岳妍，譚波濤，劉媛，伍亞民，賈功偉，蔣瑋，虞樂(lè)華，殷櫻

FTY720對(duì)急性脊髓損傷大鼠神經(jīng)功能及血脊髓屏障的影響

岳妍，譚波濤，劉媛，伍亞民，賈功偉，蔣瑋，虞樂(lè)華，殷櫻

目的 觀察新型免疫抑制劑FTY720對(duì)急性脊髓損傷后大鼠神經(jīng)功能和血脊髓屏障的影響。方法 成年SD大鼠144只，隨機(jī)分為4組(n=36)。正常對(duì)照組(NG)：不做任何處理；假手術(shù)組(SO)：?jiǎn)渭円С蛋灞┞都顾瑁挥钃p傷處理；損傷對(duì)照組(HS)：脊髓右半側(cè)橫切損傷后腹腔注射生理鹽水；FTY720治療組(FTY720)：脊髓右半側(cè)橫切損傷后30min給予腹腔注射FTY720[1mg/(kg·d)]，連續(xù)7d。術(shù)后不同時(shí)間點(diǎn)行行為學(xué)檢測(cè)，包括BBB運(yùn)動(dòng)功能評(píng)分和水平網(wǎng)格檢測(cè)；行神經(jīng)電生理運(yùn)動(dòng)誘發(fā)電位(MEP)N1波和感覺(jué)誘發(fā)電位(SEP) P1波潛伏期檢測(cè)；對(duì)脊髓組織行蘇木精-伊紅(HE)染色及Evans Blue(EB)血脊髓屏障滲透性檢測(cè)。分析FTY720治療效果。結(jié)果 半橫切損傷后，HS組和FTY720組大鼠損傷側(cè)脊髓神經(jīng)功能下降，至28d時(shí)仍未恢復(fù)至NG 組和SO組水平。行為學(xué)檢測(cè)和神經(jīng)電生理檢測(cè)結(jié)果顯示，F(xiàn)TY720組大鼠運(yùn)動(dòng)功能恢復(fù)速度比HS組更快；損傷7、14、28d時(shí)，兩組大鼠BBB評(píng)分和SEP P1波潛伏期檢測(cè)差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；損傷14、28d時(shí)，兩組大鼠水平網(wǎng)格檢測(cè)和MEP N1波潛伏期檢測(cè)差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。組織學(xué)檢測(cè)結(jié)果顯示，各時(shí)間點(diǎn)NG 組和SO組大鼠脊髓結(jié)構(gòu)均完整；損傷14d時(shí)，F(xiàn)TY720組脊髓損傷處慢性炎癥細(xì)胞浸潤(rùn)，膠質(zhì)化反應(yīng)程度小于HS組；損傷28d時(shí)，F(xiàn)TY720組損傷處空洞殘存面積小于HS組，再生纖維排列較HS組有序。血脊髓屏障滲透性檢測(cè)結(jié)果發(fā)現(xiàn)，損傷后7d內(nèi)，HS組和FTY720組大鼠血脊髓屏障EB滲出量較NG組和SO組明顯增加；各時(shí)間點(diǎn)FTY720組EB滲出面積均小于HS組(P<0.05)，其中損傷3d時(shí)差異最為明顯。結(jié)論 FTY720能顯著降低脊髓損傷急性期血脊髓屏障滲透性，有效促進(jìn)急性期后神經(jīng)功能的恢復(fù)，為損傷組織提供一定的神經(jīng)保護(hù)作用。

免疫抑制劑；FTY720；急性脊髓損傷；神經(jīng)保護(hù)藥

急性脊髓損傷是一類嚴(yán)重的中樞神經(jīng)系統(tǒng)創(chuàng)傷性疾病，可引起脊髓組織內(nèi)血管破損，神經(jīng)細(xì)胞快速死亡。局部血脊髓屏障(blood-spinal cord barrier，BSCB)結(jié)構(gòu)破壞會(huì)引發(fā)一系列細(xì)胞化學(xué)級(jí)聯(lián)反應(yīng)，加重?fù)p傷程度[1]。因此，促進(jìn)急性期BSCB結(jié)構(gòu)和功能的修復(fù)，對(duì)改善局部微環(huán)境，降低繼發(fā)性損傷程度有重要意義[2]。

