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        新生豬缺氧缺血腦損傷后基底節(jié)乳酸代謝及其轉運體表達的研究

        2017-05-12 09:31:55鄭陽王曉明
        磁共振成像 2017年1期
        關鍵詞:轉運體基底節(jié)腦損傷

        鄭陽,王曉明

        新生豬缺氧缺血腦損傷后基底節(jié)乳酸代謝及其轉運體表達的研究

        鄭陽,王曉明*

        目的通過1H-MRS在體檢測新生豬缺氧缺血腦損傷(HIBI)模型在損傷后不同時間點乳酸含量的變化并與乳酸轉運體(MCT-2、MCT-4)的表達特征進行相關研究,以期進一步明確乳酸在HI后腦損傷中的作用機制。材料與方法選用出生后3~5 d的健康新生豬,體重約為1~1.5 kg,對照組5頭,HIBI模型組30頭。通過1H-MRS成像檢測缺氧缺血后不同時間點基底節(jié)區(qū)乳酸的變化并與MCT-2、MCT-4的表達進行相關分析,P<0.05認為差異有統(tǒng)計學意義。結果(1)1H-MRS結果顯示,HIBI后Lac峰值出現(xiàn)在2~6 h,隨后Lac逐漸下降,逐步降低至與對照組水平相當。除24~48 h、48~72 h與對照組差異無統(tǒng)計學意義(P=0.86、0.26)外,其余模型組與對照組差異均有統(tǒng)計學意義(P<0.05)。(2) MCT-2、MCT-4在HI后表達先上調后降低,均在12~24 h達到高峰,與其余組均有統(tǒng)計學差異(P<0.05)。結論缺氧缺血后,乳酸含量的變化可調節(jié)神經(jīng)元及膠質細胞乳酸相關轉運體的表達。

        腦;缺氧缺血;乳酸;新生豬;磁共振波譜學

        鄭陽, 王曉明. 新生豬缺氧缺血腦損傷后基底節(jié)乳酸代謝及其轉運體表達的研究. 磁共振成像, 2017, 8(1): 45-50.

        新生腦組織需氧量較大,正常生理狀態(tài)下,腦組織內(nèi)沒有或僅存在少量的乳酸(Lac),當腦內(nèi)缺氧缺血(hypoxic ischemic,HI)時,Lac含量增多,提示無氧代謝加強,有氧能量代謝過程障礙[1-3],無氧代謝過程產(chǎn)生Lac,堆積的Lac可以使糖代謝受到抑制,使ATP耗竭,從而使細胞內(nèi)酸中毒加重[4-5]。與此同時,Lac作為HI后神經(jīng)元恢復有氧能量代謝的重要底物在HI后早期起著重要的作用?;谘芯拷Y果[6-7]顯示,中樞神經(jīng)系統(tǒng)的星形膠質細胞(AS)和神經(jīng)元之間存在著Lac轉運,即Astrocyte-neuron lactate shuttle (ANLS)。在缺氧缺血性腦損傷(hypoxic ischemic brain injury,HIBI)中,Lac及 其轉運體扮演重要角 色[8]。AS和神經(jīng)元之間通過MCTs轉運和攝取Lac,即Lac通過AS細胞膜上MCT-4轉運出胞外,聚集在細胞外腔隙中,神經(jīng)元通過自身膜上的MCT-2將Lac攝入細胞內(nèi),再通過乳酸脫氫酶將其轉化成丙酮酸,進入三羧酸循環(huán)有氧代謝[9-10],為神經(jīng)元活動提供能量代謝的底物。

        明確HI后Lac及MCTs的具體變化,可深入理解Lac在能量代謝調節(jié)中的具體作用。目前闡述Lac在能量代謝,特別是在腦HI后神經(jīng)恢復的研究還很缺乏,研究HIBI后腦內(nèi)Lac的變化以及相關轉運體的表達有助于了解HIBI后神經(jīng)元能量恢復及神經(jīng)保護機制。

