| Background and Objective:Primary hepatic carcinoma was one of the high incidence malignant tumors. The mortality of primary hepatic carcinoma was located in second place, behind lung tumor in China. With the development of the various imaging techniques in recent years, diagnostic methods of primary hepatic carcinoma are becoming multiform, detection rate is gradually increasing, and these imaging techniques are widely spread year by year. Computed tomography perfusion has a function of providing information of haemodynamics in normal tissues and tumor tissues which is a newly emerging technique of CT functional imaging. It plays an important role in the diagnosis and differential diagnosis of solid mass in liver. Meanwhile, Contrast enhanced ultrasound (CEUS) is also being a hot issue in the field of ultrasonics. By revealing the features, the degree of blood supply, and the changes of haemodynamics of the tumors, CEUS can provide a high accuracy rating and specificity of small hepatic tumors. It is useful of detection of early malignant diseases.The rabbit model in bearing VX2 hepatic tumors is the only one imaging model in studying hepatic tumors. In recent years, correlation research of CT perfusion and pathological result in different growth periods of tumor, and intervene growth of tumors by radiation therapy, chemotherapy, intervention, or comparison research of haemodynamics of normal liver and tumor by real-time contrast enhanced ultrasound have been researched by some domestic researchers. Other domestic researchers evaluated the effect of the treatment intervention of VX2 hepatic tumors by Dual-energy CT. Some foreign researchers assessed the blood flow of VX2 hepatic tumors by CT perfusion and FDG PET, and explored the function of glucose in tumor growth. Comprehension research the experiment results of domestic and foreign researches, the research of evaluating the VX2 hepatic tumors by different imaging technologies and comparison research of molecular imaging has not been reported yet. This research performed Multi-slice CT perfusion and CEUS in animal model of VX2 hepatic tumors by building the model of implantation in vivo. The objective of the research is as follows:①To assessed the diagnostic value of VX2 hepatic tumors by Multi-slice CT perfusion and CEUS;②To observed the features of blood flow perfusion of the tumor by the two imaging techniques;③Combined with pathologic and reverse transcriptase-polymerase chain reaction results, analysis the correlation of the display of blood supply feeding vessels by CEUS, CT perfusion parameters and vascularization.Materials and methods:Twenty-eight healthy New Zealand white rabbits, weighing 2.0-3.Okg, were allocated randomly in trial group with twenty-five rabbits and control group with three rabbits.The carcinoma cells were subcultured by directly implanted into the root of the rabbit legs. The VX2 hepatic carcinoma mass were implanted into the liver of rabbits in trial group via laparotomic route.The rabbits were performed by multi-slice CT enhancement and perfusion at the 1st day which was the proper time for investigation after implantment by GE 64-detector Light speed VCT. The rabbits should abrosia 12h and should be anesthetized by 10% Chloral Hydrate with the injected dose of 7-8ml/kg. The scan mode called "Toggling-table" was used to performed the CT perfusion from diaphragm to inferior border of liver. The contrast medium (320mgI/ml) was bolus injected by rabbit's auricular vein with the injected speed of 0.7ml/s and injected dose of 1ml/kg. Scan parameter:100KV,80mA, slice-thickness was 5mm,360°rotation time was 0.6s, total scan time was 50s, detectors were configured as 64×0.625mm. Two minutes later, the CT enhancement scan was performed after being injected a contrast medium at the rate of 0.5ml/s and injected dose of 4ml by a high pressure twein injector, and at the same rate 2ml of normal saline was injected, scan was started when 13s,27s,59s was postponed after injected. The regions of interest were contained the rim of the tumor, non-tumorous regions nearby the tumor and the normal liver. Time density curve, blood flow figures and the CT perfusion parameters: Blood Volume, Blood Flow, Mean Transit Time, Permeability Surface and Hepatic Arterial Fraction were measured automatically.Then, the rabbits were