FTY720(Fingolimod)是由從冬蟲(chóng)夏草中提取的免疫相關(guān)成分經(jīng)化學(xué)修飾后合成的新型免疫抑制劑，通過(guò)結(jié)合1-磷酸鞘氨醇(sphingosine 1-phosphate，S1P)受體發(fā)揮調(diào)節(jié)作用[3-4]。FTY720因其能功能性拮抗淋巴細(xì)胞膜上的1-磷酸鞘氨醇受體1(S1P1)，可在不影響免疫監(jiān)督的情況下減少外周淋巴細(xì)胞對(duì)中樞神經(jīng)系統(tǒng)的浸潤(rùn)及炎性介質(zhì)的釋放而被用于多發(fā)性硬化癥的臨床治療[5]。近年來(lái)許多研究發(fā)現(xiàn)，除免疫系統(tǒng)調(diào)節(jié)作用外，F(xiàn)TY720還能發(fā)揮上調(diào)腦源性神經(jīng)營(yíng)養(yǎng)因子表達(dá)、促髓鞘再生等更多的神經(jīng)保護(hù)作用[6-10]。

本研究采用脊髓半橫切損傷模型，觀察FTY720對(duì)大鼠急性脊髓損傷后神經(jīng)功能及BSCB的影響。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及分組 清潔級(jí)成年SD大鼠144只，雌雄各半，體重220～250g(由第三軍醫(yī)大學(xué)大坪醫(yī)院動(dòng)物中心提供)。FTY720(美國(guó)Selleckchem公司)，Evans Blue(美國(guó)Sigma公司)。實(shí)驗(yàn)大鼠隨機(jī)分為4組(n=36)。正常對(duì)照組(normal control group，NG 組)：不做任何處理；假手術(shù)組(sham-operated group，SO組)：?jiǎn)渭円С蛋灞┞都顾?，不予損傷處理；損傷對(duì)照組(hemisection group，HS組)：制作脊髓右半側(cè)橫切損傷模型后腹腔注射生理鹽水；FTY720治療組(FTY720 treated group，F(xiàn)TY720組)：脊髓右半側(cè)橫切損傷后腹腔注射FTY720治療。FTY720組大鼠腹腔注射FTY720生理鹽水溶液[1mg/ (kg·d)]，連續(xù)7d，首次給藥為術(shù)后30min；HS組大鼠按體重每日腹腔注射等量生理鹽水[11-12]。

1.2 脊髓半橫切模型建立 SD大鼠稱重，1%戊巴比妥鈉腹腔注射麻醉(40mg/kg)。麻醉大鼠俯臥位固定于手術(shù)板上，腰背部脫毛，常規(guī)消毒鋪巾。以T10棘突體表標(biāo)志點(diǎn)為中心行背部正中切口，暴露并小心咬除T9-T11椎板。打開(kāi)硬脊膜暴露脊髓后以正中動(dòng)脈為標(biāo)志，用眼科手術(shù)尖刀橫斷T10右半側(cè)脊髓，完全切斷并清除殘留的纖維組織。切割時(shí)鼠尾痙攣性擺動(dòng)、右下肢回縮撲動(dòng)后癱瘓作為造模成功的標(biāo)志。術(shù)后逐層縫合硬脊膜、肌肉、皮膚組織，肌內(nèi)注射青霉素2萬(wàn)U(2次/d)抗感染，連續(xù)3d[13]。術(shù)后各組大鼠分籠常規(guī)飼養(yǎng)，根據(jù)情況定時(shí)膀胱按壓促排尿至自主排尿功能恢復(fù)，觀察大鼠飲食及恢復(fù)情況。

1.3 檢測(cè)指標(biāo)

1.3.1 行為學(xué)檢測(cè) 各組大鼠分別于術(shù)后1、3、7、14、28d依據(jù)改良BBB評(píng)分(Basso Beattie Bresnahan locomotor rating scale，BBB)標(biāo)準(zhǔn)進(jìn)行評(píng)分[14]，以反映大鼠損傷側(cè)后肢運(yùn)動(dòng)功能的恢復(fù)情況。7、14、28d時(shí)加做水平網(wǎng)格檢測(cè)：將大鼠置于1cm×1cm大小的水平網(wǎng)格上自由活動(dòng)4min，計(jì)數(shù)損傷側(cè)后足踩空的步數(shù)與總步數(shù)，并計(jì)算踩空步數(shù)占總行走步數(shù)的比例[15]。BBB評(píng)分和網(wǎng)格計(jì)數(shù)均采用雙盲法，由非課題組的經(jīng)專業(yè)訓(xùn)練的實(shí)驗(yàn)人員負(fù)責(zé)記錄。檢測(cè)前排空大鼠膀胱。

