肖高鵬 李俞錦
【摘要】目的探討七氟醚對老齡血管性認知功能障礙大鼠認知功能的影響及作用機制。方法將60只6月齡雌性Wistar大鼠分為3組,每組各20只。A組大鼠為假手術(shù)組,B組大鼠實施右側(cè)頸動脈結(jié)扎術(shù);C組大鼠實施右側(cè)頸動脈結(jié)扎術(shù)后吸入2%七氟醚。采用穿梭箱和水迷宮試驗評價大鼠的認知功能,原位末端脫氧核苷酸轉(zhuǎn)移酶標記法檢測海馬神經(jīng)元凋亡指數(shù),流式細胞儀檢測神經(jīng)元細胞的凋亡指數(shù)和鈣離子熒光指數(shù),ELISA檢測海馬組織和外周血炎性因子[血管內(nèi)皮生長因子(VEGF)、IL1β、TNFα]水平以及海馬神經(jīng)元細胞勻漿中線粒體呼吸鏈復(fù)合體Ⅰ~Ⅳ的活性。結(jié)果 穿梭箱試驗中B、C組大鼠的電擊次數(shù)多于A組、主動逃避次數(shù)少于A組、學(xué)習(xí)和記憶潛伏期較A組延長(P均<001),而C組大鼠電擊次數(shù)較B組減少、主動逃避次數(shù)較B組增多、學(xué)習(xí)和記憶潛伏期短于C組(P均<001)。B、C組大鼠海馬組織及外周血血清VEGF水平低于A組、IL1β和TNFα水平高于A組(P均<005);與B組比較,C組VEGF水平較高、IL1β和TNFα水平較低(P均<005)。大鼠血清VEGF水平與電擊次數(shù)、學(xué)習(xí)潛伏期、記憶潛伏期均呈負相關(guān)(P均<005),與主動逃避次數(shù)呈正相關(guān)。IL1β水平和TNFα水平與電擊次數(shù)、學(xué)習(xí)潛伏期、記憶潛伏期均呈正相關(guān)(P均<005),與主動逃避次數(shù)均呈負相關(guān)(P均<005)。A、C組大鼠的海馬神經(jīng)元細胞凋亡指數(shù)和鈣離子熒光指數(shù)均低于B組(P均<001)、海馬中線粒體呼吸鏈復(fù)合物Ⅰ、Ⅱ活性高于B組(P均<005),A、C上述指標組間比較差異均無統(tǒng)計學(xué)意義(P>005)。結(jié)論七氟醚干預(yù)可減輕老齡大鼠血管性認知功能障礙,其機制可能與增加VEGF表達、減輕炎癥損傷有關(guān)。
【關(guān)鍵詞】血管性認知功能障礙;七氟醚;炎性因子;血管內(nèi)皮生長因子
Effect and mechanism of sevoflurane on the cognitive function in elderly rats with vascular cognitive impairmentXiao Gaopeng,Li Yujin Department of Anesthesiology,the First Peoples Hospital of Yunnan Province, Kunming 650000, China
Corresponding author, Li Yujin
【Abstract】ObjectiveTo investigate the effect and mechanism of sevoflurane on the cognitive function in elderly rats with vascular cognitive impairment MethodsSixty male Wistar rats aged 6 months were divided into three groups (n=20 for each group) In group A, sham operation was performed In group B, rats were treated with unilateral carotid artery ligation In group C, unilateral carotid artery ligation was performed, followed by the inhalation of 2% sevoflurane The cognitive function of rats was evaluated by shuttle box and water maze tests The apoptotic index of hippocampal neurons was detected by TUNEL The apoptotic rate and calcium ion fluorescent index of neurons were measured by flow cytometry The expression levels of inflammatory cytokines (VEGF, IL1β and TNFα) in the hippocampal tissues and peripheral blood and the activity of mitochondrial respiratory chain complex IIV in the hippocampal neurons were quantitatively detected by ELISA ResultsDuring the shuttle box test, the number of electrical shocks was significantly more, the number of active escape was considerably less and the learning and memory latency was significantly longer in groups B and C compared with those in group A (all P<001) In group C, the number of electrical shocks was significantly decreased, the number of active