磁共振弥散/张量加权及CT灌注成像对兔肝缺血再灌注损伤的诊断价值
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摘要
研究背景
肝缺血再灌注损伤(ischemia reperfusion injury, I/R)常发生于肝移植、低血容量性和心源性休克以及肝脏肿块和肿瘤的肝叶手术切除中,是肝功能急性损害的重要原因,直接影响患者的恢复和术后生存率。因而,肝I/R损伤保护研究是肝脏外科领域一项重要的课题。肝I//R病理生理过程机制复杂,涉及能量代谢障碍、钙离子超载、微循环障碍、细胞因子、中性粒细胞、氧自由基作用等相关因素。目前,对肝I/R损伤程度的评价多采用肝酶(AST、ALT)、炎性因子(TNF-a)等生化检查指标。虽然这些生化指标可作为一个非常敏感指标反映肝脏损伤程度,但是还不能反映肝I/R后肝组织的形态学特征、微循环障碍及其在空间分布等方面的信息。同时,肝酶以及炎性因子等指标还存在受肝脏术后应急性反应、创伤以及溶血等多方面因素影响,由此可能产生假象,造成不必要再手术风险。
磁共振弥散/张量成像(diffusion weighted/ tensor imaging, DWI/DTI)作为研究功能及微观影像学的重要方法之一,目前已经成为临床评估和生命科学研究重要的工具,在中枢神经系统和相关病变的基础研究已日趋成熟,但应用于腹部脏器还处于探索阶段。肝I/R的病理生理过程包括肝脏微循环的障碍、肝细胞内外水肿以及不断加重的肝细胞坏死等,这些病理过程使细胞内外水分子的运动状态、肝脏微循环的灌注量以及肝组织微观空间结构发生改变,这就使DWI/DTI监测和评估肝I/R的病理机制及发生、发展具有可行性。目前,DWI/DTI对肝I/R的研究尚处于探索阶段,各家结论尚未达成一致。
CT灌注成像(computer tomography perfusion imaging., CTP)具有很高的时间和空间分辨率,作为一种有效、非创性的检查方法,能够同时在微血管的水平上直接评估组织的灌注情况和间接反应微血管形态特征。,而肝I/R是多机制、多种因素参与复杂的一个病理过程,其中肝I/R后肝脏微循环状态的差异性分布,即血液动力学的改变及伴随的肝脏灌注失败一直被认为是其病理过程中重要的因素之一,这正是如何准确评估肝I/R后肝组织微循环障碍所面临的瓶颈所在,是对以往采用肝酶等生化方法来评估肝脏损伤程度的一个很好补充。
本实验拟采用新西兰大白兔制作部分肝I/R模型,探讨肝I/R后组织病理学、肝功能及TNF-a变化特点,探讨3.0 T DWI/DTI及CTP对兔肝I/R诊断价值,以期为肝I/R的诊断和治疗提供一种新的评价方法。
第一部分兔部分肝缺血再灌注损伤模型的建立
研究目的
采用新西兰大白兔做部分肝I/R模型,拟探讨肝I/R后大体形态、组织病理、肝酶(ALT、AST、ALP)及肿瘤坏死因子a (TNF-a)变化特点,以期为肝I/R后功能影像学研究提供病理学基础。
材料与方法
1.实验动物
健康成年、雄性新西兰大白兔,3～4月,体重2.5～3kg。所用实验兔术前均经腹部CTA检查了解肝动脉和门静脉解剖变异情况。
2.肝I/R及sham模型的制作
采用部分肝I/R模型,用无损伤性动脉夹夹闭入肝左叶管道结构(肝左动脉、门静脉左支及左肝管),肝右叶血供未阻断。阻断血供60min后,打开动脉夹恢复血流,肝左叶由暗红色变为鲜红,表示再灌注成功,关闭腹腔。I/R组按照再灌注时间,分为0.5h、2h、6h、12h、24h和48h组。sham组仅解剖肝十二指肠韧带,不阻断血供。
3.生化及病理学检查
3.1肝酶及TNF-a测定
实验兔分别于MRI、CT检查结束后立即经上腔静脉抽取静脉血4ml(肝素管),离心后,分离血清,测定ALT、AST、ALP (U/L)及TNF-a (pg/ml)含量。
3.2病理学检查
取血样后,立即处死实验动物,整体摘取肝脏,肉眼观察大体标本表面和断面解剖特点。将解剖标本制成常规HE染色切片。显微镜下观察肝组织病理学改变,根据肝脏病变程度以半定量方法积分,按各种病变程度乘以不同加权数：肝细胞坏死×(0～3),肝细胞水肿×(0～3),炎细胞浸润×(0～3),淤血×(0～3),计算每只动物肝病变的总积分,然后进行统计学处理。
4.统计学处理
采用SPSS 13.0统计学软件进行数据分析：(1)I/R与sham组间肝酶、炎性因子及形态学积分均进行单因素方差分析(one-way ANOVA);(2)组织形态学积分与肝酶、TNF-a间关系采用Pearson相关；P<0.05作为差异有显著意义的检验标准。
结果
1.兔肝I/R模型构建
成功造模并纳入实验78只,其中sham、0.5-24 h组,每组各12只；48h组6只。
2.病理表现及生化检查
2.1大体解剖
在复血、氧后早期(0.5h、2h组),肝左叶表现为显著的充血肿胀、体积略增大,在其解剖断面上显示明显充血肿胀、肝窦内充满淤积红细胞；随着I/R后时间的延长,肝脏表面颜色逐渐变淡,变成苍白色,在I/R后48 h肝表面出现不规则墨绿色样变,肝段性坏死显著。而未阻断血供的肝右叶表现为显著充血肿胀。
2.2镜下表现
在I/R早期弥漫性肝细胞肿胀,肝细胞内明显水肿,肝窦间隙变窄,中央静脉、小动脉内红细胞大量淤积、微血栓形成(0.5h),随后汇管区出现成簇中性粒细胞,肝窦稍有萎缩,肝细胞水肿略有减轻,肝窦间及汇管区红细胞淤积,并出现少量炎性细胞,部分肝细胞核肿胀、细胞形态改变(2h)；在I/R后6～12h,肝窦及肝实质聚集大量中性粒细胞,出现肝窦阻塞的现象,部分肝组织出现核固缩红染的凋亡细胞；在后期阶段(24～48h),未发生梗死的肝组织显示肝窦萎缩塌陷、解离,梗死区域细胞结构消失,出现溶解坏死的特征。
2.3组织评分结果
各I/R组镜下形态学评分值除0.5h组一过性性的升高外,总体趋势呈逐渐上升,在48h达到最峰值。各I/R组与sham组间存在显著性差异(F=172.702；P<0.001)。
2.4 AST、ALT、ALP及TNF-a
各I/R组血清ALT(F=325.531,P<0.001)、AST(F=1009.602, P<0.001)、ALP(F=1430.899,P<0.001)与sham (?)司均存在显著性差异。ALT峰值在再灌注后6h组,I/R后12～48h逐渐下降。AST在2h显著升高后,缓慢上升,在48h达到最高值；ALP在6h迅速升高,在I/R后24～48h维持峰值。
I/R组血清TNF-a在再灌注后2h迅速升高,并在I/R后12～24h维持峰值,在48h组开始回落。而sham组,TNF- a保持在一个稳定的低水平。经统计学分析,各I/R组与sham间均存在显著性差异(F=172.702,P<0.001)。
3.相关性分析
组织形态学积分与TNF-a(r=0.788, P<0.001)、ALP(r=0.841)、AST (r=0.848, P<0.001)之间存在显著相关性。
结论
1.兔肝I/R模型制作简单、成功率高,并在复血、氧后48h多个时间点观察了其病理发生、发展过程,适合功能影像评价肝I/R实验研究的需求。
2.肝I/R早期以肝细胞水肿、肝窦淤血等病理过程为主(0～2h)；晚期阶段是以肝细胞凋亡、坏死为主(6～48h)。对损伤反应的差异性是其病理病理特点这一。
3.肝细胞水肿、淤血、中性粒细胞浸润、TNF-a等因素是诱导肝I/R发生、发展的重要因素；组织形态学积分与肝酶、TNF-a具有显著相关性。
4.肝I/R后中性粒细胞聚集、内皮细胞损伤、微循环障碍、肝窦萎缩、塌陷以及局灶性坏死等的病理变化特点为其功能成像学研究提供了病理学基础。
第二部分兔部分肝缺血再灌注损伤3.0T弥散加权/张量成像
研究目的
旨在结合肝酶、肿瘤坏死因子(TNF-a)和病理学检查,探讨3.0 T DWI/DTI对兔肝I/R可行性及诊断价值。
材料与方法
1.动物模型的制作及分组
实验兔分为sham与I/R组。肝I/R模型及sham组制作同第一部分。I/R组按照再灌注时间,分为0.5h、2h、6h、12h、24h与48h组。I/R与sham,每组各6只。
2.检查方法及参数
采用GE 3.0T Signa Excite扫描仪,8通道头正交线圈,轴位扫描。扫描参数：T2WI TR/TE 3200/85ms,矩阵288×224,层厚4 mm, NEX3,FOV 20cm×15 cm；T1WI TR/TE 8.5/4 ms,矩阵288×192,层厚4 mm, NEX,3,FOV 22cm×22 cm：EPI-DWI, TR/TE 2000/49.3 ms,矩阵128×128,层厚4 mm, NEX,4,FOV20cm×10cm；弥散方向：ALL模式(X、Y、Z三个方向)。弥散梯度因子b=50、100、200、300、500、600s/mm2；EPI-DTI序列,TR/TE 2000/49.3 ms, NEX,4,FOV20cm×10 cm,层厚4mm,矩阵128×128。b=100、300、600s/mm2。弥散方向：取6个方向同时进行；增强扫描采用T1WI增强,0.2 mmol/kg顺磁性对比剂Gd-DTPA及8ml生理盐水快速手动注入。
3.MR图像分析
采用GE AW4.3 FuncTool Performance工作站进行后处理,先对原始图像采用Correction程序对图像进行校正,以减少伪影和图像变形。重建出表观扩散系数(ADC)、平均扩散系数(DCavg)及各项异性系数(FA)图。感兴趣区(ROI)：15～8mm2,分别测量ADC、DCavg及FA值,连续测量三个层面,取其平均值。
4.组织学和生化检查
同第一部分。
5.统计学分析
使用SPSS13.0软件进行统计学分析,P<0.05为差异有统计意义。多组均数差异的比较采用单因素方差分析(one-way ANOVA)。以不同b值时MR-DWI/DTI参数作为因变量,肝酶、TNF-a及组织形态学积分作为自变量行多元线性回归分析,用复相关系数R来说明MR-DWI/DTI参数与肝酶、TNF-a及组织形态学积分的相关关系。
结果
1.影像学表现
在0.5h组肝左叶T2WI信号弥漫性轻度增高,T1WI强化减低。而在2h组肝组织信号出现暂时性回复正常。再灌注后6～12h,肝脏的边缘出现T2WI上不均匀性点片状稍高信号以及相对应的强化减低区域。24h及48h组,出现明显的片状、楔形或整个肝段高信号(T2WI、DWI)及无强化的梗死区域,与未发生坏死的区域分界较为明显。随着损伤加重,梗死范围(强化减低区域)逐渐扩大。
2.ADC值变化规律
ADC的总体变化趋势是先显著下降(0.5h),再一过性升高(2h),经过6～12h缓慢上升后,24h再出现一个明显减低后,在48h明显升高。
当b≤300s/mm2时,0.5h(均P<0.001)和24h组ADC显著低于sham组(b=50s/mm2,P=0.037；b=100s/mm2,P=0.020；b=200 s/mm2,P=0.016；b=300s/mm2,P=0.001)。值得注意是,ADC于2 h组明显升高(b≤300s/mm2较为显著),虽与sham组之间无明显差异。在6h组ADC显著升高,但仍低于sham组(b=50s/mm2,P=0.029；b=200s/mm2,P=0.013；b=300s/mm2,P=0.021)。12h组与sham间均无统计学差异。与sham对比,48h组ADC显著升高(b≥300s/mm2,均P<0.001)。
3. DCavg值变化规律
各I/R组DCavg与ADC变化特点基本类似：在0.5h组显著下降,且在b=100(P<0.001)、300s/mm2(P=0.008),与sham组间差异存在统计学意义；在I/R后2h显著升高,并在b=100s/mm2时,与sham组间存在统计学意义(P=0.021)；b=100s/mm2(P=0.021)、300s/mm2(P=0.002)时,6h组较sham组显著升高；DCavg值于24 h组也出现一过性下降现象,并在b=100s/mm2(P=0.008)、300s/mm2(P=0.042)时,低于sham组；在I/R后48h显著升高,且随着b值增大,其与sham组间差异性越显著(b=300s/mm2,P=0.012；b=600s/mm2,P=0.002)。