1.3.2 神經(jīng)電生理檢測(cè) 各組大鼠分別于術(shù)后7、14、28d采用Powerlab/16SP(澳大利亞，ADI公司)多導(dǎo)生理記錄儀檢測(cè)運(yùn)動(dòng)誘發(fā)電位(MEP)和感覺(jué)誘發(fā)電位(SEP)。MEP檢測(cè)方法：將直徑1mm的銀球電極(刺激電極正極)置于感覺(jué)運(yùn)動(dòng)皮層區(qū)表面，負(fù)極置于大鼠下顎，保持濕潤(rùn)；銀質(zhì)雙保護(hù)電極(記錄電極)置于右側(cè)坐骨神經(jīng)處，正負(fù)極相距2mm；參考電極置于椎旁肌處。SEP檢測(cè)方法：刺激電極與MEP記錄電極位置相反，刺激電極負(fù)極置于顱頂切口右側(cè)皮下肌層，保持濕潤(rùn)；參考電極位置不變[16]。刺激參數(shù)：頻率4Hz，波寬0.2ms，強(qiáng)度10～40mV，信號(hào)經(jīng)前置放大器放大10萬(wàn)倍，計(jì)算機(jī)疊加256次后進(jìn)行平均[16]。記錄MEP和SEP的波形曲線并計(jì)算MEP N1波和SEP P1波的峰潛時(shí)。

1.3.3 組織學(xué)檢查 各組大鼠分別于術(shù)后14d和28d灌注取材，損傷段脊髓組織置于4%多聚甲醛中充分固定后常規(guī)石蠟包埋。行連續(xù)橫斷面石蠟切片，切片厚度5μm，標(biāo)本每隔10張取1張行HE染色。每個(gè)標(biāo)本取5張貼片備用。采用Nikon病理圖像分析采集系統(tǒng)留圖分析。

1.3.4 血脊髓屏障滲透性檢測(cè) 各組大鼠分別于術(shù)后1、3、7d檢測(cè)損傷段脊髓組織內(nèi)Evans Blue(EB)染液的滲出量。標(biāo)準(zhǔn)曲線的繪制：用二甲基甲酰胺(DMF)配制20μg/ml Evans Blue溶液，全波長(zhǎng)掃描測(cè)定其光密度(A)值，確定最大吸收峰在635nm波長(zhǎng)處。將DMF倍比稀釋配制成20、10、5、2.5、1.25、0.625、0.3175μg/ml濃度的標(biāo)準(zhǔn)液，分別檢測(cè)各梯度濃度標(biāo)準(zhǔn)液在635nm處的光密度(A635)值，計(jì)算回歸方程，繪制標(biāo)準(zhǔn)曲線。

組織切片及熒光檢測(cè)：1%戊巴比妥鈉腹腔注射麻醉大鼠，經(jīng)股靜脈緩慢推注2.5% Evans Blue生理鹽水溶液(4ml/kg)，持續(xù)10s以上，循環(huán)30min后灌注取材。稱取損傷段組織濕重后按100mg/3ml的量加入DMF勻漿，37℃避光水浴48h后，離心取上層萃取液檢測(cè)A635值，DMF作空白調(diào)零。記錄數(shù)據(jù)，根據(jù)標(biāo)準(zhǔn)曲線計(jì)算脊髓組織中的EB滲出量(μg/g)。每組另取5個(gè)組織于冰凍切片機(jī)(CM1900型，德國(guó)，Leica公司)上行連續(xù)矢狀面冰凍切片，切片厚度20μm，中性樹(shù)脂封固后于熒光顯微鏡下550nm激發(fā)光下觀察各組大鼠EB滲出情況，并采用Image-Pro Plus 6.0圖片分析軟件對(duì)目標(biāo)區(qū)域熒光強(qiáng)度進(jìn)行半定量分析。

1.4 統(tǒng)計(jì)學(xué)處理 所有數(shù)據(jù)使用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行分析。正態(tài)分布的計(jì)量數(shù)據(jù)均以±s表示，多組間比較采用單因素方差分析，進(jìn)一步兩兩比較采用LSD-t檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 行為學(xué)檢測(cè)結(jié)果

2.1.1 BBB評(píng)分結(jié)果 術(shù)后1d時(shí)，HS組死亡3只，F(xiàn)TY720組死亡2只；3d時(shí)，HS組死亡1只，F(xiàn)TY720組死亡1只；5d時(shí)，HS組死亡1只，均對(duì)各組缺失進(jìn)行補(bǔ)充。NG組大鼠在各時(shí)間點(diǎn)BBB評(píng)分為21分；術(shù)后7d內(nèi)，SO組大鼠較NG組大鼠評(píng)分有輕度下降，隨后逐漸恢復(fù)，7d時(shí)與NG組比較差異已無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。HS組及FTY720組大鼠損傷后1d，右后肢運(yùn)動(dòng)功能基本喪失，3d后評(píng)分逐漸上升，F(xiàn)TY720組上升速度大于HS組，7～28d，兩組評(píng)分差異有統(tǒng)計(jì)學(xué)意義(P<0.05，表1)。

