張小春 黑明燕 羅婭麗 李媛媛 戴津津

·論著·

高壓氧對(duì)缺氧缺血1周內(nèi)新生大鼠腦皮質(zhì)細(xì)胞線粒體膜電勢的影響

張小春1黑明燕2羅婭麗2李媛媛2戴津津2

目的 研究高壓氧(HBO)對(duì)新生大鼠缺氧缺血性腦損傷(HIBD)1周內(nèi)腦皮質(zhì)細(xì)胞線粒體功能的影響，探討HBO對(duì)HIBD可能的保護(hù)作用及其機(jī)制。方法 新生SD大鼠360只分為正常對(duì)照組、HIBD組和HIBD+HBO組，每組120只。HIBD組和HIBD+HBO組結(jié)扎左側(cè)頸總動(dòng)脈后暴露于8% O2+92% N2低氧環(huán)境中2 h制備HIBD模型。HIBD+HBO組在缺氧缺血后立即予HBO干預(yù)(壓力為2 ATA, 每次持續(xù)60 min，每日1次，連續(xù)7 d)，HIBD組不予HBO干預(yù)，正常對(duì)照組不予結(jié)扎左側(cè)頸總動(dòng)脈和HBO干預(yù)。以HIBD模型建立后設(shè)為缺氧缺血后0 h時(shí)點(diǎn)，3組于0 h、2 h、4 h、6 h、12 h、1 d、2 d、3 d、4 d、5 d、6 d和7 d時(shí)點(diǎn)斷頭處死(各組各時(shí)點(diǎn)n=10)，取損傷側(cè)腦皮質(zhì)制備單細(xì)胞懸液，予細(xì)胞線粒體膜電勢(ΔΨm)標(biāo)記物羅丹明123(Rho123)孵育，用流式細(xì)胞儀檢測Rho123的平均熒光強(qiáng)度(MFL)，并以該MFL值作為ΔΨm值。結(jié)果 ①正常對(duì)照組腦皮質(zhì)細(xì)胞ΔΨm值為(4.66±0.80)MFL，HIBD組各時(shí)點(diǎn)腦皮質(zhì)細(xì)胞ΔΨm值均低于正常對(duì)照組相應(yīng)時(shí)點(diǎn)，且最低為0 h時(shí)點(diǎn)[(2.85±0.56)MFL]，各時(shí)點(diǎn)差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；②HIBD組及HIBD+HBO組腦皮質(zhì)細(xì)胞ΔΨm均呈現(xiàn)降低-恢復(fù)-再降低的變化規(guī)律，兩組ΔΨm初次降低時(shí)間均為缺氧缺血后0 h時(shí)點(diǎn)，初次恢復(fù)時(shí)間均為缺氧缺血后2～12 h，再次降低的時(shí)間均為缺氧缺血后1～4 d，HIBD+HBO組ΔΨm的再次降低程度更明顯且最低為缺氧缺血后3 d時(shí)點(diǎn)[(2.62±1.03)MFL]；③HIBD組腦皮質(zhì)細(xì)胞ΔΨm在再次降低后未再回復(fù)，而HIBD+HBO組ΔΨm在再次降低后，于缺氧缺血后5 d時(shí)點(diǎn)后開始恢復(fù)，6和7 d時(shí)點(diǎn)ΔΨm值逐漸趨近但低于正常對(duì)照組水平，差異無統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論 HBO在HIBD后0 h至3 d內(nèi)不能改善缺氧缺血損傷側(cè)腦皮質(zhì)細(xì)胞的線粒體功能，HIBO后過早開始HBO治療可能導(dǎo)致受損腦皮質(zhì)細(xì)胞的進(jìn)一步損傷，但HBO可能在HIBD后5～7 d內(nèi)可通過改善腦皮質(zhì)線粒體功能促進(jìn)HIBD受損細(xì)胞功能恢復(fù)。

