劉玉潔 石秀玉 于新穎 胡琳燕 鄒麗萍

·論著·

劉玉潔1,3石秀玉1,3于新穎2胡琳燕1鄒麗萍1

1 方法

1.1 動物分組 生后7 d的Wistar大鼠234只任意分為13組：4種AEDs(PB，VPA，LTG，TPM)分別分為高、中、低劑量組和對照組，每組18只。分別標(biāo)記組別后母鼠與新生鼠同籠存放。

PB溶于超純水2 mL(終濃度為10 mg·mL-1)，PB高、中、低劑量組分別予PB 80、 40、20 mg·kg-1腹腔注射；VPA溶于超純水2 mL(終濃度為20 mg·mL-1)，VPA高、中、低劑量組分別予VPA 200、100、50 mg·kg-1腹腔注射；LTG溶于1%纖維素鈉2 mL(終濃度為10 mg·mL-1)，LTG高、中、低劑量組分別予LTG 80、40、20 mg·kg-1灌胃；TPM溶于1%纖維素鈉2 mL(終濃度為10 mg·mL-1)，TPM高、中、低劑量組分別予TPM 80、40、20 mg·kg-1灌胃，對照組予相同體積1%纖維素鈉，灌胃。均每日1次，共用21 d。

從第22 d始，①每組大鼠取6只斷頭取腦，迅速用冰冷的生理鹽水洗凈血液，濾紙吸干立即稱重，用于流式細(xì)胞術(shù)和RT-PCR檢測。②每組再取6只大鼠將溶于生理鹽水的Brdu(sigma公司)100 mg·kg-11次性經(jīng)腹腔注射[13]。36 h后予3%戊巴比妥60 mg·kg-1過量麻醉，4%多聚甲醛左心灌注固定取腦，在海馬部位做冠狀連續(xù)冰凍切片，切片厚度20 μm，每隔2張取1張，組成1套8～10張，行BrdU單標(biāo)免疫熒光染色。③每組余下的6只大鼠左心先后灌注硫化鈉溶液和4%多聚甲醛固定取腦，在海馬部位做冠狀冰凍連續(xù)切片，切片厚20 μm，每隔3張取1張行Timm染色和評分。

1.2 流式細(xì)胞儀Annexin-V/PI法檢測細(xì)胞凋亡 將剝離的腦組織制成每毫升1×106細(xì)胞懸液，室溫1 000 r·min-1離心5 min，去除上清，PBS清洗，加入1.25 μL 200 μg·mL-1Annexin V-FITC(晶美)室溫避光孵育15 min， 室溫1 000 r·min-1離心5 min，去除上清，加入10 μL碘化丙啶，流式細(xì)胞儀檢測。

1.3 熒光實(shí)時(shí)定量PCR法分析BDNF和NT-3的表達(dá)

1.3.1 引物設(shè)計(jì) BDNF、NT-3和β-actin的PCR擴(kuò)增引物序列由上海Invitrogen公司設(shè)計(jì)合成，β-actin sense: AAGATCCTGACCGAGCGTGG，antisense: CAGCACTGTGTT-GGCATAGAGA；BDNF sense: CGACGTCCCTGGCTGGACAC-TTTT，antisense: AGTAAGGGCCCGAACATACGATTGG； NT-3 sense: GGTCAGAATTCCAGCCGATGATTGC，antisense: CAGCGCCAGCC TACGTTTGTTGT。

1.3.2 組織總RNA的提取和逆轉(zhuǎn)錄 將稱重后的腦組織立即分離，取出兩側(cè)海馬，放入EP管中-80℃保存?zhèn)溆?，按照硫氰酸?酚-氯仿抽提法提取總RNA，溶于適量RNAase 水，立即使用或保存于-70℃。隨機(jī)引物法進(jìn)行逆轉(zhuǎn)錄反應(yīng)，按試劑說明書操作。