2.1.2 網(wǎng)格實(shí)驗(yàn)結(jié)果 各時(shí)間點(diǎn)NG 組和SO組大鼠均可在水平網(wǎng)格上協(xié)調(diào)活動(dòng)，后足能抓握于網(wǎng)格間，踩空比例小，兩組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。術(shù)后7d內(nèi)，HS組和FTY720組大鼠網(wǎng)格上協(xié)調(diào)活動(dòng)能力差，右后足踩空比例明顯增加；隨著功能的恢復(fù)，踩空比例逐漸下降，F(xiàn)TY720組大鼠下降速度大于HS組，術(shù)后14d和28d時(shí)，兩者比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05，圖1)。

表1 大鼠不同時(shí)間點(diǎn)BBB評(píng)分表(n=5，±s)Tab. 1 BBB score of rats evaluated at respective time points (n=5,±s)

表1 大鼠不同時(shí)間點(diǎn)BBB評(píng)分表(n=5，±s)Tab. 1 BBB score of rats evaluated at respective time points (n=5,±s)

(1)P<0.01 compared with NG group; (2)P<0.01 compared with SO group; (3)P<0.05 compared with HS group

?

圖1 大鼠不同時(shí)間點(diǎn)網(wǎng)格檢測(cè)情況Fig.1 Grid walking test of rats evaluated at respective time points

2.2 神經(jīng)電生理檢測(cè)結(jié)果 術(shù)后各時(shí)間點(diǎn)，NG 組和SO組大鼠MEP的N1波與SEP的P1波潛伏期的差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)；HS組大鼠和FTY720組大鼠損傷側(cè)MEP N1波和SEP P1波波幅下降，潛伏期明顯延長(zhǎng)；FTY720組大鼠較HS組大鼠MEP N1波及SEP P波潛伏期延長(zhǎng)，但幅度小，術(shù)后7d兩組P1波潛伏期差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，術(shù)后14d 和28d兩組P1波和N1波潛伏期差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，表2、3)。

2.3 脊髓組織學(xué)檢測(cè)結(jié)果 NG組及SO組大鼠脊髓組織結(jié)構(gòu)完整，神經(jīng)細(xì)胞胞體分布清楚，胞體間結(jié)構(gòu)排列有序。損傷后14d，HS組大鼠損傷周圍神經(jīng)組織大面積壞死，大量淋巴細(xì)胞和單核細(xì)胞浸潤(rùn)吞噬，壞死組織周圍膠質(zhì)化反應(yīng)明顯；FTY720組大鼠神經(jīng)組織壞死面積小于HS組，炎性細(xì)胞浸潤(rùn)數(shù)量較HS組少。損傷后28d，HS組大鼠損傷壞死處由膠質(zhì)細(xì)胞填充，組織結(jié)構(gòu)排列紊亂，灰質(zhì)殘留較大空腔；FTY720組大鼠灰質(zhì)殘留空腔較HS組小，組織結(jié)構(gòu)排列較緊密(圖2)。

表2 大鼠不同時(shí)間點(diǎn)MEP N1峰潛時(shí)檢測(cè)表(n=5，±s)Tab. 2 The N1 latency of MEP at respective time points (n=5,±s, ms)

表2 大鼠不同時(shí)間點(diǎn)MEP N1峰潛時(shí)檢測(cè)表(n=5，±s)Tab. 2 The N1 latency of MEP at respective time points (n=5,±s, ms)

(1)P<0.01 compared with NG group, (2)P<0.01 compared with SO group, (3)P<0.05 compared with HS group

?

表3 大鼠不同時(shí)間點(diǎn)SEP P1峰潛時(shí)檢測(cè)表(n=5，±s)Tab. 3 The P1 latency of SEP at respective time points (n=5,±s, ms)

表3 大鼠不同時(shí)間點(diǎn)SEP P1峰潛時(shí)檢測(cè)表(n=5，±s)Tab. 3 The P1 latency of SEP at respective time points (n=5,±s, ms)

(1)P<0.01 compared with NG group, (2)P<0.01 compared with SO group, (3)P<0.05 compared with HS group

?

圖2 大鼠脊髓組織學(xué)觀察結(jié)果(HE染色)Fig. 2 Histological results of spinal tissues of rats (HE stainning)

2.4 血脊屏障滲透性檢測(cè)結(jié)果 術(shù)后1、3、7d， NG 組及SO組大鼠脊髓內(nèi)幾乎無(wú)EB滲出，HS組和FTY720組大鼠脊髓EB滲出量明顯增加(P<0.05)；損傷前3d，HS組大鼠脊髓EB滲出量呈上升趨勢(shì)，隨后下降，而FTY720組大鼠無(wú)上升趨勢(shì)，各時(shí)間點(diǎn)兩組比較結(jié)果差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，圖3、4，表4)。

