高雪松,王自能

(暨南大學(xué)附屬第一醫(yī)院婦產(chǎn)科,廣州 510632)

研究報(bào)告

純氧或空氣環(huán)境復(fù)蘇對(duì)新生大鼠缺氧性大腦皮質(zhì)神經(jīng)元超微結(jié)構(gòu)的影響

高雪松,王自能

(暨南大學(xué)附屬第一醫(yī)院婦產(chǎn)科,廣州 510632)

目的 應(yīng)用缺氧動(dòng)物模型比較純氧環(huán)境(pure oxygen environm ent,POE,100%氧)與空氣環(huán)境(room air environm ent,RAE,21%氧)復(fù)蘇對(duì)新生大鼠缺氧性大腦皮質(zhì)神經(jīng)元超微結(jié)構(gòu)的影響。方法7日齡20只SD (Sp rague Daw ley)乳鼠建立缺氧2.5 h模型后,分為純氧環(huán)境組(POE)和空氣環(huán)境組(RAE),每組再根據(jù)復(fù)氧后時(shí)間點(diǎn)分為2個(gè)亞組,即24 h組和72 h組,每亞組5只。按時(shí)間點(diǎn)取各組乳鼠大腦半球右側(cè)額頂皮質(zhì)行電鏡樣品制備,透射電鏡觀察。結(jié)果復(fù)氧各組均可見(jiàn)神經(jīng)元、神經(jīng)氈和細(xì)胞間隙水腫。RAE 24 h組神經(jīng)元核膜結(jié)構(gòu)不清,細(xì)胞器減少,線(xiàn)粒體腫脹、空泡化、嵴斷裂;粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張,常呈空泡狀,核糖體減少;高爾基復(fù)合體囊泡擴(kuò)張;溶酶體較多。RAE 72 h組細(xì)胞器改變同前,但類(lèi)似凋亡的細(xì)胞核較多,壞死細(xì)胞亦較其他組多見(jiàn)。POE 24 h組病變較RAE 24 h組輕。POE 72 h組細(xì)胞內(nèi)線(xiàn)粒體及粗面內(nèi)質(zhì)網(wǎng)較豐富,病變亦比RAE 72 h組輕。結(jié)論P(yáng)OE復(fù)蘇較RAE復(fù)蘇可更能緩解缺氧致神經(jīng)元超微結(jié)構(gòu)的損傷、減少細(xì)胞凋亡及減輕腦水腫。提示,純氧環(huán)境對(duì)缺氧復(fù)蘇后大腦皮質(zhì)神經(jīng)元有一定的保護(hù)作用,并表明本動(dòng)物模型適合缺氧新生大鼠大腦皮質(zhì)神經(jīng)元研究。

復(fù)氧;超微結(jié)構(gòu);神經(jīng)元;大腦皮質(zhì);缺氧新生大鼠

新生兒窒息是嚴(yán)重的圍產(chǎn)醫(yī)學(xué)問(wèn)題之一,它致使每年有近一百萬(wàn)患兒死亡和同樣數(shù)目的嚴(yán)重后遺癥。近來(lái)隨著醫(yī)療技術(shù)的改進(jìn),高危兒存活率明顯提高,存活新生兒中樞神經(jīng)系統(tǒng)的發(fā)病率也相應(yīng)增加,早期干預(yù)可使高危兒中樞神經(jīng)系統(tǒng)發(fā)育障礙的狀況得到改善。對(duì)低氧致缺氧性腦損傷,復(fù)氧后腦超微結(jié)構(gòu)的研究報(bào)道甚少。本研究是在建立新生大鼠缺氧動(dòng)物模型基礎(chǔ)上,分別采用純氧環(huán)境與空氣環(huán)境復(fù)蘇,應(yīng)用透射電鏡觀察,比較純氧與空氣對(duì)缺氧性大腦皮質(zhì)神經(jīng)細(xì)胞超微結(jié)構(gòu)的影響,為臨床進(jìn)行新生兒窒息復(fù)氧方案及新生大鼠缺氧動(dòng)物模型的建立提供科學(xué)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物分組與模型制備

1.1.1 分組:取清潔級(jí)7日齡SD(Sp rague Daw ley)乳鼠20只,由南方醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供【SCXK(粵)2006-0015】,體質(zhì)量(12.3±1.18)g,雌雄不拘,在制成缺氧模型后,隨機(jī)分為純氧環(huán)境組(pure oxygen environm ent,POE,100%氧)(n=10)與空氣環(huán)境組(room air environm en t,RAE,21%氧) (n=10),每組再根據(jù)復(fù)氧后時(shí)間點(diǎn)分為2個(gè)亞組,即24 h組和72 h組,每亞組5只。

