李明 宋永周 曹舒興 王斌 童九輝 李秋童 馬維
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·論著·
氯化鎂對(duì)大鼠坐骨神經(jīng)斷端再生微結(jié)構(gòu)的作用研究
李明宋永周曹舒興王斌童九輝李秋童馬維
050000石家莊市,河北醫(yī)科大學(xué)第二醫(yī)院骨科
【摘要】目的研究氯化鎂對(duì)大鼠坐骨神經(jīng)損傷后超微結(jié)構(gòu)修復(fù)的作用。方法將90只SD大鼠隨機(jī)分為氯化鎂組(MgCl2組),神經(jīng)生長(zhǎng)因子組(NGF組),0.9%氯化鈉組(NaCl)組,每組30只。制作坐骨神經(jīng)缺損模型,分別以硅膠管橋接坐骨神經(jīng)缺損,構(gòu)成神經(jīng)再生室。分別將1 mmol/L MgCl2、NCF、0.9%氯化鈉溶液分別注入神經(jīng)再生室中。于術(shù)后4、8、12周處死動(dòng)物,取出神經(jīng)再生室中斷端再生組織,固定處理后通過光鏡觀察再生神經(jīng)纖維數(shù)目及再生纖維神經(jīng)截面積,電鏡觀察神經(jīng)再生室中雪旺細(xì)胞增殖和發(fā)育情況、髓鞘的超微結(jié)構(gòu)。結(jié)果MgCl2組和NGF組術(shù)后4、8、12周再生神經(jīng)纖維數(shù)目和再生神經(jīng)纖維截面積高于NaCl組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05),而MgCl2組與NGF組比較,差異無統(tǒng)計(jì)學(xué)意義(P<0.05)。MgCl2組和NGF組新生神經(jīng)纖維再生數(shù)量增多,雪旺細(xì)胞增生明顯,形態(tài)接近正常,髓鞘厚度均勻增加,鞘層分離不明顯,神經(jīng)膜細(xì)胞及軸漿有輕度變性,NaCl組神經(jīng)纖維再生不明顯,髓鞘結(jié)構(gòu)模糊,扭曲,雪旺細(xì)胞增生不明顯,表型幼稚。結(jié)論氯化鎂可促進(jìn)大鼠坐骨神經(jīng)損傷修復(fù)后超微結(jié)構(gòu)的修復(fù)。
【關(guān)鍵詞】氯化鎂;雪旺細(xì)胞;神經(jīng)生長(zhǎng)因子;坐骨神經(jīng)損傷
周圍神經(jīng)損傷占全身創(chuàng)傷的3%~10%[1],具有致殘率高,治療費(fèi)用高等特點(diǎn)。全球每年約發(fā)生1百萬例周圍神經(jīng)損傷。僅在美國(guó),每年需花費(fèi)約1.50億美金用于治療神經(jīng)損傷[2]。周圍神經(jīng)損傷后,局部神經(jīng)發(fā)生繼發(fā)的氧化應(yīng)激及變性,神經(jīng)斷端周圍微環(huán)境惡化,造成神經(jīng)二次損傷,導(dǎo)致神經(jīng)功能恢復(fù)緩慢。氯化鎂具有促進(jìn)脊髓損傷修復(fù)的作用,可以促進(jìn)軸突再生[2]。而且在臨床上廣泛應(yīng)用,毒副作用較小。但是對(duì)鎂離子的研究集中在中樞神經(jīng)系統(tǒng)。鎂離子對(duì)周圍神經(jīng)損傷的作用尚未能知。如果能夠證實(shí)局部應(yīng)用氯化鎂能夠穩(wěn)定神經(jīng)損傷后的微環(huán)境,促進(jìn)神經(jīng)細(xì)胞再生,今后在修復(fù)手術(shù)時(shí)加以應(yīng)用,將加速周圍神經(jīng)的恢復(fù)。本實(shí)驗(yàn)通過制作大鼠坐骨神經(jīng)缺損模型,用神經(jīng)再生室橋接缺損區(qū)域,構(gòu)成神經(jīng)再生室,將氯化鎂溶液注入再生室中,通過光鏡和電鏡觀察大鼠坐骨神經(jīng)離斷后,神經(jīng)斷端的微結(jié)構(gòu)變化,驗(yàn)證氯化鎂是否對(duì)周圍神經(jīng)損傷修復(fù)具有促進(jìn)作用。
1材料與方法
