BackgroundSpinal cord ischemia reperfusion (IR) injury causes inflammation and subsequently increases blood-spinal cord barrier leakage and Toll-like receptor 4 (TLR4) pathway activation. MicroRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, their roles during IR injury are poorly understood. We investigated miRs involvement, particularly miR-27a, in TLR4 pathway-mediated inflammatory responses after IR.MethodWe used a genomics approach to examine changed miRs of rats that had undergone 14 minutes of ischemia, followed by 24 or 72 hours of reperfusion. Quantitative RT-PCR was used to identify and confirm the miRs involved in regulating TLR4 pathway activation. We scanned miR databases for potential miR targets and confirmed these targets by quantitative RT-PCR. The miR mimic and anti-miR oligonucleotides (AMOs) were intrathecally injected at 12-hour intervals beginning three days before the ischemia. The effects of miRs on the TLR4 pathway and downstream cytokines were analyzed by PCR, western blotting, and ELISA. Double immunofluorescence staining was perfumed to determine the relationship between the targets and TLR4. Blood-spinal cord barrier (BSCB) permeability was examined using Evans blue (EB) dye.ResultsA microarray analysis revealed that at 24 hours post-injury, three miRs were upregulated (>2.0 fold) and 15 miRs were downregulated (<0.5 fold), and at 72 hours, four miRs were upregulated and 14 were downregulated compared to their levels in sham-operated controls. We focused on miR-27a, which is predicted to contain sequences complementary to the 3'-untranslated region (UTR) of Toll-like receptor adaptor molecule 2 (TICAM-2). Double immunostaining indicated that TLR4 activation correlated with changes in TICAM-2 expression. Compared to the rats in the IR and negative control groups, intrathecal infusion of the miR-27a mimic attenuated IR-induced TLR4 activation and inflammatory damage to the BSCB, which was shown as decreased EB extravasation and lower levels of nuclear factor kappa-B (NF-κB) and lnterleukin (IL)-1β at 24 and 72 hours after reperfusion, whereas pretreatment with miR-27a AMO aggravated these injuries.ConclusionsWe present the first evidence that miRs play an important role in spinal cord IR injury. We identified TICAM-2 as a novel target of miR-27a. miR-27a upregulation attenuates IR-induced inflammatory damage to the BSCB by negatively regulating TICAM-2 of the TLR4 signaling pathway and inhibiting the NF-κB/IL-1β pathway. These results provide new therapeutic targets for IR injury treatment.

BackgroundSpinal 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.MethodsRats 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.ResultsRats 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.ConclusionsThese 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.

Our results indicate that tau phosphorylation is not a direct result of anaesthesia per se, but it is due to anaesthesia-induced hypothermia and this leads to the inhibition of phosphatase activity as well as tau hyperphosphorylation. Tau hyperphosphorylation is associated with the observed deficits in spatial learning and memory following anaesthesia in hypothermic rats.