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T2 mapping動態(tài)定量監(jiān)測兔腰椎間盤退變模型

2016-05-12 04:02:14謝光友楊海濤呂富榮劉昌杰楊明放王榮品
磁共振成像 2016年3期
關(guān)鍵詞:動物實驗磁共振成像動物

謝光友,楊海濤,呂富榮,劉昌杰,楊明放,王榮品

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T2 mapping動態(tài)定量監(jiān)測兔腰椎間盤退變模型

謝光友1,楊海濤2*,呂富榮2*,劉昌杰1,楊明放1,王榮品1

[摘要]目的 提高T2 mapping技術(shù)動態(tài)定量監(jiān)測兔腰椎間盤退變模型的認(rèn)識。材料與方法 18只新西蘭大白兔,從脊柱側(cè)后方手術(shù)入路暴露腰椎間盤,18 G穿刺針抽吸12只L2~3、L3~4、L4~5髓核,各約5 mg,正常L1~2、L5~6和另6只L2~5間椎間盤分別為組內(nèi)和組間對照。術(shù)后第4、8、12周行MR矢狀面T2WI和T2-mapping序列檢查,取正中矢狀面觀察各椎間盤的信號,測量T2弛豫時間并與正常組比較。相應(yīng)各時間點取2只L1~6間椎間盤行HE和Masson染色,觀察纖維環(huán)形態(tài)、髓核細(xì)胞及基質(zhì)的變化并與正常組比較。結(jié)果 18 G穿刺針經(jīng)脊柱旁路于橫突根部可成功穿刺抽吸髓核。6只正常兔及12只實驗兔組內(nèi)對照椎間盤T2WI序列為均勻高信號,12只實驗兔椎間盤隨術(shù)后時間延長信號逐漸降低,第4周呈稍低信號,第8周明顯降低,第12周完全呈低信號。正常對照組及術(shù)后第4、8、12周組T2弛豫時間的單因素方差分析結(jié)果(F=38.82,P<0.05),造模術(shù)后第8周T2弛豫時間有顯著差異(P<0.05)。術(shù)后隨時間進(jìn)展,HE和Masson染色示椎間盤細(xì)胞和膠原逐漸減少,纖維環(huán)和髓核分界不清;第8周髓核基質(zhì)退變纖維化,幾乎被纖維組織取代;第12周椎間盤纖維軟骨化,局部形成軟骨。結(jié)論 T2 mapping技術(shù)可實時定量監(jiān)測腰椎間盤的退變進(jìn)程。

[關(guān)鍵詞]椎間盤退化;疾病模型,動物;磁共振成像;動物實驗

重慶市自然科學(xué)基金(編號:cstc2011jjA10082);國家臨床重點專科建設(shè)項目(編號:2013-544)

作者單位:

1. 貴州省人民醫(yī)院放射科,貴陽550002

2. 重慶醫(yī)科大學(xué)附屬第一醫(yī)院放射科,重慶 400016

楊海濤,E-mail: frankyang119@126.com;呂富榮,E-mail: lfr918@sina.com

謝光友, 楊海濤, 呂富榮, 等. T2 mapping動態(tài)定量監(jiān)測兔腰椎間盤退變模型. 磁共振成像, 2016, 7(3): 213-217.

腰椎間盤退行性變是導(dǎo)致腰背部疼痛最常見的原因,隨著社會老齡化日益突出,將會消耗更多醫(yī)療資源。椎間盤缺乏神經(jīng)血管,自身無再生修復(fù)能力,目前內(nèi)外科均不能逆轉(zhuǎn)椎間盤的退變進(jìn)程。磁共振成像時間、空間及組織分辨率高,可通過測量椎間盤的T2弛豫時間,無創(chuàng)性地動態(tài)監(jiān)測其退變程度,檢測其退變的起始時間及發(fā)生可逆性退變的時間窗,對指導(dǎo)臨床采取防治措施具有重要意義。

1 材料與方法

1.1 實驗動物

取3月齡健康成年新西蘭大白兔18只,平均體重2.5 kg,雌雄不拘,無脊柱畸形及椎間盤病變,肢體活動良好,分籠飼養(yǎng),由重慶醫(yī)科大學(xué)動物實驗中心提供[許可證號:SCK (渝) 2012–0001]。