1.1.2 動(dòng)物模型制作:參照經(jīng)典Rice等[1]缺血缺氧動(dòng)物模型制作及呂國(guó)蔚等[2]小鼠急性缺氧動(dòng)物模型制作,本研究對(duì)乳鼠未行頸總動(dòng)脈結(jié)扎手術(shù)操作,僅進(jìn)行缺氧模型制作,首先制做透明有機(jī)玻璃密封低氧小艙,小艙體積為2 L,艙內(nèi)底層置鈉石灰,以吸收二氧化碳和濕氣,兩側(cè)艙壁上下各有1個(gè)直徑2cm的小孔,分別接低氧氣瓶和排氣管道,排氣管道通入水瓶中,以保持容器內(nèi)壓力與大氣一致及持續(xù)低氧狀態(tài)。實(shí)驗(yàn)時(shí),將低氧小艙置于37～38℃水中水浴,使艙內(nèi)溫度控制在36℃左右,將20只乳鼠置于預(yù)熱的低氧小艙內(nèi),輸入體積分?jǐn)?shù)為8%氧和92%氮的混合氣體(廣州氣體廠提供),流速為1.5 L/m in,至2.5 h時(shí)乳鼠出現(xiàn)明顯發(fā)紺、喘呼吸、痙攣樣動(dòng)作和翻正反射難以完成,達(dá)到急性缺氧表現(xiàn)要求[2]。在制成缺氧模型后,立即將乳鼠分為POE組和RAE組。將POE組置于純氧艙,通入100%氧,流速為5 L/m in,艙內(nèi)溫度控制在36°C左右;RAE置于同一室內(nèi)空氣艙中,各30 m in。實(shí)驗(yàn)結(jié)束后,放回原母鼠籠中飼養(yǎng)。

1.2 取材、電鏡標(biāo)本制作與觀察

復(fù)氧后按時(shí)間點(diǎn),將乳鼠斷頭處死,開(kāi)顱,迅速在腦表面滴加預(yù)冷生理鹽水,在冰浴下取腦,快速切取丘腦段冠狀斷面(約厚3 mm)組織塊,于灰結(jié)節(jié)右側(cè)緣,按照與矢狀溝平行的方向,切取大腦半球右側(cè)額頂皮質(zhì)矢狀斷面,將標(biāo)本修成約1 mm3的細(xì)小組織3塊,2.5%戊二醛固定,Epon812包埋,半薄切片,甲苯胺藍(lán)染色,光鏡下定位,再修塊,取皮質(zhì)第五層,作超薄切片,醋酸雙氧鈾與硝酸鉛雙重染色。在JEOL 100CX II/T型透射電鏡下觀察,重點(diǎn)觀察各組神經(jīng)元的超微結(jié)構(gòu)變化。

2 結(jié)果

復(fù)氧各組均可見(jiàn)神經(jīng)細(xì)胞、神經(jīng)氈及間隙水腫。部分神經(jīng)細(xì)胞呈現(xiàn)不同程度的退行性病變;見(jiàn)少量壞死細(xì)胞和類(lèi)似凋亡的細(xì)胞核,后者核固縮,核形狀不規(guī)則,染色質(zhì)邊聚,核膜完整,有的形成內(nèi)褶,有的突出,且含染色質(zhì),似有凋亡小體形成趨勢(shì)。

2.1 空氣環(huán)境組

2.1.1 空氣環(huán)境24 h組:可見(jiàn)生物膜受損,核(nuc lei,N)膜結(jié)構(gòu)不清晰;細(xì)胞器減少,線(xiàn)粒體(m itochondria,M it)損傷明顯,腫脹,空泡化,膜局部模糊或缺失、嵴斷裂或缺失、有的甚至髓樣變;粗面內(nèi)質(zhì)網(wǎng)(rough endop las m ic reticulum,RER)擴(kuò)張常呈空泡狀(圖1),核糖體減少;高爾基復(fù)合體囊泡擴(kuò)張;溶酶體增多,含殘余體。

2.1.2 空氣環(huán)境72 h組:細(xì)胞器改變同上,從圖2可見(jiàn)神經(jīng)元細(xì)胞核呈現(xiàn)出類(lèi)似凋亡的改變,核形態(tài)不規(guī)則,形成內(nèi)褶,染色質(zhì)邊聚?？諝猸h(huán)境72 h組類(lèi)似凋亡的細(xì)胞核較其他組多見(jiàn)。壞死細(xì)胞也較POE 72 h組多見(jiàn)。該組細(xì)胞周間隙仍增寬,電子密度亦低。

注:N:核;M it:線(xiàn)粒體;RER:粗面內(nèi)質(zhì)網(wǎng)。圖1 空氣環(huán)境24 h組Note:N:nucleus;M it:m itochond ria;RER:rough endop las m ic reticulum.F ig.1 The u ltrastructure of a neuron in a rat of the RAE 24 h group.

