Immediate remote ischemic postconditioning reduces cerebral damage in ischemic stroke mice by enhancing leptomeningeal collateral circulation

Abstract

Remote ischemic postconditioning (RIPC) is a promising neuroprotective strategy for ischemic stroke. Here, we employed a focal ischemic stroke mouse model to test the hypothesis that poststroke collateral circulation as a potent mechanism of action underlying the therapeutic effects of immediate RIPC. During reperfusion of cerebral ischemia, the mice were randomly assigned to receive RIPC, granulocyte colony-stimulating factor (G-CSF) as a positive control, or no treatment. At 24 hr, we found RIPC and G-CSF increased monocytes/macrophages in the dorsal brain surface and in the spleen, coupled with enhanced leptomeningeal collateral flow compared with nontreatment group. Blood monocytes depletion by 5-fluorouracil (5-FU) significantly limited the neuroprotection of RIPC or G-CSF treatment. The protein expression of proangiogenic factors such as Ang-2 was increased by ischemia, but treatment with either RIPC or G-CSF showed no further upregulation. Thus, immediate RIPC confers neuroprotection, in part, by enhancing leptomeningeal collateral circulation in a mouse model of ischemic stroke.

Keywords: animal models; collateral circulation; ischemic stroke; monocytes/macrophages; remote ischemic postconditioning.

Figure 1

Infarct volume reduction and neurological function improvement by remote ischemic postconditioning. (a) At 24 hr, mice subjected to focal cerebral ischemic stroke showed asymmetry in turning preference, demonstrating impaired motor function, whereas the treatment groups reduced neurological deficit compared with nontreated animals; no further difference was observed between the two treatment groups (***p < 0.001 vs. Sham, ^### p < 0.001 vs. Stroke, n = 8). (b) Representative brain slices stained by 2,3,5‐triphenyltetrazolium‐chloride (TTC) 24 hr after reperfusion and average infarct volumes in stroke with and without treatment. Both RIPC and G‐CSF treatments significantly reduced infarct volume when compared with nontreated animals (^### p < 0.001), whereas G‐CSF exhibited a further reduction than RIPC (^$ p < 0.05; n = 8). Data were expressed as mean ± SEM. G‐CSF: granulocyte colony‐stimulating factor [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Enhanced leptomeningeal anastomoses flow by remote ischemic postconditioning as well as G‐CSF. Visualization of cerebral angioarchitecture by carbon black‐stained latex 24 hr after ischemic stroke with RIPC or G‐CSF treatment. Magnified images of the box in the upper panels were shown in the lower panels. (a) The black arrows indicate the vessels of anterior cerebral artery–middle cerebral artery anastomoses (ACA‐MCA), and the red arrows indicate the branch of distal MCA (scale bar = 200 µm). (b) The arrow heads indicate vessels of the circle of Willis (scale bar = 500 µm). (c1) RIPC as well as G‐CSF treatment promoted the leptomeningeal collateral flow after ischemic stroke (**p < 0.01, ***p < 0.001 vs. Sham, ^# p < 0.05 vs. Stroke, n = 6). (c2) Collateral flow at the circle of Willis was observed whereas there was no difference by any treatment (n = 6). (d) Macrophage staining for MAC387 in the dorsal brain surface 24 hr after 3VO stroke with RIPC or G‐CSF treatment was presented (scale bar = 100 µm). Data were expressed as mean ± SEM. G‐CSF: granulocyte colony‐stimulating factor [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

RIPC treatment induced monocytes/macrophages accumulation in the spleen similarly to G‐CSF. (a) Representative images of double immunostaining of anti‐macrophage antibody MAC387 (red) and DAPI (blue; scale bar = 100 µm). (b) Quantification of the number of MAC387 positive macrophages (data were expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs. Sham, ^## p < 0.01, ^### p < 0.001 vs. Stroke, ^$ p < 0.05 vs. Stroke&RIPC, n = 5). G‐CSF: granulocyte colony‐stimulating factor; DAPI: 4′,6‐diamidino‐2‐phenylindole; RIPC: remote ischemic postconditioning [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Monocyte depletion reversed the neuroprotective effects of RIPC. FU(+) indicates 5‐FU administration. FU(‐) indicates vehicle administration. (a) In 5‐Fu(+) monocyte depleted mice, ischemic cerebral injury promoted leptomeningeal collateral flow whereas RIPC or G‐CSF treatment exhibited no further increase (*p < 0.05 vs. Sham group, n = 4). (b) 5‐Fu treated mice of all groups showed no differences in collateral flow at the circle of Willis (n = 4). (c) Monocyte depletion by 5‐FU caused spleen shrinkage, and 3‐vessel occlusion (3VO) ischemia with or without RIPC or G‐CSF treatment aggravated the drop of spleen weight (*p < 0.05 vs. 5‐FU administrated Sham, ^# p < 0.05 vs. 5‐FU free Sham; ^$ p < 0.05, ^$$ p < 0.01 vs. 5‐FU free mice). (d) RIPC or G‐CSF treatment did not alter the cerebral infarct volume in monocyte depleted mice with ischemic stroke (n = 5). Data were expressed as mean ± SEM. FU: Fluorouracil; G‐CSF: granulocyte colony‐stimulating factor; RIPC: remote ischemic postconditioning

Figure 5

Western blot for angiopoietins Ang‐1 and Ang‐2. (a) There were no significant differences of Ang‐1 expression between all the groups (n = 5). (b) Ischemic insult upregulated Ang‐2 expression in the ischemic cortex, whereas both RIPC and G‐CSF treatment showed no further increase (1.8‐, 1.7‐, 1.8‐fold vs. Sham, *p < 0.05, n = 5). Data were expressed as mean ± SEM. Data were normalized to β‐actin. G‐CSF: granulocyte colony‐stimulating factor; RIPC: remote ischemic postconditioning

Figure 6

Enzyme‐linked immunoassay (ELISA) for mouse G‐CSF. (a) Mouse G‐CSF level in contralateral cortex of each mouse was 0, as well as the ipsilateral cortex of Sham group. There was an apparent increase of mouse G‐CSF in the ipsilateral cortex in Stroke mice, whereas remote ischemic postconditioning and G‐CSF significantly decreased the factor compared with Stroke without treatment (***p < 0.001 vs. Sham, ^## p < 0.01, ^### p < 0.001 vs. Stroke, n = 5). (b) Mouse G‐CSF level in plasma of Stroke group showed significant decrease compared with Sham group (*p < 0.05), whereas RIPC and G‐CSF treatment both attenuated the downtrend of mouse G‐CSF in plasma caused by cerebral ischemic insult (n = 5). Data were expressed as mean ± SEM. G‐CSF: granulocyte colony‐stimulating factor