4.FA值变化特点
在I/R早期(0.5h)FA显著升高(b=100s/mm2,P=0.032；b=300s/mm2,P<0.001)、2h显著减低(b=100 s/mm2,P<0.001；b=300s/mm2,P=0.004),并与sham组间差异均存在统计学意义。FA在6～2h组缓慢下降,与sham间无统计学差异。随着肝I/R损伤加重,FA在I/R后24～48h呈下降趋势,并在24 h(b=300s/mm2,P=0.018；b=600s/mm2,P=0.006)、48h(b=100s/mm2,P=0.044；b=300s/mm2,P<0.001；b=600s/mm2,P<0.001)与sham组间存在显著性差异。
5.I/R与sham组血清肝酶及TNF-a
I/R组血清ALT (F=121.404, P<0.001)、AST (F=489.615, P<0.001)、ALP(F=835.325,P<0.001)与sham对比显著升高,组间存在显著性差异。
I/R组血清TNF-a水平明显升高,与sham组间存在显著性差异(F=171.803,P<0.001)。
6.I/R及sham组组织形态学积分
各I/R组镜下形态学积分总体趋势呈逐渐上升,与sham组间存在显著性差异(F=119.350,P<0.001)。
7.多元线性回归分析
ADC (b=100 s/mm2, R=0.756; b=500/mm2, R=0.782), FA (b=300 s/mm2,R=0.781)与肝酶等生化、病理指标间存在较好的相关性。
结论
1.肝I/R功能成像参数ADC. DCavg/FA具有复杂性和多变性特点,其成像参数在I/R后2h出现一过性回复的现象,与组织形态学积分等病理指标具有类似变化特点,是其病理机制的体现。
2.在肝I/R组早期功能成像参数变化较为剧烈(b<300s/mm2),这可间接反映肝微循环障碍等病理信息；采用较大b值(b=500、600s/mm2),在I/R后期阶段功能成像参数与sham间具有较大差异,这对于准确反映I/R后肝组织损伤、坏死等病理过程具有重要意义。
3.在肝I/R早期阶段,FA与DCavg变化呈相反的趋势(0.5～12 h)；而在晚期阶段呈现逐渐下降特点(24～48h),这与肝I/R后肝组织不断溶解坏死,维持其正常微观空间结构消失相一致。
4.肝酶等生化指标与ADC. DCavg/FA参数间存在较好的相关性,证实了DTI/DWI对肝I/R诊断可行性和临床应用价值。
5. DTI/DWI可以作为一种非创性的功能成像反映肝I/R肝细胞的生理状态、微观形态学等方面信息,若结合其他功能成像方法,例如,灌注成像可促进对I/R模型探索研究,为临床的断和治疗提供了一种可行性评价方法。
第三部分兔部分肝缺血/再灌注损伤CT灌注成像
研究目的
观察兔部分肝I/R后CT灌注(CTP)参数演变规律,分析CTP评估肝I/R血流动力学病理基础,并探讨对其最敏感、有效的灌注参数。
材料与方法
1.动物模型的制作及分组
实验兔分为sham与I/R组。肝I/R模型及sham制作同第一部分。I/R组按照再灌注时间,分为0.5h、2h、6h、12h与24h组。I/R与sham,每组各6只。
2.CT灌注检查方法
采用Philips Brilliance iCT(探测器组合0.625mm×256)扫描仪,定位扫描后,确定灌注扫描范围,然后采用全肝灌注模式(JOG)对全肝及脾动态扫描。非离子型对比剂(5ml优维显,370mgI/ml)和生理盐水10ml。注射速率均为1.5ml/s,对比剂注入后延迟2s开始扫描,扫描时间1.5s,共扫描11次,扫描间隔3s。扫描参数：探测器128×0.625mm,层厚1mm,间距-0.5 mm, Pitch0.915,80Kv,100mAs,360°/0.4 s。
3.图像后处理
采用EBW4.0I Philips工作站,自动生成肝动脉灌注量(HAP)、门静脉灌注量(HPP)、总灌注量(TLP)、灌注指数(HPI)等灌注图。肝组织ROI的选取：0.5～3cm2。在I/R后2h内,ROI随机分布的肝左叶,每个层面测量3个ROI,连续测量三个层面；I/R后6-24 h在梗死区域(低灌注区,即强化减低肝组织)与未梗死区(相对正常的强化未减低肝组织)分别测量3个ROI,连续测量三个层面,计算出梗死与未梗死肝组织的平均值以及相应平均值。
4.组织学和生化检查
同第一部分。
5.统计学分析
采用SPSS13.0软件进行统计分析。多组均数差异的比较采用单因素方差分析(One Way ANOVA)。采用配对t检验比较以下CT灌注参数间有无统计学意义：(1)在6h、12h、24h组梗死与未梗死肝组织灌注参数比较；(2) sham与6h、12h、24h组肝左叶梗死肝组织灌注参数比较;(3)sham与6h、12h、24h组未梗死肝组织灌注参数比较。以梗死与未梗死肝组织中CT灌注参数为因变量,肝酶、TNF-α及组织形态学积分为自变量进行多元线性回归分析。用复相关系数(R)来说明CT灌注参数与肝酶、TNF-α及组织形态学积分的相关关系。P<0.05为差异有统计意义。
结果1.各I/R与sham组间CT灌注变化特点
与sham组相比,0.5h组表现为整个肝左叶的显著强化减低,2h组表现为明显均匀强化,在I/R后6h兔肝左叶呈现差异性灌注(低灌注区出现,即强化减低区),且随着再灌注时间的延长(12～24h),梗死的范围逐渐扩大,强化程度也呈减低趋势。
2.CT灌注参数变化特点
2.1总的变化趋势
CT灌注参数在I/R早期(0.5h组)除HPI外,均表现为显著减低；HAP于2h显著升高后,在I/R后6～24 h均表现为逐渐下降；HPP一直低于sham,尤其是在I/R后0.5-2 h；TLP变化特点与HAP基本类似。HPI作为相对性指数,各I/R组均高于sham组。
2.2梗死及未梗死肝组织CT灌注参数的变化特点
HAP于6h组梗死肝组织显著上升后,在12h、24h组呈现逐渐下降的趋势,而在未梗死的肝组织中HAP是呈现逐渐下降的趋势(6～24h)；HPP不论在梗死或未梗死的肝组织均呈现显著下降的趋势,但在梗死的肝组织内下降的幅度更大；TLP在梗死以及未梗死肝组织中变化特点与HPP基本类似；在梗死的肝组织内HPI显著升高,这也说明了肝动脉缓冲效应机制作用。
3.相关性分析
在肝I/R后梗死组织CT灌注参数与肝生化指标及组织形态学等多个自变量间的具有较好的线性相关,以梗死组织中HPP相关性最好(R=0.855)。
结论
1.在肝I/R后2h,CT强化模式以及CT灌注参数出现暂时性回复,是其“假正常化”病理机制的体现。
2.在肝I/R后6h出现了灌注性差异,且随着时间的延长,其强化程度逐渐减低,梗死的范围逐渐扩大(12～24h),在I/R后24h出现局灶性坏死。
3.肝I/R后,CT灌注参数出现了显著的差异性,在强化减低肝组织中I/R早期(2 h) HAP显著升高,而HPP明显降低,TLP保持相对稳定；HAP在梗死的肝组织(12～24h)明显高于未梗死的肝组织—肝脏动脉缓冲效应基础机制的存在。
4.HAP在梗死的肝组织中虽明显升高(12～24h),但HPP与TLP显著降低,其根本原因是肝内门体分流(末端小动静脉开放),致使肝动脉缓冲效应作用降低,最终导致门静脉的灌注量减低(24 h)。肝血流量实际上减低,进一步加剧肝实质的损害。
5.肝酶、TNF-α、形态学积分与梗死肝组织CT灌注参数间具有相关性。因此,CT灌注参数可以作为敏感、有效的指标监测肝I/R后血流动力学、肝组织的损伤及坏死程度敏感指标。
Background
Hepatic ischemia-reperfusion injury (I/R) is a problem in clinical medicine, which is a critical factor that affects organ and patient survival, especially after liver transplantation, hypovolemia, cardiac shock or massive hepatic tumor resection, and it is also responsible for acute liver failure and is usually associated with high morbidity and mortality. In short, hepatic I/R is a topic issue in the field of hepatic surgery research. Accumulating evidence suggests that energy metabolism reduction, calcium overload, microcirculation perfusion failure, cytokines disorders, neutrophils aggregation and infiltration, the effect of oxygen free radicals subsequent to I/R is a major factor in the pathogenesis of I/R-mediated liver injury. Although serum levels of aspartate and alanine transaminase (AST and ALT) activities, and the levels of tumor necrosis factor-a (TNF-a) have been used as indexes for estimating the degree of liver functional loss, they do not give morphologic information on the spatial distribution of liver injury. Moreover, laboratory parameters (liver enzymes activities and inflammatory factors levels), although valuable, could also be biased by postoperative bleeding and transfusion therapy with fresh-frozen plasma and blood, which may be lead to a pseudomorph and risk of a (resurgery) or (re-)transplantation.
Diffusion weighted/tensor imaging (DWI/DTI) has become a function and micro-imaging MRI modality that allows visual assessment of the Brownian motion of water molecules and in vivo measurement of molecular diffusion and has become an important tool in life science research and clinical practice. At present, DWI/DTI has been extensively evaluated in the central nervous system. However, DWI/DTI is not a routine approach for the characterization of liver diseases.