performed by color doppler ultrasonography and CEUS by ALOKA SSD-α10 color doppler diagnostic apparatus. Conventional detecting the liver by 2-D and recording the location, diameter, shape, color doppler flow imaging inside and outside of the tumor. The dynamic features of contrast enhanced ultrasound in the tumor and the normal liver were observed by contrast tuned imaging by bolus injection of SonoVue with injected dose of 0.1ml/kg and normal saline with injected dose of 2ml via peripheral vein. Total detection time was 50s, detector acceptance frequency was 11MHz, and mechanical index was 0.05.After executed these rabbits, the growth information of the tumors were detailed recorded and pathological sections from fresh liver specimen were got.Obtaining the fresh liver specimen of the rim of the tumor, non-tumorous regions nearby the tumor and the normal liver respectively. The expression of VEGF mRNA in the rim of the tumor, non-tumorous regions nearby the tumor and the normal liver were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The correlation between the expression of VEGF mRNA and CT perfusion parameters and features of contrast-enhanced ultrasonography were measured. SPSS 13.0 software package applied for statistical analysis. Significant level was set asα<0.05.Results:1. The CT, ultrasound and fresh liver specimen features of normal rabbit liver:From CT scan:The liver parenchyma showed smooth surface and thick left lobe. It showed insignificantly enhancement in arterial phase, and gradually increased and uniform enhancement in venous phase and decreased enhancement in delayed phase.From 2-D ultrasound:The liver parenchyma showed regulate profile and thick left lobe. It showed uniform echo in liver parenchyma and clearly detection of portal vein, inferior vena cava and hepatic veins by CDFI. CEUS showed gradually increased and then decreased enhancement in three phases with injection of contrast medium.Fresh liver specimen features:The liver parenchyma was "wedge shape" and brunneus with thick left lobe. The result of light microscope observation:Hepatocyte were seen distributing regulately with clear margin and normal morphology, showing typical structure of hepatic lobule, hepatic sinusoid was clearly and nucleous size and morphology were regular with uniform staining.2. The CT, ultrasound and pathological features of VX2 rabbit model:From CT scan:The tumors which has smooth border were demonstrated itself as the round-shaped tumors with hypodensity on plain CT scan, significantly ringe-enhancement on arterial phase, relatively hypodensity on portal phase and no enhanced in the zone of necrosis. The CT perfusion result:The HAF and HAP in trial group were higher and PVP were lower than in control group, there was significant difference, P<0.05. The increased BF,BV,PS,HAF,HAP and the decreased MTT in the rim of the tumor were differed from the non-normorous regions nearby the tumor and the normal liver, there was significant difference. Except for HAF and HAP, there was no significant difference of other perfusion parameters in the non-normorous regions nearby the tumor and the normal liver.Time density curve result:It showed high and acute time density curve in aorta, steep increased and steep decreased. In portal vein, it showed low and moderate time density curve, with steep increased and slowly decreased, the peak value in portal vein always later appearance than in aorta. The time density curve in normal liver parenchyma showed slowly increased and slowly decreased, the peak value in normal liver parenchyma also later appearance than in aorta. The curve in non-normorous regions nearby the tumor was same as the curve in normal liver parenchyma, but it's peak value was earlier appearance than normal liver parenchyma. The time density curve in the rim of the VX2 tumor showed steep increased and slowly decreased, and it's peak value was earlier appearance than in non-normorous regions nearby the tumor and normal liver parenchyma.From 2-D ultrasound:The lesions were round-shaped iso-echoic tumors with acoustic halo nearby and low level echo of necrosis in centre from 2-D. CDFI can detect spot-like or strip-like blood echo in the centre or nearby the tumors. After injecting the contrast agent, the tumors gradually showed round or branches hyperechoic enhancement during arterial