主要試劑儀器:HE和Masson染色試劑盒、15% EDTA脫鈣液、無菌PBS(武漢博士德公司),戊巴比妥鈉,青霉素、慶大霉素。T2000U倒置相差顯微鏡(日本Nikon公司),GE HD-X signa 3.0 T MRI成像系統(tǒng)、N-V Array8通道相控陣線圈、ADW 4.4工作站(美國GE公司),無菌手術(shù)包,18 G穿刺針。

1.2 構(gòu)建腰椎間盤退變模型

通過髓核針刺法建立椎間盤退變模型[1-2]。18只兔行MRI檢查,術(shù)前一天肌注青霉素20萬U/kg,術(shù)前6 h禁飲禁食。1%戊巴比妥鈉(1 ml/kg)經(jīng)耳緣靜脈注射成功麻醉后,取俯臥位固定于兔臺,以L2~5間椎體為中心,剃毛備皮、術(shù)野消毒鋪巾,以L3椎體左側(cè)橫突根部為中心,做一4~ 5 cm縱切口,逐層切開皮膚、皮下組織、胸腰筋膜,神經(jīng)剝離子沿橫突向背側(cè)剝離直至橫突根部,充分暴露L3~4椎間盤并用18 G穿刺針抽吸髓核組織,約5 mg,然后緩慢退針,同法處理L2~3、L4~5椎間盤,術(shù)中用慶大霉素生理鹽水沖洗、止血。術(shù)后逐層縫合,回籠標(biāo)準(zhǔn)條件下飼養(yǎng),連續(xù)一周消毒切口并肌注青霉素20萬U/kg。本實驗已通過生物醫(yī)學(xué)倫理審核。

1.3 MRI動態(tài)定量監(jiān)測

術(shù)前和術(shù)后4、8、12周對實驗組和正常組行腰椎MRI檢查。成功麻醉后,取俯臥位拉直脊柱后固定,以L3椎體為中心行MRI矢狀面檢查。掃描序列及參數(shù):FSE T2WI序列:TR 2620 ms,TE 102 ms,F(xiàn)OV 20 cm×20 cm,層厚3 mm,層間距0.6 mm,NEX為4,矩陣384×256;T2-maping序列:TR 1600 ms,TE 11.1、22.2、33.3、44.4、55.5、66.6、77.7、88 ms,F(xiàn)OV 20 cm×20 cm,層厚3 mm,層間距0.6 mm,NEX為1,矩陣384×256。MRI資料由本院2名骨關(guān)節(jié)疾病影像診斷醫(yī)師(工作經(jīng)驗5~10年)和1名脊柱外科醫(yī)師(工作經(jīng)驗10年)進(jìn)行雙盲評估,如有分歧,經(jīng)討論后達(dá)成一致,選腰椎正中矢狀面,觀察T2WI序列上各組L1~6間椎間盤的信號及形態(tài),參照Pfirrmann分級標(biāo)準(zhǔn)[3]。通過ADW 4.4工作站Functool軟件的時間自動測量系統(tǒng),設(shè)置橢圓形興趣區(qū)包繞椎間盤,測量T2-mapping序列上相應(yīng)椎間盤的T2弛豫時間,每個興趣區(qū)重復(fù)測量3次,取均值。L2~3、L3~4、L4~5椎間盤為實驗組,組內(nèi)L1~2、L5~6及正常兔L1~6間椎間盤為對照。

1.4 病理染色觀察

術(shù)后4、8、12周空氣栓塞法各處死實驗組及正常組兔2只,迅速游離并取出L1~6間椎間盤,生理鹽水反復(fù)沖洗,10%多聚甲醛固定24 h,15% EDTA脫鈣兩周后梯度酒精脫水,二甲苯透明處理、石蠟包埋、5 μm切片,分別行HE和Masson染色,鏡下觀察椎間盤形態(tài)、髓核細(xì)胞及膠原的變化,L2~3、L3~4、L4~5椎間盤為實驗組,組內(nèi)L1~2、L5~6及正常組L1~6間椎間盤為對照。