高壓氧； 缺氧缺血； 一周內(nèi)； 腦； 線粒體功能； 新生大鼠

新生兒缺氧缺血性腦損傷(HIBD)在許多國家依然高發(fā)且易導(dǎo)致兒童神經(jīng)系統(tǒng)后遺癥[1]，而臨床上除了亞低溫治療外還沒有其他有效治療手段[2,3]。HIBD的根本病因是腦細(xì)胞氧及能量的缺失，缺氧引發(fā)的NMDA受體被激活、細(xì)胞內(nèi)游離鈣聚集、線粒體功能障礙和其他一系列細(xì)胞內(nèi)外生化指標(biāo)的改變均參與了缺氧缺血導(dǎo)致的HIBD過程中，最終導(dǎo)致受損腦細(xì)胞凋亡或壞死[4]。理論上，可逆轉(zhuǎn)上述病理生理過程的干預(yù)手段可以用于治療HIBD，其中包括改善線粒體功能的干預(yù)手段[5]。由于HIBD與缺氧有直接的關(guān)聯(lián)，而高壓氧(HBO)可通過高壓力的氧療來影響細(xì)胞內(nèi)能量代謝，因此HBO有可能在缺氧缺血損傷后對(duì)抗缺氧所致的腦細(xì)胞線粒體功能障礙。臨床研究已證實(shí)HBO在治療急性腦水腫時(shí)可同時(shí)增加受損腦細(xì)胞氧供、增加腦血流，同時(shí)降低顱內(nèi)壓[6]；動(dòng)物實(shí)驗(yàn)研究發(fā)現(xiàn)HBO可通過增加氧彌散梯度和改善無氧代謝過程而修復(fù)受損傷后的腦細(xì)胞線粒體功能[7]。本研究假設(shè)HBO可通過改善HIBD受損部位腦細(xì)胞線粒體功能而減輕缺氧缺血所致的腦損傷。以代表線粒體功能的線粒體膜電勢(ΔΨm)為主要觀察指標(biāo)，建立HIBD新生大鼠模型，以了解HBO對(duì)HIBD后新生大鼠腦皮質(zhì)細(xì)胞線粒體功能的影響，為臨床應(yīng)用HBO治療HIBD找尋可能的實(shí)驗(yàn)室依據(jù)。

1 方法

1.1 實(shí)驗(yàn)動(dòng)物 出生后7日齡的新生清潔級(jí)SD大鼠360只，體重(12.11±1.19)g。由湖南省人民醫(yī)院動(dòng)物實(shí)驗(yàn)中心提供。本研究遵循中南大學(xué)動(dòng)物實(shí)驗(yàn)研究指南規(guī)定，并獲得中南大學(xué)湘雅三醫(yī)院醫(yī)學(xué)倫理委員會(huì)審批。

1.2 分組 將360只大鼠均分為正常對(duì)照組、HIBD組和HIBD+HBO組，每組120只。

1.3 HIBD動(dòng)物模型制備 HIBD組參照文獻(xiàn)[9,10]方法建立HIBD大鼠模型，結(jié)扎新生大鼠的左側(cè)頸總動(dòng)脈后，回母鼠身邊恢復(fù)2 h，再暴露于室溫34℃、8% O2+92% N2環(huán)境中2 h。

1.4 干預(yù) 本研究以HIBD動(dòng)物模型建立后設(shè)定為缺氧缺血后0 h時(shí)點(diǎn)(0 h)。①HIBD+HBO組干預(yù)措施：HIBD大鼠每艙10只0 h時(shí)點(diǎn)進(jìn)入特制的透明高壓氧艙，艙內(nèi)徑為 25 cm×50 cm，艙內(nèi)溫度34℃，艙內(nèi)氧濃度>85%，艙內(nèi)壓力經(jīng)過15 min 達(dá)到2個(gè)大氣壓(2ATM, 0.2 MPa)并持續(xù)60 min出艙，每日1次，連續(xù)7日(次間間隔≥24 h)；②HIBD組干預(yù)措施：HIBD大鼠10只0 h時(shí)點(diǎn)進(jìn)入特制的透明高壓氧艙，艙內(nèi)常溫、空氣和正常氣壓環(huán)境下持續(xù)60 min出艙；③正常對(duì)照組干預(yù)措施：新生大鼠每艙10只，同HIBD組干預(yù)措施。