1.1實(shí)驗(yàn)動(dòng)物及分組將90只雄性SD大鼠(280~300 g,購(gòu)于河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心,動(dòng)物合格證編號(hào):1511010),隨機(jī)分為氯化鎂組(MgCl2組)、神經(jīng)生長(zhǎng)因子組(NGF組)和0.9%氯化鈉溶液組(NaCl組),每組30只。
1.2方法以10%水合氯醛對(duì)其進(jìn)行腹腔內(nèi)注射麻醉,劑量3.5 ml/kg。制作1 cm坐骨神經(jīng)缺損模型,分別以內(nèi)徑1.5 mm硅膠管橋接坐骨神經(jīng)缺損,以9-0無創(chuàng)縫線做神經(jīng)外膜固定,構(gòu)成神經(jīng)再生室。分別將1 mmol/L氯化鎂(河北醫(yī)科大學(xué)藥理實(shí)驗(yàn)室配)、鼠神經(jīng)生長(zhǎng)因子(舒泰神北京藥業(yè)有限公司生產(chǎn))、0.9%氯化鈉溶液注入神經(jīng)再生室。分別于術(shù)后4、8、12周分批處死動(dòng)物,取出再生室中神經(jīng)組織,根據(jù)實(shí)驗(yàn)儀器要求不同,選用不同固定液固定后,觀察神經(jīng)斷端局部微結(jié)構(gòu)改變。
1.3觀察指標(biāo)
1.3.1光鏡觀察:分別在4、8、12周時(shí),處死動(dòng)物,取出神經(jīng)再生室中神經(jīng)組織,以4%多聚甲醛緩沖液中固定,脫水,石蠟包埋,60℃恒溫置于LFB液中浸泡過夜,再次以乙醇梯度脫水、HE染色,蒸餾水洗滌,連續(xù)做4 μm切片,光鏡下觀察神經(jīng)纖維及髓鞘的再生情況。3組再生神經(jīng)組織中點(diǎn)連續(xù)切取5張切片,每張切片隨機(jī)取5個(gè)面積相同視野作為測(cè)量對(duì)象, 使用自帶圖文分析系統(tǒng)計(jì)算再生神經(jīng)纖維的數(shù)目及再生纖維截面積。
1.3.2電鏡觀察:同樣取出神經(jīng)再生室中再生神經(jīng)組織,以2.5%戊二醛固定24 h后,磷酸緩沖液沖洗3次,1%鋨酸固定,蒸餾水沖洗,乙醇梯度脫水,樹脂包埋,做超薄切片后,透射電鏡觀察神經(jīng)再生室中雪旺細(xì)胞的增殖和發(fā)育情況,髓鞘的超微結(jié)構(gòu)。
2結(jié)果
2.1大體觀察在4、8、12周各個(gè)時(shí)相,MgCl2組和NGF組的神經(jīng)再生室中,神經(jīng)再生形成條索均勻,直徑較大。而對(duì)照組在各個(gè)時(shí)相新生的神經(jīng)條索纖細(xì)。
2.2光鏡觀察術(shù)后4周,MgCl2組及NGF組再生神經(jīng)纖維較少,稀疏。但分布均勻、髓鞘較厚,結(jié)締組織較少。Nacl組神經(jīng)纖維分布疏散,大小不均,髓鞘較薄。術(shù)后8周時(shí)MgCl2組及NGF組再生神經(jīng)中有髓神經(jīng)纖維均增多、增粗,髓鞘增厚,神經(jīng)纖維分布較密集。術(shù)后12周,MgCl2組和NGF組神經(jīng)纖維髓鞘腫脹消失,再生的神經(jīng)纖維明顯增多。 排列成束,軸突的直徑和髓鞘的厚度接近正常。NaCl組損傷處遠(yuǎn)端神經(jīng)神經(jīng)纖維髓鞘仍有腫脹,成纖維細(xì)胞增多,神經(jīng)纖維形成稀疏,直徑較小。MgCl2組和NGF組術(shù)后4、8、12周再生神經(jīng)纖維數(shù)目和再生神經(jīng)纖維截面積高于NaCl組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05),而MgCl2組與NGF組比較,差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表1,圖1~3。
表1 3組在術(shù)后不同時(shí)相再生纖維數(shù)目及再生神經(jīng)截面積 ±s
注:與NaCl組比較,*P<0.05
