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We recently demonstrated that limb remote preconditioning (LRP) protects against focal ischemia measured 2 days post-stroke. Here, we studied whether LRP provides long-term protection and improves neurological function. We also investigated whether LRP transmits its protective signaling via the afferent nerve pathways from the preconditioned limb to the ischemic brain and whether inflammatory factors are involved in LRP, including the novel galectin-9/Tim-3 inflammatory cell signaling pathway, which induces cell death in lymphocytes. LRP in the left hind femoral artery was performed immediately before stroke. LRP reduced brain injury size both at 2 days and 60 days post-stroke and improved behavioral outcomes for up to 2 months. The sensory nerve inhibitors capsaicin and hexamethonium, a ganglion blocker, abolished the protective effects of LRP. In addition, LRP inhibited edema formation and blood-brain barrier (BBB) permeability measured 2 days post-stroke. Western blot and immunostaining analysis showed that LRP inhibited protein expression of both galectin-9 and T-cell immunoglobulin domain and mucin domain 3 (Tim-3), which were increased after stroke. In addition, LRP decreased iNOS and nitrotyrosine protein expression after stroke. In conclusion, LRP executes long-term protective effects against stroke and may block brain injury by inhibiting activities of the galectin-9/Tim-3 pathway, iNOS, and nitrotyrosine.

The Chronic Protective Effects of Limb Remote Preconditioning and the Underlying Mechanisms Involved in Inflammatory Factors in Rat Stroke