The pathologic mechanism of ischemic tissue injury includes ischemia and induced hypoxia initially. A cascade of deterioration is further aggravated by secondary reperfusion damage resulting from intra- and extracellular edema, through membrane disintegration, microcirculation perfusion disturbances, and eventually irreversible cytolytic necrosis. Therefore, such continuing changes can be detected by DWI/DTI as more restricted water diffusion initially on account of the diffuse cellular edema and subsequent enhanced water diffusion due to cytolysis. Yet, researches related to hepatic I/R have not reached to agreement and still in the initial phase.
Multi-slice computed tomography perfusion imaging (CTP) as an effective, non-invasive technique that allows for quantitative and objective quantitative assessment of angiogenesis, blood perfusion, and vascular permeability with high temporal and spatial resolution. More evidence suggests that microcirculatory disturbances and capillary perfusion failure subsequent to I/R is a major factor in the pathogenesis of I/R-mediated liver injury, which increases in the heterogeneity of hepatic microvascular perfusion, thus, suggesting that CT together with results of laboratory tests, perfusion parameters may be well suited for the in vivo assessment of hepatic I/R.
Therefore, the subjective of this study was to establish partial liver I/R in New Zealand white rabbit models and observe the changes of histopathology, liver function loss and level of TNF-a, and to the determine whether DWI/DTI/CTP could be used to characterize hepatic I/R quantitatively, providing a valuable and practicable method for diagnosis and evaluation of hepatic I/R.
Part I Establishment of Partial Hepatic Ischemia Reperfusion in a Rabbit Model
Objective
This study was to establish partial liver I/R in New Zealand white rabbit models and observe the characteristics of gross morphology, histopathology, changes of liver function and level of TNF-a, and to provide pathological basis for functional imaging studies of hepatic I/R.
Materials and Methods
1. Animals
The experiments were performed with 3～4 month-old adult and male New Zeland rabbits, weighing 2.5 to 3.0 kg. All the experimental rabbits underwent computer tomography angiography (CTA) preoperatively to analyze anatomy variation of hepatic artery and portal vein.
2. Surgical Procedures and Experimental Groups
Seven animal groups were used. The left portal vein and hepatic artery were occluded for 60 minutes with a vascular clamp to produce ischemia. The right hepatic lobe was perfused to prevent intestinal congestion. (Thereafter, the clamp was removed to allow blood flow through the liver (reperfusion) for 0.5,2,6,12,24 and 48 h, correspondingly. Animals undergoing laparotomy in parallel without vascular occlusion served as sham-operated controls (sham group).
3. Biochemistry assays and histological studies
3.1 Serum Assays and level of TNF-a
Blood samples were collected from the supeirior mesenteric vein with heparinized capillary tubes after MRI and CT were completed at every time point. The serum was separated from the whole blood by centrifugation to determine the ALT, AST and ALP activities in the serum, and the results were expressed as international units per liter.
Using ELISA kit, serum levels of TNF-a in I/R and sham groups were detected by 3 times, and the mean value were expressed as pg per milliliter.
3.2 Histological studies
To perform biopsies, animals were sacrificed after drawing blood from the superior mesenteric vein, after that, to observe the characteristic of gross anatomy. Then, the liver was rapidly excised, fixed (formalin 10% neutral-buffered), embedded (paraffin), sectioned, and stained with H&E. Histologic analyses of tissue slices were performed randomly. The integration of liver injury using semi-quantitative method according to the degree of various diseases multiplied by different weights:liver cell necrosis×(0～3), cells swelling×(0～3), inflammatory cell infiltration×(0～3), congestion×(0～3). To calculate each animal's total scores, and then were analyzed by statistics.
4. Statistical analysis
SPSS13.0 statistical software was used for the analysis. (1):One-way analysis of variance (ANOVA) was used to determine differences between the indexes including serum levels of (AST, ALT and ALP), TNF-a and scores of histomorphologic in I/R and sham groups. (2):The correlations of histomorphologic scores with serum levels and TNF-a were analyzed using Pearson's correlation coefficient. For all tests, a P value less than 0.05 was considered to indicate a statistically significant difference.
Results
1. The result of establishment models
The study ultimately included seventy-eight experimental rabbits, which were divided into 6 I/R groups (0.5～24 h groups, n=12 each; 48 h group n=6) and one sham group (n=12 each).
2. Histologic findings and biochemistry analysis
2.1 Gross anatomy
The affected liver lobe appeared hyperemic or congestive compared to the sham group at the early stage(0.5 and 2 h group), subsequently, the liver's color gradually fades, became kermesinus, having "pattern-like" appearance(12-24 h), and infarction area present with dark green at edge of the liver(48 h). The right lobe without blocking the blood supply showed marked congestion and swelling.
2.2 Under microscope
The histopathologic changes after hepatic I/R included formation of intracellular edema and, hence, diffuse swelling of hepatocytes, narrowing of the lumen, and sinusoidal congestion. Destroyed erythrocytes were congested in and outside the sinusoidal space and the central vein (CV) in the initial phase (0.5 and 2 h group). With aggravated injury, inflammatory cells aggregated in the hepatic microvasculature and extravasated into the hepatic sinusoid lumen and hepatic parenchyma (6 and 12 h group). Some hepatocytes nuclear were staining and condensation of apoptotic cells in the affected tissues. Eventually, widespread cell necrosis/apoptosis, sinusoidal dissociation, and patchy focal necrosis were observed in the 24 and 48 h groups.
2.3 The histomorphologic scores
The general trend of histomorphologic scores in I/R groups kept on increasing except for a sharp increase of 0.5 h group, and peaked at 48 h after reperfusion. There were significant differences between sham and I/R groups(F=172.702, P<0.001).
2.4 Biochemical findings
Compared to those in the sham group, the levels of ALT (F=325.531, P<0.001), AST(F=1009.602, P<0.001) and ALP (F=1430.899, P<0.001) in the I/R group were clearly higher at every time point. The levels of ALT in the I/R groups markedly increased significantly in the 2 h and peaked at 6 h after reperfusion and decline gradually from 12 h to 48 h of reperfusion. AST increased at 2 h of reperfusion, peaking at the end of the perfusion (48 h), and ALP increased rapidly in the 6 h group and peaked at 24～48 h after reperfusion.
The levels of TNF-a in the I/R groups was increased significantly in the 2 h group and peaked at 12～24 h of reperfusion, and declined in the 48 h group. The level of TNF-a remained at stable and relatively low level in the sham group. Compared to the sham group, the levels of TNF-a in the I/R group were clearly higher at every time point (F=172.702, P<0.001).
3. Correlations
Our result demonstrated a good relationship between histomorphologic scores and serum levels (ALT, AST, and ALP) and TNF-a in the sham and I/R groups (r=0.788, r=0.841 and r=0.848, respectively, P<0.01).
Conclusions
1. Our study was to follow up and obverse the process of pathogenesis in a rabbit partial hepatic I/R model with relatively simple and high success rate during a long time window of 48 hours after reperfusion, therefore, may be suitable for functional imaging evaluation of hepatic I/R.
2. Two phases of hepatic injury followed by I/R:the initial phase (0-2 h of reperfusion), associated with the generation of toxic hepatocytes swelling, congestion, and a later phase (6-48 h post-reperfusion) associated with a rather intense state of neutrophils aggregation and subsequent to hepatocyte necrosis. One of the basic pathological features was the heterogeneity response to the injury factors after reperfusion.
3. These factors inducing in the course of the I/R, such as toxic hepatocytes swelling, congestion, neutrophil aggregation and infiltration, and release of TNF-a, were the major factors in the process of I/R pathogenesis. Morphological scores were positively correlated to the level of liver enzymes (AST, ALT, and ALP) and TNF-a.
4. A cascade of deterioration is further aggravated by secondary reperfusion damage resulting from intra-and extracellular edema, neutrophils aggregation, microcirculation perfusion disturbances, sinusoidal dissociation, and subsequent patchy focal necrosis and eventually irreversible cytolytic necrosis, thus, suggesting that function imaging methods may be well appropriate for the in vivo assessment of hepatic I/R.
Part II Diffusion weighted/tensor imaging of partial hepatic ischemia reperfusion injury in a rabbit model at 3.0T
Objective
The intention of this study was to determine whether DWI/DTI could be used to characterize hepatic I/R quantitatively in an experimental rabbit model after 48 h of reperfusion and correlate the results with histopathology, liver enzyme activities, level of TNF-a and histopathology.
Materials and Methods
1. Experimental models and Experimental Groups
Seven groups of animals (n=6 each) were used (one sham and six I/R groups). The I/R models divided into 0.5 h,2 h,6 h,12 h,24 h and 48 h groups according the reperfusion. The hepatic I/R procedures were same with the Part One.
2. Examination methods and Parameters
T2WI, T1WI, DWI, DTI, and gadolinium-enhanced T1WI were performed in a 3.0T MR scanner (Signa Excite) with an eight-channel phased-array head coil. The rabbit was placed in a supine position in the coil with head first. The scanning parameters used for morphologic imaging sequences (T1WI) were TR8.5/TE4.0 ms and spoiled gradient-recalled echo (SPGR) in T1WI, and fast spin echo(FSE)in T2WI, TR 3200/TE 85 ms,20×20 cm FOV,3 NEX,3 mm thickness layer, and 288×224 matrix. The scanning parameters used for DWI included echo-planar imaging (EPI) series using multitude b values of 50,100,200,300,500, and 600 s/mm2, repetition time (TR) 2000 ms, echo time (TE) 49.3 ms,20 cm/20 cm field of view (FOV),4 NEX,4 mm thickness layer, and 128/128 matrix. For DTI, fat-saturated coronal single-shot echo-planar imaging (EPI) sequences were performed in 6 directions with b values of 100,300, and 600 s/mm2, repetition time (TR) 2600 ms, echo time (TE) 49.3 ms,20×20 cm field of view (FOV),4 NEX,4 mm thickness layer, and 128x128 matrix. The gadolinium-enhanced T1WI was acquired after 0.2 mmol/kg body weight gadopentetate dimeglumine (Gd-DTPA) was administered manually through three-way catheters, followed by 8 ml saline manned quickly.
3. MR Image Analysis
The liver parenchyma in DWI/DTI was manually delineated at a GE AW4.3 FuncTool Performance workstation with dedicated software (GE Medical Systems) and co-registered using Automated Image Registration to correct for any misregistration caused by the body motions during DWI/DTI and the gradient eddy current related image distortions. A region of interest (ROI) was defined in the axial DWI/DTI images within 15～18 mm2 in the liver parenchyma. On DWI/DTI-derived ADC/DCavg/FA maps involving multitude b values, the left lobes were delineated by copying and pasting the ROI from the corresponding DWI/DTI images at three consecutive slices and 3 ROI were determined in the affected liver lobe in each slice.
4. Biochemical and histological studies
The same was with the Part One.
5. Statistical analysis
Statistical analyses were performed with SPSS 13.0 software. Values were expressed as means±SD. For all tests, a P value less than 0.05 was considered to indicate a statistically significant difference. One-way analysis of variance (ANOVA) was used to determine differences. Multiple linear regressions was used to compare multiple correlation coefficient (R) of histomorphologic scores, serum levels and TNF-a with DWI/DTI parameters.
