| Interstitial fibrosis (IF) refers to abnormal tubulointerstitial deposition of extracellular matrix (ECM) as a common pathological feature of all chronic kidney diseases and chronic allograft nephropathy. Complex mechanisms of IF include inflammatory response, immune cell apoptosis and intrinsic renal cell apoptosis, enhanced oxidative stress, imbalance between promoting and inhibiting fibrosis cytokines, etc. The current key point and exact pathogenesis of the occurrence and development of renal fibrosis are not completely clear yet. It has been demonstrated that glomerulosclerosis and renal interstitial fibrosis are the important factors leading to poor prognosis of kidney diseases, and both severity is closely related to renal dysfunction. Tubulointerstitial disease is more important than glomerular lesions in the progression of kidney disease as the most important prognostic marker to reflect the severity of renal dysfunction. Therefore, the severity of renal interstitial fibrosis can be determined by accurate assessment of collagen fibers which is an important prognostic marker and can guide the diagnosis and treatment.There are several methods used to determine renal fibrosis, such as Masson’s trichrome staining, Sirius red staining、immunohistochemistry, etc. But there are still many controversies in various methods for their advantages and disadvantages. Currently, traditional pathological examination is still the "gold standard" for IF assessment but with many shortcomings. The accuracy of results obtained by traditional pathological staining is not only affected by the stained methods、reagent quality、specimen saved time and technical personnel experience, also affected by the pathologist on personal experience and work condition, as well as its poor reproducibility. Therefore, international pathologists have repeatedly expressed their hope to find a method for assessment of renal interstitial fibrosis that can be easily operated, with high repeatability and reliability.In recent years, nonlinear optical microscopy provides a new method for investigation of collagen fibers in tissue, which as a multi-disciplinary research focus. It has been applied in many biomedical fields, such as imaging of collagen fibers in the cornea, skin, breast, thyroid glands, mouse kidney, rat liver and human liver, as well as determination of benign or malignant nasopharyngeal carcinoma, colon carcinoma and liver carcinoma, and determination of whether it will relapse or not. The signal strength of SHG(Second harmonic generation) is closely related to the content of collagen in biological tissue, which can specifically display the deposition and three-dimensional shape of fibrous collagen. TPEF(two-photon Microscopy) can be used to display the structures of tissue and cells. Combining both imaging technologies, the morphology of collagen fiber and its surrounding tissue can be directly revealed by compound imaging for their commutative verification and supplementation. At present, this technology has been already widely applied in the study of liver fibrosis. A fully automated quantitative system of liver fibrosis, "Fibro-C-Index", is established by Singaporean scholar Tai, et al, in which the assessment results can be directly read by the computer software. Human liver fibrosis is classified as 40 grades compared with 4 grades in traditional pathological classification. Researchers from Singapore and International nonlinear optics laboratory of our university has established a new system "qFibrosis "for assessing liver fibrosis. qFibrosis was established as a combined index based on 87 parameters of architectural features by Images acquired from 25 Thioacetamide-treated rat samples and 162 CHB core biopsies, and it could faithfully and reliably recapitulates Metavir fibrosis scores, as it can identify differences between all stages in both animal samples and human biopsies. So far, it has not been used for quantitative study of human renal fibrosis at home and abroad. In our previous studies, it has been demonstrated that SHG/TPEF can be used to assess the feasibility and sensitivity of rat renal fibrosis. In this study, SHG/TPEF will be used for quantitative analysis of human renal allograft fibrosis. And the feasibility, sensitivity and reproducibility of this technique in diagnosis of renal allograft fibrosis will be also verified.The most common and mature models of fibrosis are rat models of renal fibrosis, including the model of chronic renal failure caused by mostly radical nephrectomy, the model of renal failure caused by unilateral ureteral obstruction (UUO), the model of aristolochic acid (AA) nephropathy, the model of cyclosporin A nephropathy, the rat model of chronic rejection of renal allograft, and the rat model of acute rejection of renal allograft, etc. Rat renal models causes by different causes of disease are established and SHG/TPEF technology is used to observe collagen fibers in the disease development process, in order to understand the type, shape and deposit location of collagen fibers in the development process of different diseases, as well as the velocity of production and extension. Therefore, the present study intends to establish the following four kinds of rat models of renal fibrosis caused by different causes:the model of chronic rejection of renal allograft, the model of acute rejection of renal allograft, the model of cyclosporin A nephropathy and the model of unilateral ureteral obstruction. The dynamic changes of collagen fibers will be observed using nonlinear optical microscopy. This will provide raw data elements for the future establishment of computer-aided system