Aims

Activated microglia is known as a main mediator of inflammatory pain, but the possible mechanisms of its operation are poorly understood. Microglial cells have considered as one of the main sources of pro-inflammatory cytokines in the CNS. PTEN is one of the important targets of pro-inflammatory cytokines and the main mediator of apoptotic cell death. In this study, we investigated the possible effect of microglial activation on PTEN/PI3K/Akt signaling pathway and apoptosis in an inflammatory rat model of Complete Freund’s adjuvant (CFA).

Methods

Persistent peripheral inflammation was induced by a subcutaneous injection of CFA into the rats’ right hind paw on day 0. Minocycline (a potent selective inhibitor of microglial) was administered intraperitoneally during days 1–21 after CFA injection. Hyperalgesia was assessed on days 0, 7, and 21 using plantar test, then lumbar spinal cord segments were isolated, and the amount of spinal Iba1 (microglial marker), PTEN, P.Akt, and cleaved caspase-3 (a marker of apoptosis activation) were analyzed using Western blot. The spinal TNF-α levels were assayed by ELISA and the microglia numbers were determined using immunohistochemical technique.

Results

Results revealed that increased hyperalgesia was concurrent with an increment of Iba1 (P < 0.001), TNF-α (P < 0.001), PTEN (P < 0.01), cleaved caspase-3 (P < 0.001), and a decrement of P.Akt (P < 0.01) during the acute phase of CFA-induced inflammation, while, at the same time as decreasing hyperalgesia during the chronic phase of study, Iba1 and TNF-α expression significantly decreased and PTEN, cleaved caspase-3, and P.Akt restored to baseline on day 0. Minocycline administration reduced the elevation of spinal Iba1 (P < 0.001), TNF-α (0.001), PTEN (P < 0.01), and cleaved caspase-3 (P < 0.001) expression induced by CFA injection, and also restored Akt activity to the baseline on day 0 (P < 0.001).

Conclusions

These results suggest that microglial-mediated pain following CFA injection might be related in part to increased spinal cell apoptosis which probably is mediated by PTEN/PI3K/Akt deregulation.

中文翻译：

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小胶质细胞诱导的凋亡可能是CFA诱导的炎症过程中痛觉过敏变化的原因。

目的

活化的小胶质细胞被认为是炎性疼痛的主要介质，但对其操作的可能机制了解甚少。小胶质细胞被认为是中枢神经系统促炎细胞因子的主要来源之一。PTEN是促炎细胞因子的重要靶标之一，也是凋亡细胞死亡的主要介质。在这项研究中，我们调查了在完全弗氏佐剂（CFA）的炎性大鼠模型中，小胶质细胞激活对PTEN / PI3K / Akt信号通路和细胞凋亡的可能影响。

方法

在第0天，将CFA皮下注射到大鼠的右后爪中，引起持久性外周炎症。在注射CFA后的第1-21天，腹膜内给予米诺环素（一种有效的小胶质细胞选择性抑制剂）。在第0、7和21天使用足底试验评估痛觉过敏，然后分离腰椎脊髓节段，并测定脊髓Iba1（微胶质标记物），PTEN，P.Akt和裂解的caspase-3（凋亡标记物）的量使用蛋白质印迹分析）。用ELISA法测定脊髓的TNF-α水平，并用免疫组化技术测定小胶质细胞数。

结果

结果显示，痛觉过敏的增加与Iba1（P  <0.001），TNF-α（P  <0.001），PTEN（P  <0.01），caspase-3裂解（P  <0.001）和P的降低同时发生。 在CFA诱发的炎症急性期，Akt（P <0.01），而在慢性期研究中，随着痛觉过敏的减少，Iba1和TNF-α表达显着降低，PTEN，caspase-3和P裂解.Akt在第0天恢复到基线。Minocycline的使用降低了脊柱Iba1（P  <0.001），TNF-α（0.001），PTEN（P  <0.01）和Caspase-3裂解（P （<0.001）的CFA注射诱导表达，并在第0天将Akt活性恢复到基线（P  <0.001）。

结论

这些结果表明，注射CFA后小胶质细胞介导的疼痛可能部分与脊髓细胞凋亡的增加有关，这可能是由PTEN / PI3K / Akt失调介导的。