摘要
肾移植是终末期肾病的最佳替代治疗方法。随着配型技术的发展和新型免疫抑制剂的问世,肾移植的近期效果得到显著提高,但移植肾的长期存活却没有明显改善,影响移植肾长期存活的主要因素是慢性排斥反应。当移植肾发生慢性排斥反应时,损伤因素和修复因素不断作用,胶原纤维不断堆积和改造,导致移植肾间质纤维化。而持续进展的纤维化终将导致移植肾失功,患者面临回到透析或再移植,消耗了大量医疗资源,增加日益紧缺的器官来源压力。可见,早期诊断移植肾慢性排斥反应间质纤维化并及时采取干预措施至关重要。
目前诊断移植肾慢性排斥反应间质纤维化的金标准是病理检查,但其存在诸多不足。传统病理主要是采用特殊染色来显示纤维素沉积,其过程复杂、耗时,结果的准确性受染色方式、染色质量、染色后褪色和技术人员熟练程度等影响,很难达到高度的可重复性和统一性,不能满足临床需求。
非线性光学显微镜为观察组织纤维化提供了一种新的方法。其中二次谐波产生(Second harmonic generation, SHG)对生物组织结构的对称性变化很敏感,适合用于非中心对称性分子胶原纤维成像。双光子激发荧光(Two-photon excited fluorescence, TPEF)是生物组织在特殊激光照射时组织自身产生的一种荧光信号,可用于观察组织细胞等结构。两者结合互为补充,无需对标本染色,即可直接对组织内胶原纤维进行定性和定量分析,并获得其周围的细胞形态信息。该技术快速且对标本无损伤,获得的图片信噪比和分辨率高。目前SHG/TPEF显微成像技术已成功用于观察大鼠肝硬化纤维化,并与计算机辅助分析系统结合,建立了自动化的肝纤维化定量评估系统。国内外尚未见该技术用于移植肾纤维化的研究,故本研究先探索其用于评估大鼠移植肾慢性排斥反应间质纤维,为该技术的临床应用提供实验依据。
大鼠肾移植模型是研究肾移植术后慢性排斥反应的常用动物模型,国内外研究者一直致力于寻找一种快速、安全的建模方法。同时获取和移植供者大鼠的双侧肾脏可显著缩短建模时间,因为同时获取一只供鼠的双侧肾脏仅需实施一次术前准备和开腹手术,且两侧供肾可同时灌注,而分别获取两只供鼠的一侧肾脏则要依次对两只供鼠实施术前准备和开腹手术,且需分两次进行供肾灌注。然而绝大部分研究中仍只获取和移植供鼠的左肾,右侧供肾通常被遗弃,其主要原因在于右侧肾静脉太短,实施肾静脉的吻合难度较大。国内外许多学者对双侧供肾大鼠肾移植模型中右侧供肾静脉的吻合方式做了探讨,但其效果需要进一步的系统比较和评估,故本研究比较了大鼠双侧供肾移植中几种右肾静脉的吻合方式,寻找一种快速、稳定的双侧供肾大鼠肾移植建模方法。然后采用这种方法,以Wista大鼠为供者、SD大鼠为受者建立大鼠同种异体肾移植模型,术后给予腹腔注射CsA (2mg/kg.d-1)诱导移植肾发生慢性排斥反应,为SHG/TPEF显微成像技术应用于移植肾慢性排斥反应间质纤维化的评估提供可靠的动物模型。
本研究采用SHG/TPEF显微成像技术和传统病理染色技术获取图像和数据,并对两者的纤维化评估结果进行比较分析,从而评价该技术用于评估移植肾慢性排斥反应间质纤维化的可行性、敏感性、可靠性和可重复性等,为其应用于人移植肾间质纤维化的评估奠定基础。
第一章大鼠右侧供肾移植中肾静脉吻合方式的比较研究
目的：寻找最佳右侧供肾静脉吻合方式,并用于建立双侧供肾大鼠肾移植模型方法,以获得快速、安全的建模方法。
方法：以SD大鼠作为同系大鼠肾移植模型的供、受鼠,取45只供鼠的双侧肾脏(截取部分腔静脉作为右侧供肾静脉的一部分),采用随机数字表法将90只受鼠分为4组。A、B、C组受鼠各15只,均将右侧供肾沿纵轴旋转180度后移植于左侧肾窝,肾静脉的吻合方式分别采用端侧吻合、腔静脉搭桥、改进的端端吻合(供肾腔静脉近心端与受鼠肾静脉端端吻合,结扎供肾腔静脉远心端)；对照组45只,均将左侧供肾移植于左侧肾窝,肾静脉的吻合方式采用传统的肾静脉端端吻合。各组肾动脉和输尿管的吻合方式相同,均采用端端吻合。对各组受鼠间的手术耗时、肾动脉吻合时间、肾静脉吻合时间、输尿管吻合时间、供肾热缺血时间、供肾冷缺血时间、手术成功率及术后并发症发生情况进行比较。
结果：B组的肾静脉吻合时间显著长于对照组(P<0.001),由此导致B组的供肾热缺血时间和受鼠手术耗时也较对照组显著延长(P<0.001)。而A组和C组与对照组间的肾静脉吻合时间、供肾热缺血时间及受鼠手术耗时均无明显差异(P>0.05)。各组间的肾动脉吻合时间比较和输尿管吻合时间比较均无明显差异(P>0.05)。C组手术成功率(93.3%)与对照组(86.7%)相当(P>0.05),A组(53.3%)和B组(53.3%)手术成功率均显著低于对照组(P<0.05)。