1.3.3 PCR反應(yīng) 熒光實(shí)時(shí)定量PCR的反應(yīng)體系為20 μL，包括SYBR Premix Ex Taq(大連寶生物)10 μL，Primer1 1.2 μL，Primer 2 1.2 μL，cDNA 2 μL，dH2O 5.6 μL。PCR反應(yīng)參數(shù)：預(yù)變性95℃ 10 s, 56℃ 10 s退火、72℃ 10 s延伸，共50個(gè)循環(huán)。最后65℃ 15 s、95℃每秒改變0.1℃、40℃ 30 s進(jìn)行融解曲線分析。

1.3.4 結(jié)果分析 結(jié)果以目的基因與內(nèi)參照β-actin的比值表示。每次反應(yīng)時(shí)標(biāo)準(zhǔn)品(cDNA的PCR產(chǎn)物經(jīng)純化后做倍比稀釋：1、1×10-2、1×10-4、1×10-6、1×10-8、1×10-10)，待測樣本及陰性對照(以去離子水為模板)同時(shí)擴(kuò)增，并做溶解曲線以檢測非特異擴(kuò)增。BDNF(或NT-3)/β-actin比值代表待測樣本的BDNF(或NT-3)相對表達(dá)。

1.4 免疫組織化學(xué)分析神經(jīng)發(fā)生和苔蘚纖維發(fā)芽

1.4.1 BrdU單標(biāo)免疫熒光染色 以鼠抗Brdu單克隆抗體(Sigma公司)作為一抗，F(xiàn)ITC標(biāo)記的羊抗小鼠IgG(北京中山公司)作為二抗，熒光顯微鏡下觀察并照相，Brdu陽性者發(fā)綠色熒光。

1.4.2 Timm's 染色 將切片自然晾干，三蒸水沖洗后，放置于含有120 mL阿拉伯樹膠(50%)、60 mL氫醌(5.78%)、10 mL檸檬酸(51%)、10 mL檸檬酸鈉(47%)和212.5 mg硝酸銀的混合溶液中，暗室顯影40～60 min，流水終止染色15 min。切片晾干后，梯度乙醇脫水、透明、封片。以半定量的記分方法分析Timm染色結(jié)果，觀察CA3區(qū)和顆粒細(xì)胞上層苔蘚纖維發(fā)芽。

2 結(jié)果

2.1 腦重 表1顯示，PB高劑量組較對照組腦重下降12%，(2.03±0.16)vs(2.32±0.24) g,P<0.05。VPA中和高劑量組腦重較對照組下降最明顯，其中高劑量組腦重降低15%，(1.95±0.26)vs(2.32±0.24) g,P<0.01。LTG和TPM各劑量組腦重下降與對照組差異無顯著差異。

Notes The high, middle and low dose groups of phenobarbital (PB) were given 80, 40 and 20 mg·kg-1; the corresponding doses of valproate(VPA) in three groups were 200, 100 and 50 mg·kg-1, doses of lamotrigine(LTG) were 80, 40 and 20 mg·kg-1, doses of topiramate(TPM) were 80,40 and 20 mg·kg-1. 1)vscontrol group，P<0.05

2.2 細(xì)胞凋亡 表1顯示，4種AEDs(PB、VPA、LTG、TPM)分別的3個(gè)劑量組Annexin V+/PI -細(xì)胞的百分?jǐn)?shù)均有高于對照組的趨向，其中PB和VPA分別的3個(gè)劑量組，LTG高劑量組，TPM中、高劑量組Annexin V+/PI -細(xì)胞的百分?jǐn)?shù)與對照組的差異有統(tǒng)計(jì)學(xué)意義。

2.3 AEDs對神經(jīng)營養(yǎng)因子mRNA表達(dá)的影響 實(shí)時(shí)熒光定量PCR結(jié)果分析顯示PB 中、高劑量組, VPA中、高劑量組, LTG高劑量組和 TPM中、高劑量組可導(dǎo)致BDNF 和 NT-3 mRNA的水平較對照組顯著降低(表1)。

2.4 BrdU 染色和Timm's染色

2.4.1 神經(jīng)發(fā)生的影響 表2顯示，VPA和LTG分別的3個(gè)劑量組均可導(dǎo)致海馬門區(qū)、齒狀回以及海馬CA3區(qū)和海馬外區(qū)域 BrdU-標(biāo)記的細(xì)胞數(shù)較對照組顯著增加,P<0.05(圖1)。 PB和TPM分別的3個(gè)劑量組與對照組比較，BrdU-標(biāo)記的細(xì)胞數(shù)差異均無統(tǒng)計(jì)學(xué)意義。