1.5 統(tǒng)計學(xué)方法

利用SPSS 17.0統(tǒng)計軟件進(jìn)行數(shù)據(jù)分析,實驗數(shù)據(jù)采用表示,實驗組和正常組間T2弛豫時間采用單因素方差分析,組內(nèi)T2弛豫時間采用配對t檢驗,P<0.05作為差異有統(tǒng)計學(xué)意義的標(biāo)準(zhǔn)。

2 結(jié)果

2.1 腰椎間盤MRI

正常6只兔L2~3、L3~4、L4~5椎間盤及12只實驗兔組內(nèi)正常對照L1~2、L5~6椎間盤T2WI序列始終為均勻高信號,終板為低信號,髓核與終板分界清楚,觀察期內(nèi)無明顯變化。術(shù)后12只兔均存活,2周活動自如,無癱瘓及大小便失禁。12只實驗兔L2~3、L3~4、L4~5椎間盤信號強度隨術(shù)后時間延遲逐漸減低,椎間盤含水量減少,容積固縮變薄,第8周所有針刺椎間盤信號均明顯降低,第12周信號均進(jìn)行性下降,椎間盤突出伴硬膜囊受壓,骨性椎管繼發(fā)性狹窄(圖1)。6只正常兔及12只實驗兔椎間盤術(shù)后第4、8、12周組T2弛豫時間單因素方差分析結(jié)(F=38.82,P<0.05)(表1),造模術(shù)后組間兩兩比較(除正常組與術(shù)后第4周組間)均有統(tǒng)計學(xué)差異,說明造模術(shù)后第8周椎間盤明顯退行性變,T2弛豫時間有顯著差異(P<0.05)。

2.2 HE和Masson染色

正常椎間盤HE染色示纖維環(huán)和髓核間層次分明,含大量髓核細(xì)胞和基質(zhì),Masson染色原纖維呈藍(lán)色。實驗組隨時間延長,椎間盤含水量減少,組織容積逐漸固縮。第4周髓核區(qū)域顏色變灰暗,質(zhì)地松脆,髓核細(xì)胞稀少,內(nèi)層纖維環(huán)發(fā)生玻璃樣變性,纖維間隔模糊出現(xiàn)裂隙,纖維環(huán)軟骨樣細(xì)胞開始向成纖維細(xì)胞轉(zhuǎn)變,第8周纖維環(huán)排列明顯紊亂,質(zhì)地變硬變脆,纖維環(huán)斷裂,膠原向髓核延伸,中心的髓核區(qū)幾乎完全被纖維組織替代。第12周椎間盤呈破絮狀,出現(xiàn)大量不定形物質(zhì)和凋亡壞死細(xì)胞(圖2,3)。

圖1 正常及術(shù)后不同時間點兔腰椎間盤MR T2WI正中矢狀位圖。A:正常椎間盤MR圖,L2~3、L3~4、L4~5椎間盤均呈高信號(箭)。B、C、D分別為術(shù)后第4、8、12周椎間盤MR圖,隨術(shù)后時間延長,L2~3、L3~4、L4~5椎間盤信號均逐漸降低(箭)Fig. 1 The view of the median saggital MRI of rabbit normal and degenerative lumbar IVD at different postoperative timepoints. A: The view of normal IVD MRI, L2—3, L3—4, L4—5 discs showed high signal (arrow). B, C, D represented the MRI of degenerative IVD at postoperative 4 th, 8 th, 12 th week, respectively: the corresponding signal intensity reduced gradually and then became black with the extension of time after operation (arrow).