结论：右侧供肾移植中,截取部分腔静脉作为供肾静脉的一部分,将其近心端与受鼠肾静脉行端端吻合,并结扎其远心端,这种肾静脉的吻合方式可行性高,将其用于建立双侧供肾大鼠肾移植模型,既高效又经济,更符合伦理学要求。
第二章大鼠肾移植慢性排斥反应模型的建立和传统病理评估
目的：建立大鼠肾移植慢性排斥反应模型,为研究移植肾慢性排斥反应间质纤维化提供可靠的动物模型。
方法：获取15只供者Wista大鼠的双侧肾脏,将左、右侧供肾随机移植给30只受者SD大鼠,左侧供肾移植中肾动脉、肾静脉和输尿管均采用端端吻合方式,右侧供肾移植中肾静脉采用改进的端端吻合方式(供肾腔静脉近心端与受鼠肾静脉端端吻合,结扎供肾腔静脉远心端),肾动脉和输尿管采用端端吻合方式。术后持续给予坏孢素A注射液(2mg/kg.d-1)腹腔注射。按随机数字表法将30只受者SD大鼠分为3组,每组10只：4W组、8W组和12W组分别于肾移植术后4周、8周和12周获取大鼠移植肾。以5只正常SD大鼠为对照组,获取其双侧肾脏。比较各组受者大鼠的手术耗时、供肾冷缺血时间、供肾热缺血时间和术后情况。获取的所有肾脏进行石蜡包埋、切片、HE染色、PAS染色和Masson's三染色,光学显微镜下观察正常肾脏和各组移植肾组织内小血管、肾小管间质和肾小球的组织病理变化,以Masson纤维化染色结果为基础进行Banff纤维化评分,比较各组肾脏的间质纤维化程度。
结果：各组间受鼠手术耗时比较(F=0.245,P=0.785)、供肾热缺血时间比较(F=0.901,P=0.418)和供肾冷缺血时间比较(F=0.265,P=0.769)均无统计学差异。术后各组受者大鼠的移植肾动、静脉均未见狭窄及血栓形成。各组肾脏标本经传统病理染色后观察发现：随着肾移植术后时间的延长,大鼠移植肾的小动脉内膜向心性增生直至管腔闭塞,单核细胞浸润逐渐增多,肾小球硬化、肾小管萎缩日趋严重,间质纤维化程度进行性加重,符合移植肾慢性排斥反应的病理表现。以Masson纤维化指数为基础,根据Banff07移植肾间质纤维化评分标准对各组肾脏标本进行纤维化评分,正常组、术后4周组、术后8周组和术后12周组的Banff纤维化评分分别为：(0.10±0.32)、(0.8±0.42)、(1.9±0.32)和(3.0±0.00),术后4周纤维化评分与正常组比较无统计学差异(χ2=2.109,P>0.05),术后8周(χ2=12.651,P<0.01)和术后12周(χ2=31.604,P<0.01)的纤维化评分与正常组比较均有显著统计学差异。
结论：以Wista大鼠为供者、SD大鼠为受者进行肾移植术后,持续给予环孢素A2mg/kg.d-1腹腔注射,可诱导大鼠移植肾发生慢性排斥反应。大鼠移植肾慢性排斥反应的病理表现为持续进展的肾血管内膜向心性增生,肾小球硬化、肾小管萎缩和间质纤维化。传统病理评估移植肾慢性排斥反应间质纤维化存在染色过程复杂、耗时、主观差异大等缺点。
第三章使用非线性光学显微镜观察大鼠移植肾慢性排斥反应间质纤维化
目的：评价SHG/TPEF显微成像技术用于评估大鼠移植肾慢性排斥反应间质纤维化的可行性、敏感性、可靠性和可重复性,为其应用于人移植肾间质纤维化的评估奠定基础。
方法：以15只Wista大鼠为供者、30只SD大鼠为受者,采用双侧供肾大鼠肾移植法(右侧供肾静脉吻合方式为供肾腔静脉近心端与受鼠肾静脉端端吻合,结扎供肾腔静脉远心端)进行大鼠同种异体肾移植术,术后持续给予坏孢素A注射液(2mg/kg.d-1)腹腔注射,抑制移植肾发生急性排斥反应而诱导其发生慢性排斥反应。按随机数字表法将30只受者SD大鼠随机分为3组,每组10只：4W组、8W组和12W组分别于肾移植术后4周、8周和12周获取大鼠移植肾。为全面了解肾脏胶原纤维的分布,本研究选择每个肾脏冠状切面上覆盖肾脏上、中、下极的8个皮质区和3个髓质区为目标区域进行观察。采用非线性光学显微成像技术和传统病理Masson's三染色技术获取图像。观察未经染色的移植肾切片,评估SHG/TPEF成像用于评估大鼠移植肾间质纤维化的可行性。比较观察含胶原纤维较少的正常肾脏组织SHG/TPEF图像与传统病理染色图像,评估SHG/TPEF成像显示肾脏胶原纤维的敏感性。以视野中胶原纤维面积占视野总面积的百分比为SHG纤维化指数,采用图像分析软件测算并比较术后不同时间慢性排斥反应大鼠移植肾标本的SHG纤维化指数。对比观察不同时期慢性排斥反应大鼠移植肾标本的SHG/TPEF图像与传统病理染色图像,并采用图像分析软件测算SHG和Masson纤维化指数,比较两指数的差异,同时以这两种指数为依据分别进行Banff纤维化分级,比较分级的一致性,评价SHG/TPEF成像用于评估大鼠移植肾慢性排斥反应间质纤维化可靠性。比较同一操作者在不同时间点和不同操作者在同一时间点分别测算同批移植肾标本的SHG纤维化指数和Masson纤维化指数的差异,评估SHG/TPEF成像评估大鼠移植肾间质纤维化的可重复性。