Results
1. Morphologic MRI finding
Compared to the sham group, the slightly high signal of the whole left lobe was observed in the 0.5 h groups (T2WI) as well as the enhancement of the affected lobe decreased significantly. However, the signal of the left lobe was reappearing relatively normal on the T2WI and enhanced T1WI images in the 2 h group. As I/R progressed, the marginal areas of affected liver appeared as uneven spots of higher signal (T2WI). The patch-like or wedge-shaped high signal on T2WI had become more evident, usually at the periphery of the liver, which corresponded to a marked unenhanced necrotic area in the 24 h and 48 h groups.
2. ADC changes
The overall changes in ADC decreased significantly at the early I/R phase (0.5 h), and drastically increased in the 2 h group, and then ascended slightly from 6 h to 48 h after reperfusion, except for a transient decrease (24 h).
The ADC values in 0.5 h (all P<0.001)and 24 h groups (b=50 s/mm2, P=0.037; b=100s/mm2, P=0.020; b=200 s/mm2, P=0.016; b=300 s/mrn2, P=0.001))were significantly lower than that of the sham group when the b-values were no more than 300 s/mm2. Note that the ADC of the 2 h group rose abruptly in contrast to the sham group, especially when the b-value was lower than 300 s/mm2 at every time point, and the difference did not reach statistical significance compared to sham group. The ADC increased significantly in the 6 h group, but still lower than that of sham group (b=50 s/mm2, P=0.029; b=200 s/mm2, P=0.013; b=300 s/mm2, P=0.021). There were no significant differences between sham and 12h group. The ADC significantly increased in the 48 h group in contrast to the sham group when the b-values were 300,500 and 600 s/mm2 (all P<0.001).
3. DCavg values
The overall change in DCavg was similar to ADC's:DCavg decreased significantly at the early I/R phase (0.5 h) and was significantly lower than those in the sham group when b values of 100 (P<0.001)and 300 s/mm2(P=0.008). Then drastically increased in the 2 h group and significantly higher than sham group when b=100 s/mm2(P=0.021). The DCavg values of the 6 h group were significantly higher than that of the sham group when the b-values were 100(P=0.021) and 300 s/mm2 (P=0.002). In the 24 h group, the DCavg values were also lower in comparison with the sham group when b=100(P=0.008) and 300 s/mm2 (P=0.042). The differences in DCavg value between 48 h group and sham group were more obviously with the b values increasing(b=300 s/mm2, P=0.012; b=600 s/mm2, P=0.002).
4. FA values
Meanwhile, FA had an almost opposite trend and increased drastically in the 0.5 h group(b=100 s/mm2, P=0.032; b=300 s/mm2, P<0.001), and then declined slightly in the 2 h group(b=100 s/mm2, P<0.001; b=300 s/mm2, P=0.004). After that, FA decreased slightly in the 6 h and 12 h group, but the difference did not reach statistical significance compared to sham group. With injury progressed, the FA values were kept decreasing 24-48 h of reperfusion, and the FA values in the 24 h (b=300 s/mm2, P=0.018; b=600 s/mm2, P=0.006) and 48 h (b=100 s/mm2, P=0.044; b=300 s/mm2,P< 0.001; b=600 s/mm2, P< 0.001) were significantly decreased compared to the sham group.
5. Biochemical findings
There were significant differences between levels of ALT (F=121.404, P<0.001), AST (F=489.615, P<0.001), ALP (F=835.325, P<0.001) in I/R and sham groups.
Compared to those in the sham group, the levels of TNF-a in I/R groups were clearly higher at every time point (F=171.803, P<0.001).
6. Histomorphologic scores
The overall changes of integral scores increased significantly from 2 h to 48 h groups compared with sham group except for a transient sharply rise in the 0.5 h group (F=119.350, P<0.001).
7. Multiple linear stepwise regressions
Our data demonstrated a good relationship between ADC values (b=100 s/mm2, R=0.756; b=500/mm2, R=0.782), biochemical levels and pathologic index. The FA values (b=300 s/mm2, R=0.781) were also closely related to histomorphologic scores and hepatic biochemistry.
Conclusions
1. This study showed that complex and dramatic changes in ADC/DCavg/FA could be dynamically. We have proposed explanations for the complex mechanisms and multifactorial pathophysiologic processes related to the pathophysiology of hepatic I/R and determined the corresponding various and dramatic changes. These parameters markedly increased/decreased after 2 h of reperfusion, which make the overall trend of I/R have a transient recovery phenomenon similar to the trend of histomorphologic scores.
2.3.0T DTI/DWI can dynamically monitor the pathologic processes of liver ischemia reperfusion injury and reveal the microvascular disorder with a perfusion-sensitive DTI/DWI at the lower b values (<300 s/mm2), particularly at early stages, and can accurately reflect the injury of the liver tissue, necrosis and other pathological changes in the late phase of I/R due to the significant difference between sham and I/R groups with larger b values (b=500 and 600 s/mm2).
3. The trend of DCavg values was similar to ADC's. In the early phase of I/R, FA had an opposite trend in contrast to DCavg, and declined gradually in the late stage, which was accordance with the increasing dissolution of necrotic liver tissue and the disappearance of normal micro-spatial.
4. More importantly, there were good correlation between DTI/DWI parameters, and biochemical levels and pathologic index, which suggests that the DTI/DWI be a feasible and useful tool to prevent early postoperative liver failure, and clinical application.
5. DTI/DWI can monitor the pathologic processes of liver ischemia reperfusion injury, and reveal the micro vascular disorder and morphology changes by dramatic changes in parameters, together with perfusion parameters, might explore the mechanism of I/R, especially for potential benefits of decision making and clinical management after liver surgery.
PartⅢCT Perfusion imaging of Partial Hepatic Ischemia Reperfusion in a Rabbit Model
Objective
This experimental study was designed to evaluate changes in perfusion parameters in rabbits with partial hepatic ischemia reperfusion injury (I/R) and correlate the components of hepatic perfusion with histopathology and liver enzyme, and which hepatic perfusion parameters may be used to monitor the microvascular perfusion of hepatic I/R.
Materials and Methods
1. Experimental models and Experimental Groups
Seven groups of animals (n=6 each) were used (one sham and six I/R groups). The I/R models divided into 0.5 h,2 h,6 h,12 h and 24 h groups according the reperfusion. The hepatic I/R procedure was same with the Part One.
2. Examination methods and Parameters
Before CT scanning, rabbits were fixed on a board in a supine position and an abdominal bandage was applied to reduce movement artifact. CT perfusion imaging was performed on a 256-slice detector CT (Philips Brilliance iCT). After location scans were acquired CT perfusion examinations were performed by a JOG mode scan with a fixed 0.625 mm thick slice including the entire liver and spleen, and post injection delay 2 s before the intravenous bolus injection of contrast material (2 ml/kg, Ultravist 370 mgI/ml) and 10 ml saline at the rate of 1.5 ml/s through the auricular vein using a double power injector, and continued for 50～60 s. The CT perfusion imaging scanning parameters were as follows:detector collimation,128x0.625 mm; thickness/ increment,1 mm/-0.5 mm; rotation time,0.4 s; pitch 0.915; 80 mAs,120 kVp; and matrix,512×512; Number of scans 11; scan interval 3 s.
3. Image analysis
Data were collected and transferred into the EBW4.0I Philips workstation. Then perfusion images of HAP, HPP, TLP and HPI were produced. For every rabbit,3 consecutive axial slices were analyzed and 3 ROI (ranged in size from 0.5 to 2.5 cm2) were determined in the affected liver lobe in each slice. Within 2 h after reperfusion, ROI randomly was distributed on left lobe. Three ROI were placed into the infarcted liver tissue and 3 ROI in the non-infarcted area in the 6-24 h groups. The mean values of each 9 ROI for the viable liver tissue and nonenhancement of the necrotic tissue for every I/R model were calculated, respectively, and were expressed as relative mean values. Also, the infarcted and non-infarcted tissues in the 6,12, and 24 h groups were measured and expressed as mean values for each I/R model.
4. Biochemical and histologic studies
The same with the Part One.
5. Statistical analysis
One-way analysis of variance (ANOVA) was used to determine differences between the CT perfusion parameters. The paired t-test was used for comparison of the differences in CT perfusion parameters (1):between infarcted and non-infarcted area in the 6,12 and 24 h groups; (2):between sham group and infarcted area in the 6, 12 and 24 h groups; (3):between non-infarcted area in the 6,12,24 h groups and sham group. Multiple linear regressions was used to compare multiple correlation coefficient (R) of histomorphologic scores, serum levels and TNF-a with CT perfusion parameters. For all tests, a P value less than 0.05 was considered to indicate a statistically significant difference.
Results
1. Characteristics of CT perfusion map and perfusion parameters changes
Heterogeneity of CT perfusion patterns appeared in the 6 h group (the relatively normal enhancement of viable liver tissue or a low-enhancement of the necrotic tissue could be discriminated). As aggravated injury, the area of low perfusion was gradually expanding and showed a decreased trend of enhancement from 6 h to 24 h of reperfusion.
2 CT perfusion parameters changes
2.1 The overall trend of CT perfusion parameters
The overall changes of HAP, HPP and TLP decreased significantly at the early I/R phase (0.5 h group) except for HPI. HAP drastically increased in the 2 h of reperfusion, and then declined slightly from 6-24 h after reperfusion. HPP were always significant lower than that of sham group, especially in the 0.5 h and 2 h groups.TLP had the similar changes with HAP; However, HPI of I/R groups were always higher than that of sham group.
2.2 Changes of CT perfusion parameters in the low-enhancement of necrotic tissues and enhancement of viable liver tissues
CT perfusion parameters in the low-enhancement tissues increased significantly in the 6 h group and decreased gradually in 12, and 24 h groups, however, the HAP in enhancement of viable liver tissues was decreasing gradually. The HPP decreased significantly not only in the low-enhancement and but also in the enhancement of viable liver tissues. The TLP had a similar change with HPP. In a word, the sharply rise in the HPI implied action of "arterial buffer response"
3. Correlation
The results of multiple linear regressions showed CT perfusion parameters in the necrosis tissues were closely related to histomorphologic scores,serum levels, and TNF-a. Among them, perfusion parameters HPP had the better relationship between them(R=0.855).
Conclusions
1. The enhancement patterns and the CT perfusion parameters showed a temporary recovery in the 2 h group, which reflect the "false normalization"during the process of hepatic I/R.
2. Heterogeneity of CT perfusion patterns appeared in the 6 h group. As aggravated injury, the area of low perfusion was gradually expanding and showed a decreased trend of enhancement from 12 h to 24 h of reperfusion. Eventually, patchy focal necrosis was observed in the 24 h groups.
3. Heterogeneity of CT perfusion parameters occurred after reperfusion. Although the HPP declined in the low-enhancement tissue in the 2 h groups, the HAP increased significantly so as to the TLP remained relatively normal and HAP of infarcted tissues were higher in 6～24 h groups than that of non-infarcted tissues, thus, that may prove the "arterial buffer response".
4. In spite of HAP increased significantly in the 6-24 h groups, HPP and TLP were decreased significantly in the low-enhancement tissue which make the "arterial buffer response" out of action result in the reduction of HPP mainly because of thoroughfare channel. The perfusion in the low-enhancement tissue was actually reducing that further aggravated the liver tissues injury(24 h group).
5. Serum levels, TNF-a and morphological scores were closely correlated to the severity of liver tissue damage as determined by the CT perfusion parameters of the low-enhancement liver tissue. Our result suggested CT perfusion parameters may be a useful parameter in assessing pathologic processes of hepatic I/R.
