Data from: Down-regulation of CXCL12/CXCR4 expression alleviates ischemia-reperfusion-induced inflammatory pain via inhibiting glial TLR4 activation in the spinal cord

Toll-like receptor 4 (TLR4) is important for the pathogenesis of inflammatory reactions and the promotion of pain processing after ischemia/reperfusion (IR) in spinal cord. Recently, C-X-C chemokine ligand 12 (CXCL12) and its receptor, C-X-C chemokine receptor 4 (CXCR4), were demonstrated to be simultaneously critical for inflammatory reactions, thereby facilitating glial activation. However, whether CXCL12/CXCR4 expression can contribute to IR-induced inflammatory pain via spinal TLR4 remained unclear. A rat model was established by 8 min of aortic arch occlusion. The effects of CXCL12/CXCR4 expression and TLR4 activation on inflammatory hyperalgesia were investigated by pretreatments with CXCL12-neutralizing antibody, CXCR4 antagonist (AMD3100) and TLR4 antagonist (TAK-242) for 5 consecutive days before surgery. The results indicated that IR induced significant and sustained inflammatory pain, observed as decreases in paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), throughout the post-injury period. The increased levels of TLR4 and proinflammatory chemokine CXCL12, as well as its receptor, CXCR4, were closely correlated with the PWT and PWL trends. Double immunostaining further suggested that TLR4, which is mainly expressed on astrocytes and microglia, was closely co-localized with CXCL12 and CXCR4 in spinal dorsal horn. As expected, intrathecal pretreatment with the TLR4 antagonist, TAK-242 markedly ameliorated pain by inhibiting astrocytic and microglial activation, as shown by decreases in TLR4 immunoreactivity and the percentage of double-labeled cells. These protective effects were likely due in part to the reduced production of the downstream cytokines IL-1β and TNF-α, as well as for the recruitment of CXCL12 and CXCR4. Additionally, intrathecal pretreatment with CXCL12-neutralizing antibody and AMD3100 resulted in similar analgesic and anti-inflammatory effects as those receiving TAK-242 pretreatment. These results suggest that intrathecal blockade of CXCL12/CXCR4 expression may attenuate IR-induced pain sensation and the release of inflammatory cytokines by limiting glial TLR4 activation in spinal cord.

Toll样受体4（Toll-like receptor 4，TLR4）在脊髓缺血再灌注（ischemia/reperfusion，IR）后的炎症反应发病机制以及疼痛信号传递过程中发挥关键作用。近期研究证实，C-X-C趋化因子配体12（C-X-C chemokine ligand 12，CXCL12）及其受体C-X-C趋化因子受体4（C-X-C chemokine receptor 4，CXCR4）同样对炎症反应至关重要，可促进神经胶质细胞活化。但目前尚不明确CXCL12/CXCR4的表达是否可通过脊髓TLR4通路参与缺血再灌注诱导的炎症性疼痛。
本研究通过8分钟主动脉弓阻断术构建大鼠缺血再灌注模型。于术前连续5天分别给予CXCL12中和抗体、CXCR4拮抗剂AMD3100及TLR4拮抗剂TAK-242进行预处理，探究CXCL12/CXCR4表达与TLR4活化对炎症性痛觉超敏的调控效应。
研究结果显示，缺血再灌注可在损伤后全程诱导显著且持续的炎症性疼痛，表现为缩爪阈值（paw withdrawal threshold，PWT）与缩爪潜伏期（paw withdrawal latency，PWL）显著降低。TLR4、促炎趋化因子CXCL12及其受体CXCR4的表达水平上调，与PWT和PWL的变化趋势密切相关。双重免疫染色（double immunostaining）结果进一步提示，主要表达于星形胶质细胞（astrocyte）与小胶质细胞（microglia）的TLR4，在脊髓背角中与CXCL12、CXCR4存在紧密的共定位现象。
如预期所示，鞘内预先给予TLR4拮抗剂TAK-242可通过抑制星形胶质细胞与小胶质细胞活化，显著缓解疼痛，具体表现为TLR4免疫反应性及双标细胞占比均有所降低。该保护作用可能部分归因于下游细胞因子白细胞介素1β（IL-1β）与肿瘤坏死因子α（TNF-α）的生成减少，以及CXCL12与CXCR4的招募被抑制。此外，鞘内预先给予CXCL12中和抗体与AMD3100，可产生与TAK-242预处理相似的镇痛与抗炎效果。
上述结果表明，鞘内阻断CXCL12/CXCR4的表达，可通过限制脊髓内神经胶质细胞TLR4的活化，减轻缺血再灌注诱导的疼痛感知及炎症细胞因子的释放。