高艷，蘇壯志

(首都醫(yī)科大學(xué)宣武醫(yī)院放射科, 北京 100053)

研究報(bào)告

野百合堿性肺動(dòng)脈高壓大鼠右心功能分析

高艷，蘇壯志

(首都醫(yī)科大學(xué)宣武醫(yī)院放射科, 北京 100053)

目的研究肺動(dòng)脈壓力演變和右心室功能演變之間的關(guān)系。方法將MCT誘導(dǎo)的4組肺動(dòng)脈高壓(pulmonary artery hypertension,PAH)模型組(每小組12只)，分別在第1、2、3、4周應(yīng)用右心導(dǎo)管測量肺動(dòng)脈壓力，MRI右心功能動(dòng)態(tài)檢測，觀察肺動(dòng)脈壓力和MRI參數(shù)演變關(guān)系。比較對(duì)照組、PAH模型組各組間的相關(guān)各參數(shù)差異。采用SPSS 17.0統(tǒng)計(jì)軟件，應(yīng)用Pearson相關(guān)性分析，評(píng)價(jià)右心室射血分?jǐn)?shù), 右室舒張末期容積,右室收縮末期容積分別與平均肺動(dòng)脈壓的相關(guān)性，組間比較采用完全隨機(jī)分組的t檢驗(yàn)，P<0.05為差異有顯著性。結(jié)果注射野百合堿后1～4周，48只模型組大鼠的右心室射血分?jǐn)?shù)、右心室舒張及收縮末期容積與平均肺動(dòng)脈壓有很好的相關(guān)性(分別為rRVEF=-0.823，rRVEDV=0.732, rRVESV=0.803)。注射野百合堿前兩周，野百合堿組大鼠的平均肺動(dòng)脈壓、右心室射血分?jǐn)?shù)、右室舒張末期和收縮末期容積與對(duì)照組比較差異無顯著性(P>0.05)。3～4周后，以上各參數(shù)與對(duì)照組比較差異有顯著性(P<0.05)。結(jié)論隨著大鼠的肺動(dòng)脈壓增高，右心室射血分?jǐn)?shù)逐漸降低，右心室舒張末期及收縮末期容積逐漸增加。對(duì)于大鼠慢性肺動(dòng)脈高壓模型的監(jiān)測，MRI可以準(zhǔn)確快速測量各項(xiàng)參數(shù)變化，右心室舒張末及收縮末期容積、射血分?jǐn)?shù)等參數(shù)是提示肺動(dòng)脈高壓的敏感參數(shù)。

肺動(dòng)脈高壓；大鼠,磁共振；野百合堿；右心室功能

我國慢性阻塞性肺病(chronic obstructive pulmonary disease, COPD)的發(fā)病率達(dá)到8.2%[1]。大部分COPD患者經(jīng)慢性肺動(dòng)脈高壓過程最終發(fā)展成慢性肺源性心臟病，并最終導(dǎo)致死亡，病死率高[2]。闡明肺動(dòng)脈高壓發(fā)病機(jī)制對(duì)有效防治COPD發(fā)展成肺心病意義重大。既往對(duì)肺動(dòng)脈高壓患者右心室功能研究僅限于回顧性評(píng)價(jià),不能動(dòng)態(tài)觀察心功能的變化,心功能異常是在肺動(dòng)脈高壓進(jìn)展過程哪個(gè)階段出現(xiàn)的并不清楚。本實(shí)驗(yàn)基于野百合堿(MCT)誘導(dǎo)的大鼠肺動(dòng)脈高壓模型，研究肺動(dòng)脈高壓形成過程中，肺動(dòng)脈壓力的演變和右心功能等血流動(dòng)力學(xué)參數(shù)演變之間關(guān)系，動(dòng)態(tài)觀察右心功能變化。

圖1 MCT誘導(dǎo)的第2周，MRI左室短軸層面右心室由收縮期到舒張期形態(tài)變化過程Fig.1 MRI showing the morphological changes of left ventricular short axis from right ventricular systolic to diastolic phases at two weeks after injection of monocrotaline

1 材料和方法

1.1材料

1.1.1 實(shí)驗(yàn)動(dòng)物

6～8周齡雄性SPF級(jí)Sprague-Dawley (SD)大鼠96只,體重180～220 g,購自北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司【SCXK(京)2016-0006】。隨機(jī)分為正常對(duì)照組(n=48)，野百合堿誘導(dǎo)的PAH模型組(n=48)，實(shí)驗(yàn)大鼠飼養(yǎng)及組織取材均于宣武醫(yī)院實(shí)驗(yàn)動(dòng)物室進(jìn)行【SYXK(京)2010-0013】，室內(nèi)通風(fēng)良好，室溫(23±2)℃，相對(duì)濕度40%～70%，每日光照12 h,自由攝食飲水。常規(guī)商用鼠飼料:由宣武醫(yī)院實(shí)驗(yàn)動(dòng)物中心提供。本實(shí)驗(yàn)所有操作均符合中華人民共和國《實(shí)驗(yàn)動(dòng)物管理?xiàng)l例》。