肝缺血再灌注损伤(ischemia reperfusion injury, I/R)常发生于肝移植、低血容量性和心源性休克以及肝脏肿块和肿瘤的肝叶手术切除中,是肝功能急性损害的重要原因,直接影响患者的恢复和术后生存率。因而,肝I/R损伤保护研究是肝脏外科领域一项重要的课题。肝I//R病理生理过程机制复杂,涉及能量代谢障碍、钙离子超载、微循环障碍、细胞因子、中性粒细胞、氧自由基作用等相关因素。目前,对肝I/R损伤程度的评价多采用肝酶(AST、ALT)、炎性因子(TNF-a)等生化检查指标。虽然这些生化指标可作为一个非常敏感指标反映肝脏损伤程度,但是还不能反映肝I/R后肝组织的形态学特征、微循环障碍及其在空间分布等方面的信息。同时,肝酶以及炎性因子等指标还存在受肝脏术后应急性反应、创伤以及溶血等多方面因素影响,由此可能产生假象,造成不必要再手术风险。
磁共振弥散/张量成像(diffusion weighted/ tensor imaging, DWI/DTI)作为研究功能及微观影像学的重要方法之一,目前已经成为临床评估和生命科学研究重要的工具,在中枢神经系统和相关病变的基础研究已日趋成熟,但应用于腹部脏器还处于探索阶段。肝I/R的病理生理过程包括肝脏微循环的障碍、肝细胞内外水肿以及不断加重的肝细胞坏死等,这些病理过程使细胞内外水分子的运动状态、肝脏微循环的灌注量以及肝组织微观空间结构发生改变,这就使DWI/DTI监测和评估肝I/R的病理机制及发生、发展具有可行性。目前,DWI/DTI对肝I/R的研究尚处于探索阶段,各家结论尚未达成一致。
CT灌注成像(computer tomography perfusion imaging., CTP)具有很高的时间和空间分辨率,作为一种有效、非创性的检查方法,能够同时在微血管的水平上直接评估组织的灌注情况和间接反应微血管形态特征。,而肝I/R是多机制、多种因素参与复杂的一个病理过程,其中肝I/R后肝脏微循环状态的差异性分布,即血液动力学的改变及伴随的肝脏灌注失败一直被认为是其病理过程中重要的因素之一,这正是如何准确评估肝I/R后肝组织微循环障碍所面临的瓶颈所在,是对以往采用肝酶等生化方法来评估肝脏损伤程度的一个很好补充。
本实验拟采用新西兰大白兔制作部分肝I/R模型,探讨肝I/R后组织病理学、肝功能及TNF-a变化特点,探讨3.0 T DWI/DTI及CTP对兔肝I/R诊断价值,以期为肝I/R的诊断和治疗提供一种新的评价方法。
第一部分兔部分肝缺血再灌注损伤模型的建立
研究目的
采用新西兰大白兔做部分肝I/R模型,拟探讨肝I/R后大体形态、组织病理、肝酶(ALT、AST、ALP)及肿瘤坏死因子a (TNF-a)变化特点,以期为肝I/R后功能影像学研究提供病理学基础。
材料与方法
1.实验动物
健康成年、雄性新西兰大白兔,3～4月,体重2.5～3kg。所用实验兔术前均经腹部CTA检查了解肝动脉和门静脉解剖变异情况。
2.肝I/R及sham模型的制作
采用部分肝I/R模型,用无损伤性动脉夹夹闭入肝左叶管道结构(肝左动脉、门静脉左支及左肝管),肝右叶血供未阻断。阻断血供60min后,打开动脉夹恢复血流,肝左叶由暗红色变为鲜红,表示再灌注成功,关闭腹腔。I/R组按照再灌注时间,分为0.5h、2h、6h、12h、24h和48h组。sham组仅解剖肝十二指肠韧带,不阻断血供。
3.生化及病理学检查
3.1肝酶及TNF-a测定
实验兔分别于MRI、CT检查结束后立即经上腔静脉抽取静脉血4ml(肝素管),离心后,分离血清,测定ALT、AST、ALP (U/L)及TNF-a (pg/ml)含量。
3.2病理学检查
取血样后,立即处死实验动物,整体摘取肝脏,肉眼观察大体标本表面和断面解剖特点。将解剖标本制成常规HE染色切片。显微镜下观察肝组织病理学改变,根据肝脏病变程度以半定量方法积分,按各种病变程度乘以不同加权数：肝细胞坏死×(0～3),肝细胞水肿×(0～3),炎细胞浸润×(0～3),淤血×(0～3),计算每只动物肝病变的总积分,然后进行统计学处理。
4.统计学处理
采用SPSS 13.0统计学软件进行数据分析：(1)I/R与sham组间肝酶、炎性因子及形态学积分均进行单因素方差分析(one-way ANOVA);(2)组织形态学积分与肝酶、TNF-a间关系采用Pearson相关；P<0.05作为差异有显著意义的检验标准。
结果
1.兔肝I/R模型构建
成功造模并纳入实验78只,其中sham、0.5-24 h组,每组各12只；48h组6只。
2.病理表现及生化检查
2.1大体解剖
在复血、氧后早期(0.5h、2h组),肝左叶表现为显著的充血肿胀、体积略增大,在其解剖断面上显示明显充血肿胀、肝窦内充满淤积红细胞；随着I/R后时间的延长,肝脏表面颜色逐渐变淡,变成苍白色,在I/R后48 h肝表面出现不规则墨绿色样变,肝段性坏死显著。而未阻断血供的肝右叶表现为显著充血肿胀。
2.2镜下表现
在I/R早期弥漫性肝细胞肿胀,肝细胞内明显水肿,肝窦间隙变窄,中央静脉、小动脉内红细胞大量淤积、微血栓形成(0.5h),随后汇管区出现成簇中性粒细胞,肝窦稍有萎缩,肝细胞水肿略有减轻,肝窦间及汇管区红细胞淤积,并出现少量炎性细胞,部分肝细胞核肿胀、细胞形态改变(2h)；在I/R后6～12h,肝窦及肝实质聚集大量中性粒细胞,出现肝窦阻塞的现象,部分肝组织出现核固缩红染的凋亡细胞；在后期阶段(24～48h),未发生梗死的肝组织显示肝窦萎缩塌陷、解离,梗死区域细胞结构消失,出现溶解坏死的特征。
2.3组织评分结果
各I/R组镜下形态学评分值除0.5h组一过性性的升高外,总体趋势呈逐渐上升,在48h达到最峰值。各I/R组与sham组间存在显著性差异(F=172.702；P<0.001)。
2.4 AST、ALT、ALP及TNF-a
各I/R组血清ALT(F=325.531,P<0.001)、AST(F=1009.602, P<0.001)、ALP(F=1430.899,P<0.001)与sham (?)司均存在显著性差异。ALT峰值在再灌注后6h组,I/R后12～48h逐渐下降。AST在2h显著升高后,缓慢上升,在48h达到最高值；ALP在6h迅速升高,在I/R后24～48h维持峰值。
I/R组血清TNF-a在再灌注后2h迅速升高,并在I/R后12～24h维持峰值,在48h组开始回落。而sham组,TNF- a保持在一个稳定的低水平。经统计学分析,各I/R组与sham间均存在显著性差异(F=172.702,P<0.001)。
3.相关性分析
组织形态学积分与TNF-a(r=0.788, P<0.001)、ALP(r=0.841)、AST (r=0.848, P<0.001)之间存在显著相关性。
结论
1.兔肝I/R模型制作简单、成功率高,并在复血、氧后48h多个时间点观察了其病理发生、发展过程,适合功能影像评价肝I/R实验研究的需求。
2.肝I/R早期以肝细胞水肿、肝窦淤血等病理过程为主(0～2h)；晚期阶段是以肝细胞凋亡、坏死为主(6～48h)。对损伤反应的差异性是其病理病理特点这一。
3.肝细胞水肿、淤血、中性粒细胞浸润、TNF-a等因素是诱导肝I/R发生、发展的重要因素；组织形态学积分与肝酶、TNF-a具有显著相关性。
4.肝I/R后中性粒细胞聚集、内皮细胞损伤、微循环障碍、肝窦萎缩、塌陷以及局灶性坏死等的病理变化特点为其功能成像学研究提供了病理学基础。
第二部分兔部分肝缺血再灌注损伤3.0T弥散加权/张量成像
研究目的
旨在结合肝酶、肿瘤坏死因子(TNF-a)和病理学检查,探讨3.0 T DWI/DTI对兔肝I/R可行性及诊断价值。
材料与方法
1.动物模型的制作及分组
实验兔分为sham与I/R组。肝I/R模型及sham组制作同第一部分。I/R组按照再灌注时间,分为0.5h、2h、6h、12h、24h与48h组。I/R与sham,每组各6只。
2.检查方法及参数
采用GE 3.0T Signa Excite扫描仪,8通道头正交线圈,轴位扫描。扫描参数：T2WI TR/TE 3200/85ms,矩阵288×224,层厚4 mm, NEX3,FOV 20cm×15 cm；T1WI TR/TE 8.5/4 ms,矩阵288×192,层厚4 mm, NEX,3,FOV 22cm×22 cm：EPI-DWI, TR/TE 2000/49.3 ms,矩阵128×128,层厚4 mm, NEX,4,FOV20cm×10cm；弥散方向：ALL模式(X、Y、Z三个方向)。弥散梯度因子b=50、100、200、300、500、600s/mm2；EPI-DTI序列,TR/TE 2000/49.3 ms, NEX,4,FOV20cm×10 cm,层厚4mm,矩阵128×128。b=100、300、600s/mm2。弥散方向：取6个方向同时进行；增强扫描采用T1WI增强,0.2 mmol/kg顺磁性对比剂Gd-DTPA及8ml生理盐水快速手动注入。
3.MR图像分析
采用GE AW4.3 FuncTool Performance工作站进行后处理,先对原始图像采用Correction程序对图像进行校正,以减少伪影和图像变形。重建出表观扩散系数(ADC)、平均扩散系数(DCavg)及各项异性系数(FA)图。感兴趣区(ROI)：15～8mm2,分别测量ADC、DCavg及FA值,连续测量三个层面,取其平均值。
4.组织学和生化检查
同第一部分。
5.统计学分析
使用SPSS13.0软件进行统计学分析,P<0.05为差异有统计意义。多组均数差异的比较采用单因素方差分析(one-way ANOVA)。以不同b值时MR-DWI/DTI参数作为因变量,肝酶、TNF-a及组织形态学积分作为自变量行多元线性回归分析,用复相关系数R来说明MR-DWI/DTI参数与肝酶、TNF-a及组织形态学积分的相关关系。
结果
1.影像学表现
在0.5h组肝左叶T2WI信号弥漫性轻度增高,T1WI强化减低。而在2h组肝组织信号出现暂时性回复正常。再灌注后6～12h,肝脏的边缘出现T2WI上不均匀性点片状稍高信号以及相对应的强化减低区域。24h及48h组,出现明显的片状、楔形或整个肝段高信号(T2WI、DWI)及无强化的梗死区域,与未发生坏死的区域分界较为明显。随着损伤加重,梗死范围(强化减低区域)逐渐扩大。
2.ADC值变化规律
ADC的总体变化趋势是先显著下降(0.5h),再一过性升高(2h),经过6～12h缓慢上升后,24h再出现一个明显减低后,在48h明显升高。
当b≤300s/mm2时,0.5h(均P<0.001)和24h组ADC显著低于sham组(b=50s/mm2,P=0.037；b=100s/mm2,P=0.020；b=200 s/mm2,P=0.016；b=300s/mm2,P=0.001)。值得注意是,ADC于2 h组明显升高(b≤300s/mm2较为显著),虽与sham组之间无明显差异。在6h组ADC显著升高,但仍低于sham组(b=50s/mm2,P=0.029；b=200s/mm2,P=0.013；b=300s/mm2,P=0.021)。12h组与sham间均无统计学差异。与sham对比,48h组ADC显著升高(b≥300s/mm2,均P<0.001)。
3. DCavg值变化规律
各I/R组DCavg与ADC变化特点基本类似：在0.5h组显著下降,且在b=100(P<0.001)、300s/mm2(P=0.008),与sham组间差异存在统计学意义；在I/R后2h显著升高,并在b=100s/mm2时,与sham组间存在统计学意义(P=0.021)；b=100s/mm2(P=0.021)、300s/mm2(P=0.002)时,6h组较sham组显著升高；DCavg值于24 h组也出现一过性下降现象,并在b=100s/mm2(P=0.008)、300s/mm2(P=0.042)时,低于sham组；在I/R后48h显著升高,且随着b值增大,其与sham组间差异性越显著(b=300s/mm2,P=0.012；b=600s/mm2,P=0.002)。
4.FA值变化特点
在I/R早期(0.5h)FA显著升高(b=100s/mm2,P=0.032；b=300s/mm2,P<0.001)、2h显著减低(b=100 s/mm2,P<0.001；b=300s/mm2,P=0.004),并与sham组间差异均存在统计学意义。FA在6～2h组缓慢下降,与sham间无统计学差异。随着肝I/R损伤加重,FA在I/R后24～48h呈下降趋势,并在24 h(b=300s/mm2,P=0.018；b=600s/mm2,P=0.006)、48h(b=100s/mm2,P=0.044；b=300s/mm2,P<0.001；b=600s/mm2,P<0.001)与sham组间存在显著性差异。
