| Objective:Hepatocellular carcinoma (HCC) is one of the most malignant carcinomas among original hepatic carcinomas in our country. The incidence rate and death rate have beening increased in recent years. But the etiological factors and rationale of HCC are still unclear.Diffusion weighted image (DWI) and diffusion tenser image (DTI) have been applied by clinical diagnose and basic research succeesly in central nervous system, as developping functional magnetic resonance imaging in recent years. But there were few experimental research reports in liver diseases study.The obseration and investigation of pathology has been developed from plain pathological morphology to integrate the changes of histological types in morphology and immunohistochemistry to study pathologic changes of organics along with techniques of modern molecular biology, such as proliferating cell nuclear antigen (PCNA) and apoptosis of immunohistochemistry.We established rabbit VX2 liver tumor model and compared quantitative indices of DWI and DTI to pathologic indices of PCNA and apoptosis of immunohistochemistry during the process of rabbit VX2 liver tumor model growing,and to evaluate whether quantitative study of ADC, eADC from DWI and ADC, FA from DTI can be used to dynamically differentiate rabbit VX2 liver tumor model in their proliferating cycle, and to investigate the possibility of diagnose liver lesions by using quantitative dynamic indices of DWI and DTI, the specialties of DTI, DWI, plain MRI and contrast MRI, in order to this study would instruct to clinical application of MR DWI and DTI in theory.Materials and Methods1 Experimental Material1.1 Experimental Animals20 adult New Zealand white rabbits from medical experimental animal central of Guangdong province that ranged in weight from 2 to 3 kg (male and female not distinguished) were classified into 4 experimental groups (4 rabbits each group) and 4 contrasted groups (1 rabbit each group) in random sample.1.2 Establishment of rabbit VX2 liver tumor modelThe VX2 carcinoma was presented by Guangzhou Sun Yat-Sen memory hospital. The VX2 tumor cells had been inoculated in thigh' subcutaneous of rabbit to come into tumor.Tumor has been resected and manufactured to shivers of squre area in l~2mm2 which could be prepared for seeds.Rabbit of experimental groups were fixed supinly on CT scanning bed anaesthetised with 3% soluble pentobarbitone intravenously. The VX2 tumor cells were processed in the left lobe and right liver lobe with CT guide by paracentetic needle, respectively.1.3 Disposal method of rabbits14, 18, 22 and 26 days after tumor implanted, a experimental group anaesthetised and a contrasted group anaesthetised were performed with plain MRI, contrast MRI, DWI and DTI scan in prone position using an 8-channel phased array coil. After MRperformed, all rabbits were killed immediately by air embolism, and the sections for pathological and immunohistochemistric analysed compared with corresponding MR images.2. MR scanning methodsPlain MRI, contrast MRI, DWI and DTI were obtained at a 1.5T MR scanner (Twin Speed Excite, GE, American). All rabbits were scanned with spin-echo (SE) Tl WI and fast SE, T2WI in the axial plane.The images parameters of DWI with SE-EPI sequence were as follows: TR 4500 ms, TE 70 ms, Matrix, 128x128, FOV 16x16 cm, Section thickness 5 mm, gap 1 mm, NEX 8, b value (1000 mm2/s), acquired in three directions, and scan time 2 min 24 s.The images parameters of DTI with SE-EPI sequence were as follows: TR 3500 ms, TE 75 ms, Matrix, 128x128, FOV 16x16 cm, Section thickness 5 mm, gap 1 mm, NEX 8, b value (1000 mm2/s), acquired in nine directions, and scan time 3 min 37 s.3. Methods and standards of MRI measureAll data were measured in the control console.The functool software was Advantage Workatation AW4.005 of GE (American General Electric company).Average apparent diffusion coefficient (ADC) and exponential ADC (eADC) were measured in DWI.Average apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were measured in DTI.The standards of measurement of DWI and DTI in hepatic lesions were as follows: The largest diameter of lesions were measured; the regions of blood vessels and artifact were precluded; the edges of parenchyma in the lesion were measured; and the area should be as over 20±5 mm2 and over 50 pixels as possible; each lesion was measured twice and a mean value was acquired for calculation.4. Fabrication and analyse standard of pathological changes in HE:After MR performed, all rabbits were killed immediately by air embolism, made the rabbit liver to thick 4 mm sections and compared with corresponding MRI, naked eye observing