| Background:Low intensity ultrasound has long been used in clinic to promote bone healing and cartilage repairment,to accelerate the recovery of diabetic foot ulcers,to relieve chronic pain caused by inflammation,et al.More recently,a large number of in vitro and in vivo studies have demonstrated that low intensity ultrasound can also play a certain role in the field of tumor therapy.Low intensity ultrasound in the treatment of tumors focuses on two aspects: direct effects on the tumor cells / tissues,such as sonodynamic therapy;or facilitate other treatments to enhance the ablation of tumor tissue,such as radiotherapy,chemotherapy,and gene transfection.The enhancement was generally mediated by sonoporation and/or cavitation.In a phase I clinical trial in Norway,low intensity ultrasound combined with microbubbles(MB)has been proven able to significantly enhance the efficacy of gemcitabine chemotherapy in pancreatic cancer.MB can reduce the threshold required to induce cavitation,and strengthen the intensity of cavitation.In vivo studies have confirmed that intravascular MB in combine with low intensity ultrasound can significantly decrease tumor perfusion.Tumor microvascular dilation,interstitial edema and hemorrhage were the basic pathologically changes,which can cause cell necrosis and in consequence tumor growth inhibitation.The effect of low intensity ultrasound induced tumor perfusion decline was known as the anti-vascular effect of ultrasound.There were studies proved that,on the other hand,low intensity ultrasound combined with MB could also cause vasodilation and increase tissue blood perfusion.Theoretically,temporarily increased tumor perfusion helps to improve tumor hypoxia and hypoperfusion,to alleviate the local tumor microenvironment from low pH,high interstitial pressure,thereby can enhance the sensitivity of tumor cells to chemotherapy and radiotherapy.The diversity of vascular effects induced by low intensity ultrasound and MB is related to the heterogeneity of vascular structure and the complexity of cavitation.Change of ultrasonic pulse length,pulse repetitive frequency,and acoustic pressure will all affect the sub-types of cavitation(stable / initial cavitation)and their intensity,and result in different vascular effects.Based on the above findings,we designed this experimental study of low intensity ultrasound combined with MB in animal tumor models with different acoustic parameters,with a high amplitude therapeutic ultrasound device and a custom modified diagnostic ultrasound scanner.The aim of this study was to explore different vascular effects of different types of low intensity ultrasound excited MB cavitation,and further explore its possible application in cancer therapy.Objective:1.To investigate the enhancement effect of MB-mediated acoustic cavitation caused tumor blood flow blockage on ethanol ablation of the tumor.2.To investigate the effect and mechanism of MB-mediated acoustic cavitation on muscle and tumor tissue,and to explore the possibility of low intensity ultrasound stimulation to enhance tumor chemotherapy.Materials and Methods:1.Experimental apparatus:(1)A commercial diagnostic ultrasound system,an ACUSON S2000 scanner,equipped with a 9L4 linear array transducer,were used for conventional ultrasound and contrast-enhanced ultrasound(CEUS)assessment of tumor perfusion.(2)A pulsed high amplitude low intensity therapeutic ultrasound,CZ960,manufactured by Mianyang Sonic Electronic Ltd with a transducer operated at a central frequency of 831 kHz was used in the tumor blood flow blockage study.The ultrasonic parameters used in this study were: a pulse length of 400 cycle,a pulse repetition frequency of 10 Hz,an acoustic pressure(peak negative pressure)of 4.3 MPa,and a transmitting/intermittent time of 6s/6s,respectively.The treatment time was 5 min,and the duty cycle was approximately 0.18% with a corresponding acoustic intensity(Ispta)of 1.25w/cm2.(3)Custom modified ultrasound system,a VINNO 70 diagnostic scanner(Vinno Technology Co.Ltd,Suzhou,China)with a linear array probe(X4-12L).The modifications includes a therapeutic ultrasound mode called Vflash with a treatment area super-imposed on the low MI CEUS mode.Therapeutic impulses were emitting intermittently,for each imaging frame,an ensemble of one 4 MHz,18 cycles(4.5 ?sec)pulse was delivered line by line at line density level 4 in the treatment area.The frame rate was set at 50 Hz,the therapeutic impulses transmitting and intermittent time were set at 0.48 sec and 2.0 sec,respectively.The acoustic output power were set at 50%/100%,with an MI of approximately 0.7/1.4,respectively.(4)Varioskan Flash Multiplate Reader 3001-1723,Thermo-Fisher Scientific Inc.,USA.