5.I/R与sham组血清肝酶及TNF-a
I/R组血清ALT (F=121.404, P<0.001)、AST (F=489.615, P<0.001)、ALP(F=835.325,P<0.001)与sham对比显著升高,组间存在显著性差异。
I/R组血清TNF-a水平明显升高,与sham组间存在显著性差异(F=171.803,P<0.001)。
6.I/R及sham组组织形态学积分
各I/R组镜下形态学积分总体趋势呈逐渐上升,与sham组间存在显著性差异(F=119.350,P<0.001)。
7.多元线性回归分析
ADC (b=100 s/mm2, R=0.756; b=500/mm2, R=0.782), FA (b=300 s/mm2,R=0.781)与肝酶等生化、病理指标间存在较好的相关性。
结论
1.肝I/R功能成像参数ADC. DCavg/FA具有复杂性和多变性特点,其成像参数在I/R后2h出现一过性回复的现象,与组织形态学积分等病理指标具有类似变化特点,是其病理机制的体现。
2.在肝I/R组早期功能成像参数变化较为剧烈(b<300s/mm2),这可间接反映肝微循环障碍等病理信息；采用较大b值(b=500、600s/mm2),在I/R后期阶段功能成像参数与sham间具有较大差异,这对于准确反映I/R后肝组织损伤、坏死等病理过程具有重要意义。
3.在肝I/R早期阶段,FA与DCavg变化呈相反的趋势(0.5～12 h)；而在晚期阶段呈现逐渐下降特点(24～48h),这与肝I/R后肝组织不断溶解坏死,维持其正常微观空间结构消失相一致。
4.肝酶等生化指标与ADC. DCavg/FA参数间存在较好的相关性,证实了DTI/DWI对肝I/R诊断可行性和临床应用价值。
5. DTI/DWI可以作为一种非创性的功能成像反映肝I/R肝细胞的生理状态、微观形态学等方面信息,若结合其他功能成像方法,例如,灌注成像可促进对I/R模型探索研究,为临床的断和治疗提供了一种可行性评价方法。
第三部分兔部分肝缺血/再灌注损伤CT灌注成像
研究目的
观察兔部分肝I/R后CT灌注(CTP)参数演变规律,分析CTP评估肝I/R血流动力学病理基础,并探讨对其最敏感、有效的灌注参数。
材料与方法
1.动物模型的制作及分组
实验兔分为sham与I/R组。肝I/R模型及sham制作同第一部分。I/R组按照再灌注时间,分为0.5h、2h、6h、12h与24h组。I/R与sham,每组各6只。
2.CT灌注检查方法
采用Philips Brilliance iCT(探测器组合0.625mm×256)扫描仪,定位扫描后,确定灌注扫描范围,然后采用全肝灌注模式(JOG)对全肝及脾动态扫描。非离子型对比剂(5ml优维显,370mgI/ml)和生理盐水10ml。注射速率均为1.5ml/s,对比剂注入后延迟2s开始扫描,扫描时间1.5s,共扫描11次,扫描间隔3s。扫描参数：探测器128×0.625mm,层厚1mm,间距-0.5 mm, Pitch0.915,80Kv,100mAs,360°/0.4 s。
3.图像后处理
采用EBW4.0I Philips工作站,自动生成肝动脉灌注量(HAP)、门静脉灌注量(HPP)、总灌注量(TLP)、灌注指数(HPI)等灌注图。肝组织ROI的选取：0.5～3cm2。在I/R后2h内,ROI随机分布的肝左叶,每个层面测量3个ROI,连续测量三个层面；I/R后6-24 h在梗死区域(低灌注区,即强化减低肝组织)与未梗死区(相对正常的强化未减低肝组织)分别测量3个ROI,连续测量三个层面,计算出梗死与未梗死肝组织的平均值以及相应平均值。
4.组织学和生化检查
同第一部分。
5.统计学分析
采用SPSS13.0软件进行统计分析。多组均数差异的比较采用单因素方差分析(One Way ANOVA)。采用配对t检验比较以下CT灌注参数间有无统计学意义：(1)在6h、12h、24h组梗死与未梗死肝组织灌注参数比较；(2) sham与6h、12h、24h组肝左叶梗死肝组织灌注参数比较;(3)sham与6h、12h、24h组未梗死肝组织灌注参数比较。以梗死与未梗死肝组织中CT灌注参数为因变量,肝酶、TNF-α及组织形态学积分为自变量进行多元线性回归分析。用复相关系数(R)来说明CT灌注参数与肝酶、TNF-α及组织形态学积分的相关关系。P<0.05为差异有统计意义。
结果1.各I/R与sham组间CT灌注变化特点
与sham组相比,0.5h组表现为整个肝左叶的显著强化减低,2h组表现为明显均匀强化,在I/R后6h兔肝左叶呈现差异性灌注(低灌注区出现,即强化减低区),且随着再灌注时间的延长(12～24h),梗死的范围逐渐扩大,强化程度也呈减低趋势。
2.CT灌注参数变化特点
2.1总的变化趋势
CT灌注参数在I/R早期(0.5h组)除HPI外,均表现为显著减低；HAP于2h显著升高后,在I/R后6～24 h均表现为逐渐下降；HPP一直低于sham,尤其是在I/R后0.5-2 h；TLP变化特点与HAP基本类似。HPI作为相对性指数,各I/R组均高于sham组。
2.2梗死及未梗死肝组织CT灌注参数的变化特点
HAP于6h组梗死肝组织显著上升后,在12h、24h组呈现逐渐下降的趋势,而在未梗死的肝组织中HAP是呈现逐渐下降的趋势(6～24h)；HPP不论在梗死或未梗死的肝组织均呈现显著下降的趋势,但在梗死的肝组织内下降的幅度更大；TLP在梗死以及未梗死肝组织中变化特点与HPP基本类似；在梗死的肝组织内HPI显著升高,这也说明了肝动脉缓冲效应机制作用。
3.相关性分析
在肝I/R后梗死组织CT灌注参数与肝生化指标及组织形态学等多个自变量间的具有较好的线性相关,以梗死组织中HPP相关性最好(R=0.855)。
结论
1.在肝I/R后2h,CT强化模式以及CT灌注参数出现暂时性回复,是其“假正常化”病理机制的体现。
2.在肝I/R后6h出现了灌注性差异,且随着时间的延长,其强化程度逐渐减低,梗死的范围逐渐扩大(12～24h),在I/R后24h出现局灶性坏死。
3.肝I/R后,CT灌注参数出现了显著的差异性,在强化减低肝组织中I/R早期(2 h) HAP显著升高,而HPP明显降低,TLP保持相对稳定；HAP在梗死的肝组织(12～24h)明显高于未梗死的肝组织—肝脏动脉缓冲效应基础机制的存在。
4.HAP在梗死的肝组织中虽明显升高(12～24h),但HPP与TLP显著降低,其根本原因是肝内门体分流(末端小动静脉开放),致使肝动脉缓冲效应作用降低,最终导致门静脉的灌注量减低(24 h)。肝血流量实际上减低,进一步加剧肝实质的损害。
5.肝酶、TNF-α、形态学积分与梗死肝组织CT灌注参数间具有相关性。因此,CT灌注参数可以作为敏感、有效的指标监测肝I/R后血流动力学、肝组织的损伤及坏死程度敏感指标。
Background
Hepatic ischemia-reperfusion injury (I/R) is a problem in clinical medicine, which is a critical factor that affects organ and patient survival, especially after liver transplantation, hypovolemia, cardiac shock or massive hepatic tumor resection, and it is also responsible for acute liver failure and is usually associated with high morbidity and mortality. In short, hepatic I/R is a topic issue in the field of hepatic surgery research. Accumulating evidence suggests that energy metabolism reduction, calcium overload, microcirculation perfusion failure, cytokines disorders, neutrophils aggregation and infiltration, the effect of oxygen free radicals subsequent to I/R is a major factor in the pathogenesis of I/R-mediated liver injury. Although serum levels of aspartate and alanine transaminase (AST and ALT) activities, and the levels of tumor necrosis factor-a (TNF-a) have been used as indexes for estimating the degree of liver functional loss, they do not give morphologic information on the spatial distribution of liver injury. Moreover, laboratory parameters (liver enzymes activities and inflammatory factors levels), although valuable, could also be biased by postoperative bleeding and transfusion therapy with fresh-frozen plasma and blood, which may be lead to a pseudomorph and risk of a (resurgery) or (re-)transplantation.
Diffusion weighted/tensor imaging (DWI/DTI) has become a function and micro-imaging MRI modality that allows visual assessment of the Brownian motion of water molecules and in vivo measurement of molecular diffusion and has become an important tool in life science research and clinical practice. At present, DWI/DTI has been extensively evaluated in the central nervous system. However, DWI/DTI is not a routine approach for the characterization of liver diseases.
The pathologic mechanism of ischemic tissue injury includes ischemia and induced hypoxia initially. A cascade of deterioration is further aggravated by secondary reperfusion damage resulting from intra- and extracellular edema, through membrane disintegration, microcirculation perfusion disturbances, and eventually irreversible cytolytic necrosis. Therefore, such continuing changes can be detected by DWI/DTI as more restricted water diffusion initially on account of the diffuse cellular edema and subsequent enhanced water diffusion due to cytolysis. Yet, researches related to hepatic I/R have not reached to agreement and still in the initial phase.
