Enhanced oxidative stress response and neuroprotection of combined limb remote ischemic conditioning and atorvastatin after transient ischemic stroke in rats

Abstract

Background: Limb remote ischemic conditioning (LRIC) and atorvastatin (AtS) both provide neuroprotection in stroke. We evaluated the enhanced neuroprotective effect of combining these two treatments in preventing ischemia/reperfusion (I/R)-induced cerebral injury in a rat model and investigated the corresponding molecular mechanisms.

Materials and methods: Transient cerebral ischemia was induced in Sprague-Dawley male rats by middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion (I/R). Rats were divided into 5 groups, sham, I/R, I/R + AtS, I/R + LRIC and I/R + AtS + LRIC. Pretreatment with LRIC and/or AtS for 14 days before MCAO surgery. Infarct volume, neurological score, Western blot, immuno-histochemical analyses were performed.

Results: The combination of LRIC plus AtS pretreatment decreased infarct volume and inhibited neuronal apoptosis. Combination treatment achieved stronger neuroprotection than monotherapy with LRIC or AtS. These therapies reduced reactive oxygen species production in the peri-ischemia region, associated with significantly increased expression and activation of superoxide dismutase 1, hemeoxygenase 1 and nuclear factor erythroid 2-related factor 2.

Conclusions: Both LRIC and AtS + LRIC treatments conferred neuroprotection in ischemic stroke by reducing brain oxidative stress. AtS plus LRIC is an attractive translational research option due to its ease of use, tolerability, economical, and tremendous neuroprotective potential in stroke.

Keywords: Atorvastatin; ischemia/reperfusion; limb remote ischemic conditioning; nuclear factor erythroid 2-related factor 2 pathway; oxidative stress.

Figure 1

The representative sketches of experiment group. AtS: Atorvastatin; LRIC: limb remote ischemic conditioning; I/R: ischemia/reperfusion

Figure 2

Atorvastatin and limb remote ischemic conditioning combination protects against ischemia/reperfusion injury in rat. (a) Cerebral infarct volume evaluated by 2, 3, 5-triphenyltetrazolium chloride staining of coronal brain sections (n = 8). *P < 0.05, **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + limb remote ischemic conditioning group. (b) Neuronal apoptosis in the peri-infarct region detected by terminal deoxynucleotidyl transferase mediated nick end labeling and 4’,6-diamidino-2-phenylindole double staining (n = 4). * P < 0.05, ** P < 0.01, versus middle cerebral artery occlusion group. Scale bar = 100 μm

Figure 3

Atorvastatin and limb remote ischemic conditioning combination attenuated neurological deficiency. (a) Elevated body swing test (higher percentage correspond to more severe deficits) (n = 8). **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + limb remote ischemic conditioning group. (b) Neurological deficits were determined using neurobehavioral scoring system (higher scores correspond with more severe deficits) (n = 8). **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + limb remote ischemic conditioning group

Figure 4

Atorvastatin and limb remote ischemic conditioning combination oxidative stress after ischemia/reperfusion in mice. (a) Reactive oxygen species level in the peri-infarct region was detected by dihydroethidium and 4’,6-diamidino-2-phenylindole double staining (n = 5). (b) Relative reactive oxygen species levels in each group. *P < 0.05, **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + limb remote ischemic conditioning group. Scale bar = 100 μm

Figure 5

Atorvastatin and limb remote ischemic conditioning combination upregulates antioxidant expression and activity in the peri-ischemic region after ischemia/reperfusion injury. (a) Effect of atorvastatin and limb remote ischemic conditioning on superoxide dismutase activity (n = 4), as detected by biochemical methods (n = 4). **P < 0.01, versus ischemia/reperfusion group. (b) superoxide dismutase protein level in peri-infarct region, as determined by Western blotting (n = 4). *P < 0.05, **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + atorvastatin, or ischemia/reperfusion + limb remote ischemic conditioning group. (c) Hemeoxygenase 1 protein level in peri-infarct region, as determined by Western blotting (n = 4). *P < 0.05, **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + atorvastatin, or ischemia/reperfusion + limb remote ischemic conditioning group. (d) Nuclear factor erythroid 2-related factor 2 protein level in peri-infarct region, as determined by Western blotting (n = 4). *P < 0.05, **P < 0.01, versus ischemia/reperfusion group. #P < 0.05, versus ischemia/reperfusion + atorvastatin group