of precise diagnosis and classification of renal fibrosis.Chapter One Nonlinear optical microscopy for interstitial fibrosis assessment in human renal allograftObjective:To assess the feasibility of SHG/TPEF microscopic imaging technique in assessment of interstitial fibrosis of human renal allograft. To compare the sensitivity in assessment of early renal fibrosis between SHG/TPEF microscopic imaging technique and Masson’s staining technology. To verify the reproducibility of SHG/TPEF microscopic imaging technique in different times or different observers. To compare the difference in assessment of cortical fibrosis percentage of human renal allograft between SHG/TPEF microscopic imaging technique and Masson’s staining technology. The cortical fibrosis percentage measured and calculated by Masson-stained and SHG/TPEF images is classified according to Banff 07 standards of fibrosis grading score, in order to compare the consistency of classification, and to evaluate the reliability of SHG/TPEF imaging in assessment of interstitial fibrosis of human renal allograft.Methods:A total of 143 patients after renal transplantation were taken allograft biopsies between January 2011 to March 2013 in zhujiang hospital and 50 cases of paraffin samples were selected based on the principles of random sampling.8 cases among them were diagnosed as unqualified specimens and a total of 42 cases of specimens were included in the present study. The included paraffin specimens were taken for preparation of two consecutive slices:the first one was stained with HE and the second was not treated as a white slice. The white slice was given Masson’s Trichrome staining after direct imaging using SHG/TPEF microscopy. The unstained slice of renal allograft was observed using SHG/TPEF microscope, in order to assess the feasibility of nonlinear optical microscopy in assessment of interstitial fibrosis of human renal allograft.Using SPSS 17.0 statistical software to process data, measurement data as mean ± standard deviation (x±s); Using Reliability Analysis to test the reproducibility of the cortical fibrosis percentage of human renal allograft using SHG/TPEF microscope by different operators at the same time point.To use repeated measures data of ANOVA to test the reproducibility of the cortical fibrosis percentage of human renal allograft using SHG/TPEF microscope by the same operator at different time points. The SHG/TPEF image in mild fibrosis group was compared with that stained with Masson’s Trichrome, in order to assess the sensitivity of SHG/TPEF imaging of collagen fibers in human renal allograft. The obtained 42 cases of Masson-stained and SHG/TPEF images were used to calculate the cortical fibrosis percentage in both groups of images using Image-Pro-Plus (6.0 version) software, and Using Paired-Samples T Test to compare the difference in assessment of fibrosis index percentage between Masson staining and SHG/TPEF, With P< 0.05 for statistically significant. The fibrosis percentages measured and calculated by above two methods were classified according to BanffO7 standards of fibrosis grading score, in order to compare the consistency of classification, and using Kappa test to evaluate the reliability of SHG/TPEF imaging in assessment of interstitial fibrosis of human renal allograft, When kappa values>0.75 representative consistency,0.4≤ kappa value≤0.75 representatives better consistency, kappa values<0.4 on behalf of poor consistency.Results:The presence of nonlinear optical phenomenon in the tissue of human renal allograft was demonstrated by obtaining clear TPEF and SHG images after nonlinear optical microscopy of unstained specimen taken from human renal allograft. In RMANOVA analysis, there was no significant difference in SHG fibrosis percentage by the same observer at different time points (F=6.545, P>0.05). In a reliability analysis, there was no significant difference in SHG fibrosis percentage by different observers at the same time point (ICC=0.998, P<0.001).This indicated a high repeatability of SHG/TPEF technology operated by different observers at different time points. Masson-stained and SHG/TPEF images of specimens with mild fibrosis taken from the renal allograft were compared to find that there was not any fibrous tissue in Masson image in the early period of pathological changes, but collagen fibers were clearly distinguished in SHG/TPEF image, which demonstrated that the sensitivity of SHG/TPEF imaging was higher than Masson staining. Paired sample t-test was used to find that there was no statistical differencein assessment of cortical fibrosis percentage using two methods, SHG and Masson staining, indicating a good consistency between SHG fibrosis percentage and Masson fibrosis percentage (t=-1.703, P=0.096). Kappa test was used to compare whether Banff classification of above two fibrosis percentagees was consistent, and a kappa value of 0.845 (P<0.001) demonstrated a good consistency of Banff classification between SHG fibrosis percentage and Masson fibrosis percentage.Conclusions:SHG/TPEF microscopic imaging technique is feasible in observation of interstitial fibrosis of human renal allograft. Compared with the traditional pathological staining technique, the progression of fibrosis observed via a SHG/TPEF microscope is consistent with the traditional pathology, but it is more sensitive to early emergence of collagen fibers. SHG fibrosis percentage dose not differ with Masson’s fibrosis percentage, and there is a high consistency between Banff fibrosis-based classification percentage and Masson fibrosis percentage. SHG/TPEF microscopic imaging technology has a high reproducibility、a high