表1 兔正常腰椎間盤及術(shù)后T2弛豫時間(ms,)Tab. 1 T2 relaxation time of rabbit normal and degenerative lumbar IVD ()

表1 兔正常腰椎間盤及術(shù)后T2弛豫時間(ms,)Tab. 1 T2 relaxation time of rabbit normal and degenerative lumbar IVD ()

Note:aP<0.05, compared with normal control group

Group 0 week  4 week  8 week  12 week Normal group  128.5±3.8 127.1±2.3 123.7±2.7  126.1.4 Degeneration group 125.8±3.2 95.6±4.1  48.4±2.6a45.8±3.5a

3 討論

椎間盤退變是由基因預(yù)設(shè)多因素介導(dǎo)的進(jìn)行性病變過程,機械負(fù)荷、外傷及病毒感染等因素可導(dǎo)致椎間盤細(xì)胞凋亡、變性、壞死,細(xì)胞內(nèi)酶量及活性改變,破壞椎間盤內(nèi)環(huán)境穩(wěn)態(tài),最終退變突出并壓迫周圍神經(jīng)根及脊髓,引發(fā)一系列癥狀[4]。動物模型是研究椎間盤退變機制和檢測各種治療有效性的可靠手段,與恒河猴[5]、豬[6]、鼠[1]等相比,實驗兔的優(yōu)勢還包括經(jīng)濟實用可進(jìn)行大樣本實驗,其椎間盤大小適中取材容易,臨床應(yīng)用型3.0 T MRI儀可實時動態(tài)定量監(jiān)測椎間盤等。

3.1 針刺法制作椎間盤退變模型的機制

椎間盤內(nèi)原始細(xì)胞數(shù)量及細(xì)胞活性是維持椎間盤內(nèi)環(huán)境穩(wěn)態(tài)的關(guān)鍵。椎間盤退變始于髓核細(xì)胞功能減退和數(shù)量減少,而髓核細(xì)胞增殖力差,退變中髓核由軟骨細(xì)胞及基質(zhì)取代;伴隨髓核脫水、高度減低,于外力及機械負(fù)荷下,纖維環(huán)結(jié)構(gòu)紊亂;終板進(jìn)行性增厚伴局限性骨質(zhì)斷裂;椎間盤組織內(nèi)硫酸軟骨素和硫酸角質(zhì)素比例失調(diào),逐漸被礦化組織取代,致終板內(nèi)毛細(xì)血管硬化閉塞減少,加重髓核組織營養(yǎng)短缺;同時,基質(zhì)金屬蛋白酶量及活性進(jìn)一步增強,加速退變進(jìn)程。椎間盤失去正常解剖生理結(jié)構(gòu),脊柱出現(xiàn)異常運動,應(yīng)力軸失衡,脊柱失穩(wěn),加快加重退變進(jìn)程,進(jìn)而引起一系列臨床癥狀[7-10]?;谧甸g盤退變始于髓核細(xì)胞及基質(zhì)的病理改變,本研究通過針刺抽吸兔腰椎間盤髓核,破壞椎間盤內(nèi)環(huán)境穩(wěn)態(tài),通過定期定量監(jiān)測椎間盤的弛豫時間,并與正常組進(jìn)行對照,更好地模擬腰椎間盤的退變進(jìn)程。

圖2 兔腰椎間盤(HE × 200)。A:正常椎間盤,纖維環(huán)完整,排列整齊致密,髓核內(nèi)含有大量細(xì)胞及基質(zhì),纖維環(huán)與髓核分界清;B:術(shù)后第8周椎間盤,纖維環(huán)排列紊亂斷裂,髓核細(xì)胞和基質(zhì)減少,纖維環(huán)與髓核分界不清Fig. 2 The view of rabbit lumbar IVD HE stainning ( ×200). A: The view of normal IVD, the structure of annulus fibrosus was intact and arranged regularly, a large number of cells and matrix were obesrved in nucleus pulposus, and the boundary between annulus fibrosusand nucleus pulposus was distinct. B: The view of the IVD at the postoperative 8 th week, the annular fibrosus was in disorder and fractured, the cells and matrix in nucleus pulposus decreased, and the boundary between annulus fibrosus and nucleus pulposus was indistinct.

圖3 兔腰椎間盤膠原Masson染色( ×200)。 A:正常膠原纖維:排列規(guī)則,層次清晰;B:術(shù)后第8周,膠原膠原排列紊亂,各層間見裂隙、斷裂、溶解Fig. 3 The view of rabbit lumbar IVD collagen Masson staining ( ×200). A: The normal collagen arranged regularly and had clear boun dary. B: The view of collagen stained at the postoperative 8th week showed the collagen arranged irregularly and the interlayer fissure, frac ture and dissolution was observed.