结果：未经染色的移植肾标本经非线性光学显微镜扫描后,获得红色信号TPEF和绿色信号SHG图像。低倍镜下(20×)可清晰显示沉积于移植肾中的胶原纤维及肾小球、肾小管、肾血管等,高倍镜下(60×)可观察胶原纤维和周围组织的超微结构,两者结合可显示肾组织中胶原纤维的沉积部位及其与周围组织的关系。对比观察正常肾脏组织的SHG/TPEF图像与传统病理染色图像,发现传统病理染色仅能模糊呈现正常肾脏中存在的少量纤维组织,无法对其进行半定量分析,而SHG/TPEF成像则能清晰显示正常胶原的轮廓和结构,并能对其进行准确的定量分析。对比观察不同时期慢性排斥反应大鼠移植肾标本的SHG/TPEF图像与传统病理染色图像,发现SHG/TPEF显微镜观察到的移植肾间质纤维化进程与传统病理方法相吻合,但显示的胶原纤维轮廓、结构更清晰。肾移植术后4周、8周和12周的SHG纤维化指数分别为：(0.16±0.08)、(0.35±0.08)和(0.55±0.06),两两比较均有显著差异(P<0.05)。SHG纤维化指数与Masson纤维化指数比较,两者无显著统计学差异(z=1.613,P=0.107)。分别以SHG纤维化指数和Masson纤维化指数为基础进行Banff纤维化分级,Kappa检验发现两者具有良好的一致性(kappa=0.811>0.75, P=0.000)。同一操作者在不同时间点分别测算同批移植肾标本的SHG纤维化指数和Masson纤维化指数存在显著差异(F=7.704,P=0.004),不同操作者在同一时间点分别测算同批移植肾标本的SHG纤维化指数和Masson纤维化指数亦存在差异(F=41.422,P=0.000),进一步分析其差异主要来自于Masson纤维化指数,表明SHG/TPEF技术评估移植肾间质纤维化受评估时间和操作者的影响更小,其稳定性和可重复性较好。
结论：SHG/TPEF显微成像技术用于观察大鼠移植肾慢性排斥反应间质纤维化可行性高。与传统病理染色技术比较,SHG/TPEF显微镜观察的纤维化进程与传统病理染色相符,但对胶原纤维成像敏感性、清晰度更好。SHG纤维化指数与传统病理Masson纤维化指数无差异,用于Banff纤维化分级也与传统病理一致性良好,但结果的可重复性更好。
Kidney transplantation is the optimal replacement therapy for end-stage renal disease. As the development of matched-type technology and the advent of new immunosuppre-ssive agent, the short term therapeutic effect of kidney transplantation becomes better, but the long-term graft survival have not been improved. The primary cause is the chronic rejection. During the chronic rejection in renal allograft, damaging and repairing occur constantly, and collagen fiber piles up and transforms continuously, which leading to renal allograft interstitial fibrosis. The progressive fibrosis would result in renal allograft loss eventually. Then the patients will be faced with dialysis again or secondary kidney transplantation. This wastes a lot of medical resources, and increases the pressure of organ shortage. It is thus clear that the early diagnosis of renal allograft interstitial fibrosis with chronic rejection and take timely intervention measures is crucial.
