| BackgroundHigh intensity high intensity focused ultrasound (HIFU), has been widely applied in the clinical treatment of many kinds of benign or malignant solid tumor, including primary liver cancer, uterine fibroids, etc. The method is effective, but there are also some problems. As for deep located tumors and large tumors, complications such as skin burns, intestinal perforation, nerve injury may appear due to the high intensity and long period of therapy. What's more, when it comes to tumors near to vital location, such as brain tumor, HIFU ablation might damage normal tissue nearby and influence the brain function.Zhang et al treated tumor with two stages HIFU irradiation. They significantly improved the ablating efficiency, reduced skin injury, and improved safety, and the twice irradiation on next day is more effective than that on the third day. This modality might be applicable in the treatment of larger tumors. We previously irradiated transplanted tumor in rabbit liver with HIFU of different power, coagulation necrosis began to appear in some tumors at the power of 120 W, and all tumors can be ablated at the power of 180 W. The more the power of pre-irradiation (non-ablating HIFU, nA-HIFU) close to 120 W, the larger volume the subsequent irradiation can ablate. This phenomenon might be caused by changes of acoustic environment: pre-irradiation of nA-HIFU injured the targeted tissue, and a series of pathophysiological changes happened, and some of changes in structure or status may change acoustic related characteristics, thus improving the transformation efficiency of acoustic energy into heat energy. This study will test the changes of acoustic environment before and after nA-HIFU irradiation from both biology and acoustics aspects, to explore the mechanism.Natalia et al found apoptosis in rabbit brain irradiated with focused ultrasound at the power close to ablation threshold, and apoptosis appeared more in 48 hours after irradiation than those in 4 hours. Wu et al found a certain degree of growth inhibition of bladder cancer cells irradiated with focused ultrasound in a period of time. Zhang et al irradiated rabbit liver with nA-HIFU, and found certain pathological changes which basically disappeared in 7 days. If we can find some kind of focused ultrasound, whose intensity is not too high to injure structures nearby, but also can perish tumors, then many unnecessary risks can be avoided thus many benign or malignant tumors near to vital structures can be treated. In this study, transplanted VX2 tumors in rabbit liver were irradiated with nA-HIFU close to ablation threshold to observe the long term impact on tumor-bearing rabbits.Purpose1. Explore the biological and acoustic mechanism of the influence of nA-HIFU on the HIFU ablation efficiency.2. Explore the influence of nA-HIFU on VX2 tumor-bearing rabbits and its mechanism.Method1. Established transplanted VX2 tumor model in New Zealand rabbit liver, observed the distance from tumor to abdominal well and the adhesion situation 22 days after the transplantation on diagnosis ultrasound.2.1 20 tumor-bearing rabbits were randomly divided into 60 W group, 80 W group, 100 W group and control group, with five rabbits for each group, irradiated the rabbits respectively with corresponding acoustic power, took the targeted tumor tissue the next day. Observe the changes of enzyme activity and pathological structure though TTC dyeing, HE staining, SDH staining, and electron microscope, which reflected the changes on biological characteristics of sound environment.2.2 20 tumor-bearing rabbits were randomly divided into irradiating group and control group, irradiated tumors in irradiating group with nA-HIFU at 100 W. Took tissue samples from both groups one day after the irradiation, and measured the sound velocity and attenuation coefficient, which reflected the changes on acoustic characteristics of sound environment.3.1 25 tumor-bearing rabbits were randomly divided into 60 W group, 80 W group, 100 W group, ablating group and control group, with five rabbits for each group, irradiated the rabbits respectively with nA-HIFU at corresponding power, and observed the skin injury and survival time.3.2 54 tumor-bearing rabbits were randomly divided into 80 W group, 100 W group and control group, 6 rabbits for control group, and 24 rabbits respectively for the other two groups, irradiated the rabbits respectively with nA-HIFU at corresponding power, sacrificed the rabbits 1 h, 1 d, 3d, 5d, 7d, 14 d after the irradiation (one of control group, and four of each of the other groups each time), observed organs metastases, and observed changes on enzyme activity, pathological structure, the proliferative capacity, apoptosis and death situation by TTC dyeing, HE staining, PCNA staining and TUNEL staining.Result1. All New