Li Xiao-Qian, Lv Huang-Wei, Tan Wen-Fei, Fang Bo, Wang He, Ma Hong
Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China.
J Neuroinflammation. 2014 Mar 28;11:62. doi: 10.1186/1742-2094-11-62.
Spinal cord ischemia-reperfusion (I/R) involves two-phase injury, including an initial acute ischemic insult and subsequent inflammatory reperfusion injury, resulting in blood-spinal cord barrier (BSCB) dysfunction involving the TLR₄ pathway. However, the correlation between TLR₄/MyD₈₈-dependent and TLR₄/TRIF-dependent pathways in BSCB dysfunction is not fully understood. The aim of this study is to characterize inflammatory responses in spinal cord I/R and the events that define its clinical progression with delayed neurological deficits, supporting a bimodal mechanism of injury.
Rats were intrathecally pretreated with TAK-242, MyD₈₈ inhibitory peptide, or Resveratrol at a 12 h interval for 3 days before undergoing 14-minute occlusion of aortic arch. Evan's Blue (EB) extravasation and water content were detected at 6, 12, 18, 24, 36, 48, and 72 h after reperfusion. EB extravasation, water content, and NF-κB activation were increased with time after reperfusion, suggesting a bimodal distribution, as maximal increasing were detected at both 12 and 48 h after reperfusion. The changes were directly proportional to TLR₄ levels determined by Western blot. Double-labeled immunohistochemical analysis was also used to detect the relationship between different cell types of BSCB with TLR₄. Furthermore, NF-κB and IL-1β were analyzed at 12 and 48 h to identify the correlation between MyD₈₈-dependent and TRIF-dependent pathways.
Rats without functional TLR₄ and MyD₈₈ attenuated BSCB leakage and inflammatory responses at 12 h, suggesting the ischemic event was largely mediated by MyD₈₈-dependent pathway. Similar protective effects observed in rats with depleted TLR₄, MyD₈₈, and TRIF receptor at 48 h infer that the ongoing inflammation which occurred in late phase was mainly initiated by TRIF-dependent pathway and such inflammatory response could be further amplified by MyD₈₈-dependent pathway. Additionally, microglia appeared to play a major role in early phase of inflammation after I/R injury, while in late responding phase both microglia and astrocytes were necessary.
These findings indicate the relevance of TLR4/MyD₈₈-dependent and TLR₄/TRIF-dependent pathways in bimodal phases of inflammatory responses after I/R injury, corresponding with the clinical progression of injury and delayed onset of symptoms. The clinical usage of TLR₄ signaling inhibitors at different phases may be a therapeutic option for the prevention of delayed injury.
脊髓缺血再灌注（I/R）涉及两阶段损伤，包括初始的急性缺血性损伤和随后的炎症性再灌注损伤，导致涉及Toll样受体4（TLR₄）通路的血脊髓屏障（BSCB）功能障碍。然而，TLR₄/髓样分化因子88（MyD₈₈）依赖性通路和TLR₄/TIR结构域衔接蛋白（TRIF）依赖性通路在BSCB功能障碍中的相关性尚未完全明确。本研究的目的是明确脊髓I/R中的炎症反应以及确定其伴有延迟性神经功能缺损的临床进展的相关事件，以支持双峰损伤机制。
在大鼠接受14分钟主动脉弓阻断前3天，每隔12小时鞘内注射TAK-242、MyD₈₈抑制肽或白藜芦醇。在再灌注后6、12、18、24、36、48和72小时检测伊文思蓝（EB）外渗和含水量。再灌注后EB外渗、含水量和核因子κB（NF-κB）激活随时间增加，呈双峰分布，在再灌注后12小时和48小时均检测到最大增加。这些变化与通过蛋白质免疫印迹法测定的TLR₄水平成正比。还采用双标免疫组织化学分析来检测BSCB不同细胞类型与TLR₄之间的关系。此外，在12小时和48小时分析NF-κB和白细胞介素-1β（IL-1β），以确定MyD₈₈依赖性通路和TRIF依赖性通路之间的相关性。
缺乏功能性TLR₄和MyD₈₈的大鼠在12小时时减轻了BSCB渗漏和炎症反应，表明缺血事件主要由MyD₈₈依赖性通路介导。在48小时时，在TLR₄、MyD₈₈和TRIF受体缺失的大鼠中观察到类似的保护作用，这表明后期发生的持续炎症主要由TRIF依赖性通路启动，并且这种炎症反应可被MyD₈₈依赖性通路进一步放大。此外，小胶质细胞似乎在I/R损伤后炎症的早期阶段起主要作用，而在后期反应阶段，小胶质细胞和星形胶质细胞都是必需的。
这些发现表明TLR4/MyD₈₈依赖性通路和TLR₄/TRIF依赖性通路在I/R损伤后炎症反应的双峰阶段具有相关性，与损伤临床进展和症状延迟发作相对应。在不同阶段使用TLR₄信号抑制剂可能是预防延迟性损伤的一种治疗选择。