3.2 MRI及病理學(xué)評價椎間盤退變

評價腰椎間盤退變多從影像和病理進(jìn)行。MRI軟組織分辨率高、無骨偽影和放射性損傷,是監(jiān)測椎間盤退變的首選方法。MRI不僅能多平面顯示腰椎間盤在退變過程中的形態(tài)變化,而且能夠反映其內(nèi)部結(jié)構(gòu)的分子變化。椎間盤內(nèi)尤其是髓核的含水量決定其信號強度,退變椎間盤脫水容積縮小、信號降低[11]。

僅通過信號強度來判斷椎間盤的退變程度,由于主觀偏差較大,不能準(zhǔn)確反映椎間盤內(nèi)大分子含量及結(jié)構(gòu)的細(xì)微變化。細(xì)胞分子結(jié)構(gòu)功能的變化出現(xiàn)在形態(tài)學(xué)變化前,通過椎間盤T2弛豫時間的變化能更早、更敏感的監(jiān)測其退變程度,加深對椎間盤退變病理生理機制的認(rèn)識。本研究運用T2 mapping序列動態(tài)定量監(jiān)測椎間盤的T2弛豫時間,更加敏感的反映組織內(nèi)部水、蛋白多糖及膠原纖維的含量和排列方向。有學(xué)者通過測量T2弛豫時間可無創(chuàng)性的檢測椎間盤退變的起始時間及發(fā)生可逆性退變的時間窗[12-14],對臨床采取防治措施延緩甚至逆轉(zhuǎn)椎間盤退變具有重要的指導(dǎo)意義。基于椎間盤退變過程中生物力學(xué)和生物化學(xué)的變化,有研究應(yīng)用擴散張量成像(DTI)、擴散加權(quán)成像(DWI)及波譜分析(MRS)等定量方法來加深認(rèn)識椎間盤的退變機制[15-17]。

本研究從聯(lián)合影像與病理動態(tài)對應(yīng)分析術(shù)后兔椎間盤的退變進(jìn)程,更加深入地認(rèn)識椎間盤的退變機制。術(shù)后第4周,實驗組椎間盤纖維環(huán)逐漸出現(xiàn)裂隙、層狀纖維結(jié)構(gòu)逐漸紊亂,細(xì)胞成份逐漸減少,信號強度較正常對照組下降,弛豫時間減低。術(shù)后第8周,纖維環(huán)逐漸斷裂甚至破碎,軟骨樣細(xì)胞明顯減少,椎間隙變窄,軟骨內(nèi)形成骨贅,呈小結(jié)節(jié)狀低信號改變,T2弛豫時間明顯進(jìn)行性減低;術(shù)后第12周椎間盤細(xì)胞基質(zhì)成份變性壞死,細(xì)胞幾近消失,椎間盤信號強度進(jìn)一步下降,呈均勻低信號,T2弛豫時間較第8周變化不大,椎體邊緣形成大量軟骨及骨贅,硬脊膜囊受壓變形,部分椎管繼發(fā)性狹窄。病理染色結(jié)果與MRI信號強度及弛豫時間變化基本吻合,與國內(nèi)外研究結(jié)果相一致[11-13]。

綜上所述,針刺法可成功建立兔腰椎間盤退變模型,重復(fù)性好、可操控性強;T2 mapping技術(shù)能無創(chuàng)實時動態(tài)監(jiān)測椎間盤的退變過程,并與病理表現(xiàn)基本一致,為研究腰椎間盤退變提供了一種可靠的定量監(jiān)測方法,對指導(dǎo)臨床防治椎間盤退變具有重要意義。

參考文獻(xiàn)[References]

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[2]Sobajima S, Kompel JF, Kim JS, et al. A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology. Spine (Phila Pa 1976), 2005, 30(1): 15-24.

[3]Grunert P, Hudson KD, Macielak MR, et al. Assessment of intervertebral disc degeneration based on quantitative MRI analysis: an in vivo study. Spine, 2013, 12(9): 421-432.

