明显。这为纵向评估神经元特性提供了一个独特的机会。我们的策略将包括三种不同的方法：(1)通过逆行标记和免疫组织化学方法，确定膝关节炎过程中背根神经节(DRG)和关节中特定感觉神经元亚群的变化-关节和支配DRG的L2-L5感觉神经元亚群将被记录下来。具体地说，我们将描述肽能和非肽能C纤维、非伤害性A？纤维的数量和位置，以及包括CCR2和Nav1.8在内的标记的表达。我们将在一个独特的人类关节组织库中验证我们的发现，其中包括半月板病理患者、膝骨性关节炎的早期和晚期；(2)为了表征OA疼痛起始和维持期间感觉神经元亚群的生理特性-我们将通过电生理学分析A和C纤维的单个亚群的活性 在完整的背根节、L3/L4上记录，并测定自发活动以及对包括MCP-1、辣椒素和ATP在内的刺激的反应。我们还将使用新型Pirt-GCaMP3小鼠来对自然感觉刺激(包括膝盖上的机械刺激)做出反应时体内感觉神经元细胞内钙离子进行成像；(3)为了研究选择性沉默神经元亚群对不同OA疼痛行为的影响--我们将测试选择性阿片受体对疼痛依赖行为的影响。我们还将使用新的DREADD技术来瞬时沉默所有感觉性DRG神经元(使用PAN-DRG Advilin启动子)或部分伤害性感受器(使用Nav1.8启动子)。该提案结合了神经解剖学技术、电生理测量、活体成像和最先进的可逆性沉默神经元的方法，以评估在翻译的OA模型中导致症状的感觉神经元群体的变化。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) pain represents an enormous health burden. Patients describe different types of pain over the course of the disease, including pain with mechanical characteristics, neuropathic elements, and intermittent spontaneous pain. Signs of somatosensory disturbances such as mechanical allodynia, loss of proprioception, and vibratory deficits have been reported. Specialized sensory neurons mediate different sensations, including touch, proprioception, vibration, and different types of noxious stimuli. Therefore, we propose that "pain" in OA is more than merely a state where nociceptive neurons in the joint are activated, but rather involves widespread changes in sensory neurons, both in specialized subsets of nociceptive neurons and in non-nociceptive A¿ fibers. Our overall goal is to identify these changes in subsets of neurons in the course of progressive OA. We will use a validated murine model, destabilization of the medial meniscus (DMM) that captures the long-term progression of OA. In this model, pain-dependent behaviors change over time, with knee hyperalgesia and mechanical allodynia presenting early on, while symptoms of ongoing pain are not apparent until 8-16 weeks post DMM. This provides a unique opportunity to longitudinally assess neuronal properties. Our strategy will include three different approaches: (1) To define changes in specific subsets of sensory neurons in dorsal root ganglia (DRG) and joints in the course of knee OA - Subsets of sensory neurons in the joint and innervating DRG, L2-L5, will be documented throughout the progression of disease, through retrograde labeling and immunohistochemistry. Specifically, we will describe the number and location of peptidergic and non-peptidergic C-fibers, of non-nociceptive A¿-fibers, and their expression of markers including CCR2 and Nav1.8. We will validate our findings in a unique human joint tissue bank including patients with meniscal pathology, early and advanced stages of knee OA; (2) To characterize physiological properties of subsets of sensory neurons during initiation and maintenance of OA pain- We will analyse activity of individual subsets of A and C-fibers through electrophysiological recordings on intact DRG, L3/L4, and determine spontaneous activity as well as responses to stimuli including MCP-1, capsaicin, and ATP. We will also employ novel Pirt-GCaMP3 mice to image intracellular Ca2+ in sensory neurons in vivo in response to natural sensory stimuli, including mechanical stimulation applied to the knee; (3) To study the effect of selectively silencing subsets of neurons on different OA pain behaviors - We will test the effect of a selective ¿-opioid receptor on pain-dependent behaviors. We will also employ novel DREADD technology to transiently silence all sensory DRG neurons (using the pan-DRG Advillin promoter) or a subset of nociceptors (using Nav1.8 promoter). The proposal combines neuroanatomical techniques, electrophysiological measurements, in vivo imaging, and state-of-the-art approaches to reversibly silence neurons in order to evaluate changes in populations of sensory neurons that contribute to symptoms in a translational OA model.