王凌星, 黃紅紅, 陳雅芳, 蔡鴻潮, 錢家強(qiáng)
(福建醫(yī)科大學(xué)附屬第二醫(yī)院神經(jīng)內(nèi)科, 泉州 362000)
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產(chǎn)前應(yīng)激對(duì)子代大鼠腦缺血/再灌注后星形膠質(zhì)細(xì)胞的影響*
王凌星, 黃紅紅△, 陳雅芳, 蔡鴻潮, 錢家強(qiáng)
(福建醫(yī)科大學(xué)附屬第二醫(yī)院神經(jīng)內(nèi)科, 泉州 362000)
目的:探討產(chǎn)前應(yīng)激對(duì)雄性子代大鼠大腦中動(dòng)脈缺血/再灌注后星形膠質(zhì)細(xì)胞的影響。方法:SD孕鼠隨機(jī)分為有產(chǎn)前應(yīng)激處理(妊娠第15到21天每日3次限制活動(dòng))和無(wú)產(chǎn)前應(yīng)激處理,并對(duì)其雄性子代大鼠采用線栓法制備大腦中動(dòng)脈閉塞(MCAO)模型,共分為產(chǎn)前應(yīng)激+假手術(shù)組、MCAO模型組、產(chǎn)前應(yīng)激+MCAO組(n=10),于再灌注后第5天檢測(cè)腦梗死體積,免疫熒光雙標(biāo)染色檢測(cè)缺血灶邊緣區(qū)星形膠質(zhì)細(xì)胞形態(tài)及促紅細(xì)胞生成素肝細(xì)胞受體A4(EphA4)和膠質(zhì)纖維酸性蛋白(GFAP)的共表達(dá)情況,并采用Western blot檢測(cè)EphA4、GFAP和神經(jīng)蛋白聚糖(Neurocan)蛋白表達(dá)。結(jié)果:產(chǎn)前應(yīng)激+MCAO組子代大鼠腦梗死體積百分比、EphA4、GFAP和Neurocan蛋白表達(dá)均較MCAO組顯著增加(P均<0.05),且GFAP陽(yáng)性細(xì)胞形態(tài)學(xué)改變及EphA4/GFAP共表達(dá)也較MCAO組明顯。結(jié)論:產(chǎn)前應(yīng)激可能改變子代大鼠腦缺血/再灌注后星形膠質(zhì)細(xì)胞上EphA4受體的表達(dá),促進(jìn)星形膠質(zhì)細(xì)胞活化,產(chǎn)生神經(jīng)蛋白聚糖。
產(chǎn)前應(yīng)激;子代大鼠; 腦缺血; 星形膠質(zhì)細(xì)胞
【DOI】 10.13459/j.cnki.cjap.2016.04.004
腦缺血時(shí),會(huì)出現(xiàn)星形膠質(zhì)細(xì)胞的活化,表現(xiàn)為數(shù)量和形態(tài)的改變,并出現(xiàn)GFAP表達(dá)增加。星形膠質(zhì)細(xì)胞的早期活化、遷移和增殖可以限制缺血半暗帶的擴(kuò)展[1],所形成的膠質(zhì)瘢痕會(huì)分隔并保護(hù)未損傷組織免于損傷,抑制炎癥擴(kuò)散并調(diào)節(jié)細(xì)胞外環(huán)境[2]。但另一方面,膠質(zhì)瘢痕構(gòu)成了軸突生長(zhǎng)的物理性障礙,而且膠質(zhì)瘢痕中活化星形膠質(zhì)細(xì)胞產(chǎn)生了具有抑制作用的細(xì)胞外基質(zhì)分子,如硫酸軟骨素蛋白多糖(chondroitin sulfate proteoglycans,CSPG),對(duì)神經(jīng)軸突生長(zhǎng)形成生物化學(xué)屏障作用[3]。因此調(diào)節(jié)星形膠質(zhì)細(xì)胞的活性會(huì)改變腦缺血的轉(zhuǎn)歸和神經(jīng)功能損傷的程度。我們的前期研究表明,產(chǎn)前應(yīng)激會(huì)加重子代大鼠缺血性卒中的神經(jīng)功能損害[4],但星形膠質(zhì)細(xì)胞在這個(gè)過(guò)程中的作用尚不明確,我們推測(cè)產(chǎn)前應(yīng)激可能改變?nèi)毖笮切文z質(zhì)細(xì)胞的活性而影響神經(jīng)功能。盡管已有研究表明促紅細(xì)胞生成素肝細(xì)胞受體A4(erythropoietin-producing hepato cellular receptor A4, EphA4)受體可以調(diào)節(jié)脊髓損傷后的星形膠質(zhì)細(xì)胞活化[5,6],但關(guān)于腦缺血后EphA4受體對(duì)星形膠質(zhì)細(xì)胞的影響研究較少[7],而產(chǎn)前應(yīng)激對(duì)子代大鼠腦缺血/再灌注后星形膠質(zhì)細(xì)胞EphA4受體的影響,國(guó)內(nèi)外更是少見(jiàn)報(bào)道。