Second Harmonic Generation And Two-photon Microscopy For Cyclosporine-induced Renal Interstitial Fibrosis Assessment In Rat

BackgroundRenal transplantation operation is the optimal method as renal replacement therapy for end-stage nephropathy patients. As the development of matched-type technology, clinical application of immunosuppressive agent and optimization of immunosuppression regimen, the short term efficacy of kidney transplantation gets better, but the long-term graft survival without improving. One of the primary factors is immunosuppressant nephrotoxicity.As active substance of tolypocladium inflatum circular peptide11, cyclosporine A(CsA) has been widely applied in organ transplantation in1980s. At the same time, the adverse effect especially chronic nephrotoxicity restrict its usage. It was shown that the damage and recovery are existed in the same process, accumulation of collagen fibers, which result in renal interstitial fibrosis. The progressive renal interstitial fibrosis ultimately gives rise to renal allograft loss. Tt would be particularly important for early diagnosis of renal allograft interstitial fibrosis. Pathological examination has become the present "gold standard" for diagnosis of renal or graft interstitial fibrosis, such as Masson trichrome stain, Sirius Red, and collagen types Ⅲimmunohistochemistry. The existing pathology technique display the collagen fibers deposition with a special dyeing, then determined severity of renal fibrosis employ morphometry methods. The reason which quantitative standard of renal interstitial fibrosis are far from reaching an agreement in the academic world, complex process of stain, the result influenced by human factors, dyeing quality, specimen quality and so on, and led to the existing pathology technique lack of the accuracy, specificity and sensitivity, cannot satisfy the clinical requirements.Second harmonic generation and two-photon microscopy has been successfully used in assessing various kinds of tissues and organs fibrosis, like corneal, skin, lung, liver, etc. Second harmonic generation (SHG) is very sensitive to detect dissymmetry structure matter, and suitable for imaged collagen fibers with non-centrosymmetric structure. Organizational structure has been observed under Two-photon fluorescence imaging (TPEF), autofluorescence properties when irradiated by exogenous or endogenous laser. SHG have been combined by interactive and interdependent TPEF, could acquire the signal of collagen fibers and surrounding tissue. It reported that SHG/TPEF microscopic technology combined with computer aided diagnostic system had succeeded imaging of the liver fibrosis in rats. This technique had established a automatic system for quantitative assessment of cirrhosis of the liver. SHG/TPEF microscopic technology scan specimen without stain, can also be dectct by other histologic examinations, getting high-definition and super-resolution image. Thus far, there is no research on renal interstitial fibrosis assessment in rat induced by cyclosporine A by SHG/TPEF microscopy technology, according to domestic and overseas study. For this reason, this is the first study to investigate the SHG/TPEF microscopy for evaluation of renal interstitial fibrosis induced by cyclosporine A in rats.This study on renal interstitial fibrosis to compare how accuracy, specificity and sensitivity diagnosed based on SHG/TPEF microscopy versus existing pathological examination, and provided experimental basis for clinical medicine.Objective1. To establish a rat model of cyclosporine A-induced nephrotoxicity, and afford a reliable animal model for renal interstitial fibrosis assessment induced by cyclosporine A.2. To evaluate the accuracy, specificity and sensitivity of the SHG/TPEF technique evaluate renal interstitial fibrosis in rat induced by cyclosporine A.3. To explore a new method to quantitate fibers in interstitial fibrosis using SHG/TPEF microscopic imaging technique.3. To provided experimental basis for clinical medicine.Methods1. Establish a rat model of cyclosporine A-induced nephrotoxicity60adult male Sprague Dawley (SD) rats were divided into6groups according to randomly digital table:control group,1W,2W,4W,6W and8W group. These rats were given sodium-depletion diet(0.04%sodium).10rats of control group were sacrificed after feeding low-sodium for1week. The others were injected into the abdominal cavity at a dose of CsA at30milligram per kilogram every day. The renal and blood samples in1W,2W,4W,6W and8W group were harvested1,2,4,8and12weeks after intraperitoneally treated.2. Pathologic examination of renal in ratsAll the kidneys of rats were embedded with paraffin wax. Then do serial section and dyed them with Masson trichrome stain, Sirius Red and immunofluorescence assay respectively. Pathological changes of renal vascularity, renal interstitium and glomerulus in each group were observed. For further study, according to principles of region includes renal upper, middle