Study on Protective Effects of Moderate Ethanol Preconditioning Against Brain Injury-induced by Focal Cerebral Ischemia / Reperfusion in Rats

Abstract

Abstract:

Objective To study the protective effects of moderate ethanol preconditioning on neuronal injury induced by focal cerebral ischemic-reperfusion in rats. Methods A total of 36 male Sprague-Dawley rats were randomly divided into 3 groups (n =12): (1) sham control group; (2) ischemic-reperfusion group (I/R); (3) ethanol preconditioning group (EtOH-PC). Rats were subjected to 2 h of right middle cerebral artery occlusion (MCAO) and 24 h of reperfusion. EtOH-PC was induced by gavaging animals with a moderate dose of ethanol [volume of ethanol in microliters calculated from the equation (body weight (in g) × 0.6) + 0.3] 24 h before ischemia. This volume of ethanol (95% in μl) was mixed in 0.3 ml of sterile distilled water before gavage. Animals in the sham control group (no I/R) and the I/R-alone group (no EtOH-PC) received a similar volume of saline by gavage. Eight rats of each group were used to perform neurological score and 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining 24 h after ischemia and reperfusion. The volume of cerebral infarction was calculated based on the staining result. Another 4 rats of each group were used to perform T2-weighted imaging (T2WI) MRI 24 h after ischemia and reperfusion. Then rats were killed. After frozen section, two slices was acquired from each rat’s primary somatosensory cortex areas. One slice was used to detect the apoptotic cell by means of terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling (TUNEL); the other was used to detect the degeneration of neurons through Fluoro-Jade B. Results Compared to ischemic-reperfusion group, the neurological score (15.00 [14.25, 16.00] vs 3.50 [2.25, 4.00]) (P <0.001), cerebral infarction volume (TTC: [242.80±17.44]mm3 vs [54.83±13.43]mm3; T2WI:[296.80±8.53]mm3 vs [59.68±9.97]mm3) (P <0.001), the percentage of apoptotic cell death ([33.47±2.23]% vs [9.66±0.84]%) (P <0.001) and the percentage of degenerative neurons ([45.31±3.40]% vs [23.26±1.25]%) (P <0.001) were significantly decreased after ethanol preconditioning. Conclusion Moderate ethanol preconditioning can protect against the neuron injury induced by focal cerebral ischemic-reperfusion.

Key words: Ethanol preconditioning; Cerebral ischemia/reperfusion; Neuroprotection

摘要：

目的研究适度酒精预适应对大鼠局灶性脑缺血再灌注诱导的神经元损伤的保护作用。方法将36只雄性SD大鼠随机分为假手术组、缺血再灌注组和酒精预适应组，每组12只。局灶性脑缺血再灌注模型采用右侧大脑中动脉闭塞方式，缺血2 h，再灌注24 h。酒精预适应组在脑缺血再灌注前24 h用95%酒精与0.3 ml无菌蒸馏水混合后进行灌胃，95%酒精体积（μl）计算为：[大鼠体重（g） x0.6]+0.3。其余两组用同等剂量的生理盐水灌胃。每组取8只大鼠在缺血再灌注后24 h进行神经功能评分和氯化2，3，5-三苯基四氮唑（2，3，5-Triphenyltetrazolium chloride，TTC）染色并根据染色结果计算脑梗死体积。每组剩余的4只，在缺血再灌注后24 h进行磁共振T2加权像（T2-weighted imaging，T2WI）序列扫描，然后将大鼠处死取脑，经过冷冻切片后，每只大鼠取第一躯体感觉皮质区的2张切片，1 张用末端转移酶介导的脱氧尿嘧啶核苷三磷酸缺口末端标记（terminal deoxynucleotidyl transferasemediated 2'-deoxyuridine 5'-triphosphate nick-end labeling，TUNEL）检测其凋亡性细胞死亡情况，另一张用Fluoro-Jade B检测其神经元退化变性情况。结果相较于缺血再灌注组，经过适度酒精预适应后会显著降低大鼠的神经功能评分[15.00 （14.25，16.00）vs 3.50（2.25，4.00）（P <0.001）]、脑梗死体积[TTC：（242.80±17.44）mm3 vs （54.83±13.43）mm3；T2WI ：（296.80±8.53）mm3 vs（59.68±9.97）mm3，P均<0.001]、凋亡细胞所占百分比[（33.47±2.23）% vs（9.66±0.84）%，P <0.001]和退化变性神经元所占百分比（[ 45.31±3.40）% vs（23.26±1.25）%，P <0.001]。结论适度酒精预适应可以保护局灶性脑缺血再灌注诱导的神经元损伤。

