| Objective:To investigate the establishment and imaging characteristics of a model of hepatic ischemia reperfusion injury(HIRI)in rats,and to study the feasibility of evans blue(EB)as a necrosis-targeting probe using this model.Methods:Twelve male SD rats with a body weight of about 250g were selected and divided into two groups with 6 rats in each group.In the experimental group,the right hepatic portal vein and hepatic artery were both ligated,and the ligation line was released 3 hours later.In the sham operation group,the remaining operations were the same as the experimental group except that no ligation was performed on the right lobe vessels of the liver.MRI scans were performed 6 hours after the blood supply was restored,including flat-sweep T1-weighted image(T1WI),T2-weighted image(T2WI),diffusion weighted imaging(DWI)with b values of 0 s/mm2 and 1000 s/mm2,and contrast enhancement-T1 weighted image(CE-T1WI).After MRI scan,0.5ml of EB was injected into tail vein immediately.The rats were euthanized 24 hours later,and the major organs(brain,heart,lung,liver,spleen and kidney)were immediately removed for fluorescence imaging.After that,the content of EB in each tissue was determined by using the enzyme labelling apparatus and referring to the method of Wang(2014).Liver specimens were retained and sent to pathology for hematoxylin-eosin staining(HE)staining.The frozen section was observed and analyzed under fluorescence microscope.The accuracy of EB content in tissue was analyzed and compared by fluorescence imaging and enzyme labeling,and the distribution of EB in the rat model was studied.Results:MRI findings:MRI findings of the experimental group showed that the lesion presented high flaky signals on T2WI,high flaky signals on T1WI,and high flaky signals on CE-T1WI,with clear boundaries with surrounding hepatic lobes;as well as high signals on DWI.Vitro living fluorescence imaging:The fluorescence intensity from low to high was in order of brain,heart,spleen,kidney,lung and normal liver in the experimental group and the control group respectively,while the fluorescence intensity of diseased liver was the highest.Quantitative analysis of fluorescence intensity value found that there was no statistical difference in fluorescence intensity of each organ between the two groups(p>0.05).There was a statistically significant difference in fluorescence intensity between the pathological liver surface of the experimental group and the normal liver surface of the control group(p<0.05).There were also statistically significant differences in fluorescence intensity between the pathological liver center and normal liver center(p<0.05).The difference in fluorescence intensity between the lesion central section and its surface was also statistically significant(p<0.05).Determination of EB content in the tissues by enzyme labeling method:the lowest content of EB was found in the brain tissues of the experimental group and the control group,followed by heart,lung,normal liver,spleen and kidney tissues.The difference of EB content between the two groups was not statistically significant(p>0.05).EB content in the pathological liver was the highest among organs,and the difference of EB content between normal and damgaged liver tissues was statistically significant(p<0.05).Pathological results:HE staining of liver specimens in the control group showed normal liver structure,liver lobules were present,liver cords were arranged orderly,and cells were stained clearly.No red fluorescence was observed in the area under the cryosection fluorescence microscope.HE staining of liver specimens in the experimental group showed homogenous red staining of liver tissues,massive necrotic lesions in hepatic lobule,enlarged hepatic sinus space,inflammatory response bands and neutrophilic granulocytes at the edge of liver tissues.A large amount of red fluorescence around the central vein was observed under the fluorescence microscope of the frozen section,corresponding to the necrotic foci stained by HE staining pathologically.Conclusion:HIRI model of rats combined with dynamic monitoring at non-invasive MRI is a good platform to study necrosis-targeting probes.The content of EB in the tissues can be accurately measured by vitro living fluorescence imaging and enzyme assay.The EB has shown a necrosis-targeting characteristic in HIRI model and in vitro fluorescence. |
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