This study contains several novel findings. First, we demonstrated that LRP offers long-term protective effects against focal ischemia in rats, as evidenced by the reduced size of brain injury measured 2 months post-stroke and improved performance during behavioral tests. Second, we provided more data to support the involvement of afferent nerve pathways in transferring protective signaling from the ischemic limb to the ischemic brain, as two afferent sensory nerve inhibitors, capsaicin and hexamethonium, blocked the protective effects of LRP. A recent study also showed that hexamethonium blocked the protective effects of limb preconditioning 
. Third, we further showed that the galectin-9/Tim-3 pathway is involved in neuronal injury after stroke, and LRP blocked its overexpression. Last, we found that LRP inhibited edema formation, BBB permeability, and iNOS and nitrotyrosine production.
We offered solid evidence that LRP has chronic protective effects against stroke based on the measurement of brain injury and behavioral tests up to 2 months post-stroke. It is important to confirm this effect for clinical translation because several neuroprotectants, such as certain types of post-ischemic hypothermia 
and rapid ischemic preconditioning 
, only transiently reduced infarct size. More recently, we also found that limb ischemic postconditioning, which was performed after reperfusion, reduced infarct size measured at 2 days but not at 1 month after stroke 
. Nevertheless, in the current study, LRP not only reduced the loss of brain tissue measured 2 months after stroke, but also attenuated deficits in behavioral tests performed from 1 day to 2 months post-stroke, suggesting a long-term protective effect of LRP against stroke.
The afferent nerve systems appear to transfer the protective signaling from the preconditioned limb to the brain. Afferent neurons receive and transmit information from the peripheral organs or tissues to the central nervous system and contribute to the organism's ability to maintain homeostasis. In the case of ischemic limb preconditioning, repetitive ischemia and reperfusion resulted in the release of substances, such as adenosine and bradykinin 
, which stimulate the afferent neurons that may transmit protective signaling to the brain. The afferent nerve system consists of peripheral fibers and endings along with neuronal bodies located in the spinal sensory ganglia that ascend to the brain stem and specific nuclei in the thalamus which, in turn, send information to the cerebral cortex. It is known that information from one side of the body can be sent to the opposite cortex in the primary sensory cortex and both sides of the secondary cortex 
. In this study, both limb and brain ischemia were performed in the left side. We showed that capsaicin, which causes desensitization via its action on the peripheral fibers and endings of the afferent neurons 
, blocked the protective effects of LPR. In addition, we found that hexamethonium, which inhibits the afferent neurons by blocking the ganglion 
, also enlarged infarct size in animals treated with LRP. These two experiments suggest that protective information from the left limb can be sent to the same side of the brain cortex via the afferent neuronal pathways. Nevertheless, additional experiments should be conducted to more clearly demonstrate that remote preconditioning is transmitted to the brain by afferent innervation.
We showed that the galectin-9/Tim-3 pathway is involved in neuronal injury induced by cerebral ischemia and that LRP attenuated the expression of galectin-9 and Tim-3 in the ischemic brain, suggesting that the inhibition of this pathway may contribute to the protective effects of LRP. This is a novel pathway involved in immune modulation and inflammatory response 
. It was originally indentified in T cells and subsequently in macrophages and dendritic cells. A recent study in mice showed increased mRNA levels of Tim-3 in the brain 3 days after stroke 
, but the cell types on which it was induced was not reported. In this study, we showed that protein expression of galectin-9 was increased as early as 5 hours after stroke, but Tim-3 levels were only increased 24 hours later. It seems that Tim-3 overexpression was induced after galectin-9, which is consistent with previous studies that galectin-9 is a trigger for Tim-3 activities. Since the major role of the galectin-9/Tim-3 pathway is to induce cell death, its induction in ischemic neurons might be attributable to ischemic neuronal injury. Nevertheless, galectin-9 was induced at 1 hour after stroke in the LRP group, which is earlier than that in the control group. Whether this early induction is beneficial or detrimental is not known. Despite this, we found that LRP inhibited increases in both galectin-9 and Tim-3 expression at 24 hours post-stroke, suggesting that inhibition of the galectin-9/Tim-3 pathway may be a target for stroke therapy.
Last, we found that LRP blocked increases in iNOS and nitrotyrosine expression. The induction of iNOS is critical for neuronal injury and inflammation under oxidative stress, including stroke 
. In our study, iNOS protein expression was immediately upregulated after focal ischemia from 1 to 24 hours, and these increases were robustly blocked by LRP. Nitrotyrosine is a product of tyrosine nitration generated by ROS, a maker of NO-dependent products derived from iNOS, and its expression indicates cell damage and inflammation 
. Thus, we also measured the expression of nitrotyrosine, which was also inhibited by LRP, corroborating its effects on iNOS.
In conclusion, we found that LRP provides long-term protection against focal cerebral ischemia, and it may transmit protective signaling through afferent nerve pathways. The inhibitive effects of LRP on edema formation, BBB permeability, the galectin-9/Tim-3 pathway, and ROS activities, may contribute to its protective effects against stroke.