2.4.2 苔蘚纖維發(fā)芽的影響 表2顯示，4種AEDs分別的3個(gè)劑量組C3區(qū)和顆粒細(xì)胞上層Timm評分與對照組比較差異均無統(tǒng)計(jì)學(xué)意義(P>0.05)。

Notes The high, middle and low dose groups of phenobarbital (PB) were given 80, 40 and 20 mg·kg-1; the corresponding doses of valproate(VPA) in three groups were 200, 100 and 50 mg·kg-1, doses of lamotrigine(LTG) were 80, 40 and 20 mg·kg-1, doses of topiramate(TPM) were 80,40 and 20 mg·kg-1. 1)vscontrol group，P<0.05

圖1 海馬外神經(jīng)發(fā)生(×100)

Fig 1 Neurogenesis outside of the hippocampus (×100)

Notes A, B represented neurogenesis in the entorhinal cortex outside of the hippocampus in VPA 200mg·kg-1group and LTG 80 mg·kg-1group, respectively. The arrows pointed to newborn neurons

3 討論

既往研究[7，9，14]認(rèn)為AEDs對新生嚙齒類動物可能造成神經(jīng)毒性損害，不同AEDs對發(fā)育期大腦所造成的神經(jīng)損害差異很大。Bittigau等[7]研究發(fā)現(xiàn)苯妥英、VPA、氨己烯酸、地西泮和氯硝西泮可增加神經(jīng)細(xì)胞凋亡，而其對應(yīng)的AEDs濃度與人類控制驚厥發(fā)作的血藥濃度接近。Glier等[8]發(fā)現(xiàn)治療劑量的TPM對發(fā)育期大腦沒有毒性。本研究發(fā)現(xiàn)4種AEDs均會造成腦重下降，但VPA的不同劑量組腦重降低最明顯，LTG不同劑量組雖腦重也呈降低趨勢，但與對照組差異無統(tǒng)計(jì)學(xué)意義。AEDs可引起神經(jīng)細(xì)胞凋亡增加，但不同AEDs引起凋亡增加的閾值不同，PB為20 mg·kg-1, VPA為50 mg·kg-1, LTG為80 mg·kg-1，TPM為40mg·kg-1。神經(jīng)細(xì)胞凋亡增加的同時(shí)也伴有神經(jīng)營養(yǎng)因子BDNF和NT-3 mRNA表達(dá)的降低。

神經(jīng)細(xì)胞凋亡在大腦發(fā)育過程中起著非常重要的作用，任何影響這一過程的物質(zhì)都會導(dǎo)致神經(jīng)元凋亡的改變[15]，影響正常的腦發(fā)育，從而影響認(rèn)知。AEDs導(dǎo)致發(fā)育期大腦神經(jīng)細(xì)胞凋亡增加可能有不同的機(jī)制，其中之一為內(nèi)源性神經(jīng)營養(yǎng)物質(zhì)系統(tǒng)如BDNF和NT-3表達(dá)的降低[16]，BDNF對中樞膽堿能神經(jīng)元有刺激生長作用，可促進(jìn)背根神經(jīng)節(jié)神經(jīng)元突起向中樞生長，也可延長離體培養(yǎng)胚胎大鼠腦中隔膽堿能神經(jīng)元細(xì)胞的存活時(shí)間，并增加乙酰膽堿酶和膽堿乙酰轉(zhuǎn)移酶的酶活性。NT-3結(jié)構(gòu)上與BDNF相似，可促進(jìn)由神經(jīng)基板發(fā)生的神經(jīng)元突起的生長，也可促進(jìn)離體培養(yǎng)的背根神經(jīng)節(jié)神經(jīng)細(xì)胞突起的生長。目前認(rèn)為腦的發(fā)育、老化等改變，以及病理性損害等均與BDNF和NT-3的作用有關(guān)。其表達(dá)的降低可導(dǎo)致神經(jīng)細(xì)胞凋亡增加影響腦發(fā)育，還可直接影響了腦的正常功能，從而導(dǎo)致認(rèn)知功能的損害。這可能是本研究觀察到的AEDs導(dǎo)致腦重下降的原因之一。