本研究通過(guò)使用大鼠產(chǎn)前應(yīng)激模型和子代大鼠缺血/再灌注模型,探討產(chǎn)前應(yīng)激是否會(huì)通過(guò)EphA4受體改變子代大鼠腦缺血/再灌注后星形膠質(zhì)細(xì)胞的活化,影響CSPG的形成。本研究可為今后調(diào)節(jié)EphA4受體,改變產(chǎn)前應(yīng)激對(duì)子代大鼠腦缺血/再灌注后神經(jīng)功能的影響提供理論依據(jù)。
1.1 實(shí)驗(yàn)動(dòng)物
12周齡的健康雌性SD大鼠,體重200~250 g,共15只,12周齡健康雄性大鼠15只,體重300~350 g,均購(gòu)自上海斯萊克實(shí)驗(yàn)動(dòng)物有限公司。動(dòng)物飼養(yǎng)條件:溫度為(22±1)℃,光照7:00 am—7:00 pm,自由攝取水和食物。
1.2 實(shí)驗(yàn)方法
產(chǎn)前應(yīng)激模型:將大鼠按雌∶雄1∶1的比例隨機(jī)合籠,以發(fā)現(xiàn)陰栓當(dāng)日為妊娠第0天,次日為妊娠第1天。按隨機(jī)數(shù)字表法將孕鼠分為有產(chǎn)前應(yīng)激處理和無(wú)產(chǎn)前應(yīng)激處理。參考文獻(xiàn)方法建產(chǎn)前應(yīng)激模型[8]:于妊娠第15到21天每日3次(分別在10:00、14:00、18:00)將孕鼠限制在塑料質(zhì)地的圓柱形裝置內(nèi)(長(zhǎng)19 cm,直徑7 cm),每次45 min。無(wú)產(chǎn)前應(yīng)激處理的孕鼠在整個(gè)妊娠期均飼養(yǎng)于塑料籠內(nèi)不受干擾。除應(yīng)激處理外,孕鼠不受其他打擾。所有孕鼠均自然分娩,共獲得15窩仔鼠,為避免奶水不足對(duì)新生仔鼠生長(zhǎng)發(fā)育的影響,每窩仔鼠隨機(jī)留8只飼養(yǎng)。子代大鼠在3周齡后斷乳,僅保留雄性子代大鼠(3~4只/窩)。
MCAO模型:雄性子代大鼠于2月齡(體重約250~300 g)時(shí)采用右側(cè)頸外動(dòng)脈插入線栓法[9]制造MCAO模型,大鼠于右側(cè)大腦中動(dòng)脈缺血90 min后,向外輕輕拉出栓線,建立再灌注模型。并判定模型是否成功,以動(dòng)物清醒后有右眼Horner征,提尾時(shí)出現(xiàn)左側(cè)前肢屈曲內(nèi)收者為研究對(duì)象,并剔除蛛網(wǎng)膜下腔出血者。假手術(shù)組采取同樣的手術(shù)過(guò)程,但線栓插入較淺,并不造成大腦中動(dòng)脈閉塞。實(shí)驗(yàn)共分3組:產(chǎn)前應(yīng)激+假手術(shù)組,MCAO組,產(chǎn)前應(yīng)激+MCAO組(n=10)。
1.3 HE染色
于再灌注后第5天,每組取5只大鼠,10%水合氯醛麻醉,并使用4%中性多聚甲醛灌注內(nèi)固定,于斷頭取腦后,從額極至枕葉切成厚度為3 mm的冠狀腦切片,經(jīng)脫水、透明、浸蠟,最后包埋成蠟塊。每一蠟塊切片行常規(guī)HE染色檢測(cè)腦梗死情況。采用Image J圖像軟件(Media Cybernetics,Inc)計(jì)算總的梗死體積(MV)及左右半球體積(LV、RV)。根據(jù)公式[10]進(jìn)行計(jì)算:梗死體積(%)=[LV-(RV-MV)]/LV×100%,以糾正腦水腫對(duì)梗死面積的影響。
1.4 免疫熒光雙標(biāo)染色
將腦組織的石蠟切片經(jīng)二甲苯脫蠟、梯度酒精水化、微波修復(fù)后,加入胎牛血清封閉后,加兔抗EphA4抗體(Santa Cruz Biotechnology Inc.,1∶150)和小鼠抗GFAP抗體(北京博奧森生物制劑有限公司,1∶300),4℃過(guò)夜,加FITC熒光素標(biāo)記的羊抗兔IgG(1∶50)和CY3標(biāo)記的羊抗小鼠IgG(1∶50)混合抗體,避光室溫孵育2 h,甘油封片。熒光顯微鏡下觀察缺血灶邊緣區(qū)EphA4和GFAP的表達(dá)情況并拍照。EphA4陽(yáng)性細(xì)胞呈綠色熒光,GFAP陽(yáng)性細(xì)胞呈紅色熒光,EphA4/GFAP雙陽(yáng)性細(xì)胞呈淡黃色。