圖2 空氣環(huán)境72 h組F ig.2 The u ltrastructure of a neuron in a rat of the RAE 72 h group.

2.2 純氧環(huán)境組

2.2.1 純氧環(huán)境24h組:與空氣環(huán)境24 h組比含較多的線(xiàn)粒體,亦可見(jiàn)有的神經(jīng)元線(xiàn)粒體腫脹,嵴斷裂或消失,或髓樣變;粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張,核糖體減少。但病變較空氣環(huán)境24 h組輕。見(jiàn)圖3。

2.2.2 純氧環(huán)境72 h組:從圖4可見(jiàn)該組細(xì)胞內(nèi)線(xiàn)粒體及粗面內(nèi)質(zhì)網(wǎng)較空氣環(huán)境組豐富。有的神經(jīng)元也見(jiàn)線(xiàn)粒體腫脹、變性,還可見(jiàn)較長(zhǎng)而大的線(xiàn)粒體;有的粗面內(nèi)質(zhì)網(wǎng)可見(jiàn)輕度擴(kuò)張和核糖體減少;有的仍可見(jiàn)高爾基復(fù)合體囊泡擴(kuò)張和溶酶體。但病變比空氣環(huán)境組輕。

3 討論

注:N:核;M it:線(xiàn)粒體;RER:粗面內(nèi)質(zhì)網(wǎng)。圖3 純氧環(huán)境24 h組Note:N:nucleus,M it:m itochondria;RER:rough endop la sm ic reticu lum.F ig.3 The u ltrastructure of a neuron in a rat of the POE 24 h group.

注:N:核;M it:線(xiàn)粒體;RER:粗面內(nèi)質(zhì)網(wǎng)。圖4 純氧環(huán)境72 h組Note:N:nucleus,M it:m itochond ria;RER:rough endop la sm ic reticu lum.F ig.4 The u ltrastructure of a neuron in a rat of the POE 72 h group.

本研究選用清潔級(jí)7日齡大鼠,除保證動(dòng)物健康及動(dòng)物來(lái)源方便外,其腦組織結(jié)構(gòu)及血液供應(yīng)與人相似,更重要的是其大腦組織學(xué)發(fā)育程度與足月新生兒的大腦發(fā)育程度相似。新生兒期腦的代謝最旺盛,腦的耗氧量約占全身的一半,因此,腦缺氧時(shí)容易受損。近年來(lái),許多學(xué)者觀察到缺氧是新生兒缺氧缺血性腦病的重要原因。蔣犁等[3]在新生大鼠腦細(xì)胞凋亡與缺氧、缺血兩因素析因研究中發(fā)現(xiàn),大腦皮質(zhì)缺氧比缺血效應(yīng)大。有學(xué)者在體內(nèi)、外實(shí)驗(yàn)中,觀察到去核細(xì)胞(denuc leated cells)可誘導(dǎo)進(jìn)行性程序性細(xì)胞死亡(凋亡);在大多數(shù)核改變前呈現(xiàn)細(xì)胞器高度有序的異常,例如粗面內(nèi)質(zhì)網(wǎng)腫脹、高爾基復(fù)合體水腫及線(xiàn)粒體破壞。提出存在不依賴(lài)于細(xì)胞核的細(xì)胞質(zhì)凋亡通路[4],支持本研究結(jié)果。本實(shí)驗(yàn)采用缺氧動(dòng)物模型,在復(fù)氧后觀察到各組神經(jīng)元、神經(jīng)氈及間隙水腫;部分神經(jīng)元呈現(xiàn)不同程度的退行性變,以細(xì)胞器改變最明顯。

