Post-stroke remote limb conditioning shifts monocyte to a pro-inflammatory subset and improves functional recovery in chronic stroke

Abstract

Post-stroke remote limb conditioning (PSLC) has shown protective effects in preclinical and clinical studies. However, underlying protective mechanisms could be multiple and are not clearly defined. Although inflammation exacerbates stroke injury, studies show inflammation also plays a key role in conditioning-mediated protection. This study investigated whether PSLC induces changes in monocyte/macrophage (MM) subsets and if the changes contribute to PSLC-induced functional benefits. C57BL/6 male mice (12-weeks-old) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Either sham conditioning or 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 (N=20/group). 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-months using Rotarod and Catwalk. PSLC significantly decreased anti-inflammatory (CCR2-/Ly-6C^low) monocytes (P<0.01) without changing pro-inflammatory (CCR2+/Ly-6C^high) subset in blood, but there were no changes in the spleen. In the post-stroke brain, PSLC significantly decreased Ly-6C^low(P<0.001) and increased Ly-6C^highsubset (P<0.05) in the stroked brain. Additionally, naïve spleen cells that were treated sera obtained from PSLC animals showed reduced Ly-6C^low(P<0.01) and increased Ly-6C^high(p<0.001) subset as compared to the cells treated with sham-conditioned sera. This suggests that RLC induced monocytes to shift towards the pro-inflammatory CCR2+/Ly-6C^highsubset. RLC significantly reduced acute brain injury and swelling (P<0.05, n=20-22/group) and significantly improved motor/gait function in chronic stroke assessed up to 4-months after stroke. Adoptive transfer of CCR2-deficient monocytes to WT stroked mice abolished RLC-mediated monocyte shift and motor/gait improvement. The results demonstrate the CCR2+ pro-inflammatory monocyte shift underlies RLC-induced functional improvement in chronic stroke. This study provides a potential application of PSLC as a neuroimmune-based strategy for ischemic stroke patients.

Abstract

Post-stroke remote limb conditioning (PSLC) has shown protective effects in preclinical and clinical studies. However, underlying protective mechanisms could be multiple and are not clearly defined. Although inflammation exacerbates stroke injury, studies show inflammation also plays a key role in conditioning-mediated protection. This study investigated whether PSLC induces changes in monocyte/macrophage (MM) subsets and if the changes contribute to PSLC-induced functional benefits. C57BL/6 male mice (12-weeks-old) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Either sham conditioning or 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 (N=20/group). 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-months using Rotarod and Catwalk. PSLC significantly decreased anti-inflammatory (CCR2-/Ly-6C^low) monocytes (P<0.01) without changing pro-inflammatory (CCR2+/Ly-6C^high) subset in blood, but there were no changes in the spleen. In the post-stroke brain, PSLC significantly decreased Ly-6C^low(P<0.001) and increased Ly-6C^highsubset (P<0.05) in the stroked brain. Additionally, naïve spleen cells that were treated sera obtained from PSLC animals showed reduced Ly-6C^low(P<0.01) and increased Ly-6C^high(p<0.001) subset as compared to the cells treated with sham-conditioned sera. This suggests that RLC induced monocytes to shift towards the pro-inflammatory CCR2+/Ly-6C^highsubset. RLC significantly reduced acute brain injury and swelling (P<0.05, n=20-22/group) and significantly improved motor/gait function in chronic stroke assessed up to 4-months after stroke. Adoptive transfer of CCR2-deficient monocytes to WT stroked mice abolished RLC-mediated monocyte shift and motor/gait improvement. The results demonstrate the CCR2+ pro-inflammatory monocyte shift underlies RLC-induced functional improvement in chronic stroke. This study provides a potential application of PSLC as a neuroimmune-based strategy for ischemic stroke patients.

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