圖1 3組術(shù)后不同時(shí)相再生纖維數(shù)目
2.3電鏡觀察治療組新生神經(jīng)纖維再生數(shù)量增多,雪旺氏細(xì)胞增生明顯,形態(tài)接近正常。其髓鞘厚度均勻增加,鞘層分離不明顯。 神經(jīng)膜細(xì)胞及軸漿有輕度的變性,軸漿內(nèi)可見微絲、微管和線粒體等細(xì)胞器,仍有少量殘留的變性神經(jīng)纖維, 其余的纖維組織結(jié)構(gòu)正常。 對(duì)照組神經(jīng)纖維再生不明顯,髓鞘結(jié)構(gòu)模糊,扭曲,雪旺氏細(xì)胞增生不明顯,表型幼稚。見圖4。
圖2 3組術(shù)后不同時(shí)相再生纖維截面積
圖3 3組術(shù)后不同時(shí)相神經(jīng)斷端再生光鏡觀察(×200)
圖4 3組術(shù)后不同時(shí)相神經(jīng)斷端再生電鏡觀察(×1 200)
3討論
周圍神經(jīng)損傷約占全身創(chuàng)傷的3%~10%[1],致殘率高達(dá)1/1 000[3],治療費(fèi)用昂貴,給患者及其家屬造成很大的經(jīng)濟(jì)負(fù)擔(dān)。神經(jīng)損傷的分類較多,Sunderland分類根據(jù)組織學(xué)損傷程度,分為以下5度:Ⅰ神經(jīng)麻痹,軸索完整;Ⅱ神經(jīng)內(nèi)膜管完整,神經(jīng)纖維可以再生;Ⅲ神經(jīng)內(nèi)膜損傷,神經(jīng)移植效果不確切,形成神經(jīng)瘤或者是神經(jīng)束膜的誤接;Ⅳ僅神經(jīng)外膜完整,神經(jīng)束膜也損傷。神經(jīng)叢生,組織結(jié)構(gòu)混亂,需要手術(shù)重建;Ⅴ神經(jīng)斷裂傷。根據(jù)不同的損傷形式,選擇保守治療、直接縫合或是神經(jīng)移植治療。但是在損傷后,神經(jīng)局部發(fā)生繼發(fā)的氧化應(yīng)激及變性,神經(jīng)周圍微環(huán)境惡化,造成神經(jīng)二次損傷。因此,對(duì)神經(jīng)再生的研究熱點(diǎn)集中在移植的材料和局部微環(huán)境的改善等方面。
在材料方面,大致分為自體神經(jīng)、血管和筋膜以及各種神經(jīng)導(dǎo)管。血管、肌腱、肌肉等自體材料的缺點(diǎn):易于塌陷、形成瘢痕,供區(qū)的功能障礙[4]。與此相比,神經(jīng)再生室能橋接神經(jīng)斷端,形成局部神經(jīng)生長(zhǎng)的微環(huán)境。用于構(gòu)建神經(jīng)再生室的材料種類繁多,各具特點(diǎn)。對(duì)于羊膜等可吸收及具有生物功能的再生室,其作用可能混淆再生室中所使用藥物的作用。根據(jù)本次實(shí)驗(yàn)?zāi)康模窠?jīng)斷端的微環(huán)境需要相對(duì)封閉,構(gòu)成神經(jīng)再生室的材料通透性低,與外界體液交換較少。并且具有良好的生物相容性,可以抵抗周圍肌肉的擠壓。硅膠管具有無毒,安全,具有一定力學(xué)強(qiáng)度,通透性低的優(yōu)點(diǎn)。早在1982年,硅膠管就應(yīng)用于修復(fù)大鼠坐骨神經(jīng)6 mm缺損[5]。本實(shí)驗(yàn)選取其構(gòu)建神經(jīng)再生室可以達(dá)到本次實(shí)驗(yàn)?zāi)康摹?/p>
眾所周知,神經(jīng)損傷后,沿神經(jīng)軸索的走行,髓鞘發(fā)生華勒氏變性。雪旺細(xì)胞釋放,與巨噬細(xì)胞一起吞噬崩解的細(xì)胞碎片。繼而形成新生雪旺細(xì)胞條帶,稱之為Bungner帶。這條細(xì)胞帶伴隨斷端新生軸突生長(zhǎng),逐漸替代損傷的間隙,完成神經(jīng)的修復(fù)。神經(jīng)損傷后,斷端會(huì)分泌神經(jīng)營(yíng)養(yǎng)因子,包括神經(jīng)生長(zhǎng)因子,在斷端的梯度濃度影響下,沿此方向生長(zhǎng),保證神經(jīng)生長(zhǎng)的方向一致。一般在傷后24 h,軸突開始再生,呈圓錐形沿基膜生長(zhǎng),逐漸生長(zhǎng)至末梢器官,大約1 mm/d[6]。根據(jù)這個(gè)速度計(jì)算,所以我們選擇4、8、12周作為觀察的時(shí)相。
