Limb remote ischemic per-conditioning in combination with post-conditioning reduces brain damage and promotes neuroglobin expression in the rat brain after ischemic stroke

Abstract

Purpose: Limb remote ischemic per-conditioning or post-conditioning has been shown to be neuroprotective after cerebral ischemic stroke. However, the effect of combining remote per-conditioning with post-conditioning on ischemic/reperfusion injury as well as the underlying mechanisms are largely unexplored.

Methods: Here, adult male Sprague Dawley rats were subjected to middle cerebral artery occlusion (MCAO). The limb ischemic stimulus was immediately applied after onset of focal ischemia (per-conditioning), followed by repeated short episodes of remote ischemia 24 hr after reperfusion (post-conditioning). The infarct volume, motor function, and the expression of neuroglobin (Ngb) were measured at different durations after reperfusion.

Results: We found that a single episode of limb remote per-conditioning afforded short-term protection, but combining repeated remote post-conditioning during the 14 days after reperfusion significantly ameliorated cerebral ischemia/reperfusion injury. Interestingly, we also found that ischemic per- and post-conditioning significantly increased expression of Ngb, an oxygen-binding globin protein that has been demonstrated to be neuroprotective against stroke, at peri-infarct regions from day 1 to day 14 following ischemia/reperfusion.

Conclusion: Our results suggest that the conventional per-conditioning combined with post-conditioning may be used as a novel neuroprotective strategy against ischemia-reperfusion injury, and Ngb seems to be one of the important players in limb remote ischemia-mediated neuroprotection.

Keywords: Ischemia; neuroglobin; remote conditioning; stroke.

Fig.1

Effect of per-conditioning and post-conditioning on infarct volume and area of brain injury. A, Schematic diagram showing limb remote ischemic conditioning, which was induced during ischemia (PerC) and once daily after reperfusion (PostC). B, Representative cresyl violet staining for infarct area measured at 7 days after reperfusion. C, Quantification of average infarct volume shown in B. **P <  0.01. Error bars indicate SD. N = 5 per group. D, Representative cresyl violet staining for brain injury measured at 14 days after reperfusion. E, Quantification of average area of brain injury shown in D. **P <  0.01. Error bars indicate SD. N = 5 per group. Control: ischemic control group. PerC: per-conditioning group. PerC+PostC: per-conditioning combined with post-conditioninggroup.

Fig.2

Effect of per-conditioning and post-conditioning on DNA fragmentation and intracellular ROS levels. A, Representative TUNEL staining at 3 days after reperfusion. Scale bar = 100μm. B, Quantification of average TUNEL positive cells number. **P <  0.01, ****P <  0.001. Error bars indicate SD. N = 5 per group. C, Relative ROS levels in each group. *P <  0.05. ****P <  0.001. Error bars indicate SD. N = 4 per group.

Fig.3

Effect of per-conditioning and post-conditioning on neurobehavioral function after focal ischemia. A, Neurological deficits were determined using the neurobehavioral scoring system (higher scores correspond to more severe deficits). B, Motor function was determined by the ladder rung walking test (higher scores correspond to more severe deficits). C, Elevated Body Swing Test (higher percentage correspond to more severe deficits). *P <  0.05, **P <  0.01. Error bars indicate SD. N = 10 pergroup.

Fig.4

Western blot analysis of Ngb expression. A, Representative Western blot for Ngb protein expression in the ipsilateral peri-infarct region on days 1, 7, and 14 from each group. B, Relative Ngb protein expression level. *P <  0.05, **P <  0.01. Error bars indicate SD. N = 5 per group.

Fig.5

Ngb expression pattern determined by immohistochemistry. A, Representative images of Ngb-positive cells in the peri-infarct region of each group at 1 day after reperfusion. B, Representative images of Ngb-positive cells in the peri-infarct region of each group at 7 days after reperfusion. C, Representative images of Ngb positive cells in the peri-infarct region of each group at 14 days after reperfusion. D, left, Representative images of Ngb- and DAPI-positive cells. Right, Double-label immunohistochemistry with antibodies against Ngb and neuron marker NeurN. Scale bar = 100μm. E, Bar graphs depicting the number of Ngb-positive cells. Error bars indicate SD. *P <  0.05, **P <  0.01. N = 5 per group. Dashed line indicates the distinction between the ischemic core and peri-infarctregion.