The effects of low-frequency ultrasound and microbubbles on rabbit hepatic tumors

Abstract

High-intensity focused ultrasound in combination with microbubbles (MBs) is able to inhibit the growth of VX2 rabbit liver tumors in vivo and prolong the survival time of the animals. In this study, we attempt to investigate the feasibility of VX2 tumor growth inhibition using low-frequency ultrasound (US)-mediated MB disruption. Forty-eight New Zealand rabbits with hepatic VX2 tumors were divided into four groups: control, MBs group, low-frequency US group, and US + MB group. The parameters of the US were 20 kHz, 2 W/cm², 40% duty cycle, 5 min, and once every other day for 2 weeks. At the end of the therapy experiment, 24 rabbits were euthanized, and the cancers were collected and cut into five sections for histological examination, immunohistochemistry, laser confocal microscopy, western blotting assays, and transmission electron microscopy (TEM). Another 24 rabbits were saved, and overall survival time was recorded. The tumor volumes in control, MB, US, and US + MB groups were 6.36 ± 0.58, 5.68 ± 0.42, 5.29 ± 0.26, and 2.04 ± 0.14 cm³, respectively (US + MB versus the other three groups, P < 0.01). Tumor cells manifested coagulation necrosis with internal calcification. Hematoxylin and eosin (H–E) staining revealed interstitial hemorrhage and intravascular thrombosis. The intensity of cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) in the US + MB group in the immunohistochemical staining, laser confocal microscopy, and western blotting assays was lower than that of the other three groups (P < 0.05). TEM of the US + MB group revealed vascular endothelial cell wall rupture, widened endothelial cell gaps, interstitial erythrocyte leakage, and microvascular thrombosis, while intact vascular endothelial cells and normal erythrocytes in the tumor vessels were observed in control, MB, and US groups. Rabbits treated with US + MB had a significantly longer overall survival than those in the other three groups (χ2 = 9.328, P = 0.0242). VX2 tumor growth could be inhibited by cavitation induced using low-frequency US and MB.

Spiral computed tomography evaluation of rabbit VX2 hepatic tumors treated with 20 kHz ultrasound and microbubbles

“…The vibration time at negative pressure is longer; thus, the liquid-phase intermolecular attraction breaks more easily and more cavitation nuclei form (34). Furthermore, the longer time gap between the compression and expansion of the liquid affords the cavitation bubble with more time to grow prior to bursting, and the volume of the cavitation bubble is positively associated with the intensity of the cavitation effects when the bubble collapses (35). Thus, selecting an appropriate ultrasonic frequency is crucial to the induced cavitation effect.…”

“…During US sonication in the presence of MBs, the oscillation, collapse and cavitation of MBs in the acoustic beam produced vascular pores and disrupted the vessel wall to significantly increase the vascular permeability in the sonicated areas (35). This process may have led to the formation of smaller bubbles, which interacted with the US beam and caused the cellular bio-effects (44).…”

Ultrasound cavitation enhanced chemotherapy: In vivo research and clinical application

“…Theoretically, sound waves with lower frequencies have longer periods leading to longer MB oscillation time in the expansion phase and a larger expansion in the diameter of the MBs. 43 The greater the damage effect in the compression phase, 43 the more obvious the cavitation effect we had. In addition, while they used a single drug, either paclitaxel or docetaxel, we used three chemotherapeutic drugs, DDP, MMC, and 5-FU, which is more consistent with the clinical practice of multi-drug combination usage.…”

Selective ablation of cancer cells with low intensity pulsed ultrasound

“…Low intensity (ISPTA <5 W/cm 2 ) and low frequency (<1 MHz) pulsed ultrasound produces mechanical effects without hyperthermia, resulting in neurostimulation [15], chemotherapy uptake [16], and bone repair [16][17][18]. However, its ability to selectively ablate cancer cells has not been studied, and its mechanisms of action are not fully understood [19][20][21].…”