Liu Liping, Yin Yan, Li Fei, Malhotra Charvi, Cheng Jianguo
Departments of Pain Management and Neurosciences, Lerner Research Institute and Anaesthesiology Institute, Cleveland Clinic, Euclid Avenue, Cleveland, OH 44195, USA.
Departments of Pain Management and Neurosciences, Lerner Research Institute and Anaesthesiology Institute, Cleveland Clinic, Euclid Avenue, Cleveland, OH 44195, USA; Department of Anesthesiology, West China Hospital of Sichuan University, 610041, China.
J Neurosci Methods. 2017 Jun 1;284:47-56. doi: 10.1016/j.jneumeth.2017.04.012. Epub 2017 Apr 23.
Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations.
We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45/CD11b cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols.
We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain.
细胞对神经损伤的反应在神经性疼痛的发病机制中起核心作用。然而，对神经损伤和神经性疼痛的位点特异性细胞反应的分析仅限于免疫组织化学染色，存在诸多局限性。
我们提议应用流式细胞术来克服其中一些局限性，并开发了两种从小鼠坐骨神经和背根神经节（DRG）的小标本中分离细胞的方案。结果及与现有方法的比较：我们发现，在正常和病理条件下，非酶法和酶法在收获足够数量的细胞用于流式细胞术分析方面都非常有效。与假手术对照组或对侧对照组相比，坐骨神经慢性压迫损伤（CCI）14天后，坐骨神经及其DRG损伤部位的细胞总数显著增加。酶法获得的细胞总数和CD45阴性细胞数显著更高，这表明与非酶法相比，该方法能够收获更多的驻留细胞。坐骨神经中CD45/CD11b细胞的百分比显著增加，但DRG中未增加。这些结果在两种方案中均一致。
因此，我们提供了两种简单有效的方案，可将流式细胞术应用于神经性疼痛细胞和分子机制的研究。