1.5 Western blot檢測(cè)
于再灌注后第5天,每組均取5只大鼠,10%水合氯醛麻醉,于低溫下開(kāi)顱取缺血灶周邊區(qū),假手術(shù)組取與手術(shù)組相對(duì)應(yīng)的腦區(qū),立即放入液氮中凍存。提取總蛋白后,使用BCA法測(cè)定蛋白濃度,于10%聚丙烯酰胺凝膠電泳(SDS- PAGE)分離后,轉(zhuǎn)印到硝酸纖維素膜上,并在5%脫脂奶粉封閉后分別加入一抗:EphA4(Santa Cruz Biotechnology Inc.,1∶500);GFAP(北京博奧森生物制劑有限公司,1∶200);Neurocan(北京博奧森生物制劑有限公司,1∶200); β-肌動(dòng)蛋白(β-actin,北京博奧森生物制劑有限公司,1∶2000),4℃過(guò)夜,洗滌后與辣根過(guò)氧化酶標(biāo)記的二抗孵育。ECL顯影后掃描,使用凝膠成像處理系統(tǒng)(美國(guó)SYNGENE公司)行吸光度分析。以目的條帶與β-actin條帶吸光度之比作為目的蛋白的相對(duì)表達(dá)量。
1.6 統(tǒng)計(jì)學(xué)處理
2.1 腦梗死體積測(cè)定
各組子代大鼠腦梗死情況見(jiàn)圖1。產(chǎn)前應(yīng)激+假手術(shù)組未見(jiàn)腦梗死灶。MCAO組梗死累及尾狀殼核及蒼白球,產(chǎn)前應(yīng)激+MCAO組梗死范圍相對(duì)彌散,尚累及胼胝體和皮質(zhì)。產(chǎn)前應(yīng)激+MCAO組梗死體積百分比(30.80±2.68)%明顯大于MCAO組(16.94±1.10)%(P<0.05)。
Fig. 1 Representative photographs of hematoxylin-eosin (HE) stained brain sections obtained from three experimental groups after 5 days of transient middle cerebral artery occlusion(MCAO)
2.2 EphA4/GFAP免疫熒光雙標(biāo)染色
缺血灶邊緣區(qū)GFAP陽(yáng)性細(xì)胞呈紅色熒光,產(chǎn)前應(yīng)激+假手術(shù)組的GFAP陽(yáng)性細(xì)胞數(shù)量較少,胞體小且纖維纖細(xì)(圖2A1),MCAO組GFAP陽(yáng)性細(xì)胞數(shù)量增多且體積增大(圖2B1),產(chǎn)前應(yīng)激+MCAO組GFAP陽(yáng)性細(xì)胞分枝變長(zhǎng)且增多增粗,部分突起交織呈網(wǎng)狀(圖2C1)。缺血灶邊緣區(qū)存在大量EphA4陽(yáng)性細(xì)胞,呈綠色熒光(圖2A2、B2、C2),且免疫熒光雙標(biāo)染色提示EphA4受體與GFAP陽(yáng)性星形膠質(zhì)細(xì)胞在缺血灶邊緣區(qū)的分布有較好的一致性,兩者疊加發(fā)現(xiàn)GFAP陽(yáng)性星形膠質(zhì)細(xì)胞上均有EphA4表達(dá),信號(hào)呈淡黃色(圖2A3、B3、C3)。
2.3 Western blot檢測(cè)EphA4、GFAP和Neurocan蛋白表達(dá)情況
產(chǎn)前應(yīng)激+假手術(shù)組子代大鼠缺血灶邊緣區(qū)有EphA4和GFAP蛋白表達(dá);與產(chǎn)前應(yīng)激+假手術(shù)組比較,MCAO增加缺血灶邊緣EphA4和GFAP蛋白表達(dá),差異有統(tǒng)計(jì)學(xué)意義(P均<0.05);與MCAO組比較,產(chǎn)前應(yīng)激+MCAO顯著增加EphA4和GFAP蛋白表達(dá)(P均<0.05)。產(chǎn)前應(yīng)激+假手術(shù)組全長(zhǎng)Neurocan蛋白表達(dá)極少;與產(chǎn)前應(yīng)激+假手術(shù)組比較,MCAO增加全長(zhǎng)Neurocan蛋白表達(dá),差異有統(tǒng)計(jì)學(xué)意義(P<0.05);與MCAO組比較,產(chǎn)前應(yīng)激+MCAO顯著增加全長(zhǎng)Neurocan蛋白表達(dá)(P<0.05)。產(chǎn)前應(yīng)激+假手術(shù)組、MCAO組和產(chǎn)前應(yīng)激+MCAO組均檢測(cè)出C-末端Neurocan蛋白表達(dá),但差異無(wú)統(tǒng)計(jì)學(xué)意義(圖3,表1)。