escape was considerably increased and the latency of learning and memory was significantly longer than those in group B (all P<001) In groups B and C, the expression levels of VEGF in hippocampal tissues and peripheral blood were significantly lower, whereas the expression levels of IL1β and TNFα were significantly higher than those in group A (both P<005) Compared with group B, the expression level of VEGF was evidently higher, whereas the expression levels of IL1β and TNFα were significantly lower in group C (all P<005) The expression level of serum VEGF was negatively correlated with the number of electrical shocks, learning and memory latency (both P<005), whereas positively associated with the number of active escape The expression levels of IL1β and TNFα were positively correlated with the number of electrical shocks, learning and memory latency (all P<005), whereas negatively correlated with the number of active escape (P<005) In groups A and C, the apoptotic index of hippocampal neurons and calcium ion fluorescent index were significantly lower (both P<001), whereas the activity of mitochondrial respiratory chain complex ⅠandⅡ was considerably higher (both P<005) than those in group B No statistical significance was noted in these parameters between groups A and C (all P>005) Conclusion Sevoflurane intervention can alleviate vascular cognitive impairment in aged rats, which is probably correlated with upregulating the expression of VEGF and mitigating inflammatory injury
【Key words】Vascular cognitive impairment; Sevoflurane; Inflammatory cytokine;
Vascular endothelial growth factor
血管性認知功能障礙是血管或血管相關(guān)因素引起的認知功能障礙,與血管性炎癥性損傷密切相關(guān),常發(fā)生于麻醉和(或)手術(shù)后[12]。高齡患者耐受麻醉和手術(shù)的能力低,而且隨著肝、腎功能的減退,藥物在其體內(nèi)消除的半衰期也延長。七氟醚對心率影響不明顯、氣道刺激小,為臨床常用麻醉藥物。有學(xué)者認為,七氟醚能激活線粒體中的相關(guān)信號通路,減少炎性因子的生成,從而緩解術(shù)后腦損傷[3]。然而,4%七氟醚吸入誘導(dǎo)約2 min患者意識即可消失,麻醉過深時有誘發(fā)全身痙攣的可能。目前有關(guān)七氟醚用于血管性認知功能障礙的效果尚有爭議,因此本研究著重探討七氟醚對老年血管性認知功能障礙大鼠認知功能的影響及其作用機制,現(xiàn)報告如下。
材料與方法
一、實驗動物
60只6月齡雌性Wistar大鼠,體質(zhì)量250~300 g,購自中科院上海實驗動物中心,飼養(yǎng)于SPF級動物房,本研究對實驗動物的處置均符合中華人民共和國科學(xué)技術(shù)部頒發(fā)的《關(guān)于善待實驗動物的指導(dǎo)性意見》。
二、主要試劑和儀器
七氟醚購自江蘇恩華制藥有限公司,血管內(nèi)皮生長因子(VEGF)、IL1β和TNFα檢測試劑盒購自南京碧云天公司,戊巴比妥鈉和伊文思藍購自北京雷根生物公司,原位末端脫氧核苷酸轉(zhuǎn)移酶標記(TUNEL)試劑盒購自北京雷根生物公司;貝克曼庫爾特Cyto FLEX流式細胞儀。
三、方法
1實驗動物分組
60只大鼠按隨機數(shù)字表法分為A、B、C組,每組各20只。A組大鼠為假手術(shù)組;B組大鼠為血管性認知功能障礙模型,實施右側(cè)頸動脈結(jié)扎術(shù),結(jié)扎時間為1 h;C組大鼠實施右側(cè)頸動脈結(jié)扎術(shù)后吸入2%七氟醚(每日2 h,共計5 d)[4]。
2大鼠認知功能檢測
穿梭箱試驗:成功造模3 d后實施,穿梭箱規(guī)格為60 cm×16 cm×25 cm。將大鼠置于穿梭箱中20 s后,開啟蜂鳴器,5 s后給予30 V 50 Hz電擊,總電擊時間為1 min,統(tǒng)計電擊和主動逃避的次數(shù)。每只實驗大鼠重復(fù)3次測試,結(jié)果取平均值。