关键词: 酒精预适应; 脑缺血/再灌注; 神经保护

ZHAO Yi-Long, GUO An-Chen, SU Fang, et al.. Study on Protective Effects of Moderate Ethanol Preconditioning Against Brain Injury-induced by Focal Cerebral Ischemia / Reperfusion in Rats[J]. Chinese Journal of Stroke, 2016, 11(05): 378-385.

赵一龙，郭安臣，苏芳，李巍巍，杨华俊，孙明，王拥军，王群. 适度酒精预适应对大鼠局灶性脑缺血再灌注损伤保护作用的研究[J]. 中国卒中杂志, 2016, 11(05): 378-385.

References

1 Flynn RW，MacWalter RS，Doney AS. The cost of

cerebral ischaemia[J]. Neuropharmacology，2008，55：

250-256.

2 Collins MA，Neafsey EJ，Mukamal KJ，et al. Alcohol

in moderation，cardioprotection，and neuroprotection：

epidemiological considerations and mechanistic

studies[J]. Alcohol Clin Exp Res，2009，33：206-219.

3 Wang Q，Sun AY，Simonyi A，et al. Ethanol

precondi t ioning protect s against ischemia /

reperfusion-induced brain damage：role of NADPH

oxidase-derived ROS[J]. Free Radic Biol Med，2007，

43：1048-1060.

4 Sun M，Zhao YM，Gu Y，et al. Therapeutic window

of taurine against experimental stroke in rats[J].

Transl Res，2012，160：223-229.

5 Bederson JB，Pitts LH，Tsuji M，et al. Rat middle

cerebral artery occlusion：evaluation of the model and

development of a neurologic examination[J]. Stroke，

1986，17：472-476.

6 Chen J，Sanberg PR，Li Y，et al. Int ravenous

administration of human umbilical cord blood reduces

behavioral deficits after stroke in rats[J]. Stroke，2001，

32：2682-2688.

7 Sun K，Fan J，Han J. Ameliorat ing ef fects of

traditional Chinese medicine preparation，Chinese

materia medica and active compounds on ischemia/

reperfusion-induced cerebral microcirculatory

disturbances and neuron damage[J]. Acta Pharm Sin

B，2015，5：8-24.

8 Hu Y，Deng H，Xu S，et al. MicroRNAs regulate

mitochondr ial funct ion in cerebral ischemiareperfusion

injury[J]. Int J Mol Sci，2015，16：24895-

24917.

9 Yuan Q，Hong S，Han S，et al. Preconditioning with

physiological levels of ethanol protect kidney against

ischemia/reperfusion injury by modulating oxidative

stress[J]. PLoS One，2011，6：e25811.

10 Collins MA，Neafsey EJ，Wang K，et al. Moderate

ethanol precondit ioning of rat brain cultures

engenders neuroprotection against dementia-inducing

neuroinf lammatory proteins：possible signaling

mechanisms[J]. Mol Neurobiol，2010，41：420-425.

11 Krenz M，Korthuis RJ. Moderate ethanol ingestion

and cardiovascular protection：from epidemiologic

associations to cellular mechanisms[J]. J Mol Cell

Cardiol，2012，52：93-104.

12 Kikuchi K，Uchikado H，Morioka M，et al. Clinical

neuroprotective drugs for treatment and prevention of

stroke[J]. Int J Mol Sci，2012，13：7739-7761.

13 Fisher M，Hachinski V. European Cooperative Acute Stroke Study III：support for and questions about a truly

emerging therapy[J]. Stroke，2009，40：2262-2263.

14 Wi l l ing AE. Exper iment al model s：h e lp o r

hindrance[J]. Stroke，2009，40：S152-154.

