RIC-specific cytoprotective profile mediated by microglia in a mouse model of acute ischaemic stroke

Background: Remote ischaemic conditioning (RIC) is an endogenous neuroprotective strategy involving repeated, transient occlusion of a limb artery to reduce ischaemic injury at a distant site. We investigated the effects of RIC in a mouse model of focal cerebral ischaemia induced by distal transient middle cerebral artery occlusion (tMCAO). Animals were randomised into three groups: Stroke (tMCAO, 60 min), Stroke+remote ischaemic preconditioning (RIPerC) (RIC applied during ischaemia) and Stroke+remote ischaemic postconditioning (RIPostC) (RIC initiated 10 min after reperfusion). The RIC protocol consisted of three cycles of 5-minute hindlimb ischaemia followed by 5-minute reperfusion. Results: At 72 hours postischaemia, both RIPerC and RIPostC significantly reduced the infarct volumes in male and female mice. In males, infarct size decreased from 6.31 ± 0.28% (Stroke) to 3.77 ± 0.47% (RIPerC, p<0.0025) and 4.00 ± 0.40% (RIPostC, p<0.0061). However, the RIPerC+RIPostC group significantly increased the infarct volume compared with the Stroke group (8.41± 0.72%). In females, reductions were greater: from 6.69±0.46% (Stroke) to 2.95 ± 0.34% (RIPerC, p<0.0001) and 2.96 ± 0.32% (RIPostC, p<0.0001). Functional recovery was improved, particularly with RIPostC, correlating with infarct size reduction (Pearson’s r=0.5505 in males, r=0.7313 in females). Apoptosis was reduced by over 50% with both treatments, and microglial phagocytic activity (cluster of differentiation 68+/Iba1+ (ionised calcium binding adaptor molecule 1)) increased significantly. Microglial depletion using PLX3397 (71.3% reduction in Iba1+ cells) worsened ischaemic injury, yet RIC preserved its protective effects, suggesting additional microglia-independent mechanisms. Furthermore, RIC enhanced neurogenesis in the subventricular zone and infarct core (doublecortin marker+ cells doubled versus Stroke), with a marked proliferative response in female hippocampi (Ki67+ cells increased by 127%). Conclusion: These findings reveal sex-specific efficacy of RIC, with mechanistic insights obtained in male animals suggesting a dual mode of action via modulation of microglial function and promotion of endogenous neurorepair pathways.