摘要
目的 探讨不同剂量艾司氯胺酮预处理对脑缺血性损伤小鼠的神经保护作用及可能机制.方法 成年雄性C57BL/6小鼠44只随机分为假手术组、模型组、低剂量组和高剂量组各11只.模型组、低剂量组、高剂量组采用永久性结扎阻断右大脑中动脉和暂时性阻断双侧颈总动脉建立局灶性脑缺血模型,假手术组仅暴露动脉;低剂量组和高剂量组在夹闭双侧颈总动脉前分别腹腔注射艾司氯胺酮6、12 mg/kg,假手术组和模型组腹腔注射等体积生理盐水.造模24 h时采用Longa评分和贴纸实验观察小鼠神经行为学改变,采用实时荧光定量PCR法检测缺血脑组织诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)、Beclin1mRNA相对表达量;造模7 2 h时采用TTC染色法观察脑梗死面积.结果 (1)模型组Longa评分[(2.36±0.51)分]均高于假手术组(0分)、低剂量组[(1.27±0.47)分]、高剂量组[(1.91± 0.54)分](P＜0.05),低剂量组、高剂量组均高于假手术组(P＜0.05),高剂量组高于低剂量组(P＜0.05);模型组碰触贴纸时间[(23.18±2.36)s]、移除贴纸时间[(26.36±1.96)s]均长于假手术组[(5.27±1.42)、(6.73±1.95)s]、低剂量组[(6.73±1.42)、(8.82±1.89)s]、高剂量组[(15.65±3.08)、(18.91±3.11)s](P＜0.05),高剂量组均长于假手术组、低剂量组(P＜0.05),假手术组与低剂量组比较差异均无统计学意义(P＞0.05).(2)高剂量组脑梗死面积[(23.45± 0.84)％]均大于假手术组(0)、模型组[(19.03±0.99)％]、低剂量组[(15.98±0.97)％](P＜0.05),模型组、低剂量组均大于假手术组(P＜0.05),模型组大于低剂量组(P＜0.05).(3)假手术组缺血脑组织Arg-1、iNOS mRNA相对表达量(0.99±0.15、1.00±0.29)均低于模型组(12.37±2.99、4.17±0.76)、低剂量组(17.16±3.78、2.02±0.21)、高剂量组(5.64±0.51、1.94±0.21)(P＜0.05);模型组、高剂量组缺血脑组织Arg-1 mRNA相对表达量均低于低剂量组(P＜0.05),高剂量组低于模型组(P＜0.05);低剂量组、高剂量组缺血脑组织iNOS mRNA相对表达量均低于模型组(P＜0.05),低剂量组与高剂量组比较差异无统计学意义(P＞0.05).低剂量组缺血脑组织Beclin 1 mRNA相对表达量(0.35±0.03)均低于假手术组(0.73±0.18)、模型组(0.91±0.08)、高剂量组(0.87±0.06)(P＜0.05);模型组高于假手术组(P＜0.05),与高剂量组比较差异无统计学意义(P＞0.05);高剂量组与假手术组比较差异无统计学意义(P＞0.05).结论 低剂量艾司氯胺酮可促进局灶性脑缺血小鼠缺血区小胶质细胞从M1向M2型转化、抑制神经炎性反应和降低自噬进而发挥神经保护作用,高剂量艾司氯胺酮对脑缺血性损伤无明显神经保护作用.
Abstract
Objective To investigate the neuroprotective role of pretreatments with different doses of esketamine in mice with ischemic brain injury and explore its potential mechanisms.Methods Forty-four adult male C57BL/6 mice were randomly divided into sham-operation group,model group,low-dose group and high-dose group,with 11 mice in each group.The focal cerebral ischemia models were established in model group,low-dose group and high-dose group by permanent ligation of the right middle cerebral artery and temporary occlusion of the bilateral common carotid arteries.The mice in sham-operation group were only exposed the arteries without any occlusion treatment.The mice in low-dose group and high-dose group were respectively intraperitoneally injected with 6 and 12 mg/kg of esketamine before clamping the bilateral common carotid arteries,and the mice in sham-operation group and model group were intraperitoneally injected with equivalent volume of normal saline.At 24 h after model establishment,both the Longa score and double-sided sticker removal experiment were used to observe the neurobehavioral changes in four groups.Real-time fluorescence quantitative PCR was used to detect the relative expressions of inducible nitric oxide synthase(iNOS),arginase-1(Arg-1)and Beclin 1 mRNAs in the ischemic cerebral tissues.At 72 h after model establishment,TTC staining was performed to observe the cerebral infarct area.Results(1)The Longa score was significantly higher in model group(2.36±0.51)than that in sham-operation group(0),low-dose group(1.27±0.47)and high-dose group(1.91±0.54)(P＜0.05),higher in low-dose and high-dose groups than that in sham-operation group(P＜0.05),and higher in high-dose group than that in low-dose group(P＜0.05).The time for touching sticker and time for removing stickers were longer in model group[(23.18±2.36),(26.36±1.96)s]than those in sham-operation group[(5.27± 1.42),(6.73±1.95)s],low-dose group[(6.73±1.42),(8.82±1.89)s]and high-dose group[(15.65±3.08),(18.91±3.11)s](P＜0.05),were longer in high-dose group than those in sham-operation and low-dose groups(P＜0.05),and showed no significant difference between sham-operation and low-dose groups(P＞0.05).(2)The cerebral infarct area was larger in high-dose group[(23.45±0.84)％]than that in sham-operation group(0),model group[(19.03±0.99)％]and low-dose group[(15.98±0.97)％](P＜0.05),larger in model and low-dose groups than that in sham-operation group(P＜0.05),and larger in model group than that in low-dose group(P＜0.05).(3)The relative expressions of Arg-1 and iNOS mRNAs in the ischemic cerebral tissues were lower in sham-operation group(0.99±0.15,1.00±0.29)than those in model group(12.37±2.99,4.17±0.76),low-dose group(17.16±3.78,2.02±0.21)and high-dose group(5.64±0.51,1.94±0.21)(P＜0.05).The relative expression of Arg-1 mRNA was lower in model group and high-dose group than that in low-dose group(P＜0.05),and lower in high-dose group than that in model group(P＜0.05).The relative expression of iNOS mRNA was lower in low-dose and high-dose groups than that in model group(P＜0.05),and showed no significant difference between low-dose and high-dose groups(P＞0.05).The relative expression of Beclin 1 mRNA was lower in low-dose group(0.35±0.03)than that in sham-operation group(0.73±0.18),model group(0.91±0.08)and high-dose group(0.87±0.06)(P＜0.05),was higher in model group than that in sham-operation group(P＜0.05),and showed no significant difference between model and high-dose groups(P＞0.05)and between high-dose and sham-operation groups(P＞0.05).Conclusions A low-dose esketamine can exert neuroprotection by promoting transformation of microglia from phenotype Ml to M2,inhibiting neuroinflammatory response and decreasing autophagy in the ischemic cerebral area.However,a high-dose esketamine has no significant protection against ischemic brain injury.
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