| IntroductionChina is a highly endemic region of viral hepatitis, especially hepatitis B virus infection, and the number of patients with cirrhosis and liver cancer caused by hepatitis B is large. Operation excision is still the first choice to cure liver cancer. In the process of liver surgery, hepatic blood flow occlusion is needed more or less,which would cause hepatic ischemia reperfusion injury. The pathogenesy of HIRI is complicated, and present study suggests that HIRI is caused by many factors. The interaction of many factors lead to loss of function and even death of large numbers of hepatic parenchymal cells, which is one of the important reasons for the failure of liver function after surgery. Thus, the accurate assessment of liver function after hepatic ischemia reperfusion injury is important for clinicians to estimate the prognosis of the patients and make appropriate treatment measures, which is valuable to increase the patient’s survival rates.There are many methods evaluating hepatic function, in which the liver function imaging method based on asialoglycoprotein receptor can observe the uptake,metabolism and excretion of liver cells dynamically, and can evaluate functional hepatic cell volume, which considered to be a promising method to evaluate liver function. At present, the asialoglycoprotein receptor imaging agent of 99 m Tc GSA has been concerned. 99 m Tc GSA can specifically bind to ASGPR, which is a kind of protein expressed on liver cell membrane surface, and used for hepatic imaging. the number and function of ASGPR are positively correlated with the liver function, thus we can assess the liver function through detecting the number and activity of liver cell ASGPR. The hepatic cells could have apoptosis and necrosis more or less when HIRI occurs, and the quantity and activity of ASGPR on hepatic cell membrane surface could decrease more or less. Through ASGPR imaging, we can know the status of liver function and evaluate the damage caused by the HIRI, which will help to take timely treatment measures and estimate prognosis. In addition,99m Tc-GSA liver SPECT/CT plane and tomographic imaging can also evaluate the regional liver function. We can simulate hepatic resection by drawing lines on each SPECT/ CT transection and calculate the proportion of residual or regional liver function to the whole liver function. This ratio can evaluate the liver functional reserve and predict the operation risk, which is of great help to the clinical work.However the computation and data-operation are very complicated. As to meet the clinical needs, Peking Union Medical College Hospital has established a computer program, which would obtain 3D liver function image and simulate the surgical resection and calculate the function of each part of liver. This program will make the evaluation of liver function and regional liver function more operational, and the program is in its exploration and perfecting stage now.This study establish different degrees of HIRI models in New Zealand white rabbits, and abtain corresponding liver enzyme and pathological results. Then,perform 99 m Tc GSA liver imaging to obtain the corresponding imaging results.Analyze the correlativity between imaging results, serum liver enzyme and tissue pathological results to study the reliability of 99 m Tc GSA evaluating liver ischemia reperfusion injury. So as to provide theoretical basis and experimental evidence for clinical application of 99 m Tc-GSA in predicting hepatic ischemia reperfusion injury and helping making reasonable treatment plan.Part I Establishment of Heatic Ischemia Reperfusion in a Rabbit ModelObjectiveThis part was to establish different degrees of HIRI models in New Zealand white rabbits, and investigate the changes of the serum liver enzymes and pathomorphology before and after HIRI, and those changes in the third day and the seventh day after surgery. Thus to provide pathological basis for the research of evaluation of liver function of 99 m Tc GSA liver imaging in HIRI.Materials and Methods1. Experimental animals and groupsThe healthy adult New Zealand white rabbits with similar weight were randomly divided into four groups: sham operative group(group A),ischemia- reperfusion group(group B, C,D). In group B, the hepatic blood flow was blocked for 30 min, and in group C, the hepatic blood flow was blocked for 45 min, and the group D was blocked for 60 min. In sham operative group, only separate the hepatoduodenal ligament without blocking the blood supply of hepatic.2. Establishing rabbit model of hepatic ischemia reperfusion and obtaining hepatic tissue samplesBlock the hepatic blood flow by clamping the hepatic pedicle with a clamp for30 min, 45 min and 60 min according to the grouping. Then remove the vascular clamp and take the hepatic tissue and blood sample after restored the liver blood perfusion for 1h. In the third day and the seventh day after surgery, obtain the the hepatic tissue with the guidness of ultrasound and collect blood samples of rabbits in the four groups.3. Samples processing(1) Separate the blood serum from the blood samples to detect the content of ALT.