Yasko Jessica R, Moss Isaac L, Mains Richard E
Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, United States.
Department of Orthopedic Surgery and the Comprehensive Spine Center, University of Connecticut Health Center, Farmington, CT, United States.
Front Mol Neurosci. 2019 Nov 26;12:284. doi: 10.3389/fnmol.2019.00284. eCollection 2019.
Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently, patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons below the injury level may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15 for 1 min. Alterations in gene expression produce long-term sensory changes, therefore, we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescence-activated cell sorting (FACS). RNA was extracted from sorted sensory neurons of naïve, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed, we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.
创伤性脊髓损伤（SCI）对患者具有毁灭性影响，包括在损伤部位远端极易发生慢性疼痛。慢性疼痛在损伤数周或数月后出现，因此，患者在不可修复的变化发生后才接受治疗。伤害感受器是慢性疼痛的核心；然而，这类细胞群体的多样性给理解其机制以及将疼痛模式归因于特定细胞类型带来了挑战。为了开始探讨损伤水平以下的外周感觉神经元如何导致SCI患者报告的损伤水平以下疼痛，我们研究了损伤部位以下腰段背根神经节（DRG）中SCI诱导的基因表达变化。在T10椎体水平进行SCI，使用闭合压力为15的血管夹造成损伤，持续1分钟。基因表达的改变会产生长期的感觉变化，因此，我们感兴趣的是在慢性疼痛发作之前研究SCI诱导的转录本，这可能会触发下游信号通路的变化，并最终促进疼痛的传递。为了检查损伤部位远端DRG中伤害感受器亚群的变化，我们用荧光染料逆行标记投射到多毛后爪皮肤的感觉神经元，并在损伤后4天收集相应的腰段（L2-L6）DRG。解离后，通过荧光激活细胞分选（FACS）纯化标记的神经元。从未受伤、假手术或SCI小鼠的分选感觉神经元中提取RNA并进行测序。转录本丰度验证了所需的伤害感受器群体已被分离。与类似研究的数据集进行交叉比较证实，我们能够分离出感兴趣的细胞，并在投射到多毛后爪皮肤的神经元亚群中识别出独特的基因表达模式。差异基因表达分析显示，在与慢性疼痛发作相关的SCI细胞群体中，损伤后4天基因表达水平较高且转录本有显著变化。通路分析预测的调控相互关系表明，突触形成信号通路以及与炎症信号机制相关的网络存在变化，这表明突触可塑性在从急性疼痛向慢性疼痛转变中起作用，并与促炎信号相关。
