Animal Experiment Study Of Contrast-enhanced Ultrasound In The Diagnosis Of Hepatic Artery Stenosis

ObjectivesHepatic artery stenosis is one of common complications after liver transplantation and the clinical incidence is very high.Patency of hepatic artery is of great significance to assess of blood supply of the biliary system after transplantation, hepatic artery stenosis can cause transplant failure and receptor death.Therefore the monitoring of hepatic artery complications after transplantation is crucial.The golden standard for diagnosing hepatic artery stenosis is Still X-ray angiography,but high expense of examination,complication of the operation and nephrotoxicity contrast restrict it's clinical application.Other evaluation methods including CT,MRI and radionuclide scanning machines have some inextricable weak points like,requirement for high-tech apparatus,no bedside examination,not being repeated,lack of stability in radioactive tracers and radiological contaminations.Color Doppler flow imaging (CDFI) is the preferred method for the diagnosis of hepatic artery stenosis after liver transplantation because of the convenience,noninvasive,repeatable bedside inspecttion. But compared with contrast-enhanced ultrasound,CDFI is vulnerable with spillover,sports noise interference and other factors which may result in comparatively lower accuracy.Contrast-enhanced ultrasound is an emerging technology to evaluate substantive organ perfusion,micro-bubbles has been used as the tracer to observe organization perfusion with ultrasound.In addition,contrast-enhanced ultrasound and related contrast imaging technology development gathering the functions of all noninvasive diagnostic imaging,it enables us to achieve a small vascular imaging perfusion level observation.Therefore,avoiding the internal insufficient of CDFI,it will be more accurate in showing artery stenosis after liver transplantation and evaluating the degree of stenosis,which will help to improve the diagnostic accuracy of hepatic arterial complications after liver transplantation.This test applies contrast-enhanced ultrasound and quantitative analysis with the acoustic densitometry technique to diagnose the hepatic artery stenosis so as to find a simple, accurate,safe and noninvasive diagnostic method to diagnose hepatic artery stenosis after liver transplantation.Materials and Methods1 Animal PreparationTen healthy dogs(between 10 to 15 kg) in either sex were selected from Laboratory Animal Centre in Nanfang Hospital.2 Instrument and EquipmentThe equipment used was Philips IU22 color Doppler ultrasound with C5-2 (frequency 2.0～5.0MHz) and L9-3(frequency 3.0～9.0MHz),built-in acoustic analysis software QLAB density and select the reverse pulse harmonic imaging technology. Other settings of instrument(Gain,scanning depth,etc.) in contrast remained unchanged before and after,all the same set of applications.X-ray angiography equipment selection SIEMENS AXIOM lconos R200FL-Ccm.3 Contrast agentThe contrast agent was Perfluoropropane-albumin microsphere that provided by NangFang hospital.Diameter of micro-bubble are 2.0～5.0μm and the concentration is 1.0×10~9～2.0×10~9/ml with the distribution 90%＜10.0μm and pH 6.4～7.4.4 Experimental methods4.1 The laboratory preparation:after anesthesia by intravenous injected 3% hydrochloride pentobarbital at 30mg/kg,chest and abdominal skin preparation has been finished for every dog.During the experimental process,3%hydrochloride pentobarbital injection has been used to maintain anesthesia state;After it,separation of left femoral vein,the establishment of intravenous access and connection to the three links for infusion of contrast agent,the liquid and drugs,and the maintenance of fluid balance by normal saline have been finished step by step.The right femoral artery has been separated for the X-ray angiography intubation;Taken median abdominal incision to expose the liver and liver arteries,meanwhile to dissociate the root of hepatic artery.4.2 to establish the model of hepatic artery stenosis:to cincture the hepatic artery by surgical thread,then penetrate the ends of them into the self-made silicone tubes, clamp at the exit with vascular clamps,the wall of vessel can be pressed through thread oppression so that hepatic artery stenosis model has been established;in this way,the stenosis degree of hepatic artery can be adjusted by changing the power put on the vascular clamp.The accurate numerical values can be measured by vernier caliper and by calculating the cross section with a two-dimensional ultrasound.In the experiment,inspections were taken from the very beginning when the vessels are normal to different stages when the degree of stenosis of each dog were at 50%, 70%,90%,100%respectively.4.3 X-ray angiography:The interposition catheter will be inserted into the femoral artery guidance with X-ray fluoroscopy,sent to the liver artery through the abdominal aorta.Being injected with 20%meglumine diatrizoate 5 ml,the degree of stenosis can be confirmed by X-ray angiog- raphy.4.4 CDFI inspection:To observe the blood flow of hepatic artery of each dog and to detect its velocity peak(Vp),acceleration time(SAT) and resistive index(RI).4.5 Contrast-enhanced ultrasound inspection:A bolus injections of ultrasound contrast agent(0.025～0.03ml/kg,about 0.4 ml) with 3 ml saline to diluted to femoral vein,with 5 ml injection of saline followed to irrigate wall,then respectively observed the hepatic artery,perfusion of liver,abdominal aorta and the left