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[5]Kong J, Wang ZX, Ji Y, et al. A model of lumbar disc degeneration on the early stage in rhesus monkey with minimally invasive technique. Chin J Surgery, 2007, 23(3): 1597-1600.孔杰, 王子軒, 季玉, 等. 應(yīng)用微創(chuàng)技術(shù)建立恒河猴腰椎間盤早期退變模型. 中華外科雜志, 2007, 23(3): 1597-1600.

[6]Kwon YJ. A minimally invasive rabbit model of progressive and reproducible disc degeneration confirmed by radiology, gene expression, and histology. J Korean Neurosurg Soc, 2014, 53(5): 323-330.

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[8]Bergknut N, Smolders LA, Grinwis GC, et al. Intervertebral disc degeneration in the dog. Part 1: Anatomy and physiology of the intervertebral disc and characteristics of intervertebral disc degeneration. Vet J, 2013, 195(4): 282-291.

[9]Boubriak OA, Watson N, Sivan SS, et al. Factors regulating viable cell density in the intervertebral disc: blood supply in relation to disc height. Anat, 2013, 222(6): 341-348.

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Quantitative T2 mapping to monitor the process of lumbar intervertebral disc degeneration in a rabbit model

XIE Guang-you1, YANG Hai-tao2*, Lü Fu-rong2*, LIU Chang-jie1, YANG Mingfang1, WANG Rong-pin11Department of Radiology, Guizhou Provincial People’s Hospital, Guizhou Province, Guiyang 550002, China
2Department of Radiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

*Correspondence to: Yang HT, E-mail: frankyang119@126.com. Lü FR, E-mail: lfr918@sina.com

Received 23 Dec 2015, Accepted 30 Jan 2016

ACKNOWLEDGMENTS The paper was supported by the Natural Science Funding of Chongqing (No. cstc2011jjA10082). National Clinical Key Subject Construction Project (No. 2013-544).

AbstractObjective: To improve the recognition of quantitative T2 mapping to dynamically monitor the process of lumbar intervertebral disc degeneration in a rabbit model. Materials and Methods: The nucleus pulposus of L2—3, L3—4 and L4—5 discs of 12 rabbits were aspirated by using a 18G needle and the normal L1—2, L5—6 discs of each rabbit and the discs between L2—5 vertebral body of 6 rabbits acted as the normal intra and inter control groups, respectively. The rabbits were performed with sagittal MR T2WI and T2 mapping sequences scanning at the pre-and-postoperative 4 th, 8 th, 12 th week, respectively. The signal intensity of each disc was evaluated and T2 relaxation time was measured carefully. The discs between L1—6 were dissected and then performed with HE and Masson trichrome staining to evaluate the form of discs, content change of nucleus pulposus cells and matrix between normal and experimental groups. Results: The nucleus pulposus could be punctured successfully with a 18 G needle in the root of transverse process via spinal bypass. The normal discs manifested with uniform high signal in T2WI. The signal intensity decreased sharply at the 8 th week and became completely low signal intensity at the 12 th week post-operation. The significant difference of T2 relaxation time foundbook=214,ebook=61between normal control groups and experimental groups after 4, 8 and 12 weeks was analysed by one-way ANOVA, and the result was F=38.82 (P<0.05). The T2 relaxation time at the 8 th week post-operation was significantly different (P<0.05). The results of HE and Masson staining showed that the cells and matrix decreased gradually, moreover, the boundary between annulus fibrosus and nucleus pulposus became obscure. The nucleus pulposus displayed the process of fibrosis and was almost replaced by fibrous tissue at 8 th week. The discs was fibrocartilage and local cartilage can be observed at the 12 th week. Conclusions: T2 mapping can be used to dynamically monitor the process of lumbar intervertebral disc degeneration.

Key wordsIntervertebral disk degeneration; Disease models, animal; Magnetic resonance imaging; Animal experimentation

DOI:10.12015/issn.1674-8034.2016.03.010

文獻(xiàn)標(biāo)識碼:A

中圖分類號:R445.2;R33.2

收稿日期:2015-12-23接受日期:2016-01-30

通訊作者:

基金項目:

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