全麻/控制性降壓手術(shù)對(duì)海馬神經(jīng)元凋亡的影響*

王改梅， 劉 喆， 沈穎潔， 方劍喬

(浙江中醫(yī)藥大學(xué)第三臨床醫(yī)學(xué)院，浙江 杭州 310053)

·短篇論著·

全麻/控制性降壓手術(shù)對(duì)海馬神經(jīng)元凋亡的影響*

王改梅， 劉 喆△， 沈穎潔， 方劍喬

(浙江中醫(yī)藥大學(xué)第三臨床醫(yī)學(xué)院，浙江 杭州 310053)

目的觀察全麻/控制性降壓手術(shù)對(duì)海馬神經(jīng)元損傷的影響及其可能的機(jī)制。方法選用12只6～8月齡雄性健康比格犬，隨機(jī)分為全麻組(A組，n=6)和控壓組(C組，n=6)。全麻組采用丙泊酚注射及異氟醚吸入麻醉，控壓組在麻醉基礎(chǔ)上聯(lián)合硝普鈉行控制性降壓，達(dá)到目的血壓(基礎(chǔ)平均動(dòng)脈壓的40%)后維持60 min(2組動(dòng)物均在相應(yīng)期間行剖腹探查術(shù))，術(shù)畢使血壓回升至基礎(chǔ)水平72 h后取腦。采用免疫組織化學(xué)法檢測(cè)海馬CA1和CA3區(qū)白細(xì)胞介素 1β(IL-1β)、腫瘤壞死因子α(TNF-α)以及凋亡相關(guān)蛋白(Bcl-2、Bax和活化型caspase-3)的表達(dá)；TUNEL法檢測(cè)海馬神經(jīng)元凋亡情況。結(jié)果(1)控壓組動(dòng)物海馬CA1和CA3區(qū)IL-1β和TNF-α陽(yáng)性細(xì)胞率均較全麻組升高(P<0.01)；(2)控壓組CA1和CA3區(qū)Bcl-2/Bax陽(yáng)性細(xì)胞比值均較全麻組降低(P<0.05)；與此相應(yīng)，控壓組海馬各區(qū)caspase-3陽(yáng)性細(xì)胞率均顯著高于全麻組(P<0.01)；(3)全麻組與控壓組CA1和CA3區(qū)存在凋亡細(xì)胞，但控壓組各區(qū)細(xì)胞凋亡指數(shù)均較全麻組顯著升高(P<0.01)。結(jié)論全麻/控制性降壓下的手術(shù)可誘導(dǎo)海馬不同區(qū)域促炎細(xì)胞因子表達(dá),降低Bcl-2/Bax比值，促進(jìn)caspase-3的表達(dá)，進(jìn)而導(dǎo)致海馬神經(jīng)元的凋亡。這種術(shù)后海馬神經(jīng)元損傷可能與全麻/控壓手術(shù)誘導(dǎo)的海馬炎癥反應(yīng)有關(guān)。

神經(jīng)元凋亡； 炎癥； 全身麻醉; 控制性降壓； 海馬

藥物控制性降壓(controlled hypotension)是目前臨床根據(jù)手術(shù)及病情需要所采取的并廣泛應(yīng)用的一種麻醉方法，目的是減少術(shù)中失血和輸血，改善術(shù)野的環(huán)境，縮短手術(shù)時(shí)間[1]。但單純藥物降壓難以維持重要臟器的充分灌注，無(wú)法保證其血氧供應(yīng)而有臟器損傷之虞[2]。腦組織具有代謝率高、缺血缺氧耐受性差等特殊生理及代謝特點(diǎn)，是對(duì)缺血缺氧最敏感的器官之一，在控制性降壓期間組織灌注壓的下降有可能會(huì)造成腦血供不足和腦組織缺氧的危險(xiǎn)以及升壓后的再灌注損傷[2]。本研究用健康比格犬模擬剖腹探查術(shù)，觀察全麻/控制性降壓手術(shù)對(duì)海馬神經(jīng)元損傷的影響，并探討海馬細(xì)胞損傷的可能機(jī)制。

