Transl. Stroke Res. (2014) 5:692–700 DOI 10.1007/s12975-014-0359-5

ORIGINAL ARTICLE

Remote Ischemic Postconditioning Alleviates Cerebral Ischemic Injury by Attenuating Endoplasmic Reticulum Stress-Mediated Apoptosis Xiangrong Liu & Shangfeng Zhao & Fang Liu & Jun Kang & Ao Xiao & Fang Li & Chencheng Zhang & Feng Yan & Haiping Zhao & Mei Luo & Yumin Luo & Xunming Ji

Received: 26 March 2014 / Revised: 4 July 2014 / Accepted: 8 July 2014 / Published online: 22 July 2014 # Springer Science+Business Media New York 2014

Abstract Remote ischemic postconditioning (RIPostC) has been proved to protect the brain from stroke, but the precise mechanism remains not fully understood. In the present study, we aimed to investigate whether RIPostC attenuates cerebral ischemia-reperfusion injury by abating endoplasmic reticulum (ER) stress response. CHOP, a multifunctional transcription factor in ER stress, regulates the expression of genes related to apoptosis, such as Bim and Bcl-2. Male SD rats were subjected to right middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion, and RIPostC was induced by three cycles of 10 min ischemia and 10 min reperfusion on bilateral femoral arteries immediately after ischemia. CHOP siRNA (CHOPi) and control siRNA (Coni) were injected into the right lateral ventricle 30 min before the beginning of ischemia. X. Liu : F. Liu : A. Xiao : F. Li : C. Zhang : F. Yan : H. Zhao : M. Luo : Y. Luo : X. Ji Cerebrovascular Diseases Research Institute, Xuanwu Hospital of Capital Medical University, 45 Changchun Street, Beijing 100053, People’s Republic of China X. Liu : F. Liu : C. Zhang : F. Yan : H. Zhao : Y. Luo : X. Ji Key Laboratory of Neurodegenerative Diseases, Ministry of Education, Beijing 100053, People’s Republic of China S. Zhao : J. Kang Department of Neurosurgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100073, People’s Republic of China X. Ji (*) Department of Neurosurgery, Xuanwu Hospital of Capital Medical University, 45 Changchun Street, Beijing 100053, People’s Republic of China e-mail: [email protected] X. Liu : F. Liu : C. Zhang : F. Yan : H. Zhao : Y. Luo (*) Beijing Key Laboratory of Translational Medicine for Cerebrovascular Diseases, Beijing, China e-mail: [email protected]

RIPostC, CHOPi, or RIPostC + CHOPi application reduced infarct volume, improved the neurological function, and decreased cell apoptosis. RIPostC increased the protein level of glucose-regulated protein 78 (GRP78) and decreased the protein level of phosphorylated-EIF2α, caspase-12, and CHOP. Furthermore, the expression of CHOP, Bim and cleavedcaspase-3 was decreased, while Bcl-2 expression was increased in response to application of RIPostC, CHOPi, or RIPostC + CHOPi. In sum, RIPostC protects against ischemia-reperfusion brain injury in rats by attenuating ER stress response-induced apoptosis. Keywords Cerebral ischemia . CHOP . ER stress . Apoptosis . Ischemic postconditioning . Neuroprotection

Introduction Ischemic stroke is a leading cause of death as well as a condition responsible for most of adult long-lasting disability. Despite a large number of animal experiments and clinical studies, there is no effective therapy available in protecting against stroke in human. Ischemic postconditioning (IPostC) is an intrinsic protective phenomenon afforded by brief episodes of ischemia-reperfusion applied after the ischemic insult [1]. Some studies showed that IPostC can limit the damage induced by ischemia and reperfusion [2–7]. Remote ischemic postconditioning (RIPostC) is a special type of IPostC that applies mild intermittent short ischemic episodes to an organ remote from the main ischemic target [8]. On account of simplicity and low-risk for its application in clinical practice, RIPostC may open new perspectives for stroke treatment [1]. However, the potential of RIPostC to protect against ischemic stroke and underlying mechanisms remain elusive.