除了損傷神經(jīng)斷端的變性外,未損傷的神經(jīng)也會(huì)發(fā)生變性的原因可能是在應(yīng)激狀態(tài)下,鎂離子水平降低,鈣離子通道大量開放,鈣離子內(nèi)流,引起細(xì)胞腫脹,凋亡。從而使得神經(jīng)斷端相鄰節(jié)段的激發(fā)損傷,恢復(fù)困難。鎂離子是維持細(xì)胞正常代謝的必需的離子之一,通過調(diào)節(jié)細(xì)胞內(nèi)鉀離子的濃度,從而維持細(xì)胞正常形態(tài)。同時(shí)對(duì)鈣離子內(nèi)流起到拮抗作用,避免鈣離子大量?jī)?nèi)流造成的細(xì)胞毒性。文獻(xiàn)報(bào)道,鎂離子可以促進(jìn)腦和脊髓損傷細(xì)胞的恢復(fù)[7]。但對(duì)外周神經(jīng)研究的較少。本文旨在探討氯化鎂是否具有促進(jìn)周圍神經(jīng)損傷的作用,為臨床應(yīng)用氯化鎂促進(jìn)神經(jīng)恢復(fù)提供依據(jù)。
神經(jīng)生長(zhǎng)受神經(jīng)營(yíng)養(yǎng)物質(zhì)的調(diào)控。神經(jīng)生長(zhǎng)因子是重要的神經(jīng)營(yíng)養(yǎng)物質(zhì)。在鼠的周圍神經(jīng)損傷模型中NGF起到重要的作用[8]。其他的促神經(jīng)軸突生長(zhǎng)因子及基質(zhì),也有促進(jìn)作用。神經(jīng)生長(zhǎng)因子是已應(yīng)用于臨床的神經(jīng)營(yíng)養(yǎng)藥物,療效肯定[9]。臨床給藥途徑包括全身和局部。神經(jīng)生長(zhǎng)因子全身給藥不能通過血腦屏障,副作用大。文獻(xiàn)報(bào)道:NGF作用于軸突纖維的生長(zhǎng),而不是神經(jīng)元本身[10]。所以,在局部應(yīng)用藥物促進(jìn)神經(jīng)生長(zhǎng)是可行的。所以本實(shí)驗(yàn)選擇神經(jīng)生長(zhǎng)因子作為對(duì)照,評(píng)價(jià)鎂離子局部應(yīng)用對(duì)神經(jīng)再生微結(jié)構(gòu)的影響。本文的實(shí)驗(yàn)結(jié)果顯示:局部應(yīng)用NGF和Mgcl2后,軸突細(xì)胞再生較對(duì)照組明顯增多,表明氯化鎂可促進(jìn)神經(jīng)軸突細(xì)胞的再生。
局部用藥可以提高神經(jīng)再生室中的藥物濃度形成,直接濃度梯度,可以很快形成神經(jīng)趨向性。但是,局部藥物的濃度對(duì)神經(jīng)細(xì)胞的生長(zhǎng)也有不同的影響。查閱文獻(xiàn)未發(fā)現(xiàn)神經(jīng)再生室中可以應(yīng)用多大濃度的鎂離子。文獻(xiàn)報(bào)道,如果鎂離子濃度過高,鎂離子對(duì)神經(jīng)的保護(hù)作用反而降低[11]。其機(jī)制可能是鎂離子的濃度過高對(duì)鈣離子通道的抑制作用,使其對(duì)神經(jīng)細(xì)胞的保護(hù)作用降低。鄢開勝等[12]對(duì)體外培養(yǎng)的螺旋神經(jīng)節(jié)細(xì)胞預(yù)先給予1 mmol/L的MgCl2能增加暴露于谷氨酸的螺旋神經(jīng)節(jié)細(xì)胞的存活率,還能抑制谷氨酸引起的螺旋神經(jīng)節(jié)細(xì)胞內(nèi)游離鈣增加的效應(yīng),對(duì)螺旋神經(jīng)節(jié)細(xì)胞損傷發(fā)揮保護(hù)作用。本實(shí)驗(yàn)光鏡觀察到在實(shí)驗(yàn)的各個(gè)時(shí)相,氯化鎂組的神經(jīng)纖維再生數(shù)目和軸突面積均遠(yuǎn)高于對(duì)照組,雖然不及神經(jīng)生長(zhǎng)因子組,但是差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。結(jié)果表明,局部應(yīng)用1 mmol/L氯化鎂,可以促進(jìn)神經(jīng)軸突的生長(zhǎng)。