        本研究應用新生豬的急性缺氧缺血模型模擬新生兒HIBI病理變化,應用MRS成像LcModel (linear combination of Model in vitro spectra)軟件后處理定量分析HI后腦組織內(nèi)Lac含量,結合神經(jīng)系統(tǒng)單羧酸轉運體(Monocarboxylic acid transporter,MCT)表達,進一步理解HIBI病理生理變化。

        1 材料與方法

        1.1 實驗動物

        選用出生后3~5 d的健康新生豬共41頭(大白豬,也叫大約克夏豬),雌雄不限,體重1~1.5 kg。排除建模失敗及運動偽影等6頭,共35頭納入數(shù)據(jù)采集,隨機分配到對照組(n=5)及模型組(n=30)。模型組根據(jù)HIBI后MR掃描時間段又進一步分成6個亞組(0~2 h;2~6 h;6~12 h;12~24 h; 24~48 h;48~72 h,n=5/group)。所有實驗動物執(zhí)行《實驗動物管理條例》和《實驗動物許可證管理辦法》規(guī)定的標準。上述實驗設計通過本院倫理審核(倫理批號為:2015PS337K)。

        1.2 實驗模型制作

        1.2.1 對照組

        室溫保持在28~30℃,以0.6 ml/Kg劑量肌注麻醉藥(速眠新注射液,長春軍事醫(yī)學院獸醫(yī)研究所)。氣管插管(直徑2.5 mm),同時連接到小動物呼吸機(TKR-200C,江西特力麻醉呼吸設備中國有限公司)進行機械通氣。通氣參數(shù):100%氧氣,呼吸機參數(shù)為呼吸比(I/E)為1:1.5,呼吸頻率30次/ min,壓力:0.05~0.06 MPa;使用TuffSat手掌式脈搏血氧儀(GE,美國)監(jiān)測心率及血氧飽和度。耳緣靜脈置管針固定用于補液及注射藥物。頸部皮膚采用碘伏消毒,頸部正中切口,小心游離雙側頸總動脈。

        1.2.2 HIBI模型組

        模型組新生豬進行上述相同過程,放置保溫箱約30 min等待狀態(tài)穩(wěn)定后,用動脈夾夾閉雙側頸總動脈阻斷血流,同時機械通入濃度為6%的氧氣(大連大特氣體有限公司),計時維持該缺氧缺血狀態(tài)40 min,時間到40 min后吸入100%氧氣(大連大特氣體有限公司),同時恢復雙側頸動脈血流,最后縫合切口。全程嚴密監(jiān)控血氧飽和度和心率。若術中及術后發(fā)生休克及抽搐應及時處理[11]。待自主呼吸恢復后停用呼吸機,使其自主呼吸。注意在MR掃描時應注意保溫,避免溫度波動給實驗結果帶來偏差。術后進行MR成像時若未恢復自主呼吸可用人工抱球法進行MR檢查[11-12]。

        1.31H MRS掃描及數(shù)據(jù)處理

        采用Philips 3.0 T MRI (Achieva 3.0 T TX;Philips Healthcare Systems,Best,the Netherlands)進行掃描,筆形束,二階勻場。體線圈發(fā)射,八通道頭線圈(SENSE)接收。MRS采用SV序列,點、解析波譜(point-resolved spectroscopy,PRESS)法,單體素長TE掃描:TR 2000 ms,TE 144 ms,信號(疊加)平均次數(shù)(NSA)為64,體素(VOI)10 mm×10 mm×10 mm。感興趣區(qū)(regions of interest,ROIs)選擇右側基底節(jié)區(qū)(圖1)。對照組及模型組的ROI均為右側基底節(jié)區(qū)同一位置。掃描之前水抑制及勻場由掃描儀自動完成。MRS掃描之前進行常規(guī)MR掃描,獲得冠狀面T1WI、T2WI、DWI及矢狀面T1WI,用于觀察腦形態(tài)及MRS定位。每只新生豬在手術前均進行MRS掃描,獲取基礎數(shù)據(jù),作為自身對照,在HI后按分組中規(guī)定的時間點行MR掃描。掃描獲得的波譜數(shù)據(jù)通過(linear combination of Model in vitro spectra,LcModel)進行后處理。NAA位于2.02 ppm,Cr位于3.02 ppm,Cho位于3.2 ppm,Lac位于1.33 ppm。