圖3 4組大鼠不同時(shí)間點(diǎn)Evans Blue滲出量Fig. 3 Diffusion of fluorescent Evans Blue in injured spinal cord tissue of rats at respective time points

表4 大鼠不同時(shí)間點(diǎn)損傷段平均熒光強(qiáng)度值比較(n=5，±s)Tab. 4 Mean fluorescence intensity of Evans Blue at respective time points (n=5,±s)

表4 大鼠不同時(shí)間點(diǎn)損傷段平均熒光強(qiáng)度值比較(n=5，±s)Tab. 4 Mean fluorescence intensity of Evans Blue at respective time points (n=5,±s)

(1)P<0.01 compared with NG group, (2)P<0.01 compared with SO group, (3)P<0.05 compared with HS group

?

圖4 大鼠脊髓損傷處Evans Blue溶液的滲出情況(熒光顯微鏡)Fig. 4 Diffusion of fluorescent Evans Blue in injured spinal cord tissue of rats (Fluorescence microscope)

3 討 論

Norimatsu等[17]通過(guò)行為學(xué)檢測(cè)發(fā)現(xiàn)，F(xiàn)TY720在脊髓損傷亞急性早期(損傷后7～14d)對(duì)運(yùn)動(dòng)功能恢復(fù)有促進(jìn)作用，而Lee等[12]研究認(rèn)為FTY720對(duì)運(yùn)動(dòng)功能的影響在亞急性期后(損傷28d后)更為明顯。我們利用行為學(xué)指標(biāo)和神經(jīng)電生理技術(shù)對(duì)大鼠脊髓半橫切損傷后患肢功能恢復(fù)情況進(jìn)行檢測(cè)發(fā)現(xiàn)，半切損傷后7d，F(xiàn)TY720治療組大鼠BBB運(yùn)動(dòng)功能評(píng)分已大于損傷組，MEP N1波和SEP P1波潛伏期也較損傷組縮短，說(shuō)明急性期末，經(jīng)FTY720治療的大鼠損傷側(cè)神經(jīng)傳導(dǎo)束功能已優(yōu)于未經(jīng)治療大鼠。亞急性期內(nèi)，隨著患側(cè)肌力增加，患肢運(yùn)動(dòng)功能進(jìn)一步恢復(fù)，治療組大鼠運(yùn)動(dòng)協(xié)調(diào)性和抓握能力均明顯改善。急性脊髓損傷后，T細(xì)胞在急性期后期反應(yīng)性浸潤(rùn)，并以高水平維持到損傷后數(shù)周。持續(xù)的T細(xì)胞水平增高會(huì)對(duì)脊髓組織內(nèi)殘存神經(jīng)元造成進(jìn)一步損傷[18-19]。然而，有研究發(fā)現(xiàn)，F(xiàn)TY720對(duì)損傷處T細(xì)胞數(shù)量的抑制作用在損傷后亞急性期最明顯[17]。本研究也通過(guò)HE染色進(jìn)行了損傷脊髓的組織學(xué)觀察，結(jié)果顯示，F(xiàn)TY720治療能減輕傷后14d時(shí)損傷周圍脊髓組織內(nèi)淋巴細(xì)胞和單核細(xì)胞的浸潤(rùn)，顯著減少神經(jīng)細(xì)胞壞死面積。結(jié)合行為學(xué)和神經(jīng)電生理檢測(cè)結(jié)果，我們推測(cè)在損傷急性期內(nèi)，F(xiàn)TY720還存在其他可能的神經(jīng)保護(hù)效應(yīng)。