1.1.2 試劑與儀器

野百合堿(MCT):美國Sigma公司；采用超高場強(qiáng)小動(dòng)物磁共振儀(Bruker BioSpin MRI GmbH, PharmaScan 7.0 Tesla, Siemens, Germany)。

1.2肺動(dòng)脈高壓模型建立及實(shí)驗(yàn)分組

1.2.1 對(duì)照組

腹腔內(nèi)一次性注射與MCT等量的生理鹽水。于造模后每天注射生理鹽水l mL/kg。

1.2.2 野百合堿 (MCT) 組

腹腔內(nèi)一次性注射MCT 溶液(60 mg/kg)造模，于造模后每天注射生理鹽水l mL/kg。分別為注射MCT后1周組(l-week-PAH組，n=12)、2周組(2-week-PAH組，n=12)、3周組(3-week-PAH組，n=12)及4周組(4-week-PAH組，n=12)。于注射MCT后分別于1～4周做完檢查后安樂死。

1.3MRI右心功能成像及定量分析

將MCT誘導(dǎo)的肺動(dòng)脈高壓模型組和對(duì)照組大鼠分別隨機(jī)分為五個(gè)小組(每小組12只),分別在第1、2、3、4周進(jìn)行MRI動(dòng)態(tài)監(jiān)測右心功能參數(shù)，右心功能參數(shù)包括右心室射血分?jǐn)?shù)、右心室收縮末期容積、右心室舒張末期容積等。評(píng)價(jià)肺動(dòng)脈壓力變化和MRI參數(shù)演變間的關(guān)系。

1.3.1 MRI檢查

檢查前，隨食物口服β-blocker (5～20 mg)保證大鼠心率控制在90次/分及以下，使得MRI顯示的左、右心成像形態(tài)清晰，減少偽影。大鼠稱重后用5%～8%異氟烷吸入性麻醉后,仰位固定,連接心電裝置、呼吸門控及表面線圈,以心電極貼片連接心電門控裝置。采用Flash-cine序列,參數(shù)：TR 8 ms，TE 2.5 ms FA 15 angle。層厚1 mm，F(xiàn)OV： 6.0 × 6.0 cm，MTX 192X192, NEX 4 number of movie cycles 20。

1.3.2 實(shí)施屏氣電影法MRI

先行心電圖門控梯度回波冠狀面掃描，然后以冠狀面像定位行平行右室長軸的常規(guī)自旋回波序列掃描，最后在右室長軸像上定位，從心底至心尖逐層掃描獲得3 mm 層厚、垂直室間隔并與心電圖同步的右室各個(gè)時(shí)期的短軸電影圖像(圖1)。

1.3.3 MR圖像重建

掃描完成后的右室短軸電影圖像傳至工作站(Leonardo, Siemens, Germany)，應(yīng)用MR隨機(jī)專用心功能測量軟件(Argus, Siemens Medical Solutions, Germany)進(jìn)行右心室心功能測量，軟件自動(dòng)識(shí)別右室EDV及ESV， 手動(dòng)逐層描記舒張末期和收縮末期此兩個(gè)時(shí)相右心室心內(nèi)膜輪廓，計(jì)算心功能指標(biāo)，舒張末期容積 (end-diastolic volume，EDV)、收縮末期容積(end-systolic volume，ESV)、射血分?jǐn)?shù)(eject fraction, EF)。

1.4肺動(dòng)脈平均壓測定

PAH模型組于注射MCT后1周、2周、3周及4周末，將各組大鼠稱重后，用戊巴比妥鈉(65 mg/kg)腹腔注射麻醉大鼠后，在腹部行正中“T”形開口，將探針透過膈肌刺入右心室，探針的另一端經(jīng)壓力傳感器連接PowerLab壓力記錄分析系統(tǒng)，實(shí)時(shí)測定。

1.5統(tǒng)計(jì)方法

應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件，結(jié)果以均數(shù)±標(biāo)準(zhǔn)差表示，組間比較采用完全隨機(jī)分組的t檢驗(yàn)。應(yīng)用Pearson相關(guān)性分析，評(píng)價(jià)右心室射血分?jǐn)?shù), 右室舒張末期容積,右室收縮末期容積分別與平均肺動(dòng)脈壓的相關(guān)性，P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