tumor' conditions, such as color and luster, then made the section soak and fixed with 10% formaldehyde solution, routine paraffin embeded, serial sections for pathologic HE and immunohistochemistric analysised compared with corresponding MRI.Analysis components for pathologic HE sections: pathological histological types, pathological grade was divided into four classifications by Edmonson-steiner, conditions of tumor' border and hemorrhage, coagulation necrosis and fiber hyperplastic of tumor, et al.5. Fabrication and analyse standards of pathological changes in PCNA and apoptosisApoptotic index (AI) were measured by the terminal deoxynucleotidly transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) immunohistochemistrical technique, testing kit buied from Roche limited company, DAB staining was used to examine AI according to the manufacturer's protocol.The proliferating index (PI) of proliferating cell nuclear antigen (PCNA) were measured by two step immunohistochemistric staining (Envision System), mouse to human monoclonal antibody, testing kit buied from DAKO limited company, DAB staining, routine desiccated and hyalined.PCNA immunostaining cells showed the brown-yellow staining in the nuclei were positive cells by Envision System immunohistochemistrical technique, microscope observed by 10x40, immunostaining was calculated from the ratio of the number of positively stained tumor cells to the total number of tumor cells counted positive cells from 200 cells in 5 random sights, respectively, counting formula: PCNA PI = staining positive cells of PCNA / tumor cells.The AI was calculated from the ratio of the number of positively stained tumor cells to the total number of tumor cells counted in 5 random sights, respectively, counting formula: AI = stained cells of apoptosis / sight.6. Statistical analysis: All data in this study were expressed as mean ± standard deviation. Statistical analysis was based on a two -tailed paired Student t test with SPSS software package version 10.0. A P value of less than 0.05 was adopted to indicate a statistically significant difference.Results1. Results of pathologic changes in HE:The pathological results of 4 contrasted groups were: no tumor and other changes in pathology.All 4 experimental groups (16 rabbits) grew tumor successfully. 26 lesions were measured by MRI, contrast MRI, DWI and DTI.The diameters of the tumors ranged from 0.3 cm to 3.9 cm. 16 lesions in left lobe of liver, 10 lesions in right lobe of liver, and solitary (10 rabbits), diffuse (6 rabbits).The pathological results of experimental groups were as follows: all pathological histological types were chorda, grade III of Edmonson-Steiner cell differentiation, the occurrences of coagulation necrosis, fiber hyperplasia, envelope infiltration and metastasis having increasing trend, and the occurrences of coagulation necrosis being earlier than fiber hyperplasia.2. .Results of pathological changes in PCNAPCNA positively stained cells were distributed in den-shape within tumor cells diffusively, PCNA immunostaining cells showed the brown-yellow staining in the nuclei, cytoplasm and membrane of cellularitycytoarchitecture were negative stained.3. Results of pathological changes in apoptosisApoptosis positively stained cells were distributed in tumor cells diffusively, apoptotic immunostaining cells showed the brown-yellow staining in the nuclei and cytoplasm, and normal cells were negative stained.4. Quantitative studies of PCNA and apoptosis14, 18, 22 and 26 days after tumor implanted, the PI of the experimental groups measured were 12.212±2.561, 18.258±3.364, 23.136±3.462, 29.362±4.208, respectively; and the AI were 5.861±0.562, 5.321±0.523, 4.962±0.356, 3.761±0.625, respectively. For the contrast groups, the results for PI were 5.261 ±2.012, 7.163±3.632, 5.596±2.335, 6.963±2.596, respectively; and the results for AI were 0.261±0.041, 0.212±0.034, 0.318±0.043, 0.222±0.044, respectively.The data shows that there is a significant difference between experimental groups and contrast groups in PI and AI, and P<0.05. The mean PI of experimental groups had an increasing trend and AI of experimental groups had a descending trend.AI had a significantly negative correlation with PI, and the correlation coefficient of Spearman was -0.481, p<0.05.PCNA expression of VX2 tumor cells significantly enhanced as tumor replanted period increased, and apoptosis of VX2 tumor cells decreased as tumor replanted period increased with insignificance. Apoptosis of VX2 tumor cells significantly negative correlation with its PCNA. High-grade proliferation is associated with decrease of apoptosis in VX2 tumor cells.Cellilar homeostasis