Remote Ischemic Preconditioning-Mediated Neuroprotection against Stroke is Associated with Significant Alterations in Peripheral Immune Responses

Abstract

Aims: Remote ischemic preconditioning (RIPC) of a limb is a clinically feasible strategy to protect against ischemia-reperfusion injury after stroke. However, the mechanism underlying RIPC remains elusive.

Methods: We generated a rat model of noninvasive RIPC by four repeated cycles of brief blood flow constriction (5 min) in the hindlimbs using a tourniquet. Blood was collected 1 h after preconditioning and 3 days after brain reperfusion. The impact of RIPC on immune cell and cytokine profiles prior to and after transient middle cerebral artery occlusion (MCAO) was assessed.

Results: Remote ischemic preconditioning protects against focal ischemia and preserves neurological functions 3 days after stroke. Flow cytometry analysis demonstrated that RIPC ameliorates the post-MCAO reduction of CD3(+)CD8(+) T cells and abolishes the reduction of CD3(+)/CD161a(+) NKT cells in the blood. In addition, RIPC robustly elevates the percentage of B cells in peripheral blood, thereby reversing the reduction in the B-cell population after stroke. RIPC also markedly elevates the percentage of CD43(+)/CD172a(+) noninflammatory resident monocytes, without any impact on the percentage of CD43(-)/CD172a(+) inflammatory monocytes. Finally, RIPC induces IL-6 expression and enhances the elevation of TNF-α after stroke.

Conclusion: Our results reveal dramatic immune changes during RIPC-afforded neuroprotection against cerebral ischemia.

Keywords: Cerebral ischemia; Cytokine; Immune cells; Limb remote ischemic preconditioning.

Conflict of interest statement

The authors declare no conflict of interest.

© 2015 John Wiley & Sons Ltd.

Figures

Figure 1

Experimental protocols and model of remote limb ischemic preconditioning. A. Three groups of animals were generated: remote ischemic preconditioning (RIPC)+Sham; non‐RIPC+ middle cerebral artery occlusion (MCAO) (designated as MCAO); and RIPC+MCAO. Limb RIPC was conducted by four cycles (5 min/cycle, 40 min total) of bilateral hindlimb ischemia under pentobarbital anesthesia. Non‐RIPC animals were exposed to the same anesthesia for 40 min. Brain ischemia was induced by 90 min middle cerebral artery occlusion (MCAO). Sham‐operated animals underwent anesthesia and surgical exposure of the right MCA without occlusion. Blood was collected from the caudal tail vein at 1 h after preconditioning and 3 days after brain reperfusion. ① blood sample taken from rats in MCAO group before stroke; ② blood sample taken from rats in MCAO group after 3 days of reperfusion; ③ blood sample taken from rats in RIPC+MCAO group 1 h after preconditioning; ④ blood sample taken from rats in RIPC+MCAO group 3 days after reperfusion. Animals were sacrificed 3 days after reperfusion. B. Representative images of blood flow in the left thigh using a laser speckle contrast imager before RIPC, 3 min after one cycle of RIPC, and 1 min after reperfusion. C. Representative recordings of blood flow before RIPC, 3 min after one cycle of RIPC, and 1 min after reperfusion.

Figure 2

Remote limb preconditioning reduces infarct size and improves neurological scores after stroke. A. Representative TTC images from animals treated with or without remote ischemic preconditioning (RIPC) followed by middle cerebral artery occlusion (MCAO) or sham operation (left), and quantification of infarct volumes (right). B. Body weight in all groups. n = 12 rats per group. Data are presented as means ± SEM, *P ≤ 0.05, **P ≤ 0.01 versus MCAO.

Figure 3

The effect of preconditioning on T‐cell populations before and after middle cerebral artery occlusion (MCAO). Blood was collected from the caudal tail vein at 1 h after preconditioning and 3 days after brain reperfusion. A–C. Flow cytometry analysis of CD3+ CD4+ T cells. D–F. Flow cytometry analysis of CD3+ CD8+ T cells. G–I. Flow cytometry analysis of CD3+ CD161+ NKT cells. ②‐① reflects the change in T‐cell populations prior to and after MCAO without preconditioning; ④‐③ reflects the change in T‐cell populations prior to and after MCAO in the remote ischemic preconditioning (RIPC)+MCAO group. Data are expressed as means ± SEM for 12 independent experiments. *P ≤ 0.05, **P ≤ 0.01 versus ① or ②‐①; #P ≤ 0.05, ##P ≤ 0.01 versus ③.

Figure 4

The effect of preconditioning on B cells before and after MCAO. Blood was collected from the caudal tail vein at 1 h after preconditioning and 3 days after brain reperfusion. B cells were labeled with CD3 and CD45RA antibodies and analyzed by flow cytometry. A. Representative flow cytometry graphs of CD3+ CD45RA + B cells prior to and after MCAO. B. Statistical analysis of B‐cell percentages prior to and after MCAO. C. Statistical analysis of B‐cell changes before and after MCAO. ②‐① reflects the change in B cells before and after MCAO without preconditioning; ④‐③ reflects the change in B cells before and after MCAO in the remote ischemic preconditioning (RIPC)+MCAO group. Data are expressed as means ± SEM for 12 independent experiments, **P ≤ 0.01 versus ②‐①.

Figure 5

The effect of preconditioning on monocytes before and after middle cerebral artery occlusion (MCAO). Blood was collected from the caudal tail vein at 1 h after preconditioning and 3 days after brain reperfusion. A. Representative flow cytometry graphs of monocytes before and after MCAO. B. Statistical analysis of monocytes percentages before and after MCAO. C. Statistical analysis of monocyte changes before and after MCAO. ②‐① reflects the change in monocytes before and after MCAO without preconditioning; ④‐③ reflects the change in monocytes before and after MCAO in the remote ischemic preconditioning (RIPC)+MCAO group. Data are expressed as means ± SEM for 12 independent experiments.

Figure 6

The effect of preconditioning on NK cells before and after middle cerebral artery occlusion (MCAO). Blood was collected from the caudal tail vein at 1 h after preconditioning and 3 days after brain reperfusion. A. Representative flow cytometry graphs of CD3−/CD161a+ NK cells before and after MCAO. B. Statistical analysis of NK‐cell percentages before and after MCAO. C. Statistical analysis of NK‐cell changes before and after MCAO. ②‐① reflects the change in NK cells before and after MCAO without preconditioning; ④‐③ reflects the change in NK cells before and after MCAO in the remote ischemic preconditioning (RIPC)+MCAO group. Data are expressed as means ± SEM for 12 independent experiments.

Figure 7

Remote limb preconditioning alters cytokine expression before and after middle cerebral artery occlusion (MCAO). Blood was collected from the caudal tail vein at 1 h after preconditioning and 3 days after brain reperfusion. The levels of TNF‐α, IL‐6, and IL‐10 were measured by ELISA. A. Statistical analysis of cytokines before and after MCAO, **P ≤ 0.01 versus ①, ##P ≤ 0.01 versus ③. B. Statistical analysis of cytokine changes before and after MCAO. ②‐① reflects plasma cytokine levels before and after MCAO without preconditioning; ④‐③ reflects the change in cytokine levels before and after MCAO in the remote ischemic preconditioning (RIPC)+MCAO group. **P ≤ 0.01 versus ②‐①. Data are expressed as means ± SEM for 12 independent experiments.