BSCB是調(diào)節(jié)脊髓正常代謝，維持脊髓內(nèi)環(huán)境穩(wěn)定的重要結(jié)構(gòu)。急性脊髓損傷后，局部屏障功能直接破壞[20]，損傷周圍脊髓組織暴露于循環(huán)中的內(nèi)源性免疫炎性因子、毒性因子和外源性病原體中。內(nèi)皮細(xì)胞和神經(jīng)細(xì)胞免疫性損傷加重，使得BSCB急性期內(nèi)持續(xù)開(kāi)放，滲出增加，內(nèi)環(huán)境失衡，從而引起不可逆性損傷[21-23]。研究發(fā)現(xiàn)，F(xiàn)TY720能維持葡萄球膜炎中血-眼屏障的完整性，抑制血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor，VEGF)誘導(dǎo)的微血管滲透性增加，并能通過(guò)誘導(dǎo)血管內(nèi)皮鈣黏蛋白和β連環(huán)蛋白的整合形成新的內(nèi)皮間連接，提示其對(duì)微血管系統(tǒng)也有調(diào)節(jié)作用[24-25]。我們利用血脊髓屏障Evans Blue滲出實(shí)驗(yàn)發(fā)現(xiàn)，半切損傷后，損傷段脊髓組織內(nèi)EB滲出量明顯增加，損傷后3d達(dá)高峰，急性期后期開(kāi)始減少，F(xiàn)TY720治療能顯著減少早期EB的滲出，減輕局部水腫，熒光顯微鏡下對(duì)組織切片的觀察結(jié)果一致。該結(jié)果提示，F(xiàn)TY720對(duì)創(chuàng)傷性病理?xiàng)l件下的微血管功能也有調(diào)節(jié)作用。有研究者指出，F(xiàn)TY720能下調(diào)損傷早期脊髓神經(jīng)元上凋亡基因caspase-3的表達(dá)水平，發(fā)揮抗凋亡作用[26]。但體外實(shí)驗(yàn)發(fā)現(xiàn)FTY720并不能增加毒性因子作用下神經(jīng)元的存活數(shù)量[13]。結(jié)合本實(shí)驗(yàn)研究結(jié)果，我們認(rèn)為，F(xiàn)TY720可能是通過(guò)降低BSCB滲透性，減少炎性因子浸潤(rùn)和改善局部微循環(huán)的方式減少神經(jīng)細(xì)胞的壞死凋亡，進(jìn)而為神經(jīng)功能的恢復(fù)提供有利因素。免疫抑制劑甲潑尼松龍的使用是目前臨床常采取的治療手段之一，它可以穩(wěn)定細(xì)胞膜，抑制局部出血、水腫、微循環(huán)障礙和炎性反應(yīng)，但使用激素具有治療窗短、劑量需求大、副反應(yīng)重的限制[27-28]。FTY720為脊髓損傷早期藥物治療提供了新的選擇。我們也將繼續(xù)通過(guò)對(duì)損傷側(cè)遠(yuǎn)端脊髓運(yùn)動(dòng)神經(jīng)元自發(fā)放電檢測(cè)等實(shí)驗(yàn)途徑進(jìn)一步明確FTY720對(duì)運(yùn)動(dòng)功能的影響。

此外，本實(shí)驗(yàn)HE染色還發(fā)現(xiàn)FTY720能有效減輕亞急性期損傷周圍膠質(zhì)化反應(yīng)程度。有研究指出，F(xiàn)TY720對(duì)反應(yīng)性星形膠質(zhì)細(xì)胞增生的抑制作用與淋巴細(xì)胞抑制機(jī)制相同[29-30]。然而，星形膠質(zhì)細(xì)胞是BSCB的重要組成成分，對(duì)BSCB結(jié)構(gòu)完整性和物質(zhì)轉(zhuǎn)運(yùn)有重要作用[21]。急性期后，微血管再生和星形膠質(zhì)細(xì)胞增生明顯，因此長(zhǎng)期使用FTY720治療是否有利于BSCB結(jié)構(gòu)功能的重建和神經(jīng)功能的恢復(fù)還需進(jìn)一步研究。

[1] Oudega M. Inflammatory response after spinal cord injury[J].Exp Neurol, 2013, 250: 151-155.

[2] Yu DS, Cao Y, Mei XF, et al. Curcumin improves the integrity of blood-spinal cord barrier after compressive spinal cord injury in rats[J]. J Neurol Sci, 2014, 346(1-2): 51-59.

[3] Zheng MK, Xiao H, Li Y, et al. A novel immunosuppressant fingolimod: research advances[J]. J Int Pharm Res, 2013, 40(1): 100-104.[鄭明克, 肖鶴, 黎燕, 等. 新型免疫抑制劑芬戈莫德的研究進(jìn)展[J]. 國(guó)際藥學(xué)研究雜志, 2013, 40(1): 100-104.]

[4] Zhang CH, Li Y, Chen W, et al. Apoptosis of K562 cells induced by a new immunosuppressive agent FTY720 and its mechanism[J]. J Jilin Univ (Med Ed) 2014, 40(6): 1161-1165.[張宸豪, 李妍, 陳為, 等. 新型免疫抑制劑FTY720誘導(dǎo)K562細(xì)胞凋亡及其機(jī)制[J]. 吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版), 2014, 40(6): 1161-1165.]

[5] Pelletier D, Hafler DA. Fingolimod for multiple sclerosis[J]. N Engl J Med, 2012, 366(4): 339-347.

[6] Deogracias R, Yazdani M, Dekkers MP, et al. Fingolimod, a sphingosine-1 phosphate receptor modulator, increases BDNF levels and improves symptoms of a mouse model of Rett syndrome[J]. Proc Natl Acad Sci USA, 2012, 109(35): 14230-14235.