1.5 流式細(xì)胞儀檢測腦皮質(zhì)細(xì)胞ΔΨm

1.5.1 腦皮質(zhì)單細(xì)胞懸液制備 各組大鼠在缺氧缺血后0 h、2 h、4 h、6 h、12 h、24 h(為了解單次HBO干預(yù)后24 h內(nèi)ΔΨm的變化規(guī)律)和1 d、2 d、3 d、4 d、5 d、6 d和7 d時(shí)點(diǎn)(為了解連續(xù)多次HBO干預(yù)后1周內(nèi)ΔΨm的變化規(guī)律)斷頭處死(各組各時(shí)點(diǎn)10只大鼠)，冰上操作取左側(cè)大腦皮質(zhì)制備單細(xì)胞懸液[11]，單細(xì)胞懸液濃度為2×106·mL-1，活細(xì)胞數(shù)量采用臺(tái)盼藍(lán)染色光鏡檢測法，以保證實(shí)驗(yàn)中活細(xì)胞數(shù)量占流式細(xì)胞儀檢驗(yàn)細(xì)胞的99%以上。

1.5.2 流式細(xì)胞儀檢測ΔΨm 在大腦皮質(zhì)單細(xì)胞懸液中加入線粒體膜電勢標(biāo)記物羅丹明(Rho)123(1 mmol·L-1)(美國Sigma公司)，孵育時(shí)間為45 min，孵育溫度為37℃，孵育后將標(biāo)本置于冰上，采用流式細(xì)胞儀(Cytomics FC500，美國貝克曼庫爾特有限公司)檢測Rho123的平均綠色熒光強(qiáng)度(MFL)，并以此作為該標(biāo)本的平均腦細(xì)胞ΔΨm值，激發(fā)波長為488 nm，散發(fā)波長為525 nm。為確保大腦皮質(zhì)單細(xì)胞懸液中活細(xì)胞ΔΨm檢測有效，每次檢測時(shí)在孵育前隨意選取1份標(biāo)本加入終濃度為100 mmol·L-1的非耦合劑氫化甲基氯苯基腙(mCICCP，美國Sigma公司)作為陽性對(duì)照。

2 結(jié)果

如表1和圖1所示，缺氧缺血后0 h至7 d 各時(shí)點(diǎn)正常對(duì)照組ΔΨm值基本無變化，HIBD組和HIBD+HBO組各時(shí)點(diǎn)ΔΨm值均低于正常對(duì)照組。HIBD組和HBO+HIBD組ΔΨm值均以0 h時(shí)點(diǎn)最低；2～6 h時(shí)點(diǎn)ΔΨm值有所恢復(fù)，HIBD+HBO組均高于HIBD組(P<0.05)；之后在12 h至3 d時(shí)點(diǎn)又再次降低，且HBO+HIBD組ΔΨm值降低程度更大，兩組ΔΨm值在12 h、24 h、2 d和3 d時(shí)點(diǎn)差異均有統(tǒng)計(jì)學(xué)意義(P均<0.05)。缺氧缺血后4～7 d時(shí)點(diǎn)HIBD組ΔΨm值呈現(xiàn)平穩(wěn)趨勢，HBO+HIBD組在4～7 d時(shí)點(diǎn)ΔΨm值再次開始恢復(fù)，6和7 d時(shí)點(diǎn)ΔΨm值逐漸趨近但低于正常對(duì)照組水平，差異無統(tǒng)計(jì)學(xué)意義。

Notes HIBD: hypoxic ischemic brain damage; HBO: hyperbaric oxygenation; 1): compared with HIBD+HBO group,P<0.05.n=10 at each time point in every group

圖1 缺氧缺血后0 h至7d各時(shí)點(diǎn)正常對(duì)照組、HIBD組和HIBD+HBO組損傷側(cè)腦皮質(zhì)細(xì)胞ΔΨm的變化