Fig. 2 Immunofluorescence double staining of GFAP and EphA4 in the peri-ischemia regions of three experimental groups after 5 days of transient middle cerebral artery occlusion (MCAO)
A1-A3: Prenatal stress+Sham group; B1-B3: MCAO group; C1-C3: Prenatal Stress+MCAO group. Scale bar=25 μm
Fig. 3 Expression of Neurocan, C-Neurocan, GFAP and EphA4 in the peri-ischemia regions of three experimental groups after 5 days of transient middle cerebral artery occlusion (MCAO)
A: Prenatal stress+Sham group; B: MCAO group; C: Prenatal stress+MCAO group
產(chǎn)前應(yīng)激來(lái)自生活或工作的不良事件的影響,如自然災(zāi)害、戰(zhàn)爭(zhēng)、失業(yè)、家人或配偶的死亡、家庭和婚姻的不和諧等。本室驗(yàn)前期研究[4]表明:產(chǎn)前應(yīng)激會(huì)促進(jìn)子代大鼠缺血/再灌注后24 h的神經(jīng)細(xì)胞凋亡,提示圍產(chǎn)期不良事件會(huì)影響子代缺血性卒中的預(yù)后。與前期研究結(jié)果一致,本研究發(fā)現(xiàn)在缺血/再灌注后5 d,產(chǎn)前應(yīng)激+MCAO組較MCAO組具有更大的腦梗死體積,提示產(chǎn)前應(yīng)激會(huì)產(chǎn)生持久影響,導(dǎo)致成年子代對(duì)腦缺血/再灌注的易損性,從而加重子代腦缺血損害。
星形膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)最豐富的細(xì)胞類型,在生理環(huán)境下可以為神經(jīng)元提供許多必須的支持性活動(dòng)。在病理?xiàng)l件,如卒中、脊髓損傷、Alzheimer’s病等,星形膠質(zhì)細(xì)胞出現(xiàn)快速活化,經(jīng)歷數(shù)量和大小上的增加(增生和肥大)。活化的星形膠質(zhì)細(xì)胞不同于生理性的星形膠質(zhì)細(xì)胞,具有升高的GFAP表達(dá),已有研究表明,在腦缺血時(shí)抑制星形膠質(zhì)細(xì)胞GFAP的過(guò)度表達(dá)可以保護(hù)神經(jīng)元并減輕神經(jīng)功能缺損[11]。本實(shí)驗(yàn)觀察到MCAO組中GFAP陽(yáng)性星形膠質(zhì)細(xì)胞出現(xiàn)數(shù)量增加、體積增大和細(xì)胞突起增多,且產(chǎn)前應(yīng)激+MCAO組較MCAO組改變更明顯,而產(chǎn)前應(yīng)激+假手術(shù)組則無(wú)這種改變,提示產(chǎn)前應(yīng)激使子代的星形膠質(zhì)細(xì)胞在缺血/再灌注后出現(xiàn)更明顯的活化。我們推測(cè)星形膠質(zhì)細(xì)胞的活化及GFAP表達(dá)增加可能是產(chǎn)前應(yīng)激的子代大鼠腦梗死面積增加的原因。
Tab. 1 Quantitative results of Neurocan, C-Neurocan, EphA4 and GFAP in three experimental ±s, n=5)
MCAO: Middle cerebral artery occlusion
*P<0.05vsprenatal stress + sham group;#P<0.05vsMCAO group