Zealand rabbit were successfully established rabbit liver transplant tumor model VX2 tumors with airborne ultrasound see, without adhesion, and abdominal wall in irradiation posture on tumor abuts on abdominal wall. 2.1 Being irradiated with nA-HIFU at diffident intensity, all the tumor tissue was dyed in red by TTC. According to the increase of nA-HIFU intensity, HE staining showed swelling cells, widened cellular gaps, swelling nucleus, and the gaps around the nucleus. Hyperemia in capillary and infiltration of inflammatory cells were found in group 100 W. But homogeneous red region were not found in any group. SDH staining showed the enzyme activity was weakened gradually, but it did not disappeared in any of the samples. The electron microscope examination showed that mitochondria swelling, and the extent increased gradually. Golgi body expansion, endoplasmic relicula and apoptosis were found in 100 W group. The appearance of 60 W group was similar with that of control group, and 100 W group changed most on the enzyme activity and pathological structure.2.2 The difference of acoustic attenuation and sound velocity between irradiation group and control group were not statistically significant (P > 0.05).3.1 Survival time increased according to the increasing of nA-HIFU intensity. Apart from 60 W group, the difference of survival time between control group and the other group were statistical significant (P < 0.05), the survival time of 100 W group was similar with that of ablation group. Skin injury was only found in ablation group. 3.2 At different time after the nA-HIFU irradiation, TTC dyeing showed 80W group and control group can be dyed all the time; 100 W group can be dyed at the 1st h, 1st d, and 3rd d after the irradiation, liquefied necrotic was found in the center of some tumors at the 5th d, 14th d, but the surrounding tissue were dyed. The tumor tissue appeared similarly with those in Part one, and became more seriously according to the increasing of nA-HIFU intensity. Both the appearance was most serious at the 1st day after irradiation. The proliferation index at each time point decreased according to the increasing of nA-HIFU intensity, and increased as the time prolonged, but all of them were lower than that of control group. The apoptosis index(AI) at each time point increased according to the increasing of nA-HIFU intensity. AI of 80 W group was the highest at the 3rd day after the irradiation and decreased as the time prolonged after that. AI of 100 W group was the highest at the 7th day after the irradiation and maintain at the high level after that. The necrosis index (NI) at each time point increased according to the increasing of nA-HIFU intensity. NI of 80 W group was the highest at the 3rd day after the irradiation and decreased as the time prolonged after that. AI of 100 W group was the highest at the 5th day after the irradiation and maintain at the high level after that.Conclusion1. Transplanted VX2 tumor in rabbit liver was good animal model for studying the safety of HIFU treatment. 2.1.1 All of the HIFU used in this part could not cause coagulative necrosis in transplanted VX2 tumors in rabbit liver occur, which meant they were nA-HIFU.2.1.2 The biological mechanisms of the influence of nA-HIFU on the subsequent HIFU ablation efficiency might include the following:â‘ according to the increasing of HIFU intensity, the tumor tissue were injured more seriously, indicating injury of cells might improved the sensibility of the cells to outer stimulus (ultrasound and heat, etc) and reduce the tolerance.â‘¡Hyperemia in capillary in 100 W group reduced the energy taken away by blood flow, thus increased heat accumulation, produced larger coagulative necrosis and improving the efficiency of subsequent HIFU ablation.2.2 The changes on sound velocity and attenuation coefficient before and after nA-HIFU irradiation were not detected. Due to the limited accuracy of the equipment, it could be concluded that there was no influence of nA-HIFU on acoustic properties; However, it indicated that even though there were impacts, the impact were not very obvious, and it might not the only mechanism that improving the subsequent HIFU ablation efficiency. Further studies were need for more accurate conclusion.3. NA-HIFU at certain intensity could prolong the survival time of tumor-bearing rabbits without skin injury, and reduce organ metastases rate of tumor-bearing rabbits. The effect was improved according to the increase of acoustic intensity. The mechanisms might involve the cells injury and the decrease of enzyme activity caused directly by nA-HIFU and cell anoxia caused by hyperemia, which influenced cells'function and thus inhibit the proliferation and introduce apoptosis and subacute necrosis. |
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