Nonlinear Optical Microscopy For Interstitial Fibrosis Assessment In Rat Renal Allograft With Chronic Rejection

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 ratObjective: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 rejectionObjective: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 rejectionObjective: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.