Functional characterization of ovine dorsal root ganglion neurons reveals peripheral sensitization after osteochondral defect
Functional characterization of ovine dorsal root ganglion neurons reveals peripheral sensitization after osteochondral defect
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绵羊背根神经节神经元的功能特征揭示骨软骨缺损后的外周敏化
DOI：
10.1101/2021.02.26.432434
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发表时间：
2021
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影响因子：
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通讯作者：
Chakrabarti S
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作者：
Chakrabarti S
膝关节创伤可导致骨软骨缺损（OD），这是骨关节炎（OA）的一个风险因素，也是导致患者衰弱性疼痛的原因。啮齿动物OD模型的可平移性较差，因为其关节尺寸较小且生长板开放。这项研究提出了绵羊作为一个神经相关模型，以了解OD疼痛的神经基础。在成年雌性绵羊中诱导单侧6 mm深OD。手术后2 ~ 6周，取手术绵羊对侧（Ctrl）和OD侧的背根神经节（DRG）神经元。采用电生理学和Ca 2+成像对疼痛相关离子通道瞬时受体电位香草酸受体1（TRPV 1）和P2 X3的神经元兴奋性和活性进行功能评估。免疫组化法检测疼痛相关蛋白的表达。我们观察到OD DRG神经元的比例增加，（绵羊，N= 3;对照神经元，n= 15，OD神经元，n= 16）显示自发电兴奋性（对照组：20.33 ± 4.5%; OD：50 ± 10%; p= 0.009，未配对t检验），并且增加的比例响应于TRPV 1激动剂的应用而激发了高于基线的更多数量的尖峰（辣椒素）应用（对照：40%; OD：75%;p= 0.04，χ 2检验）。辣椒素还在增加比例的分离的OD DRG神经元中产生Ca 2+内流（对照：25%; OD：44%;p= 0.001，χ 2检验）。OD神经元中P2 X3离子通道的蛋白表达和功能均未改变。总的来说，我们提供的证据增加的DRG神经元的兴奋性（一个重要的神经相关的疼痛）和TRPV 1功能在OD绵羊模型。我们的数据表明，羊背根神经节神经元的功能评估可以提供重要的见解OD疼痛的神经基础，从而有可能防止其进展为关节炎疼痛。
Knee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis (OA) and cause of debilitating pain in patients. Rodent OD models are less translatable because of their smaller joint size and open growth plate. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain. A unilateral 6-mm deep OD was induced in adult female sheep. Two to six weeks after operation, lumbar dorsal root ganglia (DRG) neurons were collected from the contralateral (Ctrl) and OD side of operated sheep. Functional assessment of neuronal excitability and activity of the pain-related ion channels transient receptor potential vanilloid receptor 1 (TRPV1) and P2X3 was conducted using electrophysiology and Ca2+imaging. Immunohistochemistry was used to verify expression of pain-related proteins. We observed that an increased proportion of OD DRG neurons (sheep,N= 3; Ctrl neurons,n= 15, OD neurons,n= 16) showed spontaneous electrical excitability (Ctrl: 20.33 ± 4.5%; OD: 50 ± 10%;p= 0.009, unpairedttest) and an increased proportion fired a greater number of spikes above baseline in response to application of a TRPV1 agonist (capsaicin) application (Ctrl: 40%; OD: 75%;p= 0.04, χ2test). Capsaicin also produced Ca2+influx in an increased proportion of isolated OD DRG neurons (Ctrl: 25%; OD: 44%;p= 0.001, χ2test). Neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons. Overall, we provide evidence of increased excitability of DRG neurons (an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.