(5)High performance liquid chromatography(HPLC),Waters Alliance E2695,manufactured by Waters Technology(Shanghai)Co.,Ltd,were used for tissue drug concentration detection.2.Experimental reagents:(1)MB: Zhifuxian,a lipid-coated perfluoropropane MB prepared by department ultrasound,Xinqiao hospital of the Third Military Medical University.The MB had a mean bubble diameter of 2.0 ?m and a bubble concentration of 2-9×109/mL.In this study,the MB were used both as contrast agent in CEUS and as the cavitation nuclei to mediate low intensity ultrasound cavitation.(2)Total Nitric Oxide Assay Kit(S0021)and Cell lysate(S3039),Beyotime Biotechnology Co.Ltd.The concentration of NO was determined by the classical Griess method.(3)Fluorescein sodium salt(NaFI),Sigma,USA.NaFI was a small molecular fluorescent substance with a molecular weight of 376.27.(4)BCA kit(P0012S),Beyotime Biotechnology Co.Ltd.BCA was used in the calibration of protein concentration for detection of nitric oxide and fluorescein sodium concentration.3.Experimental animals:(1)? Three healthy 4-week-old SD rats,male or female,were used for primary culture of Walker-256 ascites tumor cells.(2)? Twenty eight healthy female SD rats,weighting from 160 to 180 g,established an unilateral subcutaneous Walker-256 tumor,were used in the experimental study on enhancement of ethanol ablation with MB mediated low intensity ultrasound cavitation.(3)? Forty healthy male 6 week old nude mice,weighting from 12 to 15 g,were used in the study on the effect of MB mediated diagnostic range low intensity ultrasound cavitation on muscle blood vessels.(4)? Forty-four healthy male 4 week old nude mice,weighting from 10 to 12 g,32 of the mice established bilateral lower limb subcutaneous pancreatic cancer,were used in the study on the effect and mechanism of MB mediated diagnostic range low intensity ultrasound cavitation on tumor blood vessels.The other 12 mice established a unilateral lower limb subcutaneous pancreatic cancer,were used in the experimental study of tumor chemotherapy enhanced with MB combined with diagnostic range low intensity ultrasound.4.Methods:? Enhancement of ethanol ablation with MB mediated low intensity ultrasound cavitation.(1)Establishment of animal tumor model: Walker-256 tumor cells were recovered and cultured routinely.When the tumor cells reached a concentration of 107/ml,an intraperitoneal inoculation was performed in the 4-week-old SD rats.After two weeks,The rich tumor cells containing ascite was extracted and injected subcutaneously in the 28 rats at a dosage of 0.1 ml(5-8×107/ml)to form subcutaneous Walker-256 tumor.(2)High amplitude low intensity ultrasound treatment protocal: The 28 subcutaneous Walker-256 tumor bearing rats were randomly divided into three groups.Ten rats were treated by MB enhanced therapeutic ultrasound(MEUS)in combination with Percutaneous ethanol ablation(PEA)as the combination group,10 animals were treated by PEA only and 8 were treated by MEUS only.In the combination group,the tumors were first exposed to 5 mins therapeutic ultrasound with a constant injection of a 0.5-mL dilute microbubble suspension(approximately 5×108/ml)at the same time,followed by an intratumoral slow injection of 0.3-mL absolute ethanol.The other two groups received only MEUS or PEA,respectively.(3)Tumor blood perfusion assesssment: contrast-enhanced ultrasound and quantitative analysis time intensity curve of the tumor were performed before treatment,immediately after treatment(except for PEA alone group)and 24 hours post treatment.Time intensity curves of tumor regions were calculated using software and quantitative parameters including peak intensity(PI),area under curve(AUC),time to peak(Tp)and mean transit time(MTT)were obtained for further analysis.Tumor necrosis rate was determined by histological examination of 3 equally divided sections.? Effect and mechanism of MB mediated diagnostic range low intensity ultrasound cavitation on muscle blood vessels.(1)Animal grouping and low intensity ultrasound treatment protocal: 40 healthy nude mice were randomly divided into four groups(n=10)according to the variables preset-i.e.Mechanical index(MI)and treatment time(T).MI0.7T10 min,MI0.7T20 min,MI1.4T10 min,and MI1.4T20 min.Animals received a bolus injection of 0.02 ml MB each,followed by a ultrasonic irradiation on the surface of one hind limb with corresponding ultrasound parameters(MI = 0.7/1.4,treatment time = 10min/20 min,respectively).(2)Evaluation of blood perfusion change: Bilateral leg muscles were examined with CEUS,and the time intensity curve of corresponding muscle region was quantitatively analyzed before and after treatment.(3)Nitric oxide concentration detection: Muscle samples from the treated side and the control side of 5 animals from each group were obtained and examed following the manufactor's instructions.