Experimental Study Of Diagnosis With Multiphoton Microscopy Imaging For Pancreatic And Liver Disease

BackgroundPathological analysis is the gold standard for pancreatic cancer diagnosis, but accurate diagnosis rate is low and a long period of time is needed. Traditional treatment for pancreatic carcinoma is surgical removal which is supposed to be effective, and whether operation margins are free of tumor residual or not is an important factor to determine the prognosis. Frequent examination of frozen pathological slices sent from the operative are needed before negative margin can be confirmed. Therefore, preoperative and intraoperative diagnosis of pancreatic cancer is still clinically unresolved.Carcinoma of head of pancreas tends to oppresses bile duct, therefore, obstructive jaundice occurs. Combined hepatic removal is a surgical option for the solitary liver metastasis of carcinoma of head of pancreas. Moreover, liver tumors or biliary tract neoplasm are often associated with obstructive jaundice. Surgery is the important treatment of obstructive jaundice. Anal block is needed to reduce bleeding in liver resection operation, meanwhile, hepatic ischemia‐reperfusion injury are produced after the block is lifted. Clinical method for hepatic ischemia‐reperfusion injury examination are still insufficient and intraoperative evaluation for hepatic ischemia‐reperfusion injury are clinically unresolved either.Multi‐photon microscopy imaging technology can capture the histological features as well as the metabolism of cells and the optical diagnosis by MPM for pancreatic tumor and obstructive hepatic ischemia‐reperfusion injury has not been publicly reported. For the current clinical problem in need of solution, this research focuses on feasibility of optical diagnosis by MPM for pancreatic tumor and hepatic ischemia‐reperfusion injury in liver tissues as well as mechanisms of obstructive jaundice after hepatic ischemia‐reperfusion injury. PART ONE: Multi‐photon microscopy imaging techniques for optical diagnostics of pancreatic tumorObjective: To evaluate the feasibility of using multi‐photon microscopy imaging to make diagnosis for pancreatic tumor.Methods: study pancreatic paraffin slices and fresh tissue by MPM and compare the test results with H & E staining.Results: In the pancreatic paraffin slices and fresh tissues, MPM are able to image the tumor as well as the HE staining and there are comparability between the images produced by MPM and H & E staining.Conclusion: MPM imaging technology can distinguish pancreatic tumor from normal pancreatic tissue and tells differences between benign and malignant tumors of the pancreas.PART TWO: Applications of the multi‐photon microscopy imaging in hepatic ischemia‐reperfusion injury with obstructive jaundice. Ⅰ. Establish an experimental animal model for obstructive jaundiceObjective: Establishment of animal model for obstructive jaundice.Methods: 20 male SD rats are divided into two groups randomly: simulation operation group and obstructive jaundice group.(Group SO, N = 10; Group OJ, N = 10). Obstructive jaundice group are set up to ligate rat common bile duct, but simulation operation group do not ligate rat common bile duct. Liver and kidney function are checked in two groups.Results: No significant change of liver function indexes for group SO is observed. Gradual increase of liver and kidney function indexes for group OJ is observed and the indexes of group OJ are significantly higher than group SO(P <0.05).Conclusion: The animal model of obstructive jaundice is successfully established. Ⅱ. Mechanisms of hepatic ischemia‐reperfusion injury with obstructive jaundiceObjective: Study the role of TNF‐a 、 IL‐1β and NF‐κB in hepatic ischemia‐reperfusion injury with obstructive jaundice.Methods: 80 male SD rats are divided into two groups randomly: sham operation group(group SO, N = 40) and hepatic ischemia‐reperfusion injury group(group HIRI, N = 40). By different time duration of reperfusion, either group are further divided into 4 subgroups(group 0h, 3h, 6h and 12 h, N=10). One week after the experimental animal model of obstructive jaundice was built, establish experimental model of hepatic ischemia‐reperfusion injury to detect and compare liver and kidney function, TNF‐a、IL‐1β and NF‐κB level between group SO and HIRIResults: No significant change of liver function indexes is detected for group SO, For group HIRI, The indexes of liver and kidney function, the content increase of TNF‐a and IL‐1β is higher. And, the difference detected is statistically significant. For subgroup oh, 3h, 6h and 12 h, the expression intensity of NF‐κB、TNF‐α and IL‐1β, HIRI group is higher than the corresponding SO group(P <0.01).Conclusions: In hepatic ischemia‐reperfusion injury with obstructive jaundice, the expression level of TNF‐a and IL‐1β increases and the expression of TNF‐a and IL‐1β formulates the critical factor of reperfusion injury, therefore, NF‐κB injuries liver tissue by regulating the expression of TNF‐a and IL‐1β. Ⅲ. Optical diagnosis with MPM for hepatic ischemia‐reperfusion injury with obstructive jaundiceObjective: study the feasibility of diagnosis of multi‐photon microscopy imaging technology for hepatic ischemia‐reperfusion injury with obstructive jaundice.Methods: Extract 10 slices of frozen samples of fresh liver tissues from subgroup respectively, detect them by MPM and H & E stained light microscopy,Results: No significance difference is observed among MPM images of liver tissues from Group SO. All pictures reveal cellular swelling degeneration and liver sinusoids varied by width, scattered in infiltrated inflammatory cells.The MPM images from group HIRI is observed as follows: Subgroup oh reveals significant swelling and degeneration of liver cells, hepatic sinusoid which varies in width are infiltrated by inflammatory cells and obviously collagen fibers; Subgroup 3h reveals a large area of cell necrosis, infiltration of inflammatory cells and swelling and degeneration of liver cells; Subgroup 6h reveals the infiltration of inflammatory cells, swelling and degeneration of liver cells, cell necrosis and hyperplasia of collagen fiber; Subgroup 12 h reveals infiltrated inflammatory cells infiltrated, swelling and degeneration of cells, cell necrosis and hyperplasia of collagen fiber. Each set of multi‐photon images and HE pictures are comparable. MPM reveals that light intensity of NADH/FAD from group SO is significantly higher than that of group HIRI. For each subgroup,(0h、3h、6h、12h), NADH/FAD redox ratio from group SO is higher than that of normal cells from group HIRI and the difference was significant.Conclusion: MPM imaging technology is capable of capturing hepatic ischemia‐reperfusion injury with obstructive jaundice.