隨著野百合堿注射時(shí)間的延長，與對(duì)照組比較，大鼠的血流動(dòng)力學(xué)指標(biāo)及MRI右心功能參數(shù)數(shù)值變化，結(jié)果顯示：注射野百合堿后第1、2周與對(duì)照組比較，模型組肺動(dòng)脈平均壓及右心射血分?jǐn)?shù)、右心室舒張末期容積、收縮末期容積差異均無顯著性(圖2)；第3周及第4周，模型組隨著肺動(dòng)脈壓力升高，右心射血分?jǐn)?shù)下降，右心室舒張及收縮末期容積增加 (圖3)，與對(duì)照組比較差異有顯著性，見表1～4。

注射野百合堿后1～4周，48只模型組大鼠的右心室射血分?jǐn)?shù)、右心室舒張末及收縮末期容積與平均肺動(dòng)脈壓有很好的相關(guān)性(分別為rRVEF=-0.823，rRVEDV=0.732, rRVESV=0.803)。各參數(shù)散點(diǎn)圖見圖4(A-C)。

表1 造模后第1～4周模型組與對(duì)照組的肺動(dòng)脈平均壓比較Tab.1 Comparison of mean pulmonary arterial pressure between the model group and control group at 1-4 weeks after modeling

注：模型組與正常對(duì)照組肺動(dòng)脈平均壓(mmHg)比較，#P>0.05,*P<0.01。

Note:#P>0.05,*P<0.05. compared with the normal control group.

表2 造模后第1～4周模型組與對(duì)照組右心室射血分?jǐn)?shù)的比較Tab.2 Comparison of right ventricular eject fraction between the model group and control group at 1-4 weeks after modeling

注：模型組與正常對(duì)照組右心室射血分?jǐn)?shù)(%)比較，#P>0.05,*P<0.05。

Note:#P>0.05,*P<0.05, compared with the normal control group.

表3 造模后第1～4周模型組與對(duì)照組右心室舒張末期容積的比較Tab.3 Comparison of the end-diastolic volume of right ventricle between the model group and control group at 1-4 weeks after modeling

注：模型組與正常對(duì)照組右心室舒張末期容積(mL)比較，#P>0.05,*P<0.01。

Note:#P>0.05,*P<0.05, compared with the normal control group.

表4 造模后第1～4周模型組與對(duì)照組右心室收縮末期容積的比較Tab.4 Comparison of the end-systolic volume of right ventricle between the model group and control group at 1-4 weeks after modeling

注：模型組與正常對(duì)照組右心室收縮末期容積(mL)比較，#P>0.05,*P<0.01。

Note:#P>0.05,*P<0.05, compared with the normal control group.

圖2 注射野百合堿后第2周，右心室收縮末期(A)和右心室舒張末期(B)Fig.2 The RV ESV (A) and RV EDV (B) at 2 weeks after injection of monocrotaline

圖3 注射野百合堿后第4周，右心室收縮末期(A)和右心室舒張末期(B)較注射野百合堿后第2周的右心室容積均有顯著增大，室間隔平直Fig.3 The RV ESV (A) and RV EDV (B) at 4 weeks after injection of monocrotaline

注：右心室射血分?jǐn)?shù)(A), 右室舒張末期容積 (B),右室收縮末期容積 (C)與平均肺動(dòng)脈壓的相關(guān)性的散點(diǎn)圖。圖4 注射野百合堿后1～4周肺動(dòng)脈高壓模型大鼠(n=48)Compared with the mean pulmonary arterial pressure in PAH rats at 1-4 weeks after injection of monocrotaline.Fig.4 Scatter plots of correlation of the RV EF(A), RV EDV(B) and RV ESV (C)

3 討論

慢性肺動(dòng)脈高壓的預(yù)防、監(jiān)測和治療均比較困難，及時(shí)早期診斷治療可使20%病人的病情穩(wěn)定[3]。闡明肺動(dòng)脈高壓發(fā)病機(jī)制、血流動(dòng)力學(xué)改變和尋找有效的治療手段已成為該領(lǐng)域亟待解決的重要課題。慢性肺動(dòng)脈高壓動(dòng)物模型的成功建立則是進(jìn)行基礎(chǔ)和臨床研究的平臺(tái)，目前野百合堿所致的大鼠慢性肺動(dòng)脈高壓模型己經(jīng)日趨成熟[4，5]，給藥后2～3周即形成肺動(dòng)脈高壓。

臨床上應(yīng)用MRI檢測右心室的功能來評(píng)價(jià)肺動(dòng)脈高壓(PH) 病情嚴(yán)重程度[6-18]已經(jīng)越來越受到重視并逐漸應(yīng)用于臨床，本實(shí)驗(yàn)基于MCT誘導(dǎo)的大鼠肺動(dòng)脈高壓模型，研究肺動(dòng)脈高壓形成過程中，肺動(dòng)脈壓力的演變和右心功能等血流動(dòng)力學(xué)參數(shù)演變之間關(guān)系，動(dòng)態(tài)觀察右心功能變化。