水迷宮試驗:穿梭箱試驗3 d后實施,第1~3日進行定位航行訓(xùn)練,第4日以大鼠尋找并爬上平臺的時間為逃避潛伏期;第8日進行空間探索實驗,以在原平臺Ⅰ象限游泳時間即空間探索時間為記憶潛伏期。
3大鼠海馬組織和血清標本取材
水迷宮試驗完成后,經(jīng)大鼠腹腔注射 2%戊巴比妥(50 mg/kg)進行麻醉,取尾靜脈血后斷頭處死,夾閉腹主動脈,灌注4 ℃ 4%多聚甲醛,速度先快后慢,共30 min,至右耳流出液體血色較淺且基本澄清為止,冰上分離腦組織,剝離雙側(cè)海馬。
4大鼠海馬神經(jīng)元細胞凋亡情況的檢測
每組取8只大鼠的海馬組織浸入4%的多聚甲醛溶液中,置4 ℃固定24 h,采用TUNEL染色,于顯微鏡下觀察海馬神經(jīng)元細胞的凋亡情況:每張海馬切片隨機選擇5個互相不重疊的視野,并選擇400倍熒光顯微鏡下觀察,陽性凋亡細胞的細胞核呈綠色顆粒狀,計算凋亡指數(shù),凋亡指數(shù)=陽性細胞數(shù)/(陽性細胞數(shù)+陰性細胞數(shù))×100%。
5海馬神經(jīng)元鈣離子水平檢測
每組各取6只大鼠的海馬組織制作組織勻漿,2 000~3 000轉(zhuǎn)/分離心20 min,收集上清,應(yīng)用流式細胞儀檢測,以熒光指數(shù)反映胞漿鈣離子水平。
6炎性因子及線粒體呼吸鏈復(fù)合體水平的檢測
20只大鼠的尾靜脈血加入10% 01 mol/L檸檬酸鈉混合10~20 min后,2 000~3 000轉(zhuǎn)/分離心20 min,收集上清,應(yīng)用ELISA檢測大鼠血清中TNFα、IL1β和VEGF水平。每組取6只大鼠的海馬組織剪碎后加入DMEM培養(yǎng)基制成單細胞懸液,以ELISA檢測海馬神經(jīng)元中TNFα、IL1β、VEGF和線粒體呼吸鏈復(fù)合物Ⅰ~Ⅳ水平。
四、統(tǒng)計學(xué)處理
使用SPSS 210處理數(shù)據(jù)。計量資料均先行正態(tài)性檢驗,符合正態(tài)分布者以±s表示,多組比較用方差分析,組間兩兩比較使用LSDt檢驗;老齡大鼠血清炎性因子水平與認知功能的關(guān)系采用Pearson相關(guān)性分析。P<005為差異有統(tǒng)計學(xué)意義。
結(jié)果
一、3組老齡大鼠的行為學(xué)測定結(jié)果
1穿梭箱試驗結(jié)果
B、C組大鼠的電擊次數(shù)均多于A組,主動逃避次數(shù)均少于A組(P均<001);而C組大鼠電擊次數(shù)較B組減少,主動逃避次數(shù)較B組增多(P均<001),見表1。
討論
炎癥反應(yīng)是缺血性神經(jīng)元損傷的主要機制,微血管損傷誘發(fā)的缺血再灌注損傷的核心是炎癥反應(yīng)的活化,癡呆程度和炎癥與腦血管損傷有著直接的聯(lián)系,所以抑制炎癥是治療腦血管疾病的主要策略。TNFα 與 IL1β是腦缺血損傷炎癥反應(yīng)最早出現(xiàn)的細胞因子[57]。腦室注射IL1β抑制劑可改善大鼠空間學(xué)習(xí)能力的損害[8]。TNFα水平的上調(diào)與認知功能障礙有關(guān)[9]。研究發(fā)現(xiàn),麻醉藥物能夠抑制炎性細胞因子的過度產(chǎn)生,并能減輕由缺血誘發(fā)的神經(jīng)元凋亡[1011]。VEGF能夠促進血管的生長和側(cè)支循環(huán)的建立,是腦缺血性損傷的血管修復(fù)因子。局灶腦缺血的動物實驗顯示,腦梗死后 3 d是最活躍的血管增生期,并可見明顯的VEGF表達,提示應(yīng)激反應(yīng)能夠誘發(fā)VEGF表達的上調(diào),促進神經(jīng)元功能的恢復(fù)[1213]。本研究顯示,與缺血、缺氧誘發(fā)認知功能障礙的模型大鼠相比,吸入七氟醚的認知功能障礙大鼠海馬組織及外周血VEGF水平上升,TNFα和IL1β水平下降,穿梭箱試驗中其電擊次數(shù)較少、主動逃避次數(shù)較多,水迷宮試驗中其學(xué)習(xí)和記憶潛伏期較短,提示七氟醚對改善缺血、缺氧誘發(fā)的認知功能障礙有一定的效果。
細胞凋亡是多階段且并非同步發(fā)生的進程。急性腦血管病可導(dǎo)致機體產(chǎn)生大量的自由基,并導(dǎo)致細胞內(nèi)發(fā)生鈣超載,從而引發(fā)神經(jīng)元細胞凋亡[14]。多項研究顯示,神經(jīng)元缺血、缺氧后自由基明顯增多,其能持續(xù)開放線粒體的滲透性轉(zhuǎn)換孔,導(dǎo)致線粒體膜電位發(fā)生異常、細胞凋亡[15]。研究表明,大鼠的學(xué)習(xí)和記憶能力下降,與細胞超微結(jié)構(gòu)如線粒體的腫脹和功能障礙密切相關(guān)[16]。另有研究顯示,強應(yīng)激會導(dǎo)致海馬神經(jīng)元的樹突棘發(fā)生退縮乃至消失[17]。七氟醚能夠拮抗缺血誘發(fā)的海馬神經(jīng)元凋亡及減少神經(jīng)元的鈣超載狀態(tài)[18]。本研究中,經(jīng)七氟醚干預(yù)的認知功能障礙大鼠海馬組織神經(jīng)元凋亡指數(shù)、鈣離子熒光指數(shù)、線粒體呼吸鏈復(fù)合物Ⅰ、Ⅱ活性均低于缺血、缺氧誘發(fā)認知功能障礙的模型大鼠,與假手術(shù)大鼠相近,提示七氟醚能夠緩解由于缺血、缺氧所誘發(fā)的線粒體功能失常,發(fā)揮神經(jīng)元保護作用。
綜上所述,七氟醚能夠有效減輕缺血、缺氧誘發(fā)的認知功能障礙,該保護機制與提高VEGF水平、減少IL1β和TNFα釋放、減輕炎癥損傷有關(guān)。此外七氟醚還能夠減輕海馬神經(jīng)元內(nèi)炎性反應(yīng)的程度,從而提高了線粒體內(nèi)呼吸鏈酶復(fù)合物Ⅰ、Ⅱ活性,減輕了海馬神經(jīng)元細胞凋亡,改善認知功能障礙。
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