3.1 復(fù)氧后線(xiàn)粒體改變的意義

近年來(lái)的研究發(fā)現(xiàn)線(xiàn)粒體功能障礙在缺氧缺血后神經(jīng)元損傷中起關(guān)鍵作用。缺氧時(shí)有多種促凋亡級(jí)聯(lián)轉(zhuǎn)導(dǎo)信號(hào)整合于線(xiàn)粒體通透性轉(zhuǎn)變孔道(m itochond ria per m eability transition pore,M PTP), M PTP開(kāi)放,線(xiàn)粒體跨膜電位降低,內(nèi)膜通透性突然增加,Ca2+內(nèi)流增加,膜間隙內(nèi)存儲(chǔ)的大量促凋亡分子。例如細(xì)胞色素C、凋亡誘導(dǎo)因子和各種半胱天冬酶原等,被釋放到胞質(zhì)中,引起凋亡級(jí)聯(lián)反應(yīng), DNA裂解為60 kb左右的片段,染色質(zhì)異常凝集和DNA的大規(guī)模斷片化,而致神經(jīng)細(xì)胞急性壞死或凋亡[5]。本研究發(fā)現(xiàn)新生大鼠腦缺氧復(fù)氧后,空氣環(huán)境組神經(jīng)元的線(xiàn)粒體變性顯著,線(xiàn)粒體功能損傷較嚴(yán)重,致使空氣環(huán)境72 h組細(xì)胞凋亡及細(xì)胞壞死最為明顯,提示,純氧環(huán)境復(fù)蘇可能通過(guò)改善線(xiàn)粒體呼吸功能,促使線(xiàn)粒體內(nèi)膜通透性趨于穩(wěn)定,緩解繼發(fā)性能量衰竭,使其病變有所減輕。

3.2 復(fù)氧后內(nèi)質(zhì)網(wǎng)和高爾基復(fù)合體改變的意義

粗面內(nèi)質(zhì)網(wǎng)是一個(gè)高水平的調(diào)控系統(tǒng),是蛋白質(zhì)合成、修飾及折疊的重要場(chǎng)所,也是調(diào)節(jié)細(xì)胞應(yīng)激反應(yīng)的場(chǎng)所。Q i等[6]在小鼠缺氧30 m in后大腦皮質(zhì)中觀察到缺氧造成嚴(yán)重內(nèi)質(zhì)網(wǎng)損害及ER應(yīng)激引發(fā)caspase-12的增高。有報(bào)道,在缺氧缺血刺激下,大量未折疊蛋白可在內(nèi)質(zhì)網(wǎng)中堆積,導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激(endop las m ic reticu lum stress,ERS)。近來(lái)研究發(fā)現(xiàn)過(guò)度ERS也是一條細(xì)胞凋亡信號(hào)傳導(dǎo)通路。發(fā)生過(guò)度ERS啟動(dòng)的細(xì)胞凋亡信號(hào)傳導(dǎo)通路,引起ER內(nèi)Ca2+釋放,導(dǎo)致胞質(zhì)內(nèi)Ca2+穩(wěn)態(tài)失衡,活化位于ER胞質(zhì)面的常駐分子caspase-12,激活caspase-3而誘導(dǎo)凋亡[7]。另外氧自由基還可引起P53的積累,進(jìn)而促進(jìn)內(nèi)質(zhì)網(wǎng)釋放TNF-α和Fas,也能與DNA復(fù)制蛋白A結(jié)合,阻止DNA復(fù)制,上述效應(yīng)最終激活caspase-3、6、7,促進(jìn)細(xì)胞凋亡[8]。Nakagawa等[9]觀察到高爾基復(fù)合體含有促凋亡分子神經(jīng)節(jié)苷脂GD 3結(jié)合位點(diǎn),利于caspase-12活化,介導(dǎo)特異性凋亡通路。本研究純氧環(huán)境72 h組粗面內(nèi)質(zhì)網(wǎng)和高爾基復(fù)合體病變比空氣環(huán)境72 h組輕,提示,純氧環(huán)境可隨時(shí)間進(jìn)程,減輕ER應(yīng)激性反應(yīng)和隨后的凋亡信號(hào)轉(zhuǎn)導(dǎo)通路,促進(jìn)適應(yīng)反應(yīng);促使氧應(yīng)激反應(yīng)的衰退等,對(duì)缺氧造成的傷害起到一定程度的緩解作用。