已有研究表明Eph是星形膠質(zhì)細(xì)胞活化的重要調(diào)節(jié)物,影響中樞神經(jīng)系統(tǒng)損傷后或中樞神經(jīng)系統(tǒng)疾病過(guò)程中的星形膠質(zhì)細(xì)胞增殖[12]。在Eph受體家族中,EphA4受體在損傷部位的星形膠質(zhì)細(xì)胞高度表達(dá)[5],并可在體外[7]和活體[6]調(diào)節(jié)星形膠質(zhì)細(xì)胞活性。在EphA4基因敲除的小鼠,星形膠質(zhì)細(xì)胞反應(yīng)明顯減少,GFAP表達(dá)減少,且CSPG染色也減少。在非人類的靈長(zhǎng)類動(dòng)物腦損傷模型中也發(fā)現(xiàn)EphA4受體表達(dá)在病灶部位周圍活化的星形膠質(zhì)細(xì)胞中明顯上調(diào),而在體外實(shí)驗(yàn)中EphA4受體活化介導(dǎo)星形膠質(zhì)細(xì)胞增殖和膠質(zhì)纖維酸性蛋白表達(dá)[13]。與此類似,本實(shí)驗(yàn)發(fā)現(xiàn)在MCAO組中EphA4受體表達(dá)增加且主要表達(dá)于GFAP陽(yáng)性星形膠質(zhì)細(xì)胞,提示MCAO增加缺血灶周邊EphA4受體表達(dá)且該受體可能介導(dǎo)了星形膠質(zhì)細(xì)胞的活化,實(shí)驗(yàn)還發(fā)現(xiàn)產(chǎn)前應(yīng)激+MCAO組較MCAO組EphA4受體在GFAP陽(yáng)性星形膠質(zhì)細(xì)胞的表達(dá)更明顯,提示產(chǎn)前應(yīng)激會(huì)改變?nèi)毖笞哟切文z質(zhì)細(xì)胞上EphA4受體的表達(dá),進(jìn)而調(diào)節(jié)星形膠質(zhì)細(xì)胞的活化,出現(xiàn)GFAP表達(dá)增加及細(xì)胞形態(tài)改變。
活化的星形膠質(zhì)細(xì)胞具有以損傷部位為目標(biāo)的細(xì)胞遷移增加以及隨后活躍的細(xì)胞增殖,并能產(chǎn)生CSPG,形成膠質(zhì)瘢痕[14]。Neurocan是一種主要的CSPG,在成年腦組織,神經(jīng)蛋白聚糖(275 kD)很快被分解為兩個(gè)片段,即C-端(150 kD)和N-端(130 kD)片段。因此,在正常的成年腦組織無(wú)法檢測(cè)到全長(zhǎng)的神經(jīng)蛋白聚糖,而在發(fā)育不成熟的腦組織則可以檢測(cè)出全長(zhǎng)神經(jīng)蛋白聚糖及其分解片段[15],此外,在某些病理情況下,如腦外傷、癲癇的腦組織中也可以檢測(cè)到全長(zhǎng)的神經(jīng)蛋白聚糖[15]。全長(zhǎng)神經(jīng)蛋白聚糖可以通過(guò)調(diào)節(jié)鈣粘蛋白和整聯(lián)蛋白抑制軸突延伸或再生[15]。本實(shí)驗(yàn)中,在MCAO組的缺血側(cè)額頂葉檢出全長(zhǎng)神經(jīng)蛋白聚糖,這與既往的研究一致[16],提示缺血可以誘導(dǎo)周邊腦組織表達(dá)全長(zhǎng)神經(jīng)蛋白聚糖,從而調(diào)節(jié)神經(jīng)網(wǎng)絡(luò)形成。產(chǎn)前應(yīng)激+MCAO組較MCAO組表達(dá)更多的全長(zhǎng)神經(jīng)蛋白聚糖,說(shuō)明產(chǎn)前應(yīng)激可能通過(guò)改變CSPG分泌,阻礙神經(jīng)元再生,從而影響子代卒中后神經(jīng)功能恢復(fù)障礙。
總之,產(chǎn)前應(yīng)激可能增加子代大鼠缺血/再灌注后缺血灶周邊星形膠質(zhì)細(xì)胞上EphA4受體的表達(dá),促進(jìn)星形膠質(zhì)細(xì)胞活化,分泌抑制分子,如神經(jīng)蛋白聚糖,抑制神經(jīng)元再生,可能導(dǎo)致更明顯的膠質(zhì)瘢痕形成,加重神經(jīng)損傷。本研究表明產(chǎn)前應(yīng)激會(huì)加重子代大鼠腦缺血/再灌注損害,為腦血管病的防治與優(yōu)生優(yōu)育提供新的理論依據(jù)。
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The effects of prenatal stress on the astrocytes after cerebral ischemia/reperfusion injury in adult offspring rats
WANG Ling-xing, HUANG Hong-hong△, CHEN Ya-fang, CAI Hong-chao, QIAN Jia-qiang
