Role of the TLR4 pathway in blood-spinal cord barrier dysfunction during the bimodal stage after ischemia/reperfusion injury in rats

Background: 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/MyD(88)-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. Methods: Rats were intrathecally pretreated with TAK-242, MyD(88) 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-kappa B 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-kappa B and IL-1 beta were analyzed at 12 and 48 h to identify the correlation between MyD(88)-dependent and TRIF-dependent pathways. Results: Rats without functional TLR4 and MyD(88) attenuated BSCB leakage and inflammatory responses at 12 h, suggesting the ischemic event was largely mediated by MyD(88)-dependent pathway. Similar protective effects observed in rats with depleted TLR4, MyD(88), 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 MyD(88)-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. Conclusions: These findings indicate the relevance of TLR4/MyD(88)-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.