MicroRNA-22-3p alleviates spinal cord ischemia/reperfusion injury by modulating M2 macrophage polarization via IRF5.

Cell death after spinal cord ischemia/reperfusion (I/R) can occur through necrosis, apoptosis, and autophagy, resulting in changes to the immune environment. However, the molecular mechanism of this immune regulation is not clear. Accumulating evidence indicates that microRNAs (miRs) play a crucial role in the pathogenesis of spinal cord I/R injury. Here, we hypothesized miR-22-3p may be involved in spinal cord I/R injury by interacting with interferon regulatory factor (IRF) 5. Rat models of spinal cord I/R injury were established by 12-min occlusion of the aortic arch followed by 48-hr reperfusion, with L4-6 segments of spinal cord tissues collected. MiR-22-3p agomir, a lentivirus-delivered siRNA specific for IRF5, or a lentivirus expressing wild-type IRF5 was injected intrathecally to rats with I/R injury to evaluate the effects of miR-22-3p and IRF5 on hindlimb motor function. Macrophages isolated from rats were treated with miR-22-3p mimic or siRNA specific for IRF5 to evaluate their effects on macrophage polarization. The levels of IL-1ß and TNF-α in spinal cord tissues were detected by ELISA. miR-22-3p was down-regulated, whereas IRF5 was up-regulated in rat spinal cord tissues following I/R. IRF5 was a target gene of miR-22-3p and could be negatively regulated by miR-22-3p. Silencing IRF5 or over-expressing miR-22-3p relieved inflammation, elevated Tarlov score, and reduced the degree of severity of spinal cord I/R injury. Increased miR-22-3p facilitated M2 polarization of macrophages and inhibited inflammation in tissues by inhibiting IRF5, thereby attenuating spinal cord I/R injury. Taken together, these results demonstrate that increased miR-22-3p can inhibit the progression of spinal cord I/R injury by repressing IRF5 in macrophages, highlighting the discovery of a promising new target for spinal cord I/R injury treatment.

Effects of cilostazol on oxidative stress, systemic cytokine release, and spinal cord injury in a rat model of transient aortic occlusion.

BACKGROUND: Cilostazol is a phosphodiesterase inhibitor that has anti-inflammatory potential in addition to vasodilator and antiplatelet effects. The aim of this study was to determine the influence of cilostazol on biochemical markers of oxidative damage, proinflammatory cytokine release, and spinal cord injury after transient aortic occlusion in rats. METHODS: Animals were randomized into 3 groups. Sham group rats were subjected to laparotomy without aortic occlusion. Control group rats were pretreated with intraperitoneal dimethyl sulfoxide, and cilostazol group rats received intraperitoneal cilostazol (20 mg/kg/day) for 3 days before the induction of ischemia. Ischemia was induced by clamping of the infrarenal aorta, and 48 hours after reperfusion, Tarlov grades were assessed and spinal cord conduction velocities (SCCVs) were measured using epidural electrical stimulation. Erythrocyte superoxide dismutase (SOD) and catalase activities and plasma malondialdehyde, serum tumor necrosis factor-α, interleukin-1ß, and interleukin-6 levels were analyzed. Spinal cord histopathology was examined to determine neuronal damage and tissue inflammation. RESULTS: Aortic occlusion caused significant increases in SOD, catalase activities, and malondialdehyde and cytokine levels accompanied by spinal cord injury. Cilostazol significantly reduced malondialdehyde levels but did not significantly alter the activations of antioxidant enzymes, levels of proinflammatory cytokines, or histologic severity of inflammation. The differences regarding the results of Tarlov grading, SCCVs, and neuronal viability between the ischemic and cilostazol pretreated groups were statistically nonsignificant. CONCLUSION: The present experimental study indicated that cilostazol pretreatment used in this study before aortic occlusion decreased lipid peroxidation, which may be related to the reduction of reactive oxygen species. Cilostazol did not significantly suppress systemic cytokine release and prevent spinal cord inflammation and injury; however, it did show some benefit. Additional investigations might be needed to determine the critical dose of cilostazol for clarifying the protective role of this drug in spinal cord ischemia/reperfusion injury.

Tetramethylpyrazine protects spinal cord and reduces inflammation in a rat model of spinal cord ischemia-reperfusion injury.

OBJECTIVE: Inflammation, which is known to be detrimental to the neurologic outcome during the acute phase after an ischemic stroke, provides a potential target for preventive or therapeutic approach for spinal cord ischemia-reperfusion injury. Tetramethylpyrazine (TMP), a pure compound derived from Ligusticum chuanxiong, is widely used in the treatment of ischemic stroke. The present study aimed to gain a deeper insight into the mechanism underlying the anti-inflammatory effects of TMP on spinal cord ischemia-reperfusion injury. METHODS: Spinal cord ischemia was induced in male Sprague-Dawley rats by balloon occlusion of the thoracic aorta. The experimental groups (n = 30 per group) included sham operation, control (receiving only normal saline), and TMP (30 mg/kg, 30 minutes before occlusion). Neurologic function was assessed by the Basso, Beattie, and Bresnahan (BBB) score at 1, 6, 12, 24, and 48 hours after reperfusion. Histologic changes were studied using Nissl staining. Infarct volume was analyzed using 2,3,5-triphenyltetrazolium chloride staining. Myeloperoxidase (MPO) activity was determined by using a rat MPO assay kit. Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-10 and nuclear factor (NF)-κB were examined with immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blotting. RESULTS: Compared with the control group, the TMP group showed significantly improved neurologic outcome (P < .05), decreased infarct volume (42.3% vs 17.4%), and alleviated neutrophil infiltration (0.35 vs 0.18 U/g). TMP treatment reduced the expressions of proinflammatory cytokines TNF-α (28.62 vs 15.23 pg/mg protein) and IL-1ß (13.62 vs 8.24 pg/mg protein), upregulated the expression of anti-inflammatory cytokine IL-10 (18.35 vs 31.26 pg/mg protein), and inhibited the activation of NF-κB (2.78 vs 1.22) in ischemic spinal cord. CONCLUSIONS: Treatment with TMP exerted a neuroprotective effect against spinal cord ischemia-reperfusion injury. The anti-inflammatory effect was believed to be one of the contributing mechanisms.

