Study On The Effect Of Local Hyperthermia Mediated By Fe₃O₄ Nanometer Magnetic Fluid On Rabbit VX2 Liver Tumor Model

PartⅠEstablishment and sonographic evaluation of rabbit model bearing VX2 liver tumorObjectives:To establish rabbit model bearing VX2 liver tumor. To analyze and compare its different sonographic features with color Dopple,power Doppler and contrast ultrasonography.Methods:Twenty Japanese white rabbits were implanted into the left liver lobe with fresh VX2 tumor tissues for establishment of rabbit liver models.Two and four weeks after tumor implantation,tumor diameter was monitored by ultrasound and blood flow signals were detected by color Doppler,power Doppler and contrast ultrasound respectively,and then they were compared with each other.Ten rabbits were sacrificed randomly after sonogaphy and tumor diameter was measured and compared with the results of sonography.The phathologic features of VX2 tumor were observed by HE staining.Results:All rabbits were successfully implanted with VX2 tumor. Tumor size was similar with the same growth time,and there was no significant difference between each other(P＞0.05.The maximum tumor diameters measured by ultrasound and caliper rule respectively had no significant difference(P＞0.05) In tumors,blood flow was detected sensitively by both color Doppler and power Doppler.Small liver tumors growing for two weeks were majorly charicterised by peripheral blood flow,which were dectected more by power Doppler than that by color Doppler.Blood flow of relatively large tumors growing for four weeks, was featured by peripheral large blood vessels or diffuse spot and striped abundant blood flow signals detected by both color Doppler and power Doppler,the difference of which was not significant.More integrity branches and smaller microvessels in both small and comparatively large tumors were detected by contrast ultrasound.The nested distribution of VX2 tumor cells with multinuclear division was examined by HE staining.Conclusions:The rabbit model bearing VX2 liver tumor can be duplicated by highly successful rate.Ultrasonography is useful in measuring tumor size and monitoring its change.Color Doppler ultrasound and power Doppler both can sensitively detect blood flow signals in tumors,but power Doppler is more sensitive to detecte slower blood flow,especially for small liver tumor.Distirbution of intratumoral microvessels and blood perfusion in both small and large liver tumors can detected sensitively by contrast enhanced ultrasongraphy.PartⅡFeasibility of local magnetic fluid hyperthermia(MFH) on rabbit VX2 liver tumor model and the effect on tumor growthObjectives:To assess the potential of nanometer magnetic fluid heated under alternating magnetic field in vivo and in vitro,and to evaluate the feasibility of local MFH on rabbit VX2 liver tumor model and investigate the effect of it on growth of hepatic tumor. Methods:Nanometer magnetic fluid was tested by transmission electron microscope and heated under different magetic field strength in vitro.Fourteen days after tumor implantation,the rabbits bearing VX2 liver tumor were randomly divided into five groups,including the pseudo-treatment(PT) control group,normal saline(NS) contol group, magnetic fluid(MF) control group,magnetic fluid hyperthermiaⅠ(MFH1) group and magnetic fluid hyperthermiaⅡ(MFH2) group.The rabbits were given intratumoral direct injection of magnetic fluid and immediately exposed to an alternating magnetic field and subsequently heated at 50℃for 30mins for group MFH1,and repeated hyperthermia after five days for group MFH2.CT scanning was performed at 1 and 14 days repectively after injection of magnetic fluid.Blood routine and their function of liver and kidney were tested before hyperthermia,and at 5 and 14 days after the first hyperthermia respectively.All animals were sacrificed on the 28th day after tumor implantation.Tumor size was measured,volume and growth rates of tumors were calculated.The changes of tumors were observed macroscopically and microscopically.Results:Particle diameter of magnetic fluid was uniformity about 10nm.Temperature of magnetic fluid in vitro placed under an alternating magnetic field rose rapidly.In vivo,the temperature of tumor core and rim both rose rapidly and reached the desired temperature about 50℃within the first 5 to 10mins,and then was maintained in a relatively constant range of 50℃±2℃for 30mins by manually adjusting the magnetic flied strength.The limited highter density imaging of intratumoral magnetic fluid deposits compared to the surrounding tissue was observed clearly by CT scanning.Four weeks after tumor implantation,the tumor maximal diameter was(4.25±0.68) cm.(4.70± 1.12) cm,(4.16±0.88) cm,(2.83±0.51) cm,(2.03±0.34) cm separately, and the tumor volume was(25.824±11.92) cm~3,(32.01±13.14) cm~3,(23.35±12.17) cm~3,(7.43±1.86) cm~3,(2.62±1.35) cm~3 respectively in group PT,NS,MF,MFH1 and MFH2.Tumor volume inhibition rate of group MFH1 and MFH2 compared to the three control groups was 68.19%～76.79%and 88.8%～91.87%respectively.The growth of tumors was suppressed in hyperthermia groups.The heated area was macroscopically observed obvious coagulation necrosis,which had clear boundary line with the surrounding tissues.Under the microscope,large necrotic area for group MFH1 and almost complete necrosis for group MFH2 were observed.The intratumoral distribution of magnetic nanoparticles,especially in area of necrosis,appeared much more homogenous than the untreated.Conclusions:The nanometer magntic fluid has good potential for temperature rising both in vitro and in vivo.The homogeneous high and relatively stable-state imtratumoral temperature of 50℃induced by magnetic fluid can be obtained successfully.Hyperthermia with magnetic fluid can suppress the growth of tumor and increase the tumor necrosis, and is safe and effective to treat rabbit VX2 liver tumor.CT can be served as a sensitive method to detect the distribution of intratumoral magnetic fluid. PartⅢStudy on contrast ultrasonagraphic assessment of MFH in rabbit VX2 liver tumor model and the effect on tumor vesselsObjectives:To explore the reliability of contrast enhanced ultrasonography in assessing the reponse of rabbit VX2 liver tumors after MFH compared with histopathology,and to investigate the effect of MFH on tumor vessels.Methods:Fourteen days after tumor implantaion,the rabbits bearing VX2 liver tumor were randomly divided into five groups, including group PT,NS,MF,MFH1 and MFH2.Two weeks later,tumor necrotic rate and blood flow were observed by sonography,and then all animals were sacrificed.Necrotic rate of tumors was assessed by HE staining and compared with the results of sonography;Elasticity fibre of vessel wall with Victoria blue and ponceau S histochemical staining was performed;Expression of VEGF of tumors was examined by immunohistochemistry.Results:The tumor necrotic rate determined by sonogrsphy was 17.89±7.45%,20.52±6.58%,16.78±8.92%,72.93±10.45%,94.28±3.54 %respectively for group PT,NS,MF,MFH1 and MFH2,which had no significant difference compared with the results of phathologic examination.After MFH,vascular elasticity fibrin disrupted and vascular wall was destructed.Expression of VEGF decreased after MFH.The positive rate of VEGF in group MFH1 and MFH2 was 28.57±5.66%,5.84±2.97%respectively,significantly lower than that in each control group.Conclusions:Contrast sonography can accurately discriminate the coagulation necrosis from residual tumors after MFH.MFH at 50℃can effectively injury tumor vessels and inhibit the positive expression of VEGF,which may play an important role in treatment of tumor.