Fig 1 ΔΨm changes of the ipsilateral cortex of HIBD and HBO+HIBD groups at 0 h-7 d time points after HI damage

3 討論

本研究結(jié)果顯示，HIBD組0 h 至7 d時(shí)點(diǎn)損傷側(cè)腦皮質(zhì)細(xì)胞的ΔΨm值呈現(xiàn)降低-恢復(fù)-再降低的變化規(guī)律，給予HBO干預(yù)后12 h至3 d時(shí)點(diǎn)ΔΨm值降低程度更為明顯，提示該期間HBO不能通過改善腦細(xì)胞線粒體功能而減輕HIBD，但HBO干預(yù)后4 ～7 d時(shí)點(diǎn)ΔΨm值再次恢復(fù)且可接近正常對(duì)照組的水平，提示HBO在HIBD損傷后72 h(即亞急性期)，可能通過改善線粒體功能而達(dá)到減輕HIBD的目的。

腦細(xì)胞ΔΨm是線粒體功能的標(biāo)志[12]，本研究采用Rho123檢測ΔΨm值，Rho123是一種可與活細(xì)胞線粒體膜特異性結(jié)合的陽性熒光染料，由于Rho123與線粒體膜結(jié)合的程度與ΔΨm呈正相關(guān)，因此Rho123被認(rèn)為是腦細(xì)胞線粒體功能的生物標(biāo)記[13]，可通過流式細(xì)胞儀檢測其熒光強(qiáng)度值定量測定線粒體功能[14,15]，但ΔΨm值反映的是存活細(xì)胞受損傷后線粒體功能的改變，并不能反映出所有的受損傷神經(jīng)元，特別是死亡的神經(jīng)元。HBO很可能并未糾正能量代謝衰竭，而只是促進(jìn)了損傷但尚存活細(xì)胞的線粒體功能的恢復(fù)。大鼠腦皮質(zhì)以神經(jīng)元細(xì)胞為主，本研究采用的單細(xì)胞懸液的制備方法與其他類似研究一致[11～14]，僅能控制流式細(xì)胞儀標(biāo)本的單細(xì)胞懸液濃度為2×106·mL-1，活細(xì)胞數(shù)量采用臺(tái)盼藍(lán)染色光鏡檢測法以保證實(shí)驗(yàn)中活細(xì)胞數(shù)量占流式細(xì)胞儀檢驗(yàn)細(xì)胞的99%以上，但由于本試驗(yàn)方法的限制，目前無法從形態(tài)學(xué)角度明確給出活細(xì)胞的獲得能代表多少皮質(zhì)神經(jīng)元的具體數(shù)據(jù)。