Multi-slice computed tomography perfusion imaging (CTP) as an effective, non-invasive technique that allows for quantitative and objective quantitative assessment of angiogenesis, blood perfusion, and vascular permeability with high temporal and spatial resolution. More evidence suggests that microcirculatory disturbances and capillary perfusion failure subsequent to I/R is a major factor in the pathogenesis of I/R-mediated liver injury, which increases in the heterogeneity of hepatic microvascular perfusion, thus, suggesting that CT together with results of laboratory tests, perfusion parameters may be well suited for the in vivo assessment of hepatic I/R.
Therefore, the subjective of this study was to establish partial liver I/R in New Zealand white rabbit models and observe the changes of histopathology, liver function loss and level of TNF-a, and to the determine whether DWI/DTI/CTP could be used to characterize hepatic I/R quantitatively, providing a valuable and practicable method for diagnosis and evaluation of hepatic I/R.
Part I Establishment of Partial Hepatic Ischemia Reperfusion in a Rabbit Model
Objective
This study was to establish partial liver I/R in New Zealand white rabbit models and observe the characteristics of gross morphology, histopathology, changes of liver function and level of TNF-a, and to provide pathological basis for functional imaging studies of hepatic I/R.
Materials and Methods
1. Animals
The experiments were performed with 3～4 month-old adult and male New Zeland rabbits, weighing 2.5 to 3.0 kg. All the experimental rabbits underwent computer tomography angiography (CTA) preoperatively to analyze anatomy variation of hepatic artery and portal vein.
2. Surgical Procedures and Experimental Groups
Seven animal groups were used. The left portal vein and hepatic artery were occluded for 60 minutes with a vascular clamp to produce ischemia. The right hepatic lobe was perfused to prevent intestinal congestion. (Thereafter, the clamp was removed to allow blood flow through the liver (reperfusion) for 0.5,2,6,12,24 and 48 h, correspondingly. Animals undergoing laparotomy in parallel without vascular occlusion served as sham-operated controls (sham group).
3. Biochemistry assays and histological studies
3.1 Serum Assays and level of TNF-a
Blood samples were collected from the supeirior mesenteric vein with heparinized capillary tubes after MRI and CT were completed at every time point. The serum was separated from the whole blood by centrifugation to determine the ALT, AST and ALP activities in the serum, and the results were expressed as international units per liter.
Using ELISA kit, serum levels of TNF-a in I/R and sham groups were detected by 3 times, and the mean value were expressed as pg per milliliter.
3.2 Histological studies
To perform biopsies, animals were sacrificed after drawing blood from the superior mesenteric vein, after that, to observe the characteristic of gross anatomy. Then, the liver was rapidly excised, fixed (formalin 10% neutral-buffered), embedded (paraffin), sectioned, and stained with H&E. Histologic analyses of tissue slices were performed randomly. The integration of liver injury using semi-quantitative method according to the degree of various diseases multiplied by different weights:liver cell necrosis×(0～3), cells swelling×(0～3), inflammatory cell infiltration×(0～3), congestion×(0～3). To calculate each animal's total scores, and then were analyzed by statistics.
4. Statistical analysis
SPSS13.0 statistical software was used for the analysis. (1):One-way analysis of variance (ANOVA) was used to determine differences between the indexes including serum levels of (AST, ALT and ALP), TNF-a and scores of histomorphologic in I/R and sham groups. (2):The correlations of histomorphologic scores with serum levels and TNF-a were analyzed using Pearson's correlation coefficient. For all tests, a P value less than 0.05 was considered to indicate a statistically significant difference.
Results
1. The result of establishment models
The study ultimately included seventy-eight experimental rabbits, which were divided into 6 I/R groups (0.5～24 h groups, n=12 each; 48 h group n=6) and one sham group (n=12 each).
2. Histologic findings and biochemistry analysis
2.1 Gross anatomy
The affected liver lobe appeared hyperemic or congestive compared to the sham group at the early stage(0.5 and 2 h group), subsequently, the liver's color gradually fades, became kermesinus, having "pattern-like" appearance(12-24 h), and infarction area present with dark green at edge of the liver(48 h). The right lobe without blocking the blood supply showed marked congestion and swelling.
2.2 Under microscope
The histopathologic changes after hepatic I/R included formation of intracellular edema and, hence, diffuse swelling of hepatocytes, narrowing of the lumen, and sinusoidal congestion. Destroyed erythrocytes were congested in and outside the sinusoidal space and the central vein (CV) in the initial phase (0.5 and 2 h group). With aggravated injury, inflammatory cells aggregated in the hepatic microvasculature and extravasated into the hepatic sinusoid lumen and hepatic parenchyma (6 and 12 h group). Some hepatocytes nuclear were staining and condensation of apoptotic cells in the affected tissues. Eventually, widespread cell necrosis/apoptosis, sinusoidal dissociation, and patchy focal necrosis were observed in the 24 and 48 h groups.
2.3 The histomorphologic scores
The general trend of histomorphologic scores in I/R groups kept on increasing except for a sharp increase of 0.5 h group, and peaked at 48 h after reperfusion. There were significant differences between sham and I/R groups(F=172.702, P<0.001).
2.4 Biochemical findings
Compared to those in the sham group, the levels of ALT (F=325.531, P<0.001), AST(F=1009.602, P<0.001) and ALP (F=1430.899, P<0.001) in the I/R group were clearly higher at every time point. The levels of ALT in the I/R groups markedly increased significantly in the 2 h and peaked at 6 h after reperfusion and decline gradually from 12 h to 48 h of reperfusion. AST increased at 2 h of reperfusion, peaking at the end of the perfusion (48 h), and ALP increased rapidly in the 6 h group and peaked at 24～48 h after reperfusion.
The levels of TNF-a in the I/R groups was increased significantly in the 2 h group and peaked at 12～24 h of reperfusion, and declined in the 48 h group. The level of TNF-a remained at stable and relatively low level in the sham group. Compared to the sham group, the levels of TNF-a in the I/R group were clearly higher at every time point (F=172.702, P<0.001).
3. Correlations
Our result demonstrated a good relationship between histomorphologic scores and serum levels (ALT, AST, and ALP) and TNF-a in the sham and I/R groups (r=0.788, r=0.841 and r=0.848, respectively, P<0.01).
Conclusions
1. Our study was to follow up and obverse the process of pathogenesis in a rabbit partial hepatic I/R model with relatively simple and high success rate during a long time window of 48 hours after reperfusion, therefore, may be suitable for functional imaging evaluation of hepatic I/R.
2. Two phases of hepatic injury followed by I/R:the initial phase (0-2 h of reperfusion), associated with the generation of toxic hepatocytes swelling, congestion, and a later phase (6-48 h post-reperfusion) associated with a rather intense state of neutrophils aggregation and subsequent to hepatocyte necrosis. One of the basic pathological features was the heterogeneity response to the injury factors after reperfusion.
3. These factors inducing in the course of the I/R, such as toxic hepatocytes swelling, congestion, neutrophil aggregation and infiltration, and release of TNF-a, were the major factors in the process of I/R pathogenesis. Morphological scores were positively correlated to the level of liver enzymes (AST, ALT, and ALP) and TNF-a.
4. A cascade of deterioration is further aggravated by secondary reperfusion damage resulting from intra-and extracellular edema, neutrophils aggregation, microcirculation perfusion disturbances, sinusoidal dissociation, and subsequent patchy focal necrosis and eventually irreversible cytolytic necrosis, thus, suggesting that function imaging methods may be well appropriate for the in vivo assessment of hepatic I/R.
Part II Diffusion weighted/tensor imaging of partial hepatic ischemia reperfusion injury in a rabbit model at 3.0T
Objective
The intention of this study was to determine whether DWI/DTI could be used to characterize hepatic I/R quantitatively in an experimental rabbit model after 48 h of reperfusion and correlate the results with histopathology, liver enzyme activities, level of TNF-a and histopathology.
Materials and Methods
1. Experimental models and Experimental Groups
Seven groups of animals (n=6 each) were used (one sham and six I/R groups). The I/R models divided into 0.5 h,2 h,6 h,12 h,24 h and 48 h groups according the reperfusion. The hepatic I/R procedures were same with the Part One.
2. Examination methods and Parameters
T2WI, T1WI, DWI, DTI, and gadolinium-enhanced T1WI were performed in a 3.0T MR scanner (Signa Excite) with an eight-channel phased-array head coil. The rabbit was placed in a supine position in the coil with head first. The scanning parameters used for morphologic imaging sequences (T1WI) were TR8.5/TE4.0 ms and spoiled gradient-recalled echo (SPGR) in T1WI, and fast spin echo(FSE)in T2WI, TR 3200/TE 85 ms,20×20 cm FOV,3 NEX,3 mm thickness layer, and 288×224 matrix. The scanning parameters used for DWI included echo-planar imaging (EPI) series using multitude b values of 50,100,200,300,500, and 600 s/mm2, repetition time (TR) 2000 ms, echo time (TE) 49.3 ms,20 cm/20 cm field of view (FOV),4 NEX,4 mm thickness layer, and 128/128 matrix. For DTI, fat-saturated coronal single-shot echo-planar imaging (EPI) sequences were performed in 6 directions with b values of 100,300, and 600 s/mm2, repetition time (TR) 2600 ms, echo time (TE) 49.3 ms,20×20 cm field of view (FOV),4 NEX,4 mm thickness layer, and 128x128 matrix. The gadolinium-enhanced T1WI was acquired after 0.2 mmol/kg body weight gadopentetate dimeglumine (Gd-DTPA) was administered manually through three-way catheters, followed by 8 ml saline manned quickly.
3. MR Image Analysis
The liver parenchyma in DWI/DTI was manually delineated at a GE AW4.3 FuncTool Performance workstation with dedicated software (GE Medical Systems) and co-registered using Automated Image Registration to correct for any misregistration caused by the body motions during DWI/DTI and the gradient eddy current related image distortions. A region of interest (ROI) was defined in the axial DWI/DTI images within 15～18 mm2 in the liver parenchyma. On DWI/DTI-derived ADC/DCavg/FA maps involving multitude b values, the left lobes were delineated by copying and pasting the ROI from the corresponding DWI/DTI images at three consecutive slices and 3 ROI were determined in the affected liver lobe in each slice.
4. Biochemical and histological studies
The same was with the Part One.
5. Statistical analysis
Statistical analyses were performed with SPSS 13.0 software. Values were expressed as means±SD. For all tests, a P value less than 0.05 was considered to indicate a statistically significant difference. One-way analysis of variance (ANOVA) was used to determine differences. Multiple linear regressions was used to compare multiple correlation coefficient (R) of histomorphologic scores, serum levels and TNF-a with DWI/DTI parameters.
Results
1. Morphologic MRI finding
Compared to the sham group, the slightly high signal of the whole left lobe was observed in the 0.5 h groups (T2WI) as well as the enhancement of the affected lobe decreased significantly. However, the signal of the left lobe was reappearing relatively normal on the T2WI and enhanced T1WI images in the 2 h group. As I/R progressed, the marginal areas of affected liver appeared as uneven spots of higher signal (T2WI). The patch-like or wedge-shaped high signal on T2WI had become more evident, usually at the periphery of the liver, which corresponded to a marked unenhanced necrotic area in the 24 h and 48 h groups.