(2) Fix the hepatic tissue samples with formalin and send to the pathology department for HE staining.4. Statistical analysisApply statistical package of SPSS 22.0 statistical software for data analysis.Experimental data and integral are expressed as mean ± standard deviation and repeated-measures analysis of variance is applied to process data. First, use the sphericity test to analyze whether a correlation existed between the repeated measurement datas and then apply the corresponding variance analysis model. P value less than 0.05 was considered to be of statistical significance.Result1. Serum EnzymeThe ALT level in the sham operation group stay in the normal range, and the level rose with the prolongation of ischemia time, and with significant difference. In the third day, the ALT level decreased, and in the seventh day nearly returned to normal.2. Liver histopathologyThe liver histopathology of sham operation group and ischemia group before surgery were basically normal with homogeneous cytoplasm and hyperchromatic nuclei. While the heptic tissue in ischemia and reperfusion group after surgery was damaged in varying degrees, with the clamping 60 min group damaged heavily,followed by occlusion 45 min, finally the clamped 30 min group. In the three ischemia/ reperfusion groups, the liver cells were most severely damaged at 1h after reperfusion, then recovered gradually over time, and nearly back to normal in the seventh day and the difference was statistically significant.ConclusionThe establishment of rabbit model of HIRI is simple and with high success rate.With the prolongation of ischemic time, the level of liver enzyme and the pathological injury increased. The ischemia reperfusion groups have the most severve damage at the time of T2(1h after ischemia reperfusion), and recovered to some extent over time. These gradient changes of pathology could fully meet the need of functional imaging studies in the next step.Part II Experimental study of 99 m Tc-GSA imaging for hepatic ischemia reperfusion injuryObjectiveInvestigate the value of 99 m Tc-GSA hepatic imaging for the HIRI in the basement of the above liver enzymes and pathological changes of experimental models.Materials and Methods1. Group dividation and establishent of rabbit model of hepatic ischemia reperfusionRabbits were divided into sham operation group and ischemia reperfusion group,and the ischemia reperfusion group was divided into 3 groups again according to the the duration time of ischemia(blocking the blood flow for 30 min, 45 min or 60min).Continue the follow-up experiments in the third day and the seventh day after surgery.2. 99mTc-GSA preparation and imaging parametersPrepare 99 m Tc-GSA with GSA kit according to the method described in the literature, and detect radiochemical purity of the product. Use it for imaging If qualified. Perform the imaging before and after the surgery and in the third day and seventh day. Obtain liver uptake rate(LHL15) and blood clearance rate(HH15)according to the imaging results.3. Blood biochemical and pathological examinationThe blood biochemical and pathological examination can be referred in the Part I.4. Statistical analysisAnalyze the imaging results, and compare them with the serum liver enzyme and tissue pathological results to study the correlativity between them.Result1. LHL15Among the four groups, when the time was fixed, the level of LHL15 in group D(the clamped 60 min group) is the lowest, and then the clamped 45 min group, and last the clamped 30 min group.The LHL15 in sham operation group amost maintained at a normal level. The LHL15 level in the four groups was lowest at the time of T2(1h after ischemia reperfusion), and in the third day recovered a little, and in the seventh day recovered more. LHL15 in the sham operated group amost maintained at a normal level.2. HH15Among the four groups, when the time was fixed, the level of HH15 in group D(the clamped 60 min group) is the highest, and then the clamped 45 min group, and last the clamped 30 min group.The HH15 in sham operation group amost maintained at a normal level. The HH15 level in the four groups was highest at the time of T2(1h after ischemia reperfusion), and in the third day dropped a little, and in the seventh day dropped more. HH15 in the sham operated group amost maintained at a normal level.3. Analyzing the correlation between the imaging results, pathology and liver enzymeThe Pearson correlation test of LHL15, HH15, ALT and pathologic score showed that there were four pairs of variables having highly negative correlation,the LHL15 with HH15(r =-0.967, p<0.01), LHL15 with pathologic score( r =-0.957,p<0.01), LHL15 with ALT( r =-0.934, p<0.01), and two pairs of variables having highly positive correlation, the HH15 with pathologic score( r = 0.959, p<0.01) and HH15 with ALT( r = 0.942, p<0.01).ConclusionThe parameters of LHL15 and HH15 obtained from 99 m Tc-GSA hepatic imaging have good correlations with the rusults of liver enzymes and histopathology,and could accurately reflect the changes of liver function after HIRI. |
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