(4)Detection of NaFI exudation: Five animal from each group received an injection of 0.2 ml 0.5% NaFI,the muscle samples from the treatment side and the control side were obtained after a circulation perfusion drainage.All samples were homogenized,centrifuged.The supernatant was test by the excitation wavelength of 437 nm,and the fluorescence intensity of 518 nm wavelength was received to determine the amount of NaFI exudation.? Effect and mechanism of MB mediated diagnostic range low intensity ultrasound cavitation on tumor blood vessels,and its effect of tumor chemotherapy enhancement.(1)Thirty two nude mice bearing bilateral lower limb subcutaneous pancreatic cancer were used in this study.Animal were randomly divided into 4 groups(n=8).Treatment groups and low intensity ultrasound treatment protocal,evaluation of blood perfusion change,nitric oxide concentration and NaFI exudation detection were performed in the same way as in study(2).(2)MB mediated diagnostic range low intensity ultrasound cavitation enhanced tumor chemotherapy study protocal: 12 mice bearing a unilateral lower limb subcutaneous pancreatic cancer,were randomly divided into 2 groups to receive an intravenous administration of gemcitabine only(GEM n=6),or intravenous administration of gemcitabine,followed by a low intensity ultrasound+MB treatment(US+GEM,n=6).Acoustic output power were set at MI=0.7,and the treatment time were 20 min.Tumor tissues were obtained post treatment.The concentration of gemcitabine in the tumor tissue were measured with HPLC.Results:1.Enhancement of ethanol ablation with MB mediated low intensity ultrasound cavitation.? CEUS showed that the blood perfusion almost vanished in all tumor of the MEUS group and the combination group,with a significant drop of PI and AUC(P<0.05).The contrast peak intensity dropped 84.8% in the MEUS+PEA-treated tumors,compared to 46.3% in the PEA-treated tumors and 30.9% in the MEUS-treated tumors,and the AUC dropped 90.1% in the MEUS+PEA-treated tumors,compared to 52.5% in the PEA-treated tumors and 43.4% in the MEUS-treated tumors24 h after treatment,respectively.? Large area of coagulative necrosis within all tumors.The tumor necrosis rate of the combination therapy was 97.50±1.84%,which is much higher than that of the MEUS-(66.17±12.54%)and PEA-(81.02±8.23%)treated tumors.2.Effect and mechanism of MB mediated diagnostic range low intensity ultrasound cavitation on muscle blood vessels.? The peak intensity ratio of the treated side over the control side(PIT/PIC)increased 7%,18%,16% and 9% after ultrasonic cavitation treatment in group MI0.7T10 min,MI0.7T20 min,MI1.4T10 min and MI1.4T20 min(P < 0.05).The Area under curve ratio of the treated side over the control side(AUCT/AUCC)increased 9%,28%,31% and 15% after ultrasonic cavitation treatment in group MI0.7T10 min,MI0.7T20 min,MI1.4T10 min and MI1.4T20 min,respectively(P < 0.05).? The concentration of nitric oxide in the treated side was higher than that in the control side in all groups(P < 0.05).There was no statistical difference in NaFI exudation between the treated side and the control side in all groups(P > 0.05).3.Effect and mechanism of MB mediated diagnostic range low intensity ultrasound cavitation on tumor blood vessels,and its effect of tumor chemotherapy enhancement.(1)There was no statistical difference in PIT/PIC and AUCT/AUCC of the tumor before and after treatment in all groups(P > 0.05).(2)There was no statistical difference in concentration of NO between the treated side tumor and the control side tumor in all groups(P > 0.05).(3)NaFI exudation in the treated side tumor was significantly higher than that of the control side tumor in MI0.7T20 min group(P < 0.05).There was no statistical difference in NaFI exudation between the treated side and the control side in all other groups(P > 0.05).(4)Tumor chemotherapy combined with low intensity ultrasound cavitation(MI0.7T20 min)resulted in a 2.5-folder higher concentration of gemcitabine in the tumor tissue,when compared to intravenous administration of gemcitabine alone(P < 0.05).Conclusions:1.High peak negative pressure low intensity ultrasound combined with MB can lead to a significant decrease in tumor perfusion,PEA combined with MEUS can induce a much more complete tumour necrosis.2.Diagnostic range low intensity ultrasound stimulated MB cavitation can significantly increase blood perfusion and nitric oxide synthesis in normal muscle tissue.3.Diagnostic range low intensity ultrasound(MI0.7T20min)stimulated MB cavitation can increase the permeability of small molecule drugs and increase the local drug concentration in nude mice subcutaneous pancreatic cancer. |
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