At present, the gold standard method to diagnose the renal allograft interstitial fibrosis with chronic rejection is pathological examination, but this technique haves many shortcomings. The traditional pathology technology display the cellulose deposition with a special dyeing, but the procedure is complex and time-consuming, and the result influenced by the dyeing medthods,dyeing quality, color fading and proficiency of the technical personnel. As a result, the repeatability and uniformity of the diagnosis is difficult to achieve a high agreement, and cannot meet the clinical needs.
Nonlinear optical microscopy provides a new method for observing the fibrosis in tussues. Second harmonic generation (SHG) is very sensitive to the change of symmetry structure in biological tissue, and suitable for imaging of the noncentrosymmetric collagenous fiber. Two-photon fluorescence imaging (TPEF) is an autofluorescence signal in biological tissue when a special laser irradiated, and can be used to observe the structure in cellular level. Combine the SHG with TPEF could complement each other, and can be directly used for qualitative and quantitative analysis of collagen fiber and acquire the cellular morphologic information surround the collagen without dyeing. This technology is characterized by quick, undamage to the specimen, high signal to noise ratio and resolution. Now, the SHG/TPEF microscopic imaging technology had been successfully used for observing the cirrhosis of the liver fibrosis in rats, combined with computer aided analysis system, the automation system for quantitative assessment of hepatic fibrosis had been established. However, by now, there is no research about interstitial fibrosis assessment of renal allograft with this technique at home and abroad. Therefore, this study first explored the SHG/TPEF microscopy for evaluation of renal allograft interstitial fibrosis with chronic rejection in rats, and provided experimental basis for its clinical application.
