Objective: Post-stroke remote limb conditioning (PSLC) has been shown protective effect in preclinical and clinical studies. However, underlying protective mechanisms are multiple but not clearly defined. Although inflammation exacerbates stroke injury, studies showed inflammation also plays a key role in conditioning-mediated protection. This study investigated if PSLC induces changes of monocyte/macrophage (MM) subsets and the changes contribute to PSLC-induced functional benefits.
Method: C57BL/6 male mice (12-week-old) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. PSLC was induced 2h after MCAO by applying 5 cycles of 5 min inflation (200 mmHg) and 5 min deflation with a cuff on left hind limb. Mononuclear cells were isolated from the spleen, blood and brain at 3D and analyzed by flow cytometer. Acute injury size was measured at 3D. Motor and gait functions were assessed up to 4-month using Rotarod and Catwalk. Additionally, naïve spleen cells were treated sera obtained from sham-conditioned or PSLC animals, and MM subset shifts were determined.
Results: PSLC significantly decreased anti-inflammatory (Ly-6Clow) monocytes without changing pro-inflammatory (Ly-6Chigh) subset in blood, while there were no changes in the spleen (Fig A). Similarly, PSLC decreased Ly-6Clow and increased Ly-6Chigh subset in the stroked brain (Fig B). The shift to a pro-inflammatory MM subset did not attenuate brain injury size but was associated with improved motor and gait function (Fig D). PSLC serum reduced Ly6-Clow and increased Ly-6Chigh subset in splenocytes (Fig C). There was bigger MM shift and greater behavioral benefits of PSLC in animals with bigger injury (Fig E&F).
Conclusion: The study demonstrates a contributing role of pro-inflammatory monocytes to PSLC-induced functional benefits in chronic stroke. This study provides a potential application of PSLC as a neuroimmune-based strategy for ischemic stroke patients.