Transl. Stroke Res. (2014) 5:692–700

Endoplasmic reticulum (ER) plays essential roles in folding of secreted and membrane proteins. When ER function is impaired, a pathological state known as ER stress occurs [9–11]. ER stress causes accumulation of unfolded proteins in the ER, a phenomenon termed the unfolded protein response (UPR) [9–11]. Once UPR is protracted or excessive, apoptosis is induced [9–11]. ER stress-mediated apoptosis is one of deleterious biochemical events triggered by cerebral ischemia [12, 13] which ultimately lead to the death of brain cells [14]. The chaperone glucose-regulated protein 78 (GRP78) is known to help stabilize protein-folding intermediates and as the main regulator of the major sensors including protein kinase RNA (PRKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase 1 (IRE1α), and activating transcription factor 6 (ATF6) in ER stress signaling [12]. These sensors all converge on the promoter of the gene encoding CCAAT/EBP homologous protein (CHOP) [12, 15]. As a transcription factor, CHOP regulates expression of the anti-apoptotic gene Bcl-2 and the apoptotic gene Bim [16, 17]. In addition, some proteins such as eukaryotic phosphorylation of translation initiation factor 2α (eIF2α) and caspase-12 are involved in ER stressinduced apoptosis [15, 18]. When misfolded ER proteins accumulate, PERK phosphorylates eIF2α, inhibiting translation of messenger RNA into protein [19, 20]. It has been reported that IPostC protects against cerebral ischemia injury by attenuating ER stress-induced apoptosis [7]. It is likely that RIPostC may share protective mechanisms in common with IPostC to reduce cerebral ischemic injury. The aims of the present study were as follows: (1) to determine the effects of RIPostC on cerebral ischemic injury and implication of ER stress response in the neuroprotection of RIPostC and (2) to compare the effectiveness of RIPostC and CHOP interference as a treatment used separately or in combination. Our results demonstrated that RIPostC protected against focal cerebral ischemia by attenuating ER stressdependent apoptotic signaling, suggesting it as a potential option for therapy of ischemic stroke.

Materials and Methods Animal Model All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University and in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. Male SD rats weighing 280–310 g were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion as previously described [21]. The

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rats were randomly assigned into seven groups: shamoperated group, MCAO group, MCAO + RIPostC group, MCAO + Coni group, MCAO + RIPostC + Coni group, MCAO + CHOPi group, and MCAO + RIPostC + CHOPi group. In MCAO + RIPostC group, MCAO + RIPostC + Coni group, and MCAO + RIPostC + CHOPi group, RIPostC was applied by three cycles of 10 min occlusion and 10 min reperfusion of bilateral femoral arteries immediately after ischemia. In MCAO + Coni group, MCAO + RIPostC + Coni group, MCAO + CHOPi group, and MCAO + RIPostC + CHOPi group, CHOP siRNA (CHOPi) or control siRNA (Coni) were, respectively, injected into the right ventricle at 30 min before the beginning of ischemia. To ensure the occurrence of ischemia and reperfusion in MCAO animals, regional cerebral blood flow was monitored using laser Doppler flowmetry (LDF, PeriFlux System 5000, Perimed, Stockholm, Sweden) in the following coordinates: 0.5 mm anterior and 5.0 mm lateral from the bregma. Rectal temperature was controlled at 37.0 °C during and after surgery with a temperature-regulated heating pad. Mean artery blood pressure (MABP) was monitored through an apparatus (MP100A-CE, BIOPAC Systems, Inc., CA, USA). Intracerebroventricular Injection SiRNA was purchased from Santa Cruz (Santa Cruz, CA, USA), and the concentration of which was 10 μM. The coordinates from bregma and pial surface, respectively, for the tip of the injection needle were AP=−0.8 mm, ML=+1.4 mm, and DV=−3.7 mm. The siRNA mixture (8 μl CHOP siRNA or control siRNA, and 2 μl Lipofectamine 2000) was stereotaxically delivered into the ipsilateral ventricle over 10 min. The bone wound was closed afterward with bone wax. Neurological Function Assessment Neurological functional deficits were scored at 24 h after reperfusion in a blind fashion. Two tests were used to evaluate neurological function according to the studies reported previously: the postural reflex test to examine upper body posture while the animal is suspended by the tail [22], and the forelimb placing test to examine sensorimotor integration in forelimb placing responses to visual, tactile, and proprioceptive stimuli [23]. Neurological functions were graded on a scale from 0 to 12 (normal score, 0; maximal score, 12). Determination of Infarct Volume The animals were euthanized at 24 h after reperfusion and were transcardially perfused with phosphate-buffered saline to eliminate intravascular blood. The brains were quickly

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Transl. Stroke Res. (2014) 5:692–700

Fig. 1 The effect of RIPostC on rat cerebral ischemic injury. a Representative brain slices with infarcts stained by TTC from each group at 24 h after reperfusion. b Statistical analysis of the percentage of infarct volume showing that RIPostC reduced infarct volume. c The neurological functional outcome at 24 h after reperfusion. RIPostC and/or CHOPi significantly improved neurological outcomes. d Representative TUNEL staining of brain slices from different groups at 24 h after reperfusion. Bar=100 μm. e Statistical analysis of apoptosis cells showing that RIPostC decreased cell apoptosis. *P

Remote ischemic postconditioning alleviates cerebral ischemic injury by attenuating endoplasmic reticulum stress-mediated apoptosis.

Remote ischemic postconditioning (RIPostC) has been proved to protect the brain from stroke, but the precise mechanism remains not fully understood. I...
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