Rat kidney transplantation is a commonly used animal model for chronic rejection research of renal allograft, and the domestic and foreign researchers have been looking for a quick and safe method to establish this model. The time for harvesting both kidneys from one donor rat was much shorter than that of two donor nephrectomies in two separate rats. To harvest the bilateral kidneys from one donor rat only need to implement the preoperative preparation and laparotomy once, and the two kidneys can be perfusing at the same time. But to obtain two kidneys from two separate donor rats need to perform the preoperative preparation twice and two nephrectomies, in addition, the perfusion of these two donor kidneys should be done in turn. However, only the left donor kidney is harvested and transplanted in most studies, the utilization of right donor kidney is extremely low. This is mainly due to the insufficient length of the right renal vein, which increases the difficulty of the renal vein anastomosis. Many scholars at home and abroad have tried to modify the technique for right renal vein anastomosis in both rat donor kidneys transplantation, but their effects still needs to be reconsidered and reevaluated, and requires a systematic comparison study. Therefore, our study compared several methods for right renal vein anastomosis in the bilateral donor renal transplantation in rats, aimed to find out a fast and stable technology for both donor kidneys transplantation in rat. Then we used this method to establish the rat allogeneic renal transplantation between the Wista rats and the SD rats. To induce a chronic rejection, a small dose of CsA at2milligram per kilogram everyday was injected into the abdominal cavity after transplantation. This provides a reliable animal model for interstitial fibrosis assessment research in the renal allograft with chronic rejection by the SHG/TPEF microscopic imaging technique.
This research imaged and evaluated the fibrosis in renal allografts by the SHG/TPEF microscopy and traditional pathological dyeing technology, and compared the results of these two fibrosis assessment methods, so as to evaluate the feasibility, sensitivity, reliability and repeatability of the SHG/TPEF microscopic imaging technique for renal allograft interstitial fibrosis assessment, and laid the foundation for its clinical applying.
Chapter one. A comparison study of venous anastomosis for right donor kidney transplantation in rat
Objective:Finding an optimized method for renal vein anastomosis of the rat's right donor kidney, and then using this technique for rat both donor kidneys transplantation, so as to obtain a rapid and safe modeling approach.
Methods:Sprague Dawley (SD) rats were used as donor and recipient for homologous rat kidney transplantation. Both bilateral kidneys were harvested from the donor rats (a part of vena cava is taken with the right renal vein, n=45).90rats were used as recipients and divided into4groups according to randomly digital table: In group A～C (n=15for each group), the right donor kidney were transplanted into the recipient's left side after180°rotation around the longitudinal axis, and end-to-side、vena cava bypass and modified end-to-end (donor's proximal end of vena cava was anastomosed to recipient's renal vein follow by ligating its distal end) venous anastomosis was done, respectively; In the control group (n=45), the left donor kidney were transplanted into the same side of the recipients, and the conventional end-to-end venous anastomosis was used. The end-to-end anastomosis was applied for renal artery and ureter reconstruction in each group. Then the total time of the opration on recipient rat and the time spent on renal vein, renal ratery and ureter reconstruction were recorded in the control and experimental groups, respectively, the warm and cold ischemic time were also recorded. The successful operation rates and postoperative complications were checked in each group. All of these data were compared between any two groups.
Results:The venous anastomosis time of group B was longer than that in control group (P<0.001), which significantly increased warm ischemia time of donor kidneys and operative time of recipients(P<0.001). The venous anastomosis time, warm ischemia time of donor kidneys and operative time of recipients showed no significant difference between group A or group C and control group (P>0.05). The differences of artery anastomosis time and ureter reconstruction time between any two groups were not significant (P>0.05). The successful operation rate in group C (93.3%) was similar to that in control group (86.7%)(P>0.05). The successful operation rate in group A (53.3%) and group B (53.3%) is lower than that in control group (P<0.05).
Conclusions:For right donor kidney transplantation, the method of harvesting the right donor kidney with a part of vena cava, and then anastomosing the proximal end to recipient's renal vein and ligating the distal end, is highly feasible. It is efficient and economic to use this technique for both donor kidneys transplantation in
Chapter two. The establishment and traditional pathological evaluation of rat kidney transplantation with chronic rejection
Objective:To establish a rat model of kidney transplantation with chronic rejection, and provide a reliable animal model for interstitial fibrosis assessment research in the renal allograft with chronic rejection.