哺乳動(dòng)物對(duì)氧的攝入依賴肺泡內(nèi)外的壓力梯度，給予高壓力的氧可增加這一壓力梯度并有助于提高供應(yīng)腦組織的氧，動(dòng)物實(shí)驗(yàn)證實(shí)1.5 ATA的HBO可使溶解于血漿中的氧含量提高10倍[16]、使腦組織中氧分壓達(dá)到200～300 mmHg[17]。臨床上最常采用的HBO治療方案為1.8～2.8 ATA、60～90 min[18,19]，本研究HBO干預(yù)條件采用2 ATA、60 min，2 ATA的HBO可以使得實(shí)驗(yàn)動(dòng)物血漿中氧含量增加10倍以上。HBO已在臨床上用于治療多種神經(jīng)系統(tǒng)疾病[20,21]，并有動(dòng)物實(shí)驗(yàn)報(bào)道單次HBO干預(yù)可減輕缺氧缺血導(dǎo)致的新生大鼠腦損傷[22]，其作用機(jī)制可能一方面與HBO提高腦組織內(nèi)的氧分壓、促進(jìn)氧與線粒體內(nèi)的氧化還原酶結(jié)合從而改善線粒體功能有關(guān)[7,23]，另一方面與HBO可抑制細(xì)胞凋亡過程有關(guān)[24,25]。應(yīng)用成年大鼠動(dòng)物模型實(shí)驗(yàn)研究也證實(shí)大腦中動(dòng)脈梗塞6 h內(nèi)給予HBO干預(yù)可減輕腦缺血性損傷[26]，認(rèn)為干預(yù)越早則治療效果越好。但本研究結(jié)果顯示，在新生SD大鼠HIBD后給予HBO干預(yù)并未在早期改善腦細(xì)胞的線粒體功能，而是在缺氧缺血后3 d時(shí)點(diǎn)損傷側(cè)腦細(xì)胞的線粒體功能才得到改善，在12 h至3 d時(shí)點(diǎn)HBO干預(yù)后腦細(xì)胞線粒體功能反而更進(jìn)一步降低，提示HIBD后給予HBO干預(yù)可能并不是越早效果越好，分析導(dǎo)致這一結(jié)果的原因可能與氧自由基損傷或組織再灌注損傷等機(jī)制相關(guān)。腦損傷后，氧自由基和氮自由基大量產(chǎn)生，在細(xì)胞防御系統(tǒng)功能降低的情況下自由基的升高可導(dǎo)致脂質(zhì)過氧化反應(yīng)，最終導(dǎo)致細(xì)胞功能障礙[27]，而腦組織對(duì)脂質(zhì)過氧化反應(yīng)尤其敏感，并需要更高的耗氧量來維持正常功能[28]，同時(shí)由于腦組織清除氧自由基的能力有限、腦組織的過氧化氫酶活性較低以及腦組織內(nèi)富含誘發(fā)產(chǎn)生自由基的鐵元素，故腦組織對(duì)抗自由基損傷的自然防御能力更差[23]。本研究結(jié)果顯示HIBD+HBO組在缺氧缺血后4～7 d時(shí)點(diǎn)損傷側(cè)大腦皮質(zhì)的ΔΨm值明顯高于HIBD組，由此推斷在度過HIBD最初的急性期之后，HBO可能通過改善線粒體功能而減輕HIBD。

早產(chǎn)兒視網(wǎng)膜病變和高氧肺損傷是臨床醫(yī)生高度重視的問題，因此在應(yīng)用HBO治療HIBD時(shí)，必然會(huì)考慮到HBO的安全性問題。一項(xiàng)隨機(jī)臨床試驗(yàn)結(jié)果顯示每24 h間斷給予2 ATA的HBO治療2～3次(每次60 min)對(duì)于外傷導(dǎo)致的腦損傷患者是安全的[23]，動(dòng)物實(shí)驗(yàn)結(jié)果顯示給予早產(chǎn)新生大鼠HBO治療1 h不會(huì)導(dǎo)致與視網(wǎng)膜病變相關(guān)的視網(wǎng)膜結(jié)構(gòu)改變或視網(wǎng)膜血管增生[21]，而Yoles等[29]應(yīng)用不同日齡的新生狗研究發(fā)現(xiàn)，在腦代謝、腦血流穩(wěn)定性、腦細(xì)胞電生理方面，日齡越小的新生狗對(duì)HBO導(dǎo)致的氧毒性耐受力越強(qiáng)。本研究采用的是7日齡的新生SD大鼠，HBO的壓力為2 ATA、時(shí)間為60 min，未在HBO氧毒性方面進(jìn)行相關(guān)觀察，僅根據(jù)現(xiàn)有研究報(bào)道推測，本研究采用的HBO干預(yù)方法對(duì)新生SD大鼠產(chǎn)生氧毒性的可能性較小。

本研究的局限性：①各組各時(shí)點(diǎn)樣本量較小(n=10)，數(shù)據(jù)標(biāo)準(zhǔn)差較大；②HBO對(duì)HIBD長期后遺癥(如記憶認(rèn)知能力、大運(yùn)動(dòng)功能、行為異常等)的影響沒有進(jìn)行評(píng)估；③本研究HBO干預(yù)是在HIBD后0 h時(shí)點(diǎn)開始，不能回答關(guān)于HBO治療的時(shí)間窗問題；④HBO干預(yù)后Δψm呈現(xiàn)降低-恢復(fù)-再降低-再恢復(fù)變化規(guī)律的機(jī)制尚不明確，這些機(jī)制可能牽涉到氧自由基損傷、NMDA受體激活或細(xì)胞內(nèi)游離鈣聚集。