phase, and then gradually decreased enhancement with the increased enhancement in liver parenchyma nearby.CT perfusion detected forty-nine neoplasma nodules with the detection rate of 83%(49/59), ranging from 1.0cm～3.9cm. Compare with pathological result, twenty-eight small hepatic tumors which diameters were less than or equal to 3.0cm and four tumors which diameters were less than 1.0cm could be detected by CT perfusion with a detection rate of 87.5%(28/32) and 57.1%(4/7).2-D ultrasound detected forty-six neoplasma nodules with a detection rate of 78%(46/59), ranging from 1.1cm～4.0cm.There were twenty-seven small hepatic tumors which diameters were less than or equal to 3.0cm detected by 2-D ultrasound, but three of them were more than 3.0cm proved by pathological result. One micro hepatic tumor which diameter was less than 1.0cm could be detected by 2-D ultrasound. It showed a detection rate of 78.1%(25/32) and 14.2%(1/7) with diagnosis of small hepatic tumor and micro hepatic tumor respectively by 2-D ultrasound. CEUS detected fifty-four neoplasma nodules with the detection rate of 91.5%(54/59), ranging from 0.9cm-4.0cm. There were thirty small hepatic tumors which diameters were less than or equal to 3.0cm detected by 2-D ultrasound, but only one of them was more than 3.0cm proved by pathological result. Six micro hepatic tumors which diameters were less than 1.0cm could be detected by CEUS. It showed a detection rate of 90.6%(29/32) and 85.7%(6/7) with diagnosis of small hepatic tumor and micro hepatic tumor respectively by CEUS.CT perfusion,2-D ultrasound and CEUS respectively showed a specificity of 100%,92.6% and 96.2%, positive predictive value of 100%,92.6% and 96.7%, negative predictive value of 74.2%,78.1% and 86.7%, with diagnosis of small hepatic tumor which diameters were less than or equal to 3.0cm, concordance rate of 86.4%,84.7% and 89.8%, and Younden index of 0.875,0.707 and 0.868, respectively. There was significant difference of detection rate in diagnosis of small hepatic tumor which diameters were less than or equal to 3.0cm by 2-D ultrasound and CEUS, P<0.05. There was no significant difference of specificity and concordance rate in diagnosis of small hepatic tumor by 2-D ultrasound and CEUS,P>0.05. There was significant difference of detection rate in diagnosis of micro hepatic tumor which diameters were less than 1.0cm by 2-D ultrasound and CEUS,.P<0.05. There was significant difference of detection rate in diagnosis of small hepatic tumor which diameters were less than or equal to 3.0cm by CT perfusion,2-D ultrasound and CEUS, P<0.05. CT perfusion and CEUS were better than 2-D ultrasound in detecting small hepatic tumors. There was no significant difference of specificity and concordance rate in diagnosis of small hepatic tumor by CT perfusion,2-D ultrasound and CEUS,P>0.05. There was significant difference of detection rate in diagnosis of micro hepatic tumor which diameters were less than 1.0cm by CT perfusion,2-D ultrasound and CEUS, P<0.05. CEUS was better than CT perfusion and 2-D ultrasound in detecting micro hepatic tumors.There were forty-six neoplasma nodules detected by CT enhancement and CEUS identically. Forty-two nodules showed obviously enhancement, two nodules showed moderately enhancement and two nodules showed slightly enhancement in CT arterial phase while forty-three nodules showed obviously uniform "ring form" enhancement, one nodule showed moderately heterogeneously "branches-like" enhancement and two nodules showed slightly "strip-like" enhancement in CEUS arterial phase. In portal phase, there were forty-five and forty-four nodules showed moderately enhancement, one and two nodules showed slightly enhancement by CT enhancement and CEUS, respectively. There was no significant difference of enhancement features in arterial phase and portal phase by CT enhancement and CEUS, (P=0.817 and 1.000).In the forty-six neoplasma nodules detected by CT enhancement and CEUS identically. There were forty-three and forty-two nodules showed a enhancement pattern of obviously enhancement during arterial phase, then gradually decreased enhancement in portal phase by CT enhancement and CEUS, respectively. Six and two nodules showed the other enhancement pattern of obviously enhancement during arterial phase, then continuously enhancement in portal phase; three