Bordeaux University, Bordeaux, France.
Neurocentre Magendie, INSERM U1215, Bordeaux, France.
J Neuroinflammation. 2018 Dec 20;15(1):349. doi: 10.1186/s12974-018-1378-z.
Spinal reactive astrocytes and microglia are known to participate to the initiation and maintenance of neuropathic pain. However, whether reactive astrocytes and microglia in thalamic nuclei that process sensory-discriminative aspects of pain play a role in pain behavior remains poorly investigated. Therefore, the present study evaluated whether the presence of reactive glia (hypertrophy, increased number and upregulation of glial markers) in the ventral posterolateral thalamic nucleus (VPL) correlates with pain symptoms, 14 and 28 days after unilateral L5/L6 spinal nerve ligation (SNL) in rats.
Mechanical allodynia and hyperalgesia (von Frey filament stimulation) as well as ambulatory pain (dynamic weight bearing apparatus) were assessed. Levels of nine glial transcripts were determined by quantitative real-time PCR on laser microdissected thalamic nuclei, and levels of proteins were assessed by Western blot. We also studied by immunohistofluorescence the expression of glial markers that label processes (GFAP for astrocytes and iba-1 for microglia) and cell body (S100β for astrocytes and iba-1 for microglia) and quantified the immunostained surface and the number of astrocytes and microglia (conventional counts and optical dissector method of stereological counting).
Differential, time-dependent responses were observed concerning microglia and astrocytes. Specifically, at day 14, iba-1 immunostained area and number of iba-1 immunopositive cells were decreased in the VPL of SNL as compared to naïve rats. By contrast, at day 28, GFAP-immunostained area was increased in the VPL of SNL as compared to naïve rats while number of GFAP/S100β immunopositive cells remained unchanged. Using quantitative real-time PCR of laser microdissected VPL, we found a sequential increase in mRNA expression of cathepsin S (day 14), fractalkine (day 28), and fractalkine receptor (day 14), three well-known markers of microglial reactivity. Using Western blot, we confirmed an increase in protein expression of fractalkine receptor at day 14.
Our results demonstrate a sequential alteration of microglia and astrocytes in the thalamus of animals with lesioned peripheral nerves. Furthermore, our data report unprecedented concomitant molecular signs of microglial activation and morphological signs of microglial decline in the thalamus of these animals.
已知脊髓反应性星形胶质细胞和小胶质细胞参与神经病理性疼痛的起始和维持。然而，处理疼痛感觉辨别方面的丘脑核内的反应性星形胶质细胞和小胶质细胞是否在疼痛行为中起作用仍未得到充分研究。因此，本研究评估了在大鼠单侧 L5/L6 脊神经结扎（SNL）后 14 天和 28 天，腹后外侧丘脑核（VPL）中反应性胶质细胞（肥大、数量增加和胶质标志物上调）的存在是否与疼痛症状相关。
通过 von Frey 纤维刺激评估机械性痛觉过敏和痛觉过度（痛觉）以及活动性疼痛（动态体重支撑装置）。通过定量实时 PCR 激光微切割丘脑核确定 9 种胶质转录物的水平，并通过 Western blot 评估蛋白质水平。我们还通过免疫荧光研究了标记过程（星形胶质细胞的 GFAP 和小胶质细胞的 iba-1）和细胞体（星形胶质细胞的 S100β和小胶质细胞的 iba-1）的胶质标志物的表达，并对免疫染色表面和星形胶质细胞和小胶质细胞的数量进行了量化（常规计数和立体学计数的光学切割器方法）。
观察到小胶质细胞和星形胶质细胞的差异、时间依赖性反应。具体来说，在第 14 天，与正常大鼠相比，SNL 的 VPL 中 iba-1 免疫染色面积和 iba-1 免疫阳性细胞数量减少。相比之下，在第 28 天，与正常大鼠相比，SNL 的 VPL 中 GFAP-免疫染色面积增加，而 GFAP/S100β 免疫阳性细胞数量保持不变。通过激光微切割 VPL 的定量实时 PCR，我们发现三种已知的小胶质细胞反应性标志物——组织蛋白酶 S（第 14 天）、 fractalkine（第 28 天）和 fractalkine 受体（第 14 天）的 mRNA 表达依次增加。通过 Western blot，我们证实了 fractalkine 受体蛋白表达在第 14 天增加。
我们的结果表明，在有外周神经损伤的动物的丘脑内，小胶质细胞和星形胶质细胞发生了顺序改变。此外，我们的数据报告了动物丘脑内前所未有的小胶质细胞激活的分子迹象和小胶质细胞减少的形态迹象同时出现。