本研究中AEDs導(dǎo)致BDNF和NT-3 mRNA表達(dá)下降的劑量比既往研究報(bào)道的低，Bittigau等[7]研究中發(fā)現(xiàn)PB和VPA引起B(yǎng)DNF和NT-3 mRNA表達(dá)降低的閾值分別為50和200 mg·kg-1。結(jié)果的不一致可能與發(fā)育階段不同有關(guān)，本研究大鼠在生后7 d開始應(yīng)用AEDs直至生后28 d，有研究顯示AEDs對大鼠的神經(jīng)毒性呈年齡依賴性，多集中在生后21 d內(nèi)，與腦生長高峰符合[17]。此外，本研究連續(xù)應(yīng)用AEDs 21 d，相當(dāng)于人類整個(gè)嬰幼兒時(shí)期，而Bittigau等[7]研究用藥時(shí)間僅1 d，提示AEDs治療時(shí)間越長，導(dǎo)致神經(jīng)營養(yǎng)物質(zhì)表達(dá)降低的閾值呈降低趨勢。

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(本文編輯：丁俊杰)

Effects of long-term antiepileptic treatment on the developing brain of rats

LIU Yu-jie1,3, SHI Xiu-yu1,3, YU Xin-ying2, HU Lin-yan1, ZOU Li-ping1

(1 Department of Pediatrics, Chinese PLA General Hospital, Beijing 100853; 2 Department of Pediatrics, Gaobeidian City Hospital, Gaobeidian 074000, China; 3 Co-first author)

ZOU Li-ping，E-mail:zouliping21@hotmail.com

ObjectiveTo study the effects of long-term treatment with antiepileptic drugs (AEDs) on the developing brain of rats, and to explain possible mechanisms of adverse effects of AEDs at cellular and molecular levels.MethodsA total of 234 neonatal Wistar rats(P7)were divided into 13 groups (the control group, PB, VPA, LTG and TPM with high, middle and low dosage), with 18 rats in each group. After 3-weeks treatment with AEDs the treatment groups and the controls were divided into two parts. One part was sacrificed by decapitation and the brain was removed and washed with ice-cold saline. These brains were used in the study of Annexin-V FITC/PI double staining and quantitative real-time PCR detection. The other part used in the study of BrdU staining and Timm's staining

an overdose of sodium pentobarbital (60 mg·kg-1, i.p.) and was perfused with different solution.Results①Long-term treatment with AEDs caused significant reduction in brain weight, especially in VPA groups. VPA (200 mg·kg-1) resulted in 15% decrease in brain weight. ②AEDs caused apoptotic neurodegeneration, the threshold of PB, VPA, LTG and TPM was 20, 50, 80 and 40 mg·kg-1, respectively. ③ Quantitative real-time PCR showed 4 AEDs decreased the expression of BDNF and NT-3, the threshold of PB, VPA, LTG and TPM was 40, 100, 80 and 40 mg·kg-1, respectively. Neurogenesis increased in the rats treated with valproate and lamotrigine but their effect on mossy fiber sprouting was not obvious in any rats (P>0.05).ConclusionLong-term treatment with AEDs damages developing brain of rats, PB, VPA, LTG and TPM cause apoptotic neurodegeneration in the developing brain at different dose levels. Neuronal death is associated with reduced expression of BDNF and NT-3. Interestingly, VPA and LTG cause increased neurogenesis in dentate gyrus with an absence of mossy fiber sprouting. These findings presented one possible mechanism to explain that cognitive impairment was associated with exposure of humans to antiepileptic therapy.

Antiepileptic drugs; Development ; Apoptosis; Neurotrophins; Neurogenesis

1 中國人民解放軍總醫(yī)院兒科 北京，100853；2 河北省高碑店市醫(yī)院兒科 高碑店，074000；3 共同第一作者

鄒麗萍，E-mail:zouliping21@hotmail.com

10.3969/j.issn.1673-5501.2013.05.012

2013-09-24

2013-12-12)