        1.4 免疫組化染色

        完成最后一次1H-MRS掃描后,迅速取出腦組織。分離雙側大腦半球,右側大腦半球置于10%中性甲醛中固定24~48 h,按冠狀切成4 mm厚組織片,留取含有基底節(jié)區(qū)和海馬的層面,經(jīng)脫水、二甲苯透明,石臘包埋切片,切片厚4 μm,進行MCT-2、MCT-4免疫組化染色,方法采用鏈酶菌抗生物素蛋白-過氧化物酶連結法(即S-P法),DAB顯色。切片經(jīng)37°烤箱烤干后常規(guī)脫水、透明、封片。陰性對照則用PBS代替一抗。MCT-2、MCT-4 抗體均由Abcam公司提供。

        1.5 實驗結果判斷及處理

        MCTs的免疫組化染色結果判定由兩位病理科醫(yī)師完成。以神經(jīng)元及AS細胞膜出現(xiàn)棕黃色顆粒為陽性表達。應用Nikon Edipse E 800顯微鏡及NIS-Elements F 2.30圖像采集軟件采集HE染色圖像和免疫組化圖像,應用NIS-Elements BR 2.10圖像分析軟件對免疫組化圖像進行光密度值(optical density value,OD value)分析,400倍鏡下觀察。OD值越高,表達越高。上述指標在基底節(jié)區(qū)各觀察5個視野,然后綜合5個視野的數(shù)據(jù),以此分析和判斷各組各時段MCTs的表達情況。數(shù)據(jù)以均數(shù)±標準差(x ±s)表示,應用ANOVA分析判斷各組之間有無差異。

        1.6 統(tǒng)計分析

        數(shù)據(jù)統(tǒng)計學處理采用軟件SPSS 17.0處理,計量資料以均數(shù)±標準差(x ±s)表示。采用ANOVA方差分析,比較對照組及HIBI組各個時間點基底節(jié)區(qū)Lac含量及MCTs表達是否存在統(tǒng)計學差異。P<0.05認為差異具有統(tǒng)計學意義。

        2 結果

        2.1 基底節(jié)區(qū)Lac的測量

        HI再灌注后,Lac呈現(xiàn)先上升,后降低的趨勢,在2~6 h達到最高值。達到峰值后隨時間逐步下降,直至最終與對照組水平相當或稍高于對照組(圖2)。除24~48 h、48~72 h Lac含量與對照組無差異外(P=0.86、P=0.26),其余各組與對照組均有統(tǒng)計學差異(P=0.00)。Lac含量最高時間點2~6 h與對照組及模型組其余各時間點均有統(tǒng)計學差異(P<0.05)。

        對照組及HIBI模型組部分時間點1H-MRS掃描數(shù)據(jù)經(jīng)Lcmodel擬合譜線如圖3所示。

        2.2 HI后基底節(jié)區(qū)MCT-2、MCT-4表達

        HI后,基底節(jié)區(qū)MCT-2、MCT-4表達均為先升高后降低,12~24 h表達達到高峰,而后下降,如圖4,5。MCT-2、MCT-4在12~24 h表達與對照組及模型組其他時間點均存在統(tǒng)計學差異(P<0.05)。隨著HI時間延長,二者表達降低。