在對(duì)照組也發(fā)現(xiàn)了神經(jīng)的再生現(xiàn)象。這可能是由于大鼠的神經(jīng)再生能力強(qiáng),神經(jīng)再生室形成了神經(jīng)再生的通道;雖然0.9%氯化鈉溶液不具備促神經(jīng)生長(zhǎng)的作用,但是其流動(dòng)形成了濃度梯度,間接促進(jìn)了神經(jīng)的再生[13]。
外周神經(jīng)損傷后p38 MAPK活化,雪旺氏細(xì)胞去分化[14],雪旺氏細(xì)胞衰老,同時(shí)導(dǎo)致成纖維細(xì)胞衰老。而實(shí)驗(yàn)表明:軸索再生受限與神經(jīng)斷端微環(huán)境中的衰老SCs增加有關(guān)[15]。本次實(shí)驗(yàn)電鏡觀察顯示:氯化鎂組和神經(jīng)生長(zhǎng)因子組的神經(jīng)斷端髓鞘和雪旺氏細(xì)胞增生明顯,遠(yuǎn)遠(yuǎn)高于對(duì)照組。證明局部應(yīng)用氯化鎂和神經(jīng)生長(zhǎng)因子類似,可以促進(jìn)神經(jīng)纖維再生。實(shí)驗(yàn)同時(shí)觀測(cè)到氯化鎂組和神經(jīng)生長(zhǎng)因子組成纖維細(xì)胞增多類似,肉眼觀察其形成神經(jīng)束較完整。而對(duì)照組形成神經(jīng)纖維較稀疏。成纖維細(xì)胞主要是修復(fù)斷端創(chuàng)傷中斷裂的組織,它的合成和降解,可使損傷部位形成有張力的組織。解釋了大體觀察所發(fā)現(xiàn)的氯化鎂組及神經(jīng)生長(zhǎng)因子組神經(jīng)纖維再生較好的現(xiàn)象。
通過本次實(shí)驗(yàn),我們發(fā)現(xiàn)了局部應(yīng)用氯化鎂具有促神經(jīng)軸突及雪旺氏細(xì)胞生長(zhǎng)的作用,但機(jī)制尚不明確。我們將結(jié)合進(jìn)一步體外細(xì)胞培養(yǎng)實(shí)驗(yàn),進(jìn)一步探索鎂離子對(duì)神經(jīng)細(xì)胞生長(zhǎng)、分化的作用。
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doi:10.3969/j.issn.1002-7386.2016.14.001
通訊作者:馬維,050000石家莊市,河北醫(yī)科大學(xué)第二醫(yī)院骨科;
【中圖分類號(hào)】R 424
【文獻(xiàn)標(biāo)識(shí)碼】A
【文章編號(hào)】1002-7386(2016)14-2085-04
(收稿日期:2016-02-03)
A experimental study on the effects of magnesium chloride on ultrastructure renovation after sciatic nerve injury in rats
LIMing,SONGYongzhou,CAOShuxing,etal.
DepartmentofOrthopedics,TheSecondHospitalofHebeiMedicalUniversity,Shijiazhuang050000,China