The effects of systemic hypothermia on a murine model of thoracic aortic ischemia reperfusion.

INTRODUCTION: Hypothermia is widely used to mediate ischemia-reperfusion injury associated with repair of the thoracoabdominal aorta. Experiments were designed in a murine model of thoracic aortic ischemia-reperfusion (TAR) to evaluate the effect of moderate systemic hypothermia on neurologic function, spinal cord morphology, and indices of inflammation in critical organs. METHODS: C57BL/6 mice were subjected to TAR under hypothermic (34 degrees C) or normothermic (38 degrees C) conditions, followed by 24 or 48 hours of normothermic reperfusion. Neurologic functions were assessed during reperfusion. Spinal cords were examined at 24 and 48 hours after reperfusion, and the degree of injury qualified by counting the number of viable motor neurons within the anterior horns. Keratinocyte chemokine, interleukin-6, and myeloperoxidase levels were measured from lung, liver, and kidney at 24 and 48 hours. RESULTS: Normothermic TAR resulted in a dense neurologic deficit in all mice throughout the reperfusion period. Mice subjected to TAR under hypothermic conditions had transient, mild neurologic deficit during the initial periods of reperfusion. Between 24 and 48 hours, delayed paralysis developed in half of these mice, whereas the other half remained neurologically intact. Spinal cord histology showed a graded degree of injury that correlated with neurologic function. There was no correlation between markers of inflammation in various organs and neurologic outcomes following TAR. CONCLUSION: Systemic moderate hypothermia was protective against immediate paralysis after TAR in all cases and was associated with delayed paralysis in 50% of mice. This study suggests that delayed-onset paralysis may be the result of a local insult, rather than a systemic inflammatory event, precipitating spinal cord injury.

miR-221 alleviates the inflammatory response and cell apoptosis of neuronal cell through targeting TNFAIP2 in spinal cord ischemia-reperfusion.

This study aimed to examine the role of miR-221 in inflammatory response and apoptosis of neuronal cells after spinal cord ischemia/reperfusion (I/R) injury. Blood samples were obtained from 20 I/R patients and that of 20 healthy individuals were used as a control. AGE1.HN and SY-SH-5Y neuronal cell lines subjected to oxygen-glucose deprivation (OGD) stress were used in cell experiments. Real-time PCR and western blot were used to evaluate the expression of miR-221, tumor necrosis factor-α, and TNFAIP2. TUNEL assay analyzed cell apoptosis. I/R patients had lower serum levels of miR-221 than healthy controls. In OGD-AGE1.HN and SY-SH-5Y cells, miR-221 was significantly downregulated and TNFAIP2 mRNA and protein were upregulated; meanwhile, both proinflammatory cytokine tumor necrosis factor-α and anti-inflammation cytokine interleukin-6 were elevated and the percentage of apoptotic cells was increased. This inflammatory response and cell apoptosis induced by OGD stress were attenuated by miR-221 overexpression and enhanced by miR-221 knockdown. TNFAIP2 is a target gene for miR-221 and could be regulated negatively by the miR-221 mimic or the miR-221 inhibitor with or without OGD stress. Accordingly, TNFAIP2 overexpression reversed the inflammatory response and cell apoptosis induced by miR-221 under OGD stress. Downregulation of miR-221 occurs in spinal cord I/R injury and in cell lines subjected to oxygen-glucose deprivation. miR-221 regulates the inflammatory response and apoptosis of neuronal cells through its impact on TNFAIP2.

Impact of acquired and innate immunity on spinal cord ischemia and reperfusion injury.

OBJECTIVE: The aim of this study was to clarify the impact of acquired and innate immunity on spinal cord ischemia and reperfusion injury using a mouse model of spinal cord ischemia. METHODS: To define the ischemic duration that caused paraplegia, wild-type and severe combined immunodeficiency (SCID) mice were subjected to cross-clamping of the aorta for 7, 9, 9.5, or 10.5 min with ischemic preconditioning for intestinal protection. In wild-type and SCID mice with paraplegia, histological analyses were performed to investigate viable neurons, inflammatory cells, and reactive astrocytes at 12, 24, 48, and 72 h as well as 7 days after reperfusion. RESULTS: In both wild-type and SCID mice, immediate paraplegia was induced by occlusion for 10.5 min. In both wild-type and SCID mice, no infiltration of T or B lymphocytes was observed at any point after reperfusion, but reactive astrocytes were clearly visible at 7 days after reperfusion, and the number of activated microglia peaked at 12 and 48 h after reperfusion. Although there was no significant difference, wild-type mice had a tendency to have more activated microglia than SCID mice at 12 h after reperfusion, and to have less viable neurons than SCID mice at 12, 24, 48, and 72 h after reperfusion. There was a tendency that the frequency of immediate paraplegia in wild-type mice was more than SCID mice though no statistical difference was observed. CONCLUSIONS: Innate immunity, rather than acquired immunity, may be involved in the developing immediate paraplegia in our mouse model.