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

Abstract

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.

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%).

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.

We thank Ana Martinez for her help in the preparation of the diet with the microglia inhibitor, Jose Serrano for his help in the use of the lyophiliser and Josep Esquerda for kindly providing CD68 and DkαRat 488 antibodies. This study was funded by Instituto de Salud Carlos III (ISCIII), AES2018 (FI18/00319).