15 Stetler RA，Leak RK，Gan Y，et al. Preconditioning

provides neuroprotection in models of CNS disease：

paradigms and cl inical signif icance[J ]. Prog

Neurobiol，2014，114：58-83.

16 Bonservizi WG，Koike MK，Saur im R，et al.

Ischemic preconditioning and atenolol on lung injury

after intestinal ischemia and reperfusion in rats[J].

Transplant Proc，2014，46：1862-1866.

17 Tsutsui H，Tanaka R，Yamagata M，et al. Protective

effect of ischemic preconditioning on ischemia/

reperfusion-induced acute kidney injury through

sympathetic nervous system in rats[J]. Eur J

Pharmacol，2013，718：206-212.

18 Onishi A，Miyamae M，Inoue H，et al. Sevoflurane

confers addit ive cardioprotect ion to ethanol

pre condi t ioning a s sociat ed wi t h en hanced

phosphorylation of glycogen synthase kinase-

3beta and inhibition of mitochondrial permeability

transition pore opening[J]. J Cardiothorac Vasc

Anesth，2013，27：916-924.

19 Yamag uchi T，Dayton C，Shigematsu T，et al.

Preconditioning with ethanol prevents postischemic

leukocyte-endothelial cell adhesive interactions[J]. Am

J Physiol Heart Circ Physiol，2002，283：H1019-1030.

20 Liao SL，Chen WY，Raung SL，et al. Ethanol

attenuates ischemic and hypoxic injury in rat brain

and cultured neurons[J]. Neuroreport，2003，14：

2089-2094.

21 Wang Q，Kalogeris TJ，Wang M，et al. Antecedent

ethanol attenuates cerebral ischemia/reperfusioni

nd u c e d l e u ko c y t e - e nd ot h el i a l a d h e s ive

interactions and delayed neuronal death：role of

large conductance，Ca2+-activated K+ channels[J].

Microcirculation，2010，17：427-438.

[1]	WEN Shaohong, LIU Kuan, ZHAO Shunying, DONG Wen, CHEN Qingfang, CHEN Wentao, YE Weizhen, JIANG Mingyu, LIU Xiangrong. Neuroprotective Effect and Mechanism of Salidroside in Cerebral Ischemia-reperfusion Injury in Mouse Model [J]. Chinese Journal of Stroke, 2022, 17(09): 991-1001.
[2]	ZHOU Gang, WANG Jia-Jun. Neuroprotective Effects of Trandolapril on Rat Cerebral Ischemia-reperfusion Injury [J]. Chinese Journal of Stroke, 2018, 13(04): 339-343.
[3]	LI Chuan-Wen, WANG Qing-Song. The Neuroprotective Effect of Silent Information Regulator Protein 1 in Ischemic Brain Injury [J]. Chinese Journal of Stroke, 2018, 13(01): 86-89.
[4]	DONG Wen, WANG Yong-Jun. Gene Microarray Analysis of Astrocytes in Acute Ischemic Stroke Mice Based on Gene Expression Omnibus [J]. Chinese Journal of Stroke, 2017, 12(11): 1000-1004.
[5]	ZUO Lian, LI Gang. Non-thromolytic Effect of Tissue Plasminogen Activator [J]. Chinese Journal of Stroke, 2017, 12(05): 462-467.
[6]	LI Hai-Long, BI Xiao-Ying. Progress of Studies Relevant to the Roles of Micro Ribonucleic Acids in Ischemic Stroke [J]. Chinese Journal of Stroke, 2015, 10(11): 974-979.
[7]	LI Wei-Wei*, LI Jun-Fa,GUO An-Chen, WANG Yong-Jun, WANG Qun.. Neuroprotection of Plant Polyphenols in Cerebral Ischemia [J]. Chinese Journal of Stroke, 2015, 10(02): 141-146.
[8]	ZHAO Yu-Mei;SUN Ming;XU Chao. Protection of Taurine against Transient Forebrain Ischemia in Gerbils [J]. , 2012, 7(02): 98-102.
[9]	YUAN Xia;ZHANG Shi-ming. Advances in Granulocyte Colony-Stimulating Factor for Cerebral Infarction Treatment [J]. , 2007, 2(04): 74-78.