        MCT-2、MCT-4表達與Lac含量變化趨勢一致,HI后均為先升高后降低,但MCT-2、MCT-4出現(xiàn)峰值的時間晚于Lac。

        3 討論

        正常狀態(tài)下,腦組織所需要的能量大部分來自葡萄糖的有氧代謝[13-15],生理狀態(tài)下,腦能量消耗的90%~95%發(fā)生在神經(jīng)元,但是約80%的葡萄糖利用發(fā)生在AS,這表明AS必定釋放一種葡萄糖中間代謝產(chǎn)物被神經(jīng)元攝取和利用,以滿足神經(jīng)元較高的能耗。近年來的研究[16]表明,腦葡萄糖代謝過程中,Lac是AS和神經(jīng)元能量信息交流的載體,AS攝取葡萄糖后轉變?yōu)長ac并提供給神經(jīng)元,Lac是腦內(nèi)能量代謝中重要的中間代謝物質。

        HIBI是圍產(chǎn)期多種原因導致的腦組織病變,是一種全腦的HI后再灌注腦損傷。當腦組織由低灌注轉移到再灌注時,會出現(xiàn)一系列病理生理改變。

        圖1 MRS成像ROI的定義。結合常規(guī)掃描T2WI橫斷面,選取右側基底節(jié)區(qū)作為MRS感興趣區(qū) 圖2 對照組及HIBI模型組Lac隨時間變化(橫線表示均值及標準差)。在HIBI后Lac開始上升,2~6 h內(nèi)達到峰值,繼而逐漸下降,Lac在24~48 h與對照組相當,48~72 h時Lac稍高于對照組Fig. 1 Definition of ROIs in1H-MRS. Illustration of the ROI in MRS scanning. For all animals, the right basal ganglion is selected as the ROI (T2WI image served as reference for the selection of ROIs in this study). Fig. 2 Changes in Lac content in basal ganglia within control group and HIBI group. The Lac peak increases 2—6 h after HI. The level of Lac gradually decreased and became slightly higher than the control group at 48—72 h.

        圖3 對照組及模型組部分時間段1H-MRS經(jīng)LcModel軟件處理后結果。A、B、C、D分別為對照組、HIBI后2 h、24 h、及70 h右側基底節(jié)區(qū)1H-MRS的譜線。HI后2 h、24 h Lac峰明顯升高,呈倒立單峰或雙峰改變,波峰高而尖;70 h可見Lac峰下降Fig. 3 Results of1H-MRS data at selected time points sample data analyzed by LcModel. A, B, C and D are the1H-MRS spectral curves of the right basal ganglion analyzed by LcModel in the control group and the HIBI group at 2 h, 24 h and 70 h, respectively. At 2 h and 24 h after HI insult, the Lac peaks (1.2—1.4 ppm) are markedly elevated, showing an inverted single-peak or double-peak change; at 70 h, the lactate peak was lower.

        HI再灌注后由于AS與神經(jīng)元間存在Lac穿梭,Lac作為神經(jīng)傳遞的代謝底物起著重要的作用,腦在HI狀態(tài)下,AS內(nèi)相關信號通路被激活,Lac作為重要的神經(jīng)遞質之一,通過一系列生化過程的改變,可調節(jié)能量代謝、分泌神經(jīng)保護物質等發(fā)揮神經(jīng)保護作用,并對細胞凋亡起調控作用[6,17]。AS和神經(jīng)元之間可以通過MCTs轉運 和攝取Lac為神經(jīng)元活動提供能量代謝的底物,即酵解產(chǎn)生的Lac通過細胞膜上MCT轉運出AS,聚集在細胞外腔隙中,神經(jīng)元通過自身膜上的MCT將Lac攝入,再通過乳酸脫氫酶將其轉化成丙酮酸,進入三羧酸循環(huán)有氧代謝[18-23]。在AS和神經(jīng)元中分布著不同亞型的MCTs,MCT-4主要表達于AS,而MCT-2主要表達于神經(jīng)元[22-23]。AS和神經(jīng)元之間通過MCTs轉運和攝取Lac,即Lac通過AS細胞膜上MCT-4轉運出胞外,聚集在細胞外腔隙中,神經(jīng)元通過自身膜上的MCT-2將Lac攝入細胞內(nèi),再通過乳酸脫氫酶將其轉化成丙酮酸,進入三羧酸循環(huán)有氧代謝。在HIBI中,Lac及其轉運體扮演重要角色[8]。