【Abstract】ObjectiveTo investigate the effects of magnesium chloride on ultrastructure renovation after sciatic nerve injury in rats .MethodsNinety SD rats were randomly divided into 3 groups: magnesium chloride group (MgCl2 group), nerve growth factor group (NGF group), 0.9% sodium chloride group (NaCl group), with 30 rats in each group.Firstly,the animal models with sciatic nerve injury were established,and the injury was bridged with silicone tube to set up a nerve regeneration chamber (NRC),then 1mmol/L MgCl2,NCF,0.9% sodium chloride solution was infused into the chamber,respectively. After operation the rats were sacrificed on 4,8,12 weeks,respectively. The regenerating tissues in NRC were taken out to be fixed,finally, the regenerating nerve fibers number and section area of regenerating nerve fibers were observed under light microscope,moreover, the state of proliferation and development of Schwann cells in NRC and ultrastructure of medulla sheath was observed by electron microscopy.ResultsThe regenerating nerve fibers number and section area of regenerating nerve fibers in MgCl2 group and NGF group on 4,8,12 weeks were significantly higher than those in Nacl group (P<0.05). However there were no significant differences between MgCl2 group and NGF group (P>0.05). In MgCl2 group and NGF group,the regenerating nerve fibers number was increased,the proliferation of Schwann cells was obvious and cell's shape was nearly normal,the thickness of medulla sheath was uniformly increased. in contrast to that, in NaCl group, nerve fibers regeneration was not obvious, medulla sheath structures were blurred and twisted, the proliferation of Schwann cells was not obvious, with immature phenotype.ConclusionMagnesium chloride can promote the ultrastructure renovation after sciatic nerve injury in rats.
【Key words】magnesium chloride; Schwann cell;nerve growth factor; sciatic nerve injury
項(xiàng)目來源:河北省醫(yī)學(xué)科學(xué)研究重點(diǎn)課題(編號(hào):ZL20140291)
E-mail:15803210539@163.com