and lower pole,5regions on the coronal slice of the kidney have been chosen for surveillance regions. Banff fibrosis score:0grade (5%less than the cortical area of interstitial fibrosis),1grade (6～25%),2grade (26～50%) and3grade (interstitial fibrosis is more than50%), was calculated based on image by different detected methods, and compared between any two groups.3. The study of renal interstitial fibrosis by SHG/TPEF microscopy techniqueImages of renal specimens without stain were obtained by SHG/TPEF microscopy scanning with the same5regions of traditional pathological staining images in each group. To evaluated the accuracy and specificity of SHG/TPEF imaging technique for rat renal interstitial fibrosis assessment, the renal interstitial fibrosis grades, based on Banff07standard, to be Kappa test the SHG/TPEF images and Sirius Red, immunofluorescence images respectively.The fibrosis index, was defined as the percentage of collagen fibers area in the total surveillance regions, was calculated by image based on the SHG/TPEF, Masson trichrome stain, Sirius Red and immunofluorescence in different administration time groups. The different of fibrosis index by these technologies for each group were analyzed. The sensitivity of renal interstitial fibrosis induced by cyclosporine A evaluated by SHG/TPEF microscopy technique, was assessed by comparing SHG/TPEF images on early interstitial fibrosis specimen with the existing pathological technique.4. SPSS13.0was used to for the statistical analysis of the data. The measurement data was showed as mean±SD (x±s), the difference was consider statistically significant when P<0.05.Results1. CsA concentration and The fibrosis grades CsA concentration of all5groups had no statistical difference (F=1.726, P=0.153). The fibrosis grades, according to Banff’07renal interstitial fibrosis grading standard, in each group were calculated based on Masson stain, Sirius Red and immunofluorescence. The difference of Banff fibrosis grades in control,1W,2W and4W group were not obvious based on Masson stain(χ2=6.500, P=0.09), but significant difference among these4groups based on Sirius Red (χ2=18.527, P<0.01) and immunofluorescence assay (χ2=20.797, P<0.01). The significant difference of Banff fibrosis score in4W,6W and8W group based on Masson stain(χ2=7.843, P=0.020), Sirius Red (χ2=12.974,.P=0.002) and immunofluorescence assay (χ2=14.752, P=0.001). Compared with Masson stain, Sirius Red and immunofluorescence assay of Banff fibrosis score in4W group(χ2=8.700,P=0.013) and8W group (χ2=6.235, P=0.044) had significant difference, while the other groups had no statistical difference (χ2=0.000,χ2=0.360,χ2=0.207,;χ2=4.325; P>0.05)2. Consistency and fibrosis index of SHG/TPEF microscopicThe TPEF signal is shown in red and SHG signal with a green pseudocolor, under SHG/TPEF microscopic appearance of renal specimens without staining. The SHG/TPEF picture is overlay by SHG signal and TPEF signal. SHG images images representing collagen image fibers, and TPEF images reveal red color, stand for the renal tissue. SHG/TPEF images displays collagen fibers deposited and its surrounding tissue.The renal specimens were graded based on the SHG/TPEF microscopic, Sirius Red and immunofluorescence fibrosis grade based on Banff interstitial fibrosis standard, respectively. The Kappa test gave the value of0.954>0.75(P=0.000) demonstrated a good agreement between SHG/TPEF microscopic and Sirius Red. As well a value of0.905>0.75(P=0.000) between SHG/TPEF microscopic and immunofluorescence. Compared with the fibrosis index, based on Masson stain, Sirius Red and immunofluorescence assay, in the control(F=0.428, P=0.143),1W group(F=0.821, P=0.491) and8W group (F=2.029,F=0.127) had no significant difference, while the other groups had statistical difference(F=3.424, P=0.027; F=0.241, P=0.001; F=3.939, P=0.016). Further use of the above indicators LSD-t pairwise comparison methods to compare results to fibrosis index based on SHG/TPEF microscopic the highest in2W group. Likewise, fibrosis index based on SHG/TPEF microscopic the highest in4W and8W group.Conclusions1. Appeared obvious pathologic changes of renal interstitial fibrosis, at a dose of CsA at30mg/(kg·d) after4week intraperitoneally treated in SD rats, was observed by traditional pathological staining2. These method, Sirius Red and immunofluorescence assay, had more specificity and sensitivity than Masson trichrome stain.3. Compared with Masson trichrome stain, Sirius Red and immunofluorescence assay, the renal interstitial fibrosis observed by SHG/TPEF microscopy technique with the same accuracy and specificity, but SHG/TPEF microscopy had high sensibility in the early diagnosis.4. SHG/TPEF microscopy technology can clearly show renal interstitial fibrosis and renal tissue structure. Compared with Masson stain, Sirius Red and immunofluorescence assay, SHG/TPEF microscopy technology with the advantages on specimens without dying, the images more intuitive and sensitive.