and one nodules showed another enhancement pattern of continuously enhancement from arterial phase to portal phase by CT enhancement and CEUS, respectively. There was no significant difference in the number of nodules in various enhancement patterns by CT enhancement and CEUS, P= 0.513.Pathological features:Twenty (83%) white rabbits were successfully implanted with the tumor. There were totally fifty-nine neoplasma nodules with diameters ranging from 0.2cm～4.0cm. Seven micro hepatic tumors with diameters less than 1.0cm could be find in thirty-two small hepatic tumors with diameters less than or equal to 3.0cm. Other three rabbits died from air embolus, overdose anesthesia, infection respectively and two rabbits were unsuccessful implantation. In the twenty rabbit models, two rabbits metastasis at operative incision in abdominal wall, two rabbits at greater omentum, one rabbit at base of lung and five rabbits with "satellited nodules" nearby and four rabbits have ascites, other rabbits all have solitary tumor in liver. The tumor showed round-shaped gray nodule with well-defined capsule and zone of necrosis in the centre of cross section. The result of light microscope observation:Under low magnification, the carcinoma cells have an arrangement of "nest", there was separating fibre with abundant capillary of newly developed; under high magnification, the volume of carcinoma cells were large with round or irregular morphology. Nucleus was hypertrophic with different size and shape, staining was heterogeneous, phase of nucleolus mitosis showed in carcinoma cells.3. The gene expressions of VEGF mRNA in VX2 rabbit model:By RT-PCR, the gene of VEGF in the normal liver was weakly expression, with the VEGF/β-actin of 0.200±0.115; the expression in the non-tumorous regions nearby the tumor were higher than in the normal liver, with the VEGF/β-actin of 0.384±0.140. There was significant difference of gene expression between non-tumorous regions nearby the tumor and the normal liver, P<0.05. The expression of VEGF mRNA in the rim of the tumor were higher than in the non-tumorous regions nearby the tumor and the normal liver, with the VEGF/β-actin of 0.720±0.293. There was significant difference of gene expression in the rim of the tumor, non-tumorous regions nearby the tumor and the normal liver, P<0.05.The Pearson correction test showed:There was positive correlations between VEGF mRNA expression and the data of CT perfusion BV, BF, PS, HAF and HAP in the rim of the tumor (r=0.932; r=0.935; r=0.904; r=0.951; r=0.960) and the correlations coefficient between VEGF expression and MTT was 0.527, P=0.053; and there was positive correlation between VEGF mRNA expression and the data of CT perfusion BV, BF, PS, HAF and HAP in the non-tumorous regions nearby the tumor(r=0.957; r=0.972; r=0.975; r=0.934; r=0.947) and no correlations between VEGF expression and MTT(r=-0.121, P=0.681) and there was positive correlation between VEGF mRNA expression and the data of CT perfusion BV, BF, PS, HAF and HAP in the normal liver(r=0.972; r=0.933; r=0.962; r=0.953; r=0.930) and no correlations between VEGF expression and MTT(r=0.017,P=0.953), respectively.Conclusions:1.The VX2 hepatic carcinoma mass were implanted into the liver of rabbits via laparotomic route, the biological features, pathological features, changes of blood supply and imaging features were same as hepatic cell carcinoma in human, it was useful for the research of imaging and haemodynamics in hepatic tumors.2.Multi-slice CT perfusion measured the perfusion parameters by selecting the regions of interest, real-time contrast enhanced ultrasonography diagnosis of solid mass in liver by detecting the blood features in the tumor, which could providing information of perfusion and real-time changes of vascular morphology, and detect the blood supply of hepatic tumors in vivo.3.There was positive correlation between gene expressions of VEGF mRNA and the display of blood supply feeding vessels by CEUS, the data of CT perfusion. Angiogenesis and haemodynamics could be evaluated by the two imaging technologies.4.There was high diagnostic value in detecting small hepatic tumors by Multi-slice CT perfusion and CEUS. CEUS was better than CT perfusion and 2-D ultrasound in detecting micro hepatic tumors. |
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