The spinal N-methyl-D-aspartate (NMDA) receptor, and particularly its NR2B subunit, plays a pivotal role in neuropathic pain. However, the role of peripheral NMDA receptor in neuropathic pain is less well understood. We first treated cultured human keratinocytes, HaCaT cells with NMDA or NR2B-specific antagonist, ifenprodil and evaluated the level of total and phosphorylated NR2B at 24h using Western blot. Next, using the chronic post-ischemia pain (CPIP) model, we administered NMDA or ifenprodil subcutaneously into the hind paws of male rats. Nociceptive behaviors were assessed by measuring mechanical and thermal withdrawal thresholds. Expression and phosphorylation of NR2B on keratinocyte were analyzed at 6, 12, 18, and 24 h on day 1 (initiation of pain) as well as day 2, 6, 10 and 14 (development and maintenance of pain) after the ischemia. The level of peripheral sensitization-related proteins (nuclear factor-κB (NF-κB), extracellular regulated protein kinases (ERK), and interleukin-1β (IL-1β)) in epidermis and dorsal root ganglion (DRG) were evaluated by immunofluorescence and western blot. Central sensitization-related C-fos induction, as well as astrocytes and microglia activation in the spinal cord dorsal horn (SDH) were studied using immunofluorescence. Administration of NMDA upregulated NR2B phosphorylation on HaCaT cells. CPIP-induced mechanical allodynia and thermal hyperalgesia were intensified by NMDA and alleviated by ifenprodil. CPIP resulted in an early upregulation of NR2B (peaked at 24h) and late phosphorylation of NR2B (peaked at 14d) in hindpaw keratinocytes. CPIP led to an upregulation and phosphorylation of NF-κB and ERK, as well as an increased IL-1β production in the ipsilateral skin and DRG. CPIP-associated c-fos induction in SDH persisted from acute to chronic stages after ischemia, while microglia and astrocyte activation were only observed in chronic phase. These CPIP-induced changes were also suppressed by ifenprodil administered subcutaneously in the hind paw. Our findings reveal a previously unrecognized role of keratinocyte NMDA receptor subunit 2B in peripheral and central nociceptive sensitization induced by CPIP.

中文翻译：

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角质形成细胞上的 N-甲基-d-天冬氨酸受体亚基 2B 介导雄性大鼠慢性缺血后疼痛的外周和中枢敏化

脊髓 N-甲基-D-天冬氨酸 (NMDA) 受体，特别是其 NR2B 亚基，在神经性疼痛中起关键作用。然而，外周 NMDA 受体在神经性疼痛中的作用尚不清楚。我们首先用 NMDA 或 NR2B 特异性拮抗剂艾芬地尔处理培养的人角质形成细胞 HaCaT 细胞，并使用蛋白质印迹评估 24 小时总和磷酸化 NR2B 的水平。接下来，使用慢性缺血后疼痛 (CPIP) 模型，我们将 NMDA 或艾芬地尔皮下注射到雄性大鼠的后爪中。通过测量机械和热退缩阈值来评估伤害性行为。在缺血后第 1 天（疼痛开始）的 6、12、18 和 24 小时以及第 2、6、10 和 14 天（疼痛的发展和维持）分析角质形成细胞上 NR2B 的表达和磷酸化。通过免疫荧光评估表皮和背根神经节 (DRG) 中外周致敏相关蛋白（核因子-κB (NF-κB)、细胞外调节蛋白激酶 (ERK) 和白细胞介素-1β (IL-1β)）的水平和蛋白质印迹。使用免疫荧光研究了与中枢敏化相关的 C-fos 诱导，以及脊髓背角 (SDH) 中的星形胶质细胞和小胶质细胞活化。施用 NMDA 上调 HaCaT 细胞上的 NR2B 磷酸化。CPIP 引起的机械性异常性疼痛和热痛觉过敏被 NMDA 增强，而艾芬地尔则减轻。CPIP 导致后爪角质形成细胞中 NR2B 的早期上调（在 24 小时达到峰值）和 NR2B 的晚期磷酸化（在 14 天达到峰值）。CPIP 导致 NF-κB 和 ERK 的上调和磷酸化，以及同侧皮肤和DRG中增加的IL-1β产生。SDH 中 CPIP 相关的 c-fos 诱导在缺血后从急性期持续到慢性期，而仅在慢性期观察到小胶质细胞和星形胶质细胞的活化。这些 CPIP 诱导的变化也被后爪皮下给药的艾芬地尔抑制。我们的研究结果揭示了角质形成细胞 NMDA 受体亚基 2B 在 CPIP 诱导的外周和中枢伤害性致敏中的先前未被认识的作用。