Experimental Study On Microcirculationblockage Of Normal Liver And Liver Tumor By Microbubbles Enhanced Ultrasound Cavitation

Backgrounds:Malignant tumor is a major threat to human health and life, and its treatment has long been a medical focus. The incidence, growth, and metastasis of solid tumor depend on tumor angiogenesis. New vessels in tumors grow quickly under stimulation by multiple vascular growth factors, and their structure is primitive and characterized by thin and weak vessel wall, incomplete basal membrane, hyperpermeability, and lack of the elastic fiber layer. Hence, in addition to chemotherapy and radiotherapy, anti-angiogenesis targeting at tumor new vessels has become a new therapeutic modality. However, current anti-angiogenesis methods involve attack of specific biological targets of tumor new vessels with chemical or biological drugs, and these methods have certain side effects and their efficacy is not satisfactory. No physical methods targeting at tumor new vessels have been reported yet.Over recent years, microbubble-based ultrasonic contrast media have drawn much attention from researchers because of their use in gene transfection, targeted drug release, and thrombolysis. Ultrasonic cavitation effect (note:cavitation effect refers to a series of dynamic processes of microbubbles in liquid during ultrasonication, e.g., shock, distension, contraction and implosion, with accompanying energy release behaviors such as transient high temperature, high pressure, shock wave, electrical discharge and microefflux) is a major physical effect of ultrasound besides thermal effect. Microbubble-based ultrasonic contrast media (microbubbles), as an effective cavitation core, may incur significant cavitation effect under appropriate ultrasonic impulse excitation. Cavitation released mechanical energy (non-thermal effect) has the potential of targeted tissue ablation, thus making possible damage of tumor new vessels.We demonstrated previously that after intravenous injection of microbubble-based ultrasonic contrast medium and under pulsed ultrasonic irradiation, the damage effect of ultrasonic cavitation can be controlled steadily, which may lead to cavitation-induced mechanical injury, hemorrhage, hematoma and thrombosis in small vessels of normal mesentery.Objectives: Intravenously administered microbubbles can significantly reduce the threshold of blood ultrasonic cavitation and enhance cavitation induced damage effect. The present study was based on the blockage of mesenterial microvessels by microbubbles and pulsed ultrasonication. and was intended to induce ischemic necrosis and growth inhibition through tumor microcirculation blockage by physical destruction by microbubble-based ultrasonic cavitation and blockage of new vessels in rabbit VX2 liver tumor, thus exploring a novel, non-traumatic, physical treatment aiming at tumor new vessels.Study methods:Ⅰ. Principal instruments and reagents:1. Experimental instruments:Self-made small, pulsed, focused ultrasonic cavitation treatment apparatus, with a treatment probe frequency of 1.2 MHz, an emission duty of 0.5%, a peak negative pressure of 4.6 MPa, a mean sound intensity of (ISPTA)0.89 W/cm2. Logic 9 color Doppler ultrasonic machine with ultrasonic contrasting mode (GE, USA).2.Experimental reagents:"Zhifuxian", a kind of lipid microbubbles in milky suspension developed by Xinqiao Hospital of the Third Military Medical University, with perfluoropropane as core gas at approximately 7×109 microbubbles/ml.98% microbubbles<8μm (mean,2μm) in diameter.Ⅱ. Study on blockage of normal liver blood perfusion by microbubble enhanced ultrasonic cavitation1. Experimental groups:33 healthy New Zealand rabbits were divided into the ultrasonic microbubble group, the simple ultrasonication group, and the sham group. The ultrasonic microbubble group was subjected to ultrasonic irradiation and intravenous injection of microbubbles; the simple ultrasonication group was subjected only to ultrasonic irