Experimental Study Of The Change Of CT Perfusion Parameters In VX2 Tumor Implanted In Rabbit Lung Before And After Radiotherapy And Correlation Between Perfusion Parameters And Expression Of VEGF And MMP-2

PrefaceLung cancer was one of the most common malignant tumors at present time, themorbidity of lung cancer has been the first one of malignant tumors in many citys.Even in the developed country such as USA, only 21% patients of lung cancer werediagnosed in the early stage. In these patients, most of them were non-small cell lungcancer (NSCLC). More than 50% patients of NSCLC could not be operated whendiagnosis was confirmed. For these patients, radiotherapy was one of the maintreatment methods. In clinical, CT imaging was often used in the assessment ofradiotherapy effect. But routine CT could not reflect the change of hemodynamicsbefore and after radiotherapy, which was very important not only in the target paintingand therapy plan making before radiotherapy but also in making therapy strategy afterradiotherapy. With the developing of medical imageology in recent years, the change ofhemodynamics in lung tumor could be found in early stage with the help of multi-sliceCT and some special software technology. Now people began to research utilizing CTperfusion imaging in the assessment of radiotherapy effect.In our study, we observed the change of CT perfusion parameters, the change oftumor size and the change of MVD before and after radiotherapy, investigated thecorrelative relation between CT perfusion parameters and the microvascular density, the expression of VEGF and MMP-2 in VX2 tumor on the basis of establishing animalmodel of VX2 tumor implanted in rabbit lung. All these work we have done weresupposed to provide experimental basis for CT perfusion becoming a new media in thetarget painting and therapy plan making before radiotherapy and in the assessment ofradiotherapy effect.Material and Methods1.equipment, main reagent, animals and the source of VX2 cell linesLight speed ultra 16 spiral CT (American GE company), AW4.2 workstaionperfusion software, Accelerator (American Varian 2300C/D).Main reagent: Omnipaque 320 mg I/ml, monocolon antibody of CD34 waspurchased from Maixin Company of FuZhou, antibodys of VEGF and MMP-2 werepurchased from BoShide Company of WuHan, SP9000 immunohistochemistry kit waspurchased from ZhongShan Biological Technology Company of BeiJing.healthy Newzland rabbits offered by Experimental Animal Center of HarbinMedical University, weight((2.5±0.5) kg.VX2 cell lines were provided by the Department of Radiology of the FirstAffiliated Hospital of China Medical University.2. Animal model establishmentThree methods were used in establishing animal model. In the first method, VX2single cell suspensions was injected in rabbit lung. In the second one, VX2 tumor wasimplanted in the rabbit lung by operation. In the third one, VX2 tumor was implanted inthe rabbit lung by acupuncture. The advantages and disadvantages of these threemethods were compared and summarized.3. CT perfusion techniqueCT perfusion was applied to rabbits when tumor size grew to 7～10mm. Rabbitswere fixed on examination frame. Cine full mode was adopted in perfusion scanning.3% pentobarbital sodium was used for anaesthesia. Venepuncture was made at ear edge vein. When rabbits' respiration was stable (respiratory rhythm controlled at 13～15times/min), contrast agent heating to 37℃was injected by high pressure syringe atthe speed of 0.3ml/s. Abdomen belt was used to restrict respiratory movement duringCT perfusion scanning. Scanning layer thickness was 2.5mm. After 5 second, contrastagent was injected, scanning finnished in 50 second. Scan scope: 2cm; voltage: 100kv;FOV: 11.9cm; electric current: 120mA.4. Postprocessing of CT perfusion imageScanning data was transmitted to workstation (AW 4.2), then perfusion imagewas analyzed to acquire parameters such as BV (blood flow, BF), BF (blood volume,BV), PS (permeability surface, PS) and MAV (the maximum attenuation value) whichcould reflect the hemodynamics of tumor. ROI(region of interest, ROI) was less thansix pixels and got out of the way of blood vessel. In order to avoid error, "X" type 4spot ROI were selected on the slice to be checked, then BF,BV, PS and MAV weremeasured and average value of these parameters were calculated.5. Formulating and enforcing of X-knife treatment planRabbits were fixed on examination frame at dorsal position. CT scan wasperformed when tumor size grew to 7～10mm. Image was transformed to workstationfor radiotherapy plan. Radiotherapy plan was workouted by Cadplan Treatment PlanSystem. The energy of X ray was 6 MV and the dose rate was 2.18Gy/min. Accordingto radiosurgical treatment planning, tumor received dose of 20Gy delivered by fourconverging arcs of the X-knife unit. CT perfusion was applied to rabbits before and oneweek after radiotherapy, then the change of CT perfusion parameters was observed.6. Pathology and immunohistochemistry examinationRabbits were sacrificed less than 1 hour after CT perfusion scanning. Rabbits'lung were carefully excised and fixed in 10% formalin for 1 or 2 weeks and embeddedin paraffin. Axial sections(4μm) of the tumors were obtained. Two different stainingmethods were used: hematoxylin and eosin (H&E) and immunohistochemical staining for CD34, VEGF and MMP-2. Laboratory procedure was under the direction ofimmunohistochemistry kit demonstration.MVD was defined as the number of microvessels per unit area in histologicalsections stained with CD34. Vessels were counted at low magnification (100X) in 5separate fields selected from the most vascular regions in the central non-necrotic areasand peripheral areas of the tumors. Then high magnification was used to count thenumber of microvessels stained with CD34. Microvessel density was expressed by theaverage as a number of counts/mm~2.VEGF-immunoreactive brown colored deposits were mainly located in vascularendothelial and tumor cells. VEGF expression was determined on 5 visual fields (X200)randomly. Percentage of positively stained cells in 100 cells of these 5 fields wascounted, average number of these percentage was calculated.MMP-2-immunoreactive brown colored deposits were mainly located in vascularendothelial and tumor cells. MMP-2 expression was determined on 5 visual fields(X200) randomly. Percentage of positively stained cells in 100 cells of these 5 fieldswas counted, average number of these percentage was calculated.7. Statistical analysisResults were expressed as the mean±SD. SPSS 12 for windows was used for allstatistics analysis such as one-way ANOVA, two-tailed independent T-test, Chi-squaretest and correlation of Pearson. Statistical significance was declared when p＜0.05,significant statistical significance was declared when p＜0.01.Results1.Status of rabbits after VX2 tumor implanted in lungRabbits developed abnormalities in feeding behavior or external appearance suchas fastidium and pelage glossiness descending aider 10 days to 20 days of implantation.Successful rate of implantation was 100%(13/13) in the method of suspensioninjection. The mean survival time of rabbits was (38±3)d. 5-15 tumors scattered in lung. Besides lung, tumor also appeared in pleural membrane, arcula cordis,diaphragma and lymphnode in hilum of lung after three weeks of implantation.Successful rate of implantation was 64.3%(9/14) in the method of operation. Themean survival time of rabbits was (46±5)d. Only one tumor appeared in the left lung.Besides lung, tumor only appeared in lymphnode in hilum of lung after three weeks ofimplantation.Successful rate of implantation was 84.6%(11/13) in the method of acupuncture.The mean survival time of rabbits was (54±5)d. Only one tumor appeared in the leftlung. Besides lung, tumor only appeared in lymphnode in hilum of lung after threeweeks of implantation.Compared to the method suspension injection, the mean survival time wasprolonged obviously in the other two methods. Compared to operation method, thesuccessful rate of implantation was increased and survival time of rabbits polongedmarkedly in acupuncture method.2. Results of routine CT and enhanced CTTumor appeared as the shape of oval or anomaly in routine CT scanning. CT value:43.27±5.59Hu in routine CT scanning, 112.27±22.78Hu in enhanced CT scanning.Enhancement in most tumors was uniform. Asymmetry enhancement was seen only inthree cases whose tumor size was larger than 11mm.3. Results of CT perfusionPicture quality of CT perfusion scanning for animal model was very well. Bloodvessels supplying to tumor presented red color and was very abundant in the image ofBF and BV map. Compared to normal lung tissue, blood supply to regions aroundtumor also increased obviously. Pathological changes was showed most clearly in themap of PS. PS value increased sharply in tumor. PS value in regions around tumor washigher than that in normal lung but lower than that in tumor. The extent of diseaseshowed in PS map was similar to that in the image of enhanced CT imaging, but theextent of disease showed in BF and BV map was larger than that in the image of enhanced CT imaging.4. Correlation analysis between the results of immunohistochemicalstaining, survival time of rabbits and CT perfusion parametersResults of Pearson correlation analysis: significant positive correlation wasobserved between CT perfusion parameters such as BF, BV PS and MAV in tumorregion and the expression of VEGF; significant positive correlation was observedbetween CT perfusion parameters such as BF, BV PS and MAV in tumor region andMVD; positive correlation was observed between MVD and expression of VEGF;correlation was not observed between MVD, expression of VEGF and expression ofMMP-2.5. Influence of radiotherapy to the change of CT perfusionparameters; tumor size and MVDTumor size measured one week after radiotherapy in treatment group was smallerthan that in non-treatment group without radiotherapy, P＜0.01.CT perfusion parameters such as MAV, BF, BV and PS measured one week afterradiotherapy in treatment group was obviously lower than that in non-treatment groupwithout radiotherapy, P＜0.01.The value of MVD in non-treatment group was 73±8, while the value of MVDmeasured one week after radiotherapy in treatment group was 48±9. The value ofMVD in these two groups was different with statistical significance, P＜0.01.Results of Pearson correlation analysis: positive correlation was observedbetween the change of CT perfusion parameters such as MAV, BF and BV in tumorregion and the change of MVD after radiotherapy, correlation was not observedbetween the change of PS in tumor region and the change of MVD after radiotherapy;positive correlation was observed between the change of tumor size and the change of BF and MAV after radiotherapy, correlation was not observed between the change oftumor size and the change of PS and BV after radiotherapy.Conclusion1. Compared to the method suspension injection and operation, animal model ofVX2 tumor implanted in rabbits' lung established by the method of acupuncture wasmore stable and convenient for experiment. Animal model of VX2 tumor in rabbits'lung established by acupuncture method can provide a reliable experimental andresearch basis for the diagnosis and treatment of lung tumor.2. Not only can CT perfusion parameters reflect the circumstance of angiogenesisof microvascular but also can reflect the expression of VEGF and MMP-2, whichindicated VEGF and MMP-2 play an important role in the angiogenesis and metastasisof VX2 tumor implanted in rabbits' lung.3. Compared to non-treatment group, CT perfusion parameters in treatment groupsuch as BF, BV and MAV in tumor region and MVD decreased obviously withstatistical significance after radiotherapy. Positive correlation was observed betweenthe change of CT perfusion parameters such as BF, BV and MAV in tumor region andthe change of MVD after radiotherapy. All these above indicated that we could use CTperfusion parameters as an index corresponding to MVD to evaluate capillaryangiogenesis of tumor in vivo.4. Compared to non-treatment group, tumor size of rabbits in treatment groupdecreased obviously with statistical significance after radiotherapy. Positive correlationwas observed between the change of tumor size and the change of BF, MAV and MVDafter radiotherapy. All these above indicated that we could predict the effect ofradiotherapy through the change of CT perfusion parameters before and afterradiotherapy, meanwhile our study results also indicated that it was necessary toevaluate the role of CT perfusion technique in radiotherapy plan making andradiotherapy effect monitoring further.