致謝：真誠地感謝中南大學(xué)湘雅醫(yī)院兒科楊于嘉教授為本研究無償提供動(dòng)物實(shí)驗(yàn)專用高壓氧艙！

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(本文編輯：張萍)

Effect of hyperbaric oxygenation on mitochondrial membrane potential of cortex neuronal cells of neonatal rats in the first week after hypoxic ischemic brain damage

ZHANG Xiao-chun1, HEI Ming-yan2, LUO Ya-li2, LI Yuan-yuan2, DAI Jin-jin2

(1 Department of Pediatrics, Central Hospital of Loudi, Loudi 417000, China; 2 Department of Pediatrics, The Third Xiangya Hospital of Central South University, Changsha 410013, China)

HEI Ming-yan，E-mail:heiming_yan@aliyun.com

ObjectiveThe initial insult of hypoxic-ischemic (HI) brain damage (HIBD) is the deprivation of oxygen (O2) to the brain cells, followed by a cascade of brain cell damage including mitochondrial dysfunction. Theoretically, hyperbaric oxygenation (HBO) could affect the recovery of mitochondrial function in HIBD by greatly increasing the O2delivery diffusion gradient. The objective of this study was to prove the hypothesis that HBO may reduce HI-induced brain injury via affecting brain cell mitochondrial function, and to understand the changing patterns of mitochondrial function following HBO treatment in the first week after HI.MethodsIn the present study, HIBD rat model and flow cytometer were used to explore the change of ΔΨm, the indicator of mitochondrial function of cortex neuronal cells of neonatal rats after HIBD. Neonatal Sprague Dawley (SD) rat pups were randomly divided into normal control, HIBD, and HIBD+HBO groups. The end of HI was considered to be 0 h time point. The HBO treatment was given at 0h time point, and then once a day for consecutive 7 days (in 24 h intervals). Animals were euthanized at 0, 2, 4, 6, 12 h time points (in order to study the ΔΨm changes at the very early stage after a single dose of HBO treatment), and at 2, 3, 4, 5, 6, and 7 d time points (in order to study the ΔΨm changes after a series of HBO treatment). ResultsThe change of ΔΨm of the ipsilateral cortex in both HIBD and HIBD+HBO groups showed fluctuating change pattern. Within 2 h to 12 h after HI insult, ΔΨm of HIBD group recovered to some extent, but ΔΨm of HIBD+HBO group recovered to almost normal level. A secondary drop of ΔΨm was observed in both groups at 1-4 d after HI insult. The secondary drop of HIBD+HBO group was more severe than that of HIBD group. There was a secondary recovery of ΔΨm observed in HIBD+HBO group in 5-7 d after HI insult, but not in HIBD group. The ΔΨm of HIBD+HBO group recovered again to almost normal level at 6 d time point. The ΔΨm of HIBD group in 2-7 d after HI stayed at low level, showing slowly decreasing tendency.ConclusionHBO in the early stage after HI might not be a good therapy to improve the mitochondrial function in the cerebral cortex. The secondary recovery observed in HIBD+HBO group indicated that HBO treatment may protect HI-induced brain damage by improving neural cell mitochondrial function in the cerebral cortex during sub-acute stage after HI.

Hyperbaric oxygenation; Hypoxic-ischemic; The first week; Brain; Mitochondrial function; Neonatal rat

湖南省自然科學(xué)基金課題：11JJ6067

1 湖南省婁底市中心醫(yī)院兒科 婁底，417000；2 中南大學(xué)湘雅三醫(yī)院兒科 長沙，410013

黑明燕，E-mail:heiming_yan@aliyun.com

10.3969/j.issn.1673-5501.2014.03.011

2014-03-17

2014-05-11)