2. ADC changes
The overall changes in ADC decreased significantly at the early I/R phase (0.5 h), and drastically increased in the 2 h group, and then ascended slightly from 6 h to 48 h after reperfusion, except for a transient decrease (24 h).
The ADC values in 0.5 h (all P<0.001)and 24 h groups (b=50 s/mm2, P=0.037; b=100s/mm2, P=0.020; b=200 s/mm2, P=0.016; b=300 s/mrn2, P=0.001))were significantly lower than that of the sham group when the b-values were no more than 300 s/mm2. Note that the ADC of the 2 h group rose abruptly in contrast to the sham group, especially when the b-value was lower than 300 s/mm2 at every time point, and the difference did not reach statistical significance compared to sham group. The ADC increased significantly in the 6 h group, but still lower than that of sham group (b=50 s/mm2, P=0.029; b=200 s/mm2, P=0.013; b=300 s/mm2, P=0.021). There were no significant differences between sham and 12h group. The ADC significantly increased in the 48 h group in contrast to the sham group when the b-values were 300,500 and 600 s/mm2 (all P<0.001).
3. DCavg values
The overall change in DCavg was similar to ADC's:DCavg decreased significantly at the early I/R phase (0.5 h) and was significantly lower than those in the sham group when b values of 100 (P<0.001)and 300 s/mm2(P=0.008). Then drastically increased in the 2 h group and significantly higher than sham group when b=100 s/mm2(P=0.021). The DCavg values of the 6 h group were significantly higher than that of the sham group when the b-values were 100(P=0.021) and 300 s/mm2 (P=0.002). In the 24 h group, the DCavg values were also lower in comparison with the sham group when b=100(P=0.008) and 300 s/mm2 (P=0.042). The differences in DCavg value between 48 h group and sham group were more obviously with the b values increasing(b=300 s/mm2, P=0.012; b=600 s/mm2, P=0.002).
4. FA values
Meanwhile, FA had an almost opposite trend and increased drastically in the 0.5 h group(b=100 s/mm2, P=0.032; b=300 s/mm2, P<0.001), and then declined slightly in the 2 h group(b=100 s/mm2, P<0.001; b=300 s/mm2, P=0.004). After that, FA decreased slightly in the 6 h and 12 h group, but the difference did not reach statistical significance compared to sham group. With injury progressed, the FA values were kept decreasing 24-48 h of reperfusion, and the FA values in the 24 h (b=300 s/mm2, P=0.018; b=600 s/mm2, P=0.006) and 48 h (b=100 s/mm2, P=0.044; b=300 s/mm2,P< 0.001; b=600 s/mm2, P< 0.001) were significantly decreased compared to the sham group.
5. Biochemical findings
There were significant differences between levels of ALT (F=121.404, P<0.001), AST (F=489.615, P<0.001), ALP (F=835.325, P<0.001) in I/R and sham groups.
Compared to those in the sham group, the levels of TNF-a in I/R groups were clearly higher at every time point (F=171.803, P<0.001).
6. Histomorphologic scores
The overall changes of integral scores increased significantly from 2 h to 48 h groups compared with sham group except for a transient sharply rise in the 0.5 h group (F=119.350, P<0.001).
7. Multiple linear stepwise regressions
Our data demonstrated a good relationship between ADC values (b=100 s/mm2, R=0.756; b=500/mm2, R=0.782), biochemical levels and pathologic index. The FA values (b=300 s/mm2, R=0.781) were also closely related to histomorphologic scores and hepatic biochemistry.
Conclusions
1. This study showed that complex and dramatic changes in ADC/DCavg/FA could be dynamically. We have proposed explanations for the complex mechanisms and multifactorial pathophysiologic processes related to the pathophysiology of hepatic I/R and determined the corresponding various and dramatic changes. These parameters markedly increased/decreased after 2 h of reperfusion, which make the overall trend of I/R have a transient recovery phenomenon similar to the trend of histomorphologic scores.
2.3.0T DTI/DWI can dynamically monitor the pathologic processes of liver ischemia reperfusion injury and reveal the microvascular disorder with a perfusion-sensitive DTI/DWI at the lower b values (<300 s/mm2), particularly at early stages, and can accurately reflect the injury of the liver tissue, necrosis and other pathological changes in the late phase of I/R due to the significant difference between sham and I/R groups with larger b values (b=500 and 600 s/mm2).
3. The trend of DCavg values was similar to ADC's. In the early phase of I/R, FA had an opposite trend in contrast to DCavg, and declined gradually in the late stage, which was accordance with the increasing dissolution of necrotic liver tissue and the disappearance of normal micro-spatial.
4. More importantly, there were good correlation between DTI/DWI parameters, and biochemical levels and pathologic index, which suggests that the DTI/DWI be a feasible and useful tool to prevent early postoperative liver failure, and clinical application.
5. DTI/DWI can monitor the pathologic processes of liver ischemia reperfusion injury, and reveal the micro vascular disorder and morphology changes by dramatic changes in parameters, together with perfusion parameters, might explore the mechanism of I/R, especially for potential benefits of decision making and clinical management after liver surgery.
PartⅢCT Perfusion imaging of Partial Hepatic Ischemia Reperfusion in a Rabbit Model
Objective
This experimental study was designed to evaluate changes in perfusion parameters in rabbits with partial hepatic ischemia reperfusion injury (I/R) and correlate the components of hepatic perfusion with histopathology and liver enzyme, and which hepatic perfusion parameters may be used to monitor the microvascular perfusion of hepatic I/R.
Materials and Methods
1. Experimental models and Experimental Groups
Seven groups of animals (n=6 each) were used (one sham and six I/R groups). The I/R models divided into 0.5 h,2 h,6 h,12 h and 24 h groups according the reperfusion. The hepatic I/R procedure was same with the Part One.
2. Examination methods and Parameters
Before CT scanning, rabbits were fixed on a board in a supine position and an abdominal bandage was applied to reduce movement artifact. CT perfusion imaging was performed on a 256-slice detector CT (Philips Brilliance iCT). After location scans were acquired CT perfusion examinations were performed by a JOG mode scan with a fixed 0.625 mm thick slice including the entire liver and spleen, and post injection delay 2 s before the intravenous bolus injection of contrast material (2 ml/kg, Ultravist 370 mgI/ml) and 10 ml saline at the rate of 1.5 ml/s through the auricular vein using a double power injector, and continued for 50～60 s. The CT perfusion imaging scanning parameters were as follows:detector collimation,128x0.625 mm; thickness/ increment,1 mm/-0.5 mm; rotation time,0.4 s; pitch 0.915; 80 mAs,120 kVp; and matrix,512×512; Number of scans 11; scan interval 3 s.
3. Image analysis
Data were collected and transferred into the EBW4.0I Philips workstation. Then perfusion images of HAP, HPP, TLP and HPI were produced. For every rabbit,3 consecutive axial slices were analyzed and 3 ROI (ranged in size from 0.5 to 2.5 cm2) were determined in the affected liver lobe in each slice. Within 2 h after reperfusion, ROI randomly was distributed on left lobe. Three ROI were placed into the infarcted liver tissue and 3 ROI in the non-infarcted area in the 6-24 h groups. The mean values of each 9 ROI for the viable liver tissue and nonenhancement of the necrotic tissue for every I/R model were calculated, respectively, and were expressed as relative mean values. Also, the infarcted and non-infarcted tissues in the 6,12, and 24 h groups were measured and expressed as mean values for each I/R model.
4. Biochemical and histologic studies
The same with the Part One.
5. Statistical analysis
One-way analysis of variance (ANOVA) was used to determine differences between the CT perfusion parameters. The paired t-test was used for comparison of the differences in CT perfusion parameters (1):between infarcted and non-infarcted area in the 6,12 and 24 h groups; (2):between sham group and infarcted area in the 6, 12 and 24 h groups; (3):between non-infarcted area in the 6,12,24 h groups and sham group. Multiple linear regressions was used to compare multiple correlation coefficient (R) of histomorphologic scores, serum levels and TNF-a with CT perfusion parameters. For all tests, a P value less than 0.05 was considered to indicate a statistically significant difference.
Results
1. Characteristics of CT perfusion map and perfusion parameters changes
Heterogeneity of CT perfusion patterns appeared in the 6 h group (the relatively normal enhancement of viable liver tissue or a low-enhancement of the necrotic tissue could be discriminated). As aggravated injury, the area of low perfusion was gradually expanding and showed a decreased trend of enhancement from 6 h to 24 h of reperfusion.
2 CT perfusion parameters changes
2.1 The overall trend of CT perfusion parameters
The overall changes of HAP, HPP and TLP decreased significantly at the early I/R phase (0.5 h group) except for HPI. HAP drastically increased in the 2 h of reperfusion, and then declined slightly from 6-24 h after reperfusion. HPP were always significant lower than that of sham group, especially in the 0.5 h and 2 h groups.TLP had the similar changes with HAP; However, HPI of I/R groups were always higher than that of sham group.
2.2 Changes of CT perfusion parameters in the low-enhancement of necrotic tissues and enhancement of viable liver tissues
CT perfusion parameters in the low-enhancement tissues increased significantly in the 6 h group and decreased gradually in 12, and 24 h groups, however, the HAP in enhancement of viable liver tissues was decreasing gradually. The HPP decreased significantly not only in the low-enhancement and but also in the enhancement of viable liver tissues. The TLP had a similar change with HPP. In a word, the sharply rise in the HPI implied action of "arterial buffer response"
3. Correlation
The results of multiple linear regressions showed CT perfusion parameters in the necrosis tissues were closely related to histomorphologic scores,serum levels, and TNF-a. Among them, perfusion parameters HPP had the better relationship between them(R=0.855).
Conclusions
1. The enhancement patterns and the CT perfusion parameters showed a temporary recovery in the 2 h group, which reflect the "false normalization"during the process of hepatic I/R.
2. Heterogeneity of CT perfusion patterns appeared in the 6 h group. As aggravated injury, the area of low perfusion was gradually expanding and showed a decreased trend of enhancement from 12 h to 24 h of reperfusion. Eventually, patchy focal necrosis was observed in the 24 h groups.
3. Heterogeneity of CT perfusion parameters occurred after reperfusion. Although the HPP declined in the low-enhancement tissue in the 2 h groups, the HAP increased significantly so as to the TLP remained relatively normal and HAP of infarcted tissues were higher in 6～24 h groups than that of non-infarcted tissues, thus, that may prove the "arterial buffer response".
4. In spite of HAP increased significantly in the 6-24 h groups, HPP and TLP were decreased significantly in the low-enhancement tissue which make the "arterial buffer response" out of action result in the reduction of HPP mainly because of thoroughfare channel. The perfusion in the low-enhancement tissue was actually reducing that further aggravated the liver tissues injury(24 h group).
5. Serum levels, TNF-a and morphological scores were closely correlated to the severity of liver tissue damage as determined by the CT perfusion parameters of the low-enhancement liver tissue. Our result suggested CT perfusion parameters may be a useful parameter in assessing pathologic processes of hepatic I/R.
引文
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