ventricular cavity.Interval time of repeated injection should be over 10 min.All contrast images were stored in CD-ROMs for offline analysis.5 Image analysisAfter the inspection,analysis software of acoustic densitometry QLAB has been applied to analyze.Selected region-of-interest(ROI) should be in appropriate size and shape and placed respectively in liver parenchyma,abdominal aorta and the left ventricular cavity(ROI from the edge within envelope 2 mm),avoiding larger vessels. The size and shape of ROI were the same to reduce sampling error.TIC curve will be automatically drawn by the machine,and the parameters including PI,AUC,TTP and Alpha of every stenosis level in liver parenchyma,abdominal aorta and the left ventricular cavity can be captured according to the curves.6 Pathological examinationSmall liver tissues of all animals have been obtained immediately after the experiment,then they were fixed up by10%formalin-fixed and were processed into HE stained sections for pathologic examination.Meanwhile 5ml blood of each dogs have been taken for biochemical tests.After ligated hepatic artery and delt with the wounds and sutured incision,dogs would be sent back to the experimental animal center.They would be executed with injection of air after feeding 24 hours,then the liver tissues would be conducted both pathologically and biochemically,the experimental results of the two groups would be compared.7 Statistical analysisData were expressed as mean±standard deviation.Statistical analysis is conducted with SPSS13.0.Means were compared by ANOVA,multiple comparison between means(LSD),correlation analysis with spearman.The P value(bilateral) lower than 0.05 was considered statistically significant.ResultsUltrasound contrast image shows the normal liver parenchyma echo were enhanced after administration of contrast agents.The TIC curve with an early and steep increase of similar magnitude,contrast agent were perfused in lumen of hepatic artery and fine continuity with complete filling.X-ray angiography can display filling integrity of hepatic artery.Liver parenchyma and hepatic artery contrast images displayed(degree of hepatic artery stenosed at 50%,70%and 90%):liver parenchyma showed enhanced imaging,but the enhanced level were lower than that of hepatic artery at basic state,and enhancement were less and less with the increase of stenosis. The increase of the TIC curve has slowed and the peak reduced.Hepatic artery stenosis were partially visible after contrast agent,and its significance increased with the enhanced of stenosis degree;X-ray angiography images also clearly confirmed the results.Statistical analysis showed that:Compared with the normal,the PI to PI ratio of liver tissue was decreased in four type stenosis(P＜0.05),and the ratio reduced with the increase of the stenosis level(P＜0.05).Comparison of AUC,TTP and Alpha between the four type stenosis and the the basic state showed no statistical significance.The 50 cases of canine hepatic artery were respectively inspeeted by color Doppler and contrast-enhanced ultrasound,and the result indicated:CDFI and PW checks to narrow peak flow＞2.0 m / s,SAT＞0.08s and RI＜0.5 blood flow parameters as a standards to diagnose the hepatic artery stenosis,found 45 times abnormal parameters of blood flow,the sensitivity,specificity and positive predictive value were 100%,50%and 89%.Contrast enhanced ultrasound can clearly showing the course of hepatic artery and 39 times hepatic artery stenosis,performance for the partial filling of contrast agent and same show the location and degree with X-ray angiography.The sensitivity,specificity and positive predictive value were 100%, 91%and 98%,There were statistically significant of specificity and positive predictive values between color Doppler ultrasound and contrast enhanced ultrasound.ConclusionsContrast-enhanced ultrasound is a new technology to evaluate perfusion of substantive organs.Micro-bubbles are tracers to observe organization perfusion with ultrasound.Pulse inversion harmonic contrast ultrasound imaging technology allows wider bandwidth,which improves resolution and increases sensitivity of contrast agent.The experimental indicates that combination of the two technologies can avoid the inherent insufficient of CDFI,direct observation of stripe-shaped imaging resulted from the bubbles flowing in hepatic artery has been possible.Though direct observation of local perfusion deficiency,the stenosis in hepatic artery after transplantation and its degree can be diagnosed,therefore the accuracy of diagnosis on hepatic artery stenosis can be significantly increased.It has been shown in our research that the result provided by ultrasound matched with that by X-ray angiography.This study also take the advantage of QLAB quantitative analysis technology. The grayscale intensity in ROI can be described by TIC,which enables the quantitative parameters of liver tissue perfusion.Quantitative analysis through TIC curve can further provide a parameter,PI to PI ratio of liver tissue,to evaluate normal and abnormal hepatic blood flow,which is sensitive index to evaluate degree of hepatic arterial stenosis.The application of the new technique will enable the detection of hepatic artery anastomosis possible,in this way,it will become a sensitive index to evaluate the abnormal blood flow of hepatic artery besides detecting hepatic artery.This will provide valuable experience of diagnosis on hepatic artery stenosis after liver transplanted using contrast enhanced ultrasound.