[7] Doi Y, Takeuchi H, Horiuchi H, et al. Fingolimod phosphate attenuates oligomeric amyloid beta-induced neurotoxicity via increased brain-derived neurotrophic factor expression in neurons[J]. PLoS One, 2013, 8(4): e61988.

[8] Miron VE, Ludwin SK, Darlington PJ, et al. Fingolimod (FTY720) enhances remyelination following demyelination of organotypic cerebellar slices[J]. Am J Pathol, 2010, 176(6): 2682-2694.

[9] Zhou H, Li S, Niu X, et al. Protective effect of FTY720 against sevoflurane-induced developmental neurotoxicity in rats[J]. Cell Biochem Biophys, 2013, 67(2): 591-598.

[10] Asle-Rousta M, Kolahdooz Z, Oryan S, et al. FTY720 (fingolimod) attenuates beta-amyloid peptide (Abeta42)-induced impairment of spatial learning and memory in rats[J]. J Mol Neurosci, 2013, 50(3): 524-532 .

[11] Huang P, Yu Y, Wang YT, et al. The establishment and certification of animal model of hemisected spinal cord injury in rats[J]. J Pract Med, 2011, 27(6): 954-956. [黃鵬, 余瑛, 王永堂, 等. 大鼠脊髓半橫切損傷動(dòng)物模型的建立與神經(jīng)功能評(píng)價(jià)[J]. 實(shí)用醫(yī)學(xué)雜志, 2011, 27(6): 954-956.]

[12] Lee KD, Chow WN, Sato-Bigbee C, et al. FTY720 reduces inflammation and promotes functional recovery after spinal cord injury[J]. J Neurotrauma, 2009, 26(12): 2335-2344.

[13] Wei Y, Yemisci M, Kim HH, et al. Fingolimod provides longterm protection in rodent models of cerebral ischemia[J]. Ann Neurol, 2011, 69(1): 119-129.

[14] Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma, 1995, 12(1): 1-21.

[15] Wang YT, Lu XM, Zhu F, et al. The use of a gold nanoparticlebased adjuvant to improve the therapeutic efficacy of hNgRFc protein immunization in spinal cord-injured rats[J]. Biomaterials, 2011, 32(31): 7988-7998.

[16] Hou T, Wu Y, Wang L, et al. Cellular prostheses fabricated with motor neurons seeded in self-assembling peptide promotes partial functional recovery after spinal cord injury in rats[J]. Tissue Eng Part A, 2012, 18(9-10): 974-985.

[17] Norimatsu Y, Ohmori T, Kimura A, et al. FTY720 improves functional recovery after spinal cord injury by primarily nonimmunomodulatory mechanisms[J]. Am J Pathol, 2012, 180(4): 1625-1635.

[18] Donnelly DJ, Popovich PG. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury[J]. Exp Neurol, 2008, 209(2): 378-388.

[19] Zhang N, Yin Y, Xu SJ, et al. Inflammation & apoptosis in spinal cord injury[J]. Indian J Med Res, 2012, 135(3): 287.

[20] Wang ZQ, Xie P, Zhang S. Clinical research on the effect of neurotropin combined with methylprednisolone in the treatment of acute spinal cord injury[J]. J Logist Univ PAPF (Med Sci), 2014, 23(8): 664-666. [王志強(qiáng), 謝鵬, 張賽. 神經(jīng)妥樂(lè)平聯(lián)合甲基潑尼松龍治療急性脊髓損傷的臨床研究[J]. 武警后勤學(xué)院學(xué)報(bào)(醫(yī)學(xué)版), 2014, 23(8): 664-666.]

[21] Whetstone WD, Hsu JY, Eisenberg M, et al. Blood-spinal cord barrier after spinal cord injury: relation to revascularization and wound healing[J]. J Neurosci Res, 2003, 74(2): 227-239.

[22] Bartanusz V, Jezova D, Alajajian B, et al. The blood-spinal cord barrier: morphology and clinical implications[J]. Ann Neurol, 2011, 70(2): 194-206.

[23] Figley SA, Khosravi R, Legasto JM, et al. Characterization of vascular disruption and blood-spinal cord barrier permeability following traumatic spinal cord injury[J]. J Neurotrauma, 2014, 31(6): 541-552.

[24] Copland DA, Liu J, Schewitz-Bowers LP, et al. Therapeutic dosing of fingolimod (FTY720) prevents cell infiltration, rapidly suppresses ocular inflammation, and maintains the blood-ocular barrier[J]. Am J Pathol, 2012, 180(2): 672-681.

[25] Patel CB, Cohen DM, Ahobila-Vajjula P, et al. Effect of VEGF treatment on the blood-spinal cord barrier permeability in experimental spinal cord injury: dynamic contrast-enhanced magnetic resonance imaging[J]. J Neurotrauma, 2009, 26(7): 1005-1016.