Methods:Wista and SD rats were used as donor and recipient, respectively, for allogeneic rat kidney transplantation. Both bilateral kidneys were harvested from the donor rats (n=15), and transplated into the recipient rats (n=30). In left donor kidney transplantation, the end-to-end anastomosis was used for renal artery, renal vein and ureter reconstruction. In right donor kidney transplantation, the renal vein was reconstructed with modified end-to-end anastomosis (donor's proximal end of vena cava was anastomosed to recipient's renal vein follow by ligating its distal end), and the renal artery and ureter was reconstructed by traditional end-to-end anastomosis. A small dose of CsA at2milligram per kilogram everyday was injected into the abdominal cavity after transplantation to induce a chronic rejection.30recipients were divided into3groups (n=10for each group) according to randomly digital table: the renal allografts in4W,8W and12W group were harvested4,8and12weeks after transplantation. SD rats without operation were used as control (n=5), and their bilateral kidneys were harvested. Then the total time of the opration on recipient rat, the warm and cold ischemic time of donor kidneys and the condition postoperation in3experimental groups were recorded and compared. All of the kidneys were embedded with paraffin and sliced. Some of these slices were dyed with HE, PAS and Masson trichrome stain. Then histopathological changes of blood vessels, renal tubule interstitial and glomerular within normal and transplanted kidneys were observed by an optical microscopy. Banff fibrosis score was calculated based on the Masson trichrome stain, and compared between any two groups.
Results:The operative time of recipients (F=0.245, P=0.785), warm ischemia time (F=0.901, P=0.418) and cold ischemia time (F=0.265,P=0.769) of donor kidneys showed no significant difference between any two groups. Complications of stenosis and thrombosis related to renal arterial and venious anastomosis were not observed in any of the three groups. The kidney specimens were observed under an optical microscopy after traditional pathological dyeing:as time goes on after kidney transplantation, the small artery intima proliferates centrally and eventually resulting in vessel blocking, mononuclear cell infiltration increases, glomerular sclerosis and renal tubular atrophy aggravate gradually and interstitial fibrosis progressed constantly, which corresponds with the pathological changes of renal allograft with chronic rejection. Then fibrosis score in each group was calculated based on the Masson fibrosis index, according to Banff07renal allograft interstitial fibrosis grading standard. The Banff fibrosis score of normal group,4W group,8W group and12W group were (0.10±0.32),(0.8±0.42),(1.9±0.32) and (3.0±0.00). The difference of Banff fibrosis score in4W and normal groups was not obvious (χ2=2.109, P>0.05), but significant in8W (χ2=12.651, P<0.01) or12W (χ2=31.604, P<0.01) groups and the normal group.
Conclusions:A chronic rejection of rat renal allograft can be induced by establishing a kidney transplantation model between Wista and SD rat, which were used as donor and recipients respectively, followed by intraperitoneal injection of a small dose of CsA at2milligram per kilogram everyday. The pathology of renal allograft with chronic rejection is characterized by progressive small artery intima hyperplasia, glomerular sclerosis, renal tubular atrophy and interstitial fibrosis. For interstitial fibrosis assessment in renal allograft with chronic rejection, the traditional pathological techinique has many disadvantages, such as complicated and time-consuming dyeing procedure, subjective differences, et al.
Chapter three. Nonlinear optical microscopy for observing interstitial fibrosis in rat renal allograft with chronic rejection
Objective:To evaluate the feasibility, sensitivity, reliability and repeatability of the SHG/TPEF microscopic imaging technique for interstitial fibrosis assessment in rat renal allograft with chronic rejection, and laid the foundation for its clinical applying.