        本研究結果顯示,在HIBI早期,即出現(xiàn)Lac增加(圖3),Lac水平增高是腦HI的重要標志,這與之前研究相一致[24]。這是由于腦在缺氧情況下大量丙酮酸被還原成Lac。再灌注后Lac逐漸減少,是由于部分有氧代謝的恢復,以及再灌注后部分Lac被排出。在48~72 h Lac水平稍高于對照組,可能有以下原因:HI后繼發(fā)能量衰竭致線粒體損傷,有氧代謝障礙;修復期病損處巨噬細胞浸潤、AS增生,該兩種細胞活動均可使Lac增高[25],由于膠質細胞堿化,將導致糖酵解速率增加[26]。同時,大量的Lac能夠幫助神經(jīng)存活,對神經(jīng)起到保護的作用[27],因此,Lac作為HI后神經(jīng)恢復的有氧能量代謝的重要底物在HI后早期起著重要的作用。Lac作為HI的標志,出現(xiàn)最早、且最早達到高峰。Lac峰值出現(xiàn)早于MCTs,高濃度Lac或缺氧使部分糖酵解酶激活[28-29],繼而上調MCTs。免疫組化染色結果顯示,HI后MCT-2、MCT-4表達也增高,于12~24 h達到高峰,而后降低。

        圖4 對照組與HI模型組基底節(jié)區(qū)MCT-2、MCT-4表達。A~C為對照組、24 h、72 h基底節(jié)區(qū)MCT-2的表達。MCT-2主要表達于神經(jīng)元細胞膜,呈棕黃色。與對照組(A)相比較,HI后24 h (B)神經(jīng)元表達的MCT-2顏色加深,陽性細胞數(shù)目增多。MCT-2在72 h表達減低(C)。D~F為對照組、HI后24 h、72 h 基底節(jié)區(qū)MCT-4的表達。MCT-4主要表達于神經(jīng)膠質細胞膜(D)。HI后24 h (E) MCT-4染色較對照組(D)加深。MCT-4在72 h表達減低(F)Fig. 4 Expression of MCT-2 and MCT-4 in the basal ganglia in control and HI model groups. A—C: Expression of MCT-2 in the control group and at 24 h, and 72 h of the HI model group. MCT-2 was mainly expressed in the membrane of neurons seen in brown (A). Compared with the control group (A), MCT-2 staining at 24 h after HI (B) was darker and greater numbers of positive cells was observed. The expression of MCT-2 was reduced at 72 h (C). D—F: Expression of MCT-4 in basal ganglia of the control group and 24 h, and 72 h of the model group. MCT-4 was mainly expressed in the membranes of astrocytes (D). MCT-4 staining was darker at 24 h after HI (E) compared to the control group (D). The expression of MCT-4 was reduced 72 h after HI (F).

        本研究中,1H-MRS成像選擇基底節(jié)區(qū)作為ROI,這是由于基底節(jié)是神經(jīng)元細胞集中地,在缺氧缺血性腦損傷后,最易導致Lac與谷氨酸在細胞外大量堆積[30],一旦發(fā)生神經(jīng)元損傷則后果嚴重,可導致神經(jīng)系統(tǒng)障礙及腦癱[31-33],同時基底節(jié)區(qū)也是新生豬腦解剖中最容易分辨的區(qū)域,所以將基底節(jié)區(qū)作為感興趣區(qū)進行研究。選擇此區(qū)域作為感興趣區(qū)可以增加生化指標檢測的敏感性;另一方面也可以避免因腦邊緣部脂肪波的干擾而影響Lac波的準確測定。