材 料 和 方 法

1動(dòng)物分組與處理

本研究選用12只6～8月齡健康雄性比格犬，體重8～12 kg(購(gòu)自上海新岡實(shí)驗(yàn)動(dòng)物養(yǎng)殖場(chǎng))。采用隨機(jī)法先將實(shí)驗(yàn)動(dòng)物分為2組：?jiǎn)渭內(nèi)榻M(全麻組，A組)、控制性降壓組(控壓組，C組)。每組6只。全麻組麻醉前肌注阿托品，靜脈注射丙泊酚4 mg/kg進(jìn)行誘導(dǎo)麻醉，靜脈注射肌松藥維庫(kù)溴銨，行氣管插管，氧氣流量1 L/min, 維持呼吸頻率15 min-1，潮氣量150～300 mL，繼以3%異氟醚吸入維持麻醉；血?dú)獗O(jiān)測(cè)控制pH值在7.35～7.45范圍?？貕航M以硝普納靜脈輸注行控制性降壓；維持目的血壓為基礎(chǔ)平均動(dòng)脈壓(mean arterial pressure，MAP)的40% 60 min，其間(全麻組亦在相應(yīng)時(shí)間內(nèi))行剖腹探查術(shù)，術(shù)畢停止硝普鈉輸注，調(diào)異氟醚濃度至1%，回升血壓，恢復(fù)自主呼吸后，停止異氟醚吸入，撤除所有監(jiān)測(cè)性插管及輸液。

2組織取材

術(shù)后72 h 30%水合氯醛腹腔麻醉，將動(dòng)物經(jīng)股動(dòng)脈灌注，取腦置4%多聚甲醛溶液固定，取含海馬組織區(qū)域行石蠟包埋。

3免疫組織化學(xué)(SP法)染色

取組織切片(厚5 μm)常規(guī)脫蠟至水。PBS漂洗5 min×3次，0.01 mol/L pH6.0檸檬酸緩沖液抗原修復(fù)，高火5 min×3次；0.3%H2O2-80%甲醇溶液室溫處理15 min，PBS沖洗5 min×3次；滴加山羊血清封閉，室溫1 h，甩去多余液體；滴加Ⅰ抗(Bax 1∶250；Bcl-2 1∶350；活化型caspase-3 1∶200；IL-1β 1∶250；TNF-α 1∶100; 均購(gòu)自北京博奧森)，放入濕盒，37 ℃孵育1 h，移至4 ℃冰箱過(guò)夜；次日，置于37 ℃水浴箱復(fù)溫1 h，PBS沖洗5 min×3次；滴加生物素Ⅱ抗，37 ℃孵育1 h，PBS沖洗5 min×3次；滴加S-A/HRP，37 ℃孵育1 h，PBS沖洗5 min×3次；DAB顯色，自來(lái)水沖洗，以終止反應(yīng)；蘇木素復(fù)染，脫水、透明，封片，光鏡觀察。選5個(gè)視野，計(jì)數(shù)陽(yáng)性細(xì)胞并計(jì)算其陽(yáng)性比率，取均值。

4TUNEL(Roche)法檢測(cè)神經(jīng)元凋亡

石蠟切片常規(guī)脫蠟至水，200 mL 0.01 mol/L pH 6.0的檸檬酸緩沖液進(jìn)行修復(fù)，750 W微波修復(fù)1 min，PBS漂洗5 min×3次，小牛血清室溫封閉30 min；PBS漂洗5 min×3次，加50 μL TdT 酶反應(yīng)液，加蓋玻片37 ℃避光濕潤(rùn)反應(yīng)60 min；滴加50 μL streptavidin-HRP工作液，加蓋玻片濕潤(rùn)避光反應(yīng)30 min；PBS漂洗5 min×3次，滴加50 μL DAB工作液，室溫顯色20 s；自來(lái)水沖洗，蘇木素復(fù)染，脫水、透明，封片，光鏡觀察。選5個(gè)視野，計(jì)數(shù)海馬CA1區(qū)和CA3區(qū)陽(yáng)性細(xì)胞數(shù)和細(xì)胞總數(shù)，計(jì)算凋亡指數(shù)(apoptotic index，AI)，AI(%)=TUNEL陽(yáng)性細(xì)胞數(shù)/細(xì)胞總數(shù)×100%。