Methods:15Wista and30SD rats were used as donor and recipient, respectively, for allogeneic rat kidney transplantation. Both bilateral kidneys were harvested from the donor rats, and then transplated into the recipient rats (the right renal vein was reconstructed by anastomosing the proximal end of vena cava to recipient's renal vein and ligating its distal end). A small dose of CsA at2milligram per kilogram everyday was injected into the abdominal cavity after transplantation to suppress the acute rejection and induce a chronic rejection.30recipients were divided into3groups (n=10for each group) according to randomly digital table:the renal allografts in4W,8W and12W group were harvested4,8and12weeks after transplantation. For a comprehensive understanding of the renal fibrosis,11regions on the coronal slice of the kidney, including8cortical areas and3medulla areas in the upper, middle and lower poles, were selected to be scanned, according to the renal anatomy. Images of renal allografts in each group were obtained by nonlinear optical microscopy scanning and Masson trichrome staining. The feasibility of SHG/TPEF imaging technique for rat renal allograft interstitial fibrosis assessment was evaluated by observing the renal specimen of moderate fibrosis without staining. The sensitivity of SHG/TPEF imaging to show the collagen fibers was assessed by comparing its imaging effect on normal renal specimen with that of traditional pathological staining images. The SHG fibrosis index was defined as the percentage of collagen fiber area in the total area of view. Then the SHG fibrosis of the renal specimens at different time points after kidney transplantation was calculated by image analysis software and compared. To estimate the reliability of SHG/TPEF microscopic imaging for rat renal allograft interstitial fibrosis assessment, the imaging effect of SHG/TPEF and traditional pathological staining on renal specimens with different degree of fibrosis was observed, the SHG and Masson fibrosis index was graded by image analysis software, and their difference was evaluated. Meanwhile, the renal specimens were graded based on the SHG/TPEF and Masson fibrosis index according to the Banff standard, respectively, and their agreement was analysed. In order to evaluate the repeatability of SHG/TPEF imaging technique for rat renal allograft interstitial fibrosis assessment, the SHG and Masson fibrosis index of these renal allograft specimens were calculated repeatedly by the same operator at different time points, respectively, then the intra-group and inter-group differences were analyzed by repeated measures analysis of variance.
Results:The TPEF signal is displayed in red pseudocolor and SHG signal is shown in green pseudocolor after nonlinear optical microscope scanned the renal specimen without sraining. The collagen fibers deposition, glomerular, renal tubules and vessels within the kidneys were clearly showed at the low magnification (20×), and the ultrastructure of collagen fiber and its surrounding tissue was displayed at the high magnification (60×). Simultaneous TPEF and SHG image clearly revealed the fibrosis deposited and the tissue surround the collagen. Comparing the SHG/TPEF images of normal renal specimen with the traditional pathological staining images, the latter one only fuzzily displayed a small amount of fibrous tissue within the normal kidney and the semi-quantitative analysis was impracticable, while the former one clearly showed the outline and structure of the normal collagen and an accurate quantitative analysis is feasible. Comparing the SHG/TPEF images of renal specimen at different time points after kidney transplantation with the traditional pathological staining images, the interstitial fibrosis progress observed by SHG/TPEF microscopy was in conformity with that of traditional pathological method, but the outline and structure of collagen fiber was clearer than the latter. The SHG fibrosis index of renal allograft4,8and12weeks after kidney transplantation were (0.16±0.08),(0.35±0.08) and (0.55±0.06), respectively. There was significant difference between any two groups (P<0.05). Compared the SHG fibrosis index with the Masson fibrosis index, no difference was found (z=1.613, P=0.107). The renal specimens were graded based on the SHG/TPEF and Masson fibrosis index according to the Banff standard, respectively. And a kappa test gave a value of0.811>0.75(P=0.000). This result demonstrates a good agreement between these two methods. The SHG and Masson fibrosis index of the renal allograft specimens were calculated repeatedly by the same operator at different time points, respectively. The SHG and Masson fibrosis index of the same specimens evaluated by one operator at different time points (F=7.704, P=0.004) and estimated by different operators at the same time point (F=41.422,.P=0.000) was both significantly different, and further analyzed found that these differences mainly resulted from the diversity of Masson fibrosis index. This indicated smaller influence of temporal and human diversity and better stability and repeatability of SHG/TPEF for interstitial fibrosis assessment in renal allograft.
Conclusions:The SHG/TPEF microscopic imaging technique using for interstitial fibrosis assessment in rat renal allograft with chronic rejection is highly feasible. Compared with traditional pathological dyeing technology, the interstitial fibrosis progress observed by SHG/TPEF microscopy was in conformity with that of traditional pathological method, but the sensibility, definition of SHG/TPEF microscopy for cillagenour fiber imaging was better. The SHG fibrosis index was coincided with the Masson fibrosis index, and the Banff fibrosis grading based on SHG fibrosis index was in agreement with that based on Masson fibrosis index, but the repeatability of the SHG fibrosis index was better.
引文
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