有報(bào)道指出在缺氧和酸中毒時(shí),溶酶體穩(wěn)定性降低,可導(dǎo)致溶酶體膜破裂,溶酶體酶釋放,釋放出的酸性水解酶,能直接破壞細(xì)胞本身的成分,引起細(xì)胞內(nèi)消化、自溶而壞死[10]。本研究觀察到空氣環(huán)境組比純氧環(huán)境組溶酶體增多,可能是由于空氣環(huán)境組細(xì)胞器病理改變較重,激活溶酶體功能,導(dǎo)致自身消化能力增強(qiáng)。

以上不難看出,本研究純氧環(huán)境復(fù)蘇72 h對(duì)缺氧引起的新生大鼠大腦皮質(zhì)神經(jīng)細(xì)胞損害改善效應(yīng)大于空氣環(huán)境復(fù)蘇,提示,純氧環(huán)境較空氣環(huán)境可更能緩解缺氧致神經(jīng)細(xì)胞超微結(jié)構(gòu)的損傷、減少細(xì)胞凋亡及減輕腦水腫,純氧環(huán)境對(duì)缺氧復(fù)蘇后大腦皮質(zhì)神經(jīng)元有一定的保護(hù)作用,為臨床選擇使用純氧環(huán)境或空氣環(huán)境復(fù)蘇及新生大鼠缺氧動(dòng)物模型的建立提供了科學(xué)依據(jù)。

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Effects of Resusc ita tion a t 100%O xygen Env ironm en t or Room A ir Env ironm en t on the U ltrastructure of Neuron s in the Cerebra l Cor tex of Hypox ic Neona ta l Ra t

GAO Xue-Song,WANG Zi-neng
(Departm ent ofObstetrics&Gyneco logy,The FirstA ffiliated Hosp ital of Jinan University,Guangzhou 510630,China)

O b jective To compare the effects of resuscitation at pure oxygen environm ent(100%oxygen)or room air environm ent(21%oxygen)on the u ltrastructure of cerebral cortical neurons in hypoxic neonatal rat.M ethods Twenty Sp rague-Daw ley neonatal rats aged 7 days were divided into the pure oxygen environm ent(POE)and room air environm ent groups(RAE)after hypoxia,and each group was divided again into 24 h and 72 h subgroups.The pupswere p laced in an incubato r containing 8%O2to be at acute hypoxia continued for 2.5 h.The pups were reoxygenated imm ed iately after the above m entioned hypoxia.The pup s of POE group were then p laced in an incubato r containing 100% oxygen and the flow tim e was 30 m in.The pups of RAE group were reoxygenated w ith room air in an incubator for 30 m in. A cco rding to the p lanned t im e points the pup swere sacrificed and the b rain was removed at 24 h and 72 h after trea tm en t, respectively.The tissue of fron toparietal co rtex of the righ t cereb ral hem isphere was p repared fo r transm ission electron m icroscop ic exam ination.ResultsIn each reoxygenation group,edem a was found in the neurons,neurop il and in tercellu lar space.In the RAE 24 h group,the nuc learm em b rane in neu rons was unc lear,the amoun t of cell o rganelles was reduced,the m itochond ria were s wo llen w ith dam ages of cristae.The rough endop lasm ic reticulum was dilatated or vacuo lized and w ith reduction of ribosom es.The Go lgi com p lex was vacuo lized.The num ber of lysosom es was obviously increased.In the RAE 72 h group s,the changes were s im ilar to those of the fo rm er group,bu t apop to tic-like nuc lei andnecro tic neuronswere mo re frequen tly seen.The cellu lar dam ages of POE 24 h group were m ilder than those of RAE 24 h group.The m itochond ria and rough endop lasm ic reticu lum were more abundant and showed less patho logical changes in the POE 72 h group as compared w ith those of RAE 72 h group.Conc lusion The rats of POE reoxygenation disp lay m ilder ultrastructural dam ages,less apop tosis,necrosis and edem a in cerebral neurons than those in rats after RAE reoxygenation. The p rotective effect of POE resuscitation on cerebral dam age of hypoxic neonate rats is superior to that after RAE resuscitation.This hypoxic neonatal ratmodelm ay serve as a suitab le anim almodel for research on cerebral cortex neurons caused by hypoxia.

Reoxygenation;U ltrastructure;Neuron,cerebral cortex;Hypoxia;Neonatal rat

R714

A

1005-4847(2010)01-0074-04

2009-03-11

高雪松(1972-),女,碩士,暨南大學(xué)附屬第一醫(yī)院婦產(chǎn)科主治醫(yī)師,研究方向:圍產(chǎn)醫(yī)學(xué)神經(jīng)生物學(xué)。E-m ail:tgxs@jnu. edu.cn