5統(tǒng)計(jì)學(xué)處理

數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。用SPSS 13.0統(tǒng)計(jì)軟件分析。數(shù)據(jù)均進(jìn)行正態(tài)性檢驗(yàn),組間均數(shù)比較采用配對(duì)樣本t檢驗(yàn)，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1海馬CA1和CA3區(qū)IL-1β和TNF-α表達(dá)情況

海馬IL-1β與TNF-α陽(yáng)性細(xì)胞為棕褐色?？貕航M海馬CA1和CA3區(qū)IL-1β和TNF-α陽(yáng)性細(xì)胞率均較全麻組顯著升高(P<0.01),見(jiàn)圖1和表1。

2海馬CA1和CA3區(qū)Bcl-2和Bax表達(dá)情況

海馬Bcl-2和Bax陽(yáng)性細(xì)胞為棕褐色?？貕航MCA1和CA3區(qū)Bcl-2/Bax比值均較全麻組降低(P<0.05),見(jiàn)圖2和表2。

Figure 1. Representative immunohistochemical staining (brown) of IL-1β and TNF-α in hippocampal CA1 and CA3 areas.Group A:general anesthesia group；group C: controlled hypotension group.Bar: 20 μm.

圖1海馬CA1和CA3區(qū)IL-1β與TNF-α免疫組化染色情況

表1海馬CA1和CA3區(qū)IL-1β和TNF-α陽(yáng)性細(xì)胞率

Table 1. IL-1β and TNF-α positive cell rates in hippocampal CA1 and CA3 areas (%.Mean±SD.n=6)

GroupCA1CA3IL-1βTNF-αIL-1βTNF-αA9.42±0.598.14±1.187.75±0.637.31±0.39C17.40±0.76▲▲13.63±0.53▲▲14.06±0.82▲▲13.49±0.64▲▲

Group A:general anesthesia; group C:controlled hypotension.▲▲P<0.01vsgroup A.

3海馬CA1區(qū)和CA3區(qū)活化型caspase-3表達(dá)及TUNEL染色情況

海馬活化型caspase-3陽(yáng)性細(xì)胞為棕褐色，控壓組CA1和CA3區(qū)caspase-3陽(yáng)性細(xì)胞率均較全麻組顯著升高(P<0.01)。TUNEL陽(yáng)性表達(dá)為細(xì)胞核呈棕黃色，CA1區(qū)神經(jīng)元細(xì)胞稀疏，陽(yáng)性細(xì)胞散在分布；控壓組CA1和CA3區(qū)細(xì)胞凋亡指數(shù)均較全麻組顯著升高(P<0.01),見(jiàn)圖3和表3。

Figure 2. Representative immunohistochemical staining (brown) of Bax and Bcl-2 in hippocampal CA1 and CA3 areas.Bar: 20 μm.

圖2海馬CA1和CA3區(qū)Bax與Bcl-2免疫組化染色情況

表2海馬CA1和CA3區(qū)凋亡蛋白Bcl-2/Bax比值

Table 2. Bcl-2/Bax ratio in hippocampal CA1 and CA3 areas (mean±SD.n=6)

GroupCA1CA3A1.01±0.021.15±0.04C0.70±0.04△0.82±0.03△

△P<0.05vsgroup A.

