Peripheral and central nociceptive sensitization is a critical pathogenetic component in osteoarthritis (OA) chronic pain. T-type calcium channel 3.2 (CaV3.2) regulates neuronal excitability and plays important roles in pain processing. We previously identified that enhanced T-type/CaV3.2 activity in the primary sensory neurons (PSNs) of dorsal root ganglia (DRG) is associated with neuropathic pain behavior in a rat model of monosodium iodoacetate (MIA)-induced knee OA. PSN-specific T-type/CaV3.2 may therefore represent an important mediator in OA painful neuropathy. Here, we test the hypothesis that the T-type/CaV3.2 channels in PSNs can be rationally targeted for pain relief in MIA-OA. MIA model of knee OA was induced in male and female rats by a single injection of 2 mg MIA into intra-knee articular cavity. Two weeks after induction of knee MIA-OA pain, recombinant adeno-associated viruses (AAV)-encoding potent CaV3.2 inhibitory peptide aptamer 2 (CaV3.2iPA2) that have been characterized in our previous study were delivered into the ipsilateral lumbar 4/5 DRG. Effectiveness of DRG-CaV3.2iPA2 treatment on evoked (mechanical and thermal) and spontaneous (conditioned place preference) pain behavior, as well as weight-bearing asymmetry measured by Incapacitance tester, in the arthritic limbs of MIA rats were evaluated. AAV-mediated transgene expression in DRG was determined by immunohistochemistry. AAV-mediated expression of CaV3.2iPA2 selective in the DRG-PSNs produced significant and comparable mitigations of evoked and spontaneous pain behavior, as well as normalization of weight-bearing asymmetry in both male and female MIA-OA rats. Analgesia of DRG-AAV-CaV3.2iPA1, another potent CaV3.2 inhibitory peptide, was also observed. Whole-cell current-clamp recordings showed that AAV-mediated CaV3.2iPA2 expression normalized hyperexcitability of the PSNs dissociated from the DRG of MIA animals, suggesting that CaV3.2iPA2 attenuated pain behavior by reversing MIA-induced neuronal hyperexcitability. Together, our results add therapeutic support that T-type/CaV3.2 in primary sensory pathways contributes to MIA-OA pain pathogenesis and that CaV3.2iPAs are promising analgesic leads that, combined with AAV-targeted delivery in anatomically segmental sensory ganglia, have the potential for further development as a peripheral selective T-type/CaV3.2-targeting strategy in mitigating chronic MIA-OA pain behavior. Validation of the therapeutic potential of this strategy in other OA models may be valuable in future study.

中文翻译：

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选择性阻断感觉神经元 T 型/Cav3.2 活性可减轻大鼠骨关节炎疼痛模型中的神经性疼痛行为

外周和中枢伤害性致敏是骨关节炎 (OA) 慢性疼痛的关键致病成分。T 型钙通道 3.2 (CaV3.2) 调节神经元兴奋性并在疼痛处理中起重要作用。我们之前发现，背根神经节 (DRG) 的初级感觉神经元 (PSN) 中增强的 T 型/CaV3.2 活性与碘乙酸钠 (MIA) 诱导的膝关节 OA 大鼠模型中的神经性疼痛行为相关。因此，PSN 特异性 T 型/CaV3.2 可能代表 OA 疼痛性神经病变的重要介质。在这里，我们检验了 PSN 中的 T 型/CaV3.2 通道可以合理地靶向 MIA-OA 疼痛缓解的假设。在雄性和雌性大鼠的膝关节腔内单次注射 2 mg MIA，可诱导膝关节 OA 的 MIA 模型。膝关节 MIA-OA 疼痛诱导两周后，将我们先前研究中已表征的编码有效 CaV3.2 抑制肽适体 2 (CaV3.2iPA2) 的重组腺相关病毒 (AAV) 递送到同侧腰椎 4/ 5根病根。评估了 MIA 大鼠关节炎肢体中 DRG-CaV3.2iPA2 治疗对诱发（机械和热）和自发（条件性位置偏好）疼痛行为以及失能测试仪测量的负重不对称性的有效性。通过免疫组织化学测定 DRG 中 AAV 介导的转基因表达。AAV 介导的 CaV3.2iPA2 在 DRG-PSN 中的选择性表达在雄性和雌性 MIA-OA 大鼠中对诱发和自发性疼痛行为产生了显着且相当的缓解，以及负重不对称的正常化。还观察到另一种有效的 CaV3.2 抑制肽 DRG-AAV-CaV3.2iPA1 的镇痛作用。全细胞电流钳记录显示，AAV 介导的 CaV3.2iPA2 表达使与 MIA 动物的 DRG 分离的 PSN 的过度兴奋性正常化，这表明 CaV3.2iPA2 通过逆转 MIA 诱导的神经元过度兴奋性来减轻疼痛行为。总之，我们的结果增加了治疗支持，即初级感觉通路中的 T 型/CaV3.2 有助于 MIA-OA 疼痛发病机制，并且 CaV3.2iPA 是有希望的镇痛药，与解剖节段性感觉神经节中的 AAV 靶向递送相结合，具有作为减轻慢性 MIA-OA 疼痛行为的外周选择性 T 型/CaV3.2 靶向策略进一步发展的潜力。