高艷等[19]研究報(bào)道，隨著肺動(dòng)脈壓持續(xù)升高，右心排出量下降，右心室收縮末期殘留血量會(huì)逐漸增加，舒張末壓增高，超過右心室的代償能力，促使右心室擴(kuò)大和右心功能衰竭,右室射血分?jǐn)?shù)(RVEF)值進(jìn)一步下降，發(fā)展成為肺心病，臨床特征為RVEF下降，但每搏輸出量保持正常以代償RVEF的減小。而右室EDV、ESV均增大。

本研究發(fā)現(xiàn)，野百合堿所致的肺動(dòng)脈高壓大鼠在第1～4周內(nèi)肺動(dòng)脈平均壓逐漸升高，右心室射血分?jǐn)?shù)逐漸減低，右心室收縮末期和舒張末期容積逐漸增加，但是這種變化在第1～2周時(shí)與對(duì)照組比較差異無顯著性，當(dāng)?shù)搅说?周時(shí)這種異常變化與對(duì)照組比較差異有顯著性，且隨著肺動(dòng)脈壓升高時(shí)間延長而變化明顯，即到第4周，隨著肺動(dòng)脈平均壓顯著升高，與對(duì)照組比較，右心室射血分?jǐn)?shù)顯著減低，右心室收縮末期和舒張末期容積顯著增加。說明右心功能能參數(shù)的變化是隨著肺動(dòng)脈高壓的形成而逐漸變化的。通過相關(guān)性結(jié)果分析，也可以看出肺動(dòng)脈高壓模型組大鼠的右心室射血分?jǐn)?shù)、右心室收縮末期和舒張末期容積等參數(shù)與肺動(dòng)脈平均壓有很好的相關(guān)性，說明隨著肺動(dòng)脈高壓的形成，右心功能是逐漸受損。右心室射血分?jǐn)?shù)、右心室收縮末期和舒張末期容積等右心功能參數(shù)是反映肺動(dòng)脈高壓程度的敏感影像學(xué)參數(shù)。

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MRIanalysisofrightventricularfunctioninratmodelofpulmonaryarteryhypertensioninducedbymonocrotaline

GAO Yan, SU Zhuang-zhi

(Xuanwu Hospital of Capital Medical University, Beijing 100053, China)

ObjectiveThe aim of this study was to establish a rat models of pulmonary artery hypertention with monocrotaline, and to study the relationship between the evolution of right ventricular function and the evolution of pulmonary artery pressure (PAP) by magnetic resonance (MR) imaging of the right ventricular function.MethodsRat models of pulmonary artery hypertension were established by monocrotaline (MCT). The model rats were divided into 4 groups: the 1-week-PAH group, 2-week-PAH group, 3-week-PAH group, and 4-week-PAH group, and pulmonary artery pressure in the rats was measured by right heart catheterization. After injection of MCT, we used MRI to evaluate the ventricular function of the rats every week. All the measurement data of right ventricular function in the model group were compared with the average pulmonary pressure using Pearson’s correlation test.ResultsThere were strong correlations between the parameters of RV function in model group with the average pulmonary pressure (r=-0.823 for RV EF,r=0.732 and 0.803 for RV EDV and RV ESV). At 2 weeks after injection of monocrotaline, the mean pulmonary pressure, right ventricular eject fraction (RVEF), the end-diastolic volume (EDV) and the end-systolic volume (ESV)of right ventricle between rats in PAH and the control group showed no significant difference (P>0.05). But three-four weeks after MCT injection, all these parameters were significantly different in the PAH rats than in control rats (P<0.05).ConclusionsAs the pulmonary arterial pressure is increased in the rats, the right ventricular function is gradually impaired. For the monitoring of chronic pulmonary artery hypertension in rats, MRI can be used to accurately measure the changes of parameters. The PAH can be indicated by looking at the changes of parameter such as RV EF, RV EDV and RV ESV.

Pulmonary artery hypertension, PAH; Rat; MRI; Monocrotaline; Right ventricular function

GAO Yan. E-mail： wsggy518@sina.com

Q95-33

A

1005-4847(2017) 05-0534-05

10.3969/j.issn.1005-4847.2017.05.012

北京市自然科學(xué)基金(No.7163217)。

高艷(1970-)，女，博士，主任醫(yī)師，副教授。研究方向:心胸、神經(jīng)系統(tǒng)影像診斷。 E-mail： wsggy518@sina.com