討 論

控制性降壓是目前廣泛應(yīng)用的臨床麻醉方法?；颊咴诼樽硎中g(shù)期間由于麻醉藥物和方法的影響，?？刹l(fā)心率減慢、血壓下降及呼吸抑制，難以保證對(duì)重要臟器組織的血流灌注及血氧供應(yīng)而極易發(fā)生組織器官的缺血/缺氧性損傷，加之麻醉藥物誘導(dǎo)性降壓及手術(shù)創(chuàng)口失血更加重了這種風(fēng)險(xiǎn)。在易損器官中，由于大腦結(jié)構(gòu)功能復(fù)雜，能量及氧儲(chǔ)備低、組織代謝率高，對(duì)缺血缺氧十分敏感且耐受性差等特性[3-4]，故腦缺血/缺氧成為控制性降壓的最大顧慮之一[2]。來(lái)自臨床和基礎(chǔ)實(shí)驗(yàn)的研究均證實(shí)[5-6]，單純麻醉或手術(shù)均可造成中樞神經(jīng)的損傷并產(chǎn)生術(shù)后認(rèn)知功能障礙、譫妄等并發(fā)癥。本研究發(fā)現(xiàn)麻醉可誘導(dǎo)海馬CA1和CA3區(qū)神經(jīng)元發(fā)生凋亡，而控制性降壓更加重這種損傷，進(jìn)一步證實(shí)了上述認(rèn)識(shí)。

Figure 3. Representative immunohistochemical staining (brown) of cleaved caspase-3 and TUNEL staining of apoptosis in hippocampal CA1 and CA3 areas.Bar: 20 μm.

圖3海馬CA1和CA3區(qū)活化型caspase-3與TUNEL染色情況

表3海馬CA1區(qū)和CA3區(qū)活化型caspase-3陽(yáng)性細(xì)胞率和細(xì)胞凋亡指數(shù)

Table 3. Cleaved caspase-3 positive cell rate and apoptotic index (AI) in hippocampal CA1 and CA3 areas (%.Mean±SD.n=6)

GroupCA1CA3Caspase-3AICaspase-3AIA13.76±0.4327.94±0.8210.92±0.3021.53±1.00C 34.48±0.49▲▲ 45.12±2.07▲▲ 26.21±0.53▲▲ 37.47±2.86▲▲

▲▲P<0.01vsgroup A.

麻醉和控制性降壓條件下的手術(shù)過(guò)程導(dǎo)致的腦缺血/缺氧誘導(dǎo)海馬細(xì)胞因子表達(dá)升高，可能是造成海馬神經(jīng)損傷的誘因。腦缺血等多種傷害性刺激可直接刺激中樞產(chǎn)生過(guò)多的促炎細(xì)胞因子(正常情況下因其表達(dá)極低而難以檢測(cè))，并在神經(jīng)損傷初始階段起著誘導(dǎo)和加劇損傷的關(guān)鍵作用，與神經(jīng)系統(tǒng)損傷關(guān)系最為密切的是IL-1β、TNF-α等促炎細(xì)胞因子[7-8]。海馬是對(duì)缺血/缺氧極為敏感和易損的腦區(qū)[3, 9]，大量研究證實(shí)腦缺血[10]或缺氧/缺血[3]均可誘導(dǎo)相關(guān)細(xì)胞因子表達(dá)的升高而導(dǎo)致海馬神經(jīng)損傷。本研究采用免疫組化染色觀察到全麻組動(dòng)物海馬CA1、CA3區(qū)均存在IL-1β和TNF-α陽(yáng)性細(xì)胞，而控壓組CA1、CA3區(qū)陽(yáng)性細(xì)胞數(shù)均較全麻組升高(P<0.01)。此外，麻醉劑本身也是誘導(dǎo)其增高的直接原因[11]；各類應(yīng)激是大腦炎癥反應(yīng)的條件[12]并可加重中樞神經(jīng)系統(tǒng)(central nervous system，CNS)炎癥反應(yīng)[13]，誘導(dǎo)海馬的易損性[14]，圍手術(shù)期的心理緊張/焦慮、麻醉、手術(shù)創(chuàng)傷及疼痛均是強(qiáng)烈或持久的應(yīng)激原，可導(dǎo)致CNS損傷及術(shù)后行為障礙[5]。手術(shù)創(chuàng)傷可導(dǎo)致組織局部和循環(huán)內(nèi)促炎細(xì)胞因子的異常改變[15]，外周感染和炎性反應(yīng)所刺激的促炎細(xì)胞因子均可通過(guò)多種途徑影響CNS神經(jīng)元、膠質(zhì)細(xì)胞及血管內(nèi)皮細(xì)胞的活性，誘發(fā)中樞炎性反應(yīng)導(dǎo)致神經(jīng)損傷[16]。盡管本研究未能進(jìn)行中樞內(nèi)外應(yīng)激激素及循環(huán)內(nèi)促炎細(xì)胞因子變化的觀測(cè)，但不能否認(rèn)圍手術(shù)期應(yīng)激和手術(shù)創(chuàng)傷也可能是造成海馬炎性損傷的原因。

