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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain.
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Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain.

机译：大鼠神经性疼痛模型中脊髓小胶质细胞和星形细胞激活标记的实时定量RT-PCR评估。

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摘要

Activated spinal glial cells have been strongly implicated in the development and maintenance of persistent pain states following a variety of stimuli including traumatic nerve injury. The present study was conducted to characterize the time course of surface markers indicative of microglial and astrocytic activation at the transcriptional level following an L5 nerve transection that results in behavioral hypersensitivity. Male Sprague-Dawley rats were divided into a normal group, a sham surgery group with an L5 spinal nerve exposure and an L5 spinal nerve transected group. Mechanical allodynia (heightened response to a non-noxious stimulus) of the ipsilateral hind paw was assessed throughout the study. Spinal lumbar mRNA levels of glial fibrillary acidic protein (GFAP), integrin alpha M (ITGAM), toll-like receptor 4 (TLR4) and cluster determinant 14 (CD14) were assayed using real-time reverse transcription polymerase chain reaction (RT-PCR) at 4 h, 1, 4, 7, 14 and 28 days post surgery. The spinal lumbar mRNA expression of ITGAM, TLR4, and CD14 was upregulated at 4 h post surgery, CD14 peaked 4 days after spinal nerve transection while ITGAM and TLR4 continued to increase until day 14 and returned to almost normal levels by postoperative day 28. In contrast, spinal GFAP mRNA did not significantly increase until postoperative day 4 and then continued to increase over the duration of the study. Our optimized real-time RT-PCR method was highly sensitive, specific and reproducible at a wide dynamic range. This study demonstrates that peripheral nerve injury induces an early spinal microglial activation that precedes astrocytic activation using mRNA for surface marker expression; the delayed but sustained expression of mRNA coding for GFAP implicates astrocytes in the maintenance phase of persistent pain states. In summary, these data demonstrate a distinct spinal glial response following nerve injury using real-time RT-PCR.
机译：激活的脊髓神经胶质细胞与包括创伤性神经损伤在内的各种刺激后的持续性疼痛状态的发展和维持密切相关。进行本研究以表征指示L5神经横断后导致行为超敏反应的转录水平上小胶质细胞和星形胶质细胞活化的表面标志物的时程。将雄性Sprague-Dawley大鼠分为正常组，具有L5脊神经暴露的假手术组和L5脊神经横断组。在整个研究中评估了同侧后爪的机械性异常性疼痛（对非有害刺激的增强反应）。使用实时逆转录聚合酶链反应（RT-PCR）测定了神经胶质纤维酸性蛋白（GFAP），整联蛋白αM（ITGAM），通行费样受体4（TLR4）和簇决定簇14（CD14）的腰椎mRNA水平）在手术后4小时，1、4、7、14和28天。术后4 h，ITGAM，TLR4和CD14的腰椎mRNA表达上调，脊髓神经横断后4天CD14达到峰值，而ITGAM和TLR4持续增加直到第14天，并在术后第28天恢复到几乎正常水平。相反，直到手术后第4天，脊髓GFAP mRNA才显着增加，然后在研究期间继续增加。我们优化的实时RT-PCR方法在广泛的动态范围内具有很高的灵敏度，特异性和可重现性。这项研究表明，周围神经损伤可诱导早期的脊髓小胶质细胞活化，先于星形胶质细胞活化而使用mRNA表达表面标记。编码GFAP的mRNA的延迟表达但持续表达，提示星形胶质细胞处于持续性疼痛状态的维持阶段。总之，这些数据证明了使用实时RT-PCR的神经损伤后脊髓神经胶质的独特反应。

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