within VX2 tumor depends on the balance between apoptosis and cell proliferation.5. Quantitative study of DWI and DTIAll experimental group and contrasted group rabbits were performed with MRI, contrast MRI, DWI and DTI scan.14, 18, 22 and 26 days after tumor implanted, the ADCs of the experimental groups measured by DWI were (1.227 ± 0.069)xl0"3mm2/s, (1.291±O.287)xlO~3mm2/s, (1.082 ± 0.284)xl0~3mm2/s, (1.014 ± 0.208)xl0"3mm2/s , respectively; and the eADCs were 0.295 ± 0.020, 0.285 ± 0.075, 0.352 ± 0.103, 0.370 ± 0.082, respectively. For the contrast groups, the results for ADCs were (0.861 ± 0.112) xlO~3mm2/s, (0.823 ± 0.062) xlO~3mm2/s, (0.803 ± 0.231) xlO"3mm2/s, (0.816 ± 0.118) xlO"3mm2/s, respectively; and the results for FAs were 0.419 ± 0.041, 0.423 ± 0.014, 0.408 ± 0.043, 0.431± 0.038, respectively.The data shows that there is a significant difference between experimental groups and contrast groups in ADCs and eADCs in DWI, and P<0.05. The mean ADCs of experimental groups had an increasing to descending trend and eADCs of experimental groups had a descending to increasing trend.The occurrences of coagulation necrosis and fiber hyperplasia had an increasing trend in pathological results.And there is a significant effect between PI and ADCs and eADCs in DWI, correlation coefficients of Spearman were -0. 381, 0. 353, /?<0. 05.14, 18, 22 and 26 days after tumor implanted, the ADCs of the experimental groups measured by DTI were (1.269 ± 0.248)xl0~3mm2/s, (1.379 ± 0.227)xl0"3mm2/s, (1.226 ± 0.447)xl0"3mm2/s, (1.225 ± 0.448)xl0'3mm2/s, respectively; and the FAs were 0.229 ± 0.079, 0.210 ± 0.078, 0.163 ± 0.114, 0.233 ± 0.107, respectively. For the contrast groups, the results for ADCs were (1.167 ± 0.123) xlO~3mm2/s, (1.171 ± 0.162) xl0"3mm2/s, (1.098 ± 0.031) xlO"3mm2/s, (1.125 ± 0.126) xl0'3mm2/s, respectively; and the results for FAs were 0.142 ± 0.031, 0.147 ± 0.024, 0.138 ± 0.033, 0.136 ± 0.028, respectively.The data shows that there is a significant difference between experimental groups and contrast groups in ADCs and FAs in DTI, and P<0.05. The mean ADCs of experimental groups had an increasing to descending trend and mean FAs of experimental groups had a descending to increasing trend.The occurrences of coagulation necrosis and fiber hyperplasia had an increasing trend in pathologicalresults. And there is a significant effect between PI and ADCs and FAs in DTI, correlation coefficients of Spearman were -0.483,-0.516, /?<0.05.In Correlation statistics, the absolute value of PI and ADC, FA greater than the absolute value of PI and ADC, eADC, so DTI could reflect the pathologic changes in accurately.Conclusions1. Results of pathology:All pathological histological types were chorda types, grade III of Edmonson-Steiner cell differentiation, the occurrences of coagulation necrosis, fiber hyperplasia, envelope infiltration and metastasis having increasing trend, and the occurrences of coagulation necrosis being earlier than fiber hyperplasia.PCNA expression of VX2 tumor cells significantly enhanced as tumor replanted period increased, and apoptosis of VX2 tumor cells decreased as tumor replanted period increased with insignificance. Apoptosis of VX2 tumor cells significantly negative correlation with its PCNA. High-grade proliferation is associated with decrease of apoptosis in VX2 tumor cells.Cellilar homeostasis within VX2 tumor depends on the balance between apoptosis and cell proliferation.2. Results of MRI:The mean ADCs of experimental groups in DWI had an increasing to descending trend and eADCs of experimental groups had a descending to increasing trend.The mean ADCs of experimental groups in DTI had an increasing to descending trend and mean FAs of experimental groups had a descending to increasing trend. The occurrences of coagulation necrosis and fiber hyperplasia had an increasing trend results in above changes of DWI and DTI. PI of experimental groups had significantly effect to DWI and DTI. DTI was sensitive to tumor cells than plain MRI, contrast MRI and DWI. DTI could reflect the pathologic changes inaccurately.Our preliminary study showed that measurements and quantitative study of ADC and eADC in DWI and ADC and FA in DTI can be used to dynamically differentiate rabbit VX2 liver tumor model in their proliferation cycle, and dynamic MR DWI and DTI quantitative analysis had significant values in differentiate some characters of rabbit VX2 liver tumor model, DTI could reflect the pathologic changes in accurately than DWI, plain MRI, contrast MRI, so this study could instruct to clinical application of MR DWI and DTI in theory.
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