| Background and objectiveHigh Intensity Focused Ultrasound (HIFU) is a promising new technique that provides the possibility of producing tissue destruction selectively and noninvasively. Biological effects of HIFU include thermal effect, mechanical effect and cavitational effect. Non-thermal effects (mechanical effect and cavitational effect) play roles that some people believe we should restraint cavitational effects as possiblely, the others think cavitation can assist thermal damage and monitoring. It's true that we are able to produce predictable tissue lesion, however, individual contribution of each effect is poorly defined. Non-thermal effects (mechanical effect and cavitational effect) were rarely studied and applied.In the first stage of this study, we exposed ox liver to Pulsed High Intensity Focused Ultrasound (PHIFU) in vitro so as to screen out parameter set that produce non-thermal effects. In the second stage PHIFU with the screened non-thermal parameter sets was then combined with Ultrasound Contrast Agent (UCA)to investigate the possibility and the biological effects of tumor tissue in vivo by non-thermal effects. The study aims to give theory support and a new method of enhancing HIFU efficiency clinically.MethodsFirst stage (non-thermal parameter set screening stage): There were 7 parameter sets which were grouped based on different duty cycles: 1%, 5%,15%, 25%, 50%,75% and 100% .Total energy was held constant for each parameter set, a thermal needle (temperature sensor) was inserted into the liver tissue under guidance of B-mode ultrasound, and move HIFU focal point to the hyperechoic region, and then temperature,gray scale and histological changes of the irradiated point were observed.Second stage: A VX2 liver tumor model was established in 36 rabbits. The rabbits were divided randomly and averagely into three groups: sham PHIFU group, PHIFU group and PHIFU+UCA group. Both PHIFU group and PHIFU+UCA group were given PHIFU exposure of non-thermal parameters screened out in the first stage of our study (ultrasound frequency 0.87 MHz, total acustic power 150 W, focal length 150 mm, pulse repetition frequency 100 Hz, and duty cycle 15%). One day after PHIFU exposure, the liver tumors were sectioned and observed. histo-pathological and ultra-structural changes were examined. Proliferating cell nuclear antigen (PCNA) expression was detected by immunohistochemical staining, and apoptosis was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL). Observed with light microscope, apoptosis index (AI) and proliferating index (PI) were recorded and calculated.ResultsFirst stage: The mean values of temperature difference after PHIFU irradiation were 63.4±9.2℃, 65.0±11.5℃, 66.6±9.9℃and 79.6±10.6℃for group 25%, 50%, 75% and 100%, respectively. In these four groups, the highest temperature was over 85℃and obvious coagulated necrosis was found. In contrast, for group 1%, 5% and 15%, the mean values were 29.2±1.9℃, 30.0±2.8℃, and 31.0±2.4℃, respectively, and the highest temperature was 56℃or even lower. In addition, no necrosis could be seen with naked eye. In group25%,50%,75%,and 100%, we observed blanched cytoplasm, nucleole disappearance,karyopyknosis and chromatin margination and increased with duty cycle prolongation.In group 15% light microscopic observation found that intercellular space was widened and many vacuoles of different sizes were found in the cytoplasm. But no significant changes were found in group 1% and 5%.Second stage: After PHIFU exposure, we found grossly that liver tumors of all the three groups were uniformly stained red by Triphenyl tetrazolium chloride (TTC) staining, suggesting that none of the animals had coagulated necrotic changes. In group PHIFU, histopathological examination demonstrated that eosinophilic staining is weak in the cytoplasm of tumour cells; we observed cellular swelling, cytoplasm looseness, cytoplasm vacuoles, and in a few tumor cells we found karyopyknosis and chromatin margination. In group PHIFU+UCA, light microscope displayed presence of abundant vacuoles of different sizes in the cytoplasm; we also found chromatin margination and karyopyknosis. Under transmission electron microscope (TEM), we discovered mitochondrion swelling and endocytoplasmic reticulum expanding in PHIFU group; while in PHIFU+UCA group, we observed condensation of the nuclear chromatin with margination. Both PHIFU group and PHIFU+UCA group had apoptotic body and cytoplasm vacuoles. TUNEL method detect few positive staining tumor cells in the sham PHIFU group, but in the PHIFU group and PHIFU+UCA group we found a lot of brow-colored positive apoptotic cells. PCNA detection showed more brow-colored positive cells in the sham PHIFU group than in the PHIFU group and PHIFU+UCA group. AI of the tumor cells in the PHIFU group and PHIFU+UCA group was higher than that of the tumor cells in the sham-HIFU group, while PI expression of the tumor cells in the PHIFU group and PHIFU+UCA group was lower than that of the tumor cells in the sham-HIFU group (P<0.05). AI of the tumor cells in the PHIFU+UCA group was higher than that of the tumor cells in the PHIFU group, while PI expression of the tumor cells in the PHIFU+UCA group was lower than that of the tumor cells in the PHIFU group(P<0.05).Conclusions: 1. Long duty cycle PHIFU induces tissue necrosis mainly by thermal effects, while short duty cycle may damage tissues by nonthermal effect. 2. Tumor tissue in vivo was destroyed mainly by nonthermal effects under the following HIFU parameters (ultrasonic frequency 0.87 MHz, focal length 150 mm, acustic power 150 W, pulse repetition frequency 100 Hz, duty cycle 15%). 3. UCA may enhance nonthermal damage of PHIFU. 4. We regulated duty cycle of PHIFU to nonthermal damage and provided a new method to therapy.
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