細(xì)胞凋亡是各種傷害性刺激造成CNS神經(jīng)變性、損傷的主要形式[17]。TUNEL染色是一種被廣泛接受的判定細(xì)胞凋亡與否的形態(tài)學(xué)方法，其可直接檢測(cè)DNA的斷裂碎片即所謂的“凋亡小體”和染色質(zhì)凝集[18]。本研究采用TUNEL染色顯示，麻醉組與控壓組海馬CA1和CA3區(qū)均有細(xì)胞凋亡出現(xiàn)，且控壓組細(xì)胞凋亡指數(shù)明顯高于麻醉組(P<0.01)。Caspase家族擁有14個(gè)成員，在中樞損傷的細(xì)胞凋亡級(jí)聯(lián)反應(yīng)中具有重要作用，其中caspase-3是最關(guān)鍵的凋亡執(zhí)行蛋白酶[19]。本實(shí)驗(yàn)觀察到，全麻組動(dòng)物海馬CA1和CA3區(qū)均有caspase-3陽(yáng)性細(xì)胞表達(dá)，控壓組相關(guān)區(qū)域的陽(yáng)性細(xì)胞數(shù)均較全麻組升高(P<0.01)，表明全麻和控制性降壓下手術(shù)所致caspase-3表達(dá)升高，進(jìn)而導(dǎo)致神經(jīng)細(xì)胞凋亡。

Bcl-2家族成員多達(dá)20余種，其中主要的抗凋亡成員Bcl-2和促凋亡蛋白Bax位于線粒體膜，對(duì)線粒體通透性轉(zhuǎn)換孔的開放具有調(diào)節(jié)作用，調(diào)控細(xì)胞色素C(cytochrome C，Cyt C)的釋放，而釋放的Cyt C和凋亡蛋白酶激活因子(apoptotic protease-activating factor，Apaf)結(jié)合，引起caspase-9和caspase-3的順序激活，最終導(dǎo)致細(xì)胞凋亡[18-19]。因此，Bcl-2與Bax比例的變化可促進(jìn)或抑制下游信號(hào)caspase-3的表達(dá)和活化，從而決定細(xì)胞在受到凋亡信號(hào)刺激時(shí)是否發(fā)生凋亡[20]。研究表明，腦缺氧/缺血刺激及促炎細(xì)胞因子IL-1β和TNF-α的上調(diào)均可導(dǎo)致Bcl-2表達(dá)減少而Bax表達(dá)上調(diào)，直接或間接地導(dǎo)致caspase-3的表達(dá)和活性增高[8,17,21]；異氟醚等麻醉劑具有神經(jīng)毒性作用，可導(dǎo)致腦神經(jīng)凋亡[7]，研究證實(shí)其與對(duì)Bcl-2和Bax的差異性影響導(dǎo)致caspase-3的表達(dá)和活性改變有關(guān)[11,20]。本實(shí)驗(yàn)結(jié)果顯示，麻醉組動(dòng)物海馬相關(guān)區(qū)域均可見(jiàn)到Bcl-2和Bax的表達(dá)，而控壓組相關(guān)區(qū)域的Bcl-2/Bax比值小于麻醉組(P<0.05)；下游caspase-3表達(dá)在2組動(dòng)物海馬也呈現(xiàn)相應(yīng)趨勢(shì)，并與觀測(cè)的凋亡結(jié)果相符。