(Department of Neurology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, China)
Objective: To investigate the effects of prenatal stress on astrocytes after ischemia/reperfusion of cerebral middle artery in adult offspring rats. Methods: Pregnant rats were randomly assigned to prenatal stress treatment group, which was exposed to restraint three times daily in the last week of pregnancy, and no prenatal stress treatment group. Adult male offspring rats were subjected to transient focal cerebral ischemia by middle cerebral artery occlusion (MCAO). There were three groups: prenatal stress + sham group, MCAO group and prenatal stress + MCAO group (n=10). After 5 days of reperfusion, the infarct size was evaluated. The morphology of astrocytes, co-localization of erythropoietin-producing hepatocellular receptor A4 (EphA4) and glial fibrillary acidic protein (GFAP) were detected by double immunofluorescent staining. And the protein expressions of EphA4, GFAP and Neurocan in peri-ischemic regions were detected by Western blot. Results: The infarct size and the expression of EphA4, GFAP and Neurocan were significantly increased in prenatal stress + MCAO group compared with MCAO group (allP<0.05). And the morphological changes of GFAP-positive astrocytes and co-localization of EphA4/GFAP were more obvious in prenatal stress + MCAO group compared with MCAO group. Conclusion: Prenatal stress may upregulate the expression of EphA4 on astrocytes in the offspring rats after cerebral ischemia/reperfusion, which promotes the reactivity of astrocyte and increases the expression of neurocan.
prenatal stress; offspring rats; cerebral ischemia; astrocyte
福建省自然科學(xué)基金項(xiàng)目(2015J01449);福建省教育廳項(xiàng)目(JB13067);泉州市技術(shù)研究與開(kāi)發(fā)項(xiàng)目(2012Z35);院苗圃基金(2012MP65)
2015-11-24
2016-04-04
R741.02
A
1000-6834(2016)04-301-05
△【通訊作者】Tel: 13600766682; E-mail: minghuaxin@139.com