Dynamic changes of autophagy activity of cortical neurons in rats with cerebral ischemia and cerebral ischemia/reperfusion

doi:10.12047/j.cjap.5627.2018.065

Abstract

Abstract: Objective: To investigate the changes of autophagy in cerebral cortex neurons at different times after cerebral ischemia and ischemia/reperfusion (I/R) in rats.Methods: Healthy male SD rats (n=60) were randomly assigned into sham-operation group (sham, n=10), cerebral ischemia and ischemia/reperfusion model group (I/R, n=50). In the model group, 10 rats were randomly sampled at five different time points:ischemia 30 min, 2 h, and reperfusion 1 h, 6 h and 24 h after ischemia 2 h. Cerebral infarction volume and cerebral water content were measured. Autophagy of cerebral cortical neurons was examined by transmission electron microscopy (TEM). The protein levels of microtubule associated protein light chain 3-Ⅱ (LC3-Ⅱ) in cerebral cortex of the rats in each group were determined by Western blot.Results: LC3-Ⅱ/Ⅰ ratio had no obvious rise at ischemia 30min,and it began to increase at ischemia 2 h point which was significantly higher than that in sham group (P<0.01).LC3-Ⅱ/Ⅰ ratio was still higher at ischemia/reperfusion 1h and 6h points than that in sham group (P<0.05),although it was decreased comparison with that at ischemia 2 h point. LC3-Ⅱ/Ⅰ ratio reached a peak at ischemia/reperfusion 24 h point, which was significantly higher than that in sham group (P<0.01). This phenomenon was further confirmed by the transmission electron microscopy. The infarction volume at ischemia/reperfusion 6h and 24 h points was increased significantly, and there was a statistical difference compared with sham group (P<0.01). The content of water in the brain tissue of rats at ischemia/reperfusion 24 h was increased obviously, which was significantly higher than that in sham group (P<0.05). Edema, rarefaction, some cell degeneration and apoptosis only in the cortical tissue of rats at ischemia/reperfusion 24 h were showed,and a large number of neurons nuclei in the hippocampus was shrunken,hyperchromatic showing degeneration and apoptosis by HE staining.Conclusion: Neurons autophagy in cerebral cortices of rats with focal cerebral ischemia and ischemia/reperfusion was obviously activated at ischemia 2 h point, it was increased continuously at ischemia/reperfusion 1 h and 6 h points and reached the peak at ischemia/reperfusion 24 h point.

Key words: brain, ischemic/reperfusion injury, LC3, autophagy, cortical neurons, rat

TAO Hong-miao, SHAN Xiao-yun, LI Xu-sheng, CHEN Hao-hao, MAO Yu-fei, HE Zhong-ping. Dynamic changes of autophagy activity of cortical neurons in rats with cerebral ischemia and cerebral ischemia/reperfusion[J]. CJAP, 2018, 34(3): 277-282.

References

[1] Donnan GA, Fisher M, Macleod M, et al. Stroke[J]. Lancet, 2008, 371(9624):1612-1623.
[2] Carloni S, Buonocore G, Balduini W. Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury[J]. Neurobiol Dis, 2008, 32(3):329-339.
[3] Tian F, Deguchi K, Yamashita T, et al. In vivo imaging of autophagy in a mouse stroke model[J]. Autophagy, 2010, 6(8):1107-1114.
[4] Wang JY, Xia Q, Chu KT, et al. Severe global cerebral ischemia-induced programmed necrosis of hippocampal CA1 neurons in rat is prevented by 3-methyladenine:a widely used inhibitor of autophagy[J]. J Neuropathol Exp Neurol, 2011, 70(4):314-322.
[5] Yu J, Bao C, Dong Y, et al. Activation of autophagy in rat brain cells following focal cerebral ischemia-reperfusion through enhanced expression of Atg1/pULK and LC3[J]. Mol Med Rep, 2015, 12(3):3339-3344.
[6] 胡中慧, 马慧萍, 樊鹏程, 等. PC12细胞在缺氧条件下的自噬现象[J]. 中国应用生理学杂志, 2017, 33(1):65-68.
[7] Joo SP, Xie W, Xiong X, et al. Ischemic postconditioning protects against focal cerebral ischemia by inhibiting brain inflammation while attenuating peripheral lymphopenia in mice[J]. Neuroscience, 2013, 243(30):149-157.
[8] Zhang X, Yan H, Yuan Y, et al. Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance[J]. Autophagy, 2013, 9(9):1321-1333.
[9] Gao L, Jiang T, Guo J, et al. Inhibition of autophagy contributes to ischemic postconditioning-induced neuroprotection against focal cerebral ischemia in rats[J]. PLoS One, 2012, 7(9):1-13.
[10] 彭志锋, 孟健, 张继红. 热休克蛋白A5介导的自噬在小鼠脑缺血/再灌注损伤中的作用[J]. 中国应用生理学杂志, 2017, 33(3):234-238.
[11] Su J, Zhang T, Wang K, et al. Autophagy activation contributes to the neuroprotection of remote ischemic perconditioning against focal cerebral ischemia in rats[J]. Neurochem Res, 2014, 39(11):2068-2077.
[12] Sheng R, Zhang LS, Han R, et al. Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning[J]. Autophagy, 2010, 6(4):482-494.
[13] Qi Z, Dong W, Shi W, et al. Bcl-2 phosphorylation triggers autophagy switch and reduces mitochondrial damage in limb remote ischemic conditioned rats after ischemic stroke[J]. Transl Stroke Res, 2015, 6(3):198-206.
[14] Shao ZQ, Liu ZJ. Neuroinflammation and neuronal autophagic death were suppressed via Rosiglitazone treatment:New evidence on neuroprotection in a rat model of global cerebral ischemia[J]. J Neurol Sci, 2015, 349(1-2):65-71.
[15] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1):84-91.
[16] Ren C, Yan Z, Wei D, et al. Limb remote ischemic postconditioning protects against focal ischemia in rats[J]. Brain Res, 2009, 1288(9):88-94.
[17] Klionsky DJ, Abdelmohsen K, Abe A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[J]. Autophagy, 2016, 12(1):1-222.
[18] Wang J, Han D, Sun M, et al. Cerebral ischemic postconditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model[J]. Mol Med Rep, 2016, 14(5):4162-4172.
[19] Deng YH, He HY, Yang LQ, et al. Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke[J]. Neural Regen Res, 2016, 11(7):1108-1114.
[20] 乜全民, 张世明. 大鼠局灶性脑缺血再灌注损伤后自噬的初步研究[J]. 中国临床神经科学, 2010, 18(1):14-18.