        本研究1H-MRS掃描數(shù)據(jù)采用LcModel軟件包后處理,基線糾正、譜線分解以及精確獲取代謝物絕對濃度等問題得到有效解決[34]。LcModel能夠自動完成基線校正、渦流校正、相位校正等,可以在基線不平、信噪比較低時得到較理想的擬合譜線和較準確的代謝物濃度(圖4)。研究表明LcModel計算出的物質絕對濃度的變異系數(shù)要低于代謝物比值[35-36]。

        本研究采用新生豬制作急性HIBI模型,該模型先阻斷雙側頸內(nèi)動脈血供,維持一定缺氧缺血狀態(tài)后,恢復血供,有利于模擬再灌注的研究[37-39]。本研究建立模型過程中采用完全夾閉雙側頸內(nèi)動脈,而臨床新生兒HI時發(fā)病原因較為復雜,因此該模型可能和臨床病例有一定的病理生理差別。該模型制作方法雖然有些復雜,但重復性高,實驗結果可靠。

        總之,本研究1H-MRS成像結合LcModel軟件分析Lac絕對濃度,結合其相關轉運體的表達情況,分析了HI再灌注后部分能量代謝調節(jié)的機制,提示Lac的變化對其轉運體的表達具有調節(jié)作用。

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        Changes in lactate and associated transporters expression in basal ganglia following hypoxic-ischemic brain injury in piglets

        ZHENG Yang, WANG Xiao-ming*
        Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, China
        *

        Wang XM, E-mail: wangxm024@163.com
        Received 15 Nov 2016, Accepted 8 Dec 2016
        ACKNOWLEDGMENTSThis study was supported by National Natural Science Foundation of China (NO. 30570541, 30770632, 81271631).

        Objective:To investigate the expression characteristics of lactate and associated transporters in basal ganglia following hypoxic-ischemic reperfusion brain injury in a piglet model.Materials and Methods:A total of 35 healthy piglets (3—5 days old, 1.0—1.5 kg) were selected. They were divided into control (n=5) and hypoxic-ischemic (HI) model groups (n=30). The HI model group was further divided into six groups according to1H-magnetic resonance spectroscopy (1H-MRS) scan times after HI (0—2 h, 2—6 h, 6—12 h, 12—24 h, 24—48 h and 48—72 h; n=5/ group). The HI model was established by bilateral common carotid artery occlusion and simultaneous hypoxia treatment for 40 min. Piglets in the control group received the same surgical procedure without the hypoxia-ischemia process.1H-MRS imaging was performed at various time points after HI. The right basal ganglia was the region of interest (ROI) in1H-MRS imaging for which data was processed by LcModel software. Animals were euthanized immediately after the last scan and the whole brain was quickly removed and bilateral hemispheres separated. The right hemisphere was used for the pathological examination and immunohistochemical staining of monocarboxylate transporters (MCTs). ANOVA analyses were conducted. P<0.05 represented statistical significance.Results:(1) The lactate level became reduced after an initial increase, with the maximal level occurring around 2—6 h following HI. (2) The expression of both MCT-2 and MCT-4 in the basal ganglia initially reached a peak value at 12—24 h and decreased thereafter and they were significantly different at 12—24 h after HI compared to the control group and the other time points of the HI model group (P<0.05).Conclusions:These results indicate that lactate content has potential to regulate the expression of its related transporters in neuronal and glial cell and they have a synergistic effect on the energy metabolism following hypoxic-ischemic reperfusion brain injury.

        Brain; Hypoxia-ischemia; Lactate; Piglet; Magnetic resonance spectroscopy

        國家自然科學基金(編號:30570541、30770632、81271631)

        中國醫(yī)科大學附屬盛京醫(yī)院放射科,沈陽 110004

        王曉明,E-mail:wangxm024@163. com

        2016-11-15

        R445.2;R743

        A

        10.12015/issn.1674-8034.2017.01.011

        接受日期:2016-12-08

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