[26] Yang L, Lü DC, Zheng LJ, et al. Neuroprotective effects of the immunodepressant FTY720 on caspase-3 expression and neural apoptosis in a rat model of acute spinal cord injury [J]. Neural Regen Res, 2010, 5(9): 700-705.

[27] Qian T, Guo X, Levi AD, et al. High-dose methylprednisolone may cause myopathy in acute spinal cord injury patients[J]. Spinal Cord, 2005, 43(4): 199-203.

[28] Miekisiak G, Kloc W, Janusz W, et al. Current use of methylprednisolone for acute spinal cord injury in Poland: survey study[J]. Eur J Orthop Surg Traumatol, 2014, 24(Suppl 1): S269-S273.

[29] Choi JW, Gardell SE, Herr DR, et al. FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation[J]. Proc Natl Acad Sci U S A, 2011, 108(2): 751-756.

[30] Brunkhorst R, Kanaan N, Koch A, et al. FTY720 treatment in the convalescence period improves functional recovery and reduces reactive astrogliosis in photothrombotic stroke[J]. PLoS One, 2013, 8(7): e70124 .

Effects of FTY720 on neurological function and blood-spinal cord barrier of rats with acute spinal cord injury

YUE Yan1, TAN Bo-tao1, LIU Yuan2, WU Ya-min2, JIA Gong-wei1, JIANG Wei1, YU Le-hua1, YIN Ying1*1Department of Rehabilitation Medicine,Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
2Third Department of Institute of Field Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China
*

, E-mail: yinying@cqmu.edu.cn
This work was supported by the National Natural Science Foundation of Chongqing (cstc2012jjA10058)

ObjectiveTo investigate the effects of a novel immunomodulator FTY720 (Fingolimod) on nerve function and blood-spinal cord barrier (BSCB) of rats with acute spinal cord injury.MethodsOne hundred and forty-four adult Sprague-Dawley (SD) rats were randomly divided into four groups with 36 each: normal control group (NG Group): rats without any treatment; sham-operated group (SO Group): rats' spinal cords were exposed by laminectomy without injury; hemisection group (HS Group): rats underwent spinal cord hemisection followed by intraperitoneal injection of normal saline; FTY720 treatment group (FTY720 Group): rats underwent spinal cord hemisection followed by intraperitoneal injection of FTY720 [1mg/(kg.d)] for 7 days. The neurological function was assessed by Basso Beatlie Bresnahan (BBB) scores, grid walking, N1 and P1 delay of motor evoked potential (MEP) and somatosensory evoked potentials (SEP), histological evaluation with light microscope with HE staining, and determination of blood-spinal cord barrier permeability with EB at different time points after injury.ResultsThe nerve function of rats in HS group and FTY720 group was impaired after hemisection injury without signs of recovery up to Day 28 after damage as compared with NG group or SO group. The recovery of motor function in FTY720 treatment group was earlier than in HS group. The BBB scores, the results of grid walking test, and the latent period of SEP-P1 showed statistically significant difference between FTY720 group and HS group from Day 7 to 28 after injury (P<0.05). Comparing the pathological picture at Day 14 and Day 28 afterinjury, the number of chronic inflammatory cells, the degree of glial cell reaction, and the size of syringomyelia cavities in gray matter in FTY720 group were significantly less than those in HS group. In addition, the leakage of EB from the damaged BSCB increased in HS group and FTY720 group than in NG group and SO group through 7 days after injury (P<0.01), while at each time point the leakage of EB was less in FTY720 group than in HS group (P<0.05), and the most significant difference was observed on Day 3 after injury.ConclusionFTY720 can lower the permeability of blood-spinal cord barrier at acute phase of spinal cord injury, effectively promotes the recovery of nerve function after acute injury phase, and it provides certain potential neuroprotective effects.

immunosuppressive agents; FTY720; spinal cord injuries; neuroprotective agents

R651.2

A

0577-7402(2013)03-0200-06

10.11855/j.issn.0577-7402.2015.03.06

2014-11-07；

2015-02-13)

(責(zé)任編輯：沈?qū)?

重慶市自然科學(xué)基金計(jì)劃項(xiàng)目(cstc2012jjA10058)

岳妍，碩士研究生。主要從事神經(jīng)康復(fù)方面的研究

400010 重慶 重慶醫(yī)科大學(xué)附屬第二醫(yī)院康復(fù)醫(yī)學(xué)科(岳妍、譚波濤、賈功偉、蔣瑋、虞樂(lè)華、殷櫻)；400042 重慶第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所三室，創(chuàng)傷、燒傷與復(fù)合傷國(guó)家重點(diǎn)實(shí)驗(yàn)室(劉媛、伍亞民)

殷櫻，E-mail: yinying@cqmu.edu.cn