綜上所述，麻醉、控壓性麻醉及手術(shù)創(chuàng)傷等因素均可誘發(fā)中樞促炎細(xì)胞因子表達(dá)的增高，誘導(dǎo)海馬相關(guān)區(qū)域Bcl-2/Bax比值失衡,caspase-3表達(dá)升高，導(dǎo)致海馬神經(jīng)元的凋亡，推測(cè)其與術(shù)后神經(jīng)功能障礙的發(fā)生密切相關(guān)。本研究結(jié)果提示，全麻手術(shù)可導(dǎo)致海馬神經(jīng)損傷，而全麻/控壓手術(shù)以及手術(shù)創(chuàng)傷應(yīng)激等因素可加重這種損傷趨勢(shì)。

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Effectofsurgicaloperationwithgeneralanesthesia/controlledhypotensiononneuronalapoptosisinhippocampus

WANG Gai-mei, LIU Zhe, SHEN Ying-jie, FANG Jian-qiao

(TheThirdClinicalCollege,ZhejiangChineseMedicalUniversity,Hangzhou310053,China.E-mail:ssrsliu@163.com)

AIM: To observe the influence and the possible mechanism of surgical operation with general anesthesia/controlled hypotension on neuronal injury in hippocampus.METHODSTwelve healthy male Beagle dogs aged 6～8 months were randomly divided into general anesthesia group (group A,n=6) and controlled hypotension group (group C,n=6). The dogs in group A were anesthetized with propofol injection and isoflurane inhalation. The dogs in group C were combined with intravenous infusion of sodium nitroprusside to induce hypotension based on the anesthesia, and the target mean arterial pressure (MAP) was maintained at 40% of the baseline level for 1 h.The animals in both groups underwent exploratory laparotomy in the corresponding period. MAP was controlled to return to baseline level after surgery, and then the brain tissues were taken out 72 h later. The levels of IL-1β, TNF-α, Bcl-2, Bax and cleaved caspase-3 were measured by the method of immunohistochemistry, and neuronal apoptosis was detected by TUNEL in CA1 and CA3 regions of hippocampus.RESULTSThe ratios of IL-1β and TNF-α positive cells in CA1 and CA3 regions of hippocampus were significantly increased in group C than those in group A. The ratio of Bcl-2/Bax in CA1 and CA3 regions of hippocampus was lower in group C than that in group A. In contrast, the ratio of caspase-3 positive cells in group C was significantly higher than that in group A. The apoptotic index of hippocampal neurons in group C was significantly higher than that in group A.CONCLUSIONThe exaggerated expression of proinflammatory cytokines in hippocampus may be induced by surgical operation with general anesthesia/controlled hypotension, leade to reduce the Bcl-2/Bax ratio, and to promote the expression of caspase-3, so as to resulting in neuronal apoptosis in hippocampus. These results suggest that postoperative neuronal damage may be related to hippocampal neuro-inflammation caused by surgical operation with general anesthesia/controlled hypotension.

Neuronal apoptosis; Inflammation; General anesthesia; Controlled hypotension; Hippocampus

R363

A

10.3969/j.issn.1000- 4718.2013.01.025

1000- 4718(2013)01- 0145- 05

2012-06-28

2012-11-14

國(guó)家基礎(chǔ)研究重點(diǎn)項(xiàng)目(973計(jì)劃)課題(No.2007CB512506)；浙江省“重中之重”學(xué)科(針灸推拿學(xué))資助項(xiàng)目(No.浙教高科[2008]255號(hào))

△ 通訊作者Tel：0571-86613516；E-mail: ssrsliu@163.com