Mild Therapeutic Hypothermia Protects the Brain from Ischemia/Reperfusion Injury through Upregulation of iASPP
Liu Xiangrong1,2,3, Wen Shaohong1,2,3, Zhao Shunying1, Yan Feng2,3, Zhao Shangfeng4, Wu Di1,2, Ji Xunming1,2,3,5,*
1China-America Joint Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing, China. 2 Beijing Key Laboratory of Translational Medicine for Cerebrovascular Diseases, Beijing, China. 3Cerebrovascular Diseases Research Institute, Xuanwu Hospital of Capital Medical University, Beijing, China. 4Department of Neurosurgery, Beijing Tongren Hospital, Capital University of Medical Sciences, Beijing, China. 5Department of Neurosurgery, Xuanwu Hospital of Capital Medical University, Beijing, China.
Mild therapeutic hypothermia, a robust neuroprotectant, reduces neuronal apoptosis, but the precise mechanism is not well understood. Our previous study showed that a novel inhibitor of an apoptosis-stimulating protein of p53 (iASPP) might be involved in neuronal death after stroke. The aim of this study was to confirm the role of iASPP after stroke treated with mild therapeutic hypothermia. To address this, we mimicked ischemia/reperfusion injury in vitro by using oxygen-glucose deprivation/reperfusion (OGD/R) in primary rat neurons. In our in vivo approach, we induced middle cerebral artery occlusion (MCAO) for 60 min in C57/B6 mice. From the beginning of ischemia, focal mild hypothermia was applied for two hours. To evaluate the role of iASPP, small interfering RNA (siRNA) was injected intracerebroventricularly. Our results showed that mild therapeutic hypothermia increased the expression of iASPP and decreased the expression of its targets, Puma and Bax, and an apoptosis marker, cleaved caspase-3, in primary neurons under OGD/R. Increased iASPP expression and decreased ASPP1/2 expression were observed under hypothermia treatment in MCAO mice. iASPP siRNA (iASPPi) or hypothermia plus iASPPi application increased infarct volume, apoptosis and aggravated the neurological deficits in MCAO mice. Furthermore, iASPPi downregulated iASPP expression, and upregulated the expression of proapoptotic effectors, Puma, Bax and cleaved caspase-3, in mice after stroke treated with mild therapeutic hypothermia. In conclusion, mild therapeutic hypothermia protects against ischemia/reperfusion brain injury in mice by upregulating iASPP and thus attenuating apoptosis. iASPP may be a potential target in the therapy of stroke.
Liu Xiangrong,Wen Shaohong,Zhao Shunying, et al. Mild Therapeutic Hypothermia Protects the Brain from Ischemia/Reperfusion Injury through Upregulation of iASPP[J]. Aging and disease,
2018, 9(3): 401-411.
Figure 1. Mild therapeutic hypothermia regulates the expression of iASPP and its targets in primary neurons treated with OGD/R. (A) Representative protein in bands of iASPP, Puma, Bax and cleaved caspase-3 at 24 h after OGD from the Western blot. Actin served as a loading control. (B) Densitometric quantification of iASPP, Puma, Bax and cleaved caspase-3 from the Western blot, n = 4 per group. (C) Representative images showing expression of iASPP (green) in primary neurons (red) treated with OGD at 37°C or 33°C. DAPI was used as a nuclear marker. Bar, 50 μm.*p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA and Tukey’s test.
Figure 2. Mild therapeutic hypothermia augmented expression of iASPP after stroke. (A) Representative protein bands of iASPP from the Western blot. Actin served as a loading control. Mice subjected to normothermic or mild hypothermic stroke were euthanized at 4, 12, 24 and 72 h later. (B) Densitometric quantification of iASPP from the Western blot. *p < 0.05, ***p < 0.001, vs sham group under normothermia; # p < 0.05, ### p < 0.001, vs sham group under mild therapeutic hypothermia; ♦♦ p < 0.01, ♦♦♦ p < 0.01, vs corresponding groups under normothermia, by two-way ANOVA and Sidak’s test, n = 4 per group. (C) Representative images showing localization of iASPP (red) in neurons (green) of the cortex at 24 h after stroke. Bar, 50 μm.
Figure 3. Mild therapeutic hypothermia decreased expression of ASPP1 and ASPP2 after stroke. (A) Representative protein bands of ASPP1 and ASPP2 from the Western blot. Actin served as a loading control. Mice subjected to normothermic or mild hypothermic stroke were euthanized at 4, 12, 24 and 72 h later. (B) Densitometric quantification of ASPP1 and ASPP2 in Western blot, n = 4 per group. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, vs sham group under normothermia; # p < 0.05 vs sham group under mild therapeutic hypothermia; ♦ p < 0.05, vs. corresponding group under normothermia, by two-way ANOVA and Sidak’s test. (C) Representative images showing expression of ASPP1 and ASPP2 (red) in neurons (green) of the cortex at 12 h after cerebral ischemia. DAPI was used as a nuclear marker.
Figure 4. Effect of iASPPi and iASPPi plus hypothermia on mice cerebral ischemic injury. (A) Representative brain slices with infarcts stained by 2, 3, 5-triphenyltetrazolium chloride from each group at 24 h after reperfusion. (B) Statistical analysis of the percentage of infarct volume in different treated groups, n = 10-12 per group. (C) Statistical analysis of neurological severity scores (NSS) in different treated groups, n = 13-16 per group. (D) Representative TUNEL staining (green) of brain slices from different groups at 24 h after reperfusion. DAPI was used as a nuclear marker. (E) Statistical analysis of apoptosis cells showing that mild hypothermia decreased cell apoptosis, n = 4-6 per group. 37°C, normothermia. 33°C, mild therapeutic hypothermia. Small interfering RNA (siRNA) was intracerebroventricularly injected. Coni, control siRNA; iASPP siRNA (iASPPi). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA and Tukey’s test.
Figure 5. Effect of iASPPi and iASPPi plus hypothermia on the expression of iASPP and its targets in MCAO mice. (A) Representative protein in bands of iASPP, Puma, Bax and cleaved caspase-3 at 24 h after cerebral ischemia from the Western blot. Actin served as a loading control. (B) Densitometric quantification of iASPP, Puma, Bax and cleaved caspase-3 measured by Western blot. C-casp3, cleaved caspase-3, n = 4-6 per group. 37°C, normothermia. 33°C, mild therapeutic hypothermia. Small interfering RNA (siRNA) was intracerebroventricularly injected. Coni, control siRNA; iASPP siRNA (iASPPi). Data are expressed as mean ± SEM. N.S., not significant. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA and Tukey’s test.
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