Application Of ^99mTc-PVLA And ^99mTc-GSA In Evaluating The Hepatic Ischemia-Reperfusion Injury In Rabbit

BackgroundHepatic Ischemia Reperfusion Injury（IRI） refers to the phenomenon that the injury of liver which lacks both blood and oxygen, accentuates after recovering blood and oxygen supplying. Because of the liver’s particular blood supplying system as well as its particular position, hepatic IRI seldomly happens.However, surgical resection of hepatocellular carcinoma, liver transplantation and traumas can inevitably lead to IRI of liver. How to evaluate the liver’s storage function in IRI acuratly and appropriately will be of great challenge not only for diagnosing and curing liver diseases but also for evaluating the curing effects and prognosis. The most commonly used methods for evaluating liver functions in clinic are the liver biochemistry, the Child-Pugh grading score, Model for end-stage liver disease score（MELD）, and the indocyanine green（ICG）excretion experiments, but all have some disadvantages, which limit their clinical application. This study is designed to find a new way to evaluate the hepatic function when hepatic IRI occurs.Asialoglycoprotein receptor（ASGPR） is also called liver lectin or Ashwell-Morell receptor and is mainly scattered on the surface of the parenchymal heptic cells in hepatic sinusoid, also seldom expressed in some extrahepatic tissues. When inflammation, hardening or malignant lesions appear on liver, ASGPR on the surface will be down-regulated accordingly. Many researches have already verified that ^99mTc-GSA is of great significance in evaluating the liver’s function quantitatively and in liver targeted therapy.^99mTc-PVLA, as a new liver image scope, belongs to the lactose colloid category compared with GSA.There is still no report about the relationship between the ischemia reperfusion injury and the level of ASGPR on liver as well as the ^99mTc-GSA and ^99mTc-PVLA application value in ischemia reperfusion injury.This experiment, through the imaging of blood biochemistry, immunohistochemistry, ^99mTc-PVLA and^99mTc-GSA, evaluates the liver’s functions and ASGPR’s expressing conditions in different blood lacking conditions and different reperfusion time so as to further evaluate the applicable value of ^99mTc-PVLA and^99mTc-GSA in the ischemia reperfusion of liver.ObjectivesEstablish the liver ischemia reperfusion injury model by using New Zealand white rabbits; meassure the rabbit liver’s functions and the ASGPR’s expressing conditions in different lacking conditions and different reperfusion time; compare and evaluate the significance of ^99mTc-PVLA and ^99mTc-GSA SPECT/CT in evaluating the liver IRI.Methods:1. The rabbit liver’s ASGPR’s expression level in ischemia reperfusion.Group division in this experiment: divide randomly 32 male and female Newland rabbits with no obvious distinctions in weight into Group A, Group B, Group C and Group D（N=8）. Group A is the negative control group, Group B: lacking blood for 30 minutes, Group C: lacking blood for 45 minutes,Group D: lacking blood for 60 minutes. Divide each group randomly into two small groups（N=4）: A1 A2;B1 B2; C1 C2; D1 D2 among which ^99mTc-GSA SPCET/CT imaging is used for Group A1 B1 C1 D1 while 99m Tc-PLVA SPCET/CT imaging is used for the rest groups.Experiment method: In the first seven days（T1） after the first open laparotomy, biopsy the liver through abdomen to get pathological samples under the introduction of supersonic ware. After seven days,adopt the renewed Pringle method to establish the ischemia reperfusion model of the rabbit liver. Group A:only open laparotomy and no porta hepatis clipping; Group B: open laparotomy and clipped the porta hepatis for 30 minutes; Group C: open laparotomy and clipped the porta hepatis for 45 minutes; Group D:open laparotomy and clipped the porta hepatis for 60 minutes. After loosing the scatheless vascular clamp for an hour（T2）, take the pathological samples from the liver and close the abdomen; take the pathological samples by piercing the liver through abdomen under the introduction of supersonic ware after three days（T3） and seven days（T4） respectively. Image processing: mark out the ROI of heart and liver respectively,Mark the corresponding compound according to the references and instructions and test the radioactive chemical purity and chemical purity by using TLC and HPLC. Sketch out the corresponding TAC, calculate the HH15 and LHL15 and MRI, then adopt SPSS for statistical analysis.After getting the pathological samples, conduct HE dyeing and ASGPR and HIF-1α immunohistochemical testing. Before anaesthesia,blood samples will be taken to test the white blood cell and ALT. Examine closely the rabbits’ mental state,eating and drinking water conditions as well as the nature calling conditions before and after the operation.Apply SPSS 22 for statistical analysis.Results: Group A: no obvious change in the rabbits’ mental state, eating and drinking water conditions as well as the nature conditions before and after the operation; Group B C D: the rabbits’ mental state becomes worse to varying degrees, they moved less often, had less food and water, pissed less often with the colour of their urine being yellow, defecated less often with the excrement being drier and the size being smaller. The order of severity of the phenomenon is as follows: D, C, B. Additionally, compared with the rabbits in other groups, the Group D rabbits have obvious inclination to bleed after being pierced and given an acupuncture treatment. For the blood biochemical index, there is no obvious distinction in the white blood cell at different time of a certain group or among different groups. The variation tendency of ALT is basically the same. Comparison within a group: for Group A, there is no obvious change at different time points and for other groups, B: T2> T3 = T4 = T1, Group C: T2> T3> T4 = T1, D group: T2> T3>T4> T1（“=” means there is no obvious distinctions between Group A and Group B and there exist obvious distinctions among all the other groups p<0.05,and the former mean larger, the same below）; T3:A<B<C<D（obvious distinctions exist among all the groups p<0.05）. Comparison among different groups:T1: no significant difference between the groups, T2: D> C> B> A; T3: D> C> B = A; T4: D> C= B= A.Statistical analysis which is performed first between data from ^99mTc-PVLA and ^99mTc-GSA imaging parameters shows no significant statistical difference. Therefore put on the analysis of imaging parameters from ^99mTc-PVLA of ^99mTc-GSA together to compare. Comparison within a group: A: no difference at all time points, B: T2> T1 = T4> T3, C: T2> T3> T1> T4, D: T2> T3> T1> T4; Comparison among different groups : no significant difference between the groups; T2: D> C> B> A; T3: D> C> A> B; T4: A = B> C>D. Trends of LHL15 and MRI are basically the same:reduce with the extension of time of clamping, after surgery gradually recover, as slower as the longer of clamping. HE: edema and inflammation of the liver cell worsen with the extension of ischemic cell infiltration time, T2 the heaviest, recovery graduals with time prolonges. ASGPR in normal liver and postoperative liver function expresses normal and early postoperative T2 the minimum. HIF-1 α restore normal liver and normal liver functions almost no expression, T2 expression the most, and gradually reduced.Conclusion:1 The ASGPR expression level declined during the ischemia reperfusion to the rabbits’ livers and isin negetive correlation to liver’s injuring degree.2. The data obtained between from ^99mTc-GSA and ^99mTc-PLVA SPCET/CT in liver ischemia reperfusion show no significant differences. ^99mTc-GSA and ^99mTc-PLVA SPCET/CT imaging methods are clinically applicable for evaluating hepatic function in hepatic IRI.