specificity of collagen fibers and it will be less impacted by the differences in the time and the observer. SHG/TPEF microscopic imaging technology is a promising new technology in diagnosis of renal interstitial fibrosis.Chapter Two Establishing four different kinds of rat model of renal fibrosis and using SHG/TPEF technique to observe the collagens in the renalsObjective:To establish the following four kinds of rat models of renal fibrosis caused by different causes of disease:chronic rejection to renal allograft model, acute rejection to renal allograft model, cyclosporine nephropathy model and unilateral ureteral obstruction (UUO) model, and to observe the type, occurrence, development and distribution of collagen fibers in renals of different models by SHG/TPEF microscopic imaging technique.Methods:To establish the rat model of chronic rejection, both kidneys of 12 Wista rats as donors were randomly transplanted into 24 SD (Sprague Dauley) rats as receptors. They were given continuous intraperitoneal injection of cyclosporine A (2 mg/kg.d-1) after the procedures. The receptors were then randomly divided into six groups (n=4). The left allograft kidneys were respectively removed at2 w,4 w,6 w, 8 w,10 w and 12 w postoperatively and take the left and right kidney of 2 normal SD rats as the control group. To establish the rat model of acute rejection, the left kidneys of 16 Wista rats as donors were transplanted into 16 SD rats as receptors(orthotopic transplantation). The receptors were randomly divided into group A (acute rejection) not given postoperative immunosuppressants and group B (control) given postoperative intraperitoneal injection of cyclosporine A (2 mg/kg.d-1). According to the principle of random, the renal allograft and contralateral original kidneys were respectively removed at 1 d,3 d,5 d and 7 d postoperatively. To establish the rat model of cyclosporine nephropathy,5 of 25 SD rats fed with low-sodium diet for one week were randomly executed for removal of left kidneys as the control group, and other 20 SD rats fed with low-sodium diet were given intraperitoneal injection of cyclosporine A (30 mg/kg.d’1). According to the principle of random, these 20 SD rats were divided into 4 groups (n= 5), and their left kidneys were respectively removed at 1 w,2 w,4 w and 6 w after administration. To establish the rat model of UUO,18 SD rats were divided into UUO group (n= 9) with ligation of the left ureter and sham-operated group (n= 9) with separation of the ureter according to the principle of random. Their left kidneys were respectively removed at 3 d,7 d and 14 d after modeling. To observe the histopathological features of kidneys in four kinds of rat models and of the control groups for testing whether the establishment of models are successfully and to compare the difference of cortical fibrosis percentage between each group of the four models respectiyely.Using SHG/TPEF microscopy to observe the type、occurrence、development and distribution of collagen fibers in each period of kidneys in all models.Results:Four kinds of models were all successfully established. In One-Way ANOVA analysis, there was significant difference of cortical fibrosis percentage in each group in chronic rejection model (F=104.53, P=0.000)、cyclosporine nephropathy model (F=76.191,P=0.000)、UUO model (F=27.23,P=0.001) respectively,and there was on significant difference of cortical fibrosis percentage in each group in acute rejection model (F=4.722, P=0.084). UUO model of renal fibrosis made the most rapid progress, because a large amount of renal interstitial collagen fibers were observed 7 days after the procedures and severe renal interstitial fibrosis was observed 14 days after the procedures. There was no progression interstitial fibrosis in the model of acute rejection to renal allograft. A slower progression of fibrosis was observed in the model of chronic rejection to renal allograft for its long period of modeling. In the model of cyclosporine A nephropathy fed with low-sodium diet, a large amount of distribution of collagens were observed within 6 weeks. In these four kinds of models, Masson-stained images showed blue-stained collagen fibers on Bowman’s capsule wall and glomerular basilar membrane, but SHG/TPEF images showed no green-stained collagen signal on Bowman’s capsule wall and glomerular basilar membrane. The green-stained collagen fibers were mainly concentrated at the junction between the cortex and medulla. Fewer collagen fibers in the medulla area mainly contained coarse and fine forms of collagen fibers in the process of fibroplasia. In the SHG/TPEF images, the morphological characteristics of coarse fibers are similar to collagen I and The morphological characteristics of fine fibers are similar to collagen III. The collagen fiber formation and development process is similar to each other in the three different models, and the deposit area is the same, only the time of fiber deposition is different.Conclusion:Four kinds of rat models of renal fibrosis caused by different causes of disease are all successfully established, in which UUO and CsA nephropathy models are more ideal models of renal fibrosis, chronic rejection model is an ideal model of renal fibrosis, and acute rejection model has a poor effectiveness as a fibrosis model. SHG/TPEF microscopic imaging can specifically display type-I collagens other than type-IV collagens in the kidney. In the three different models, collagen fiber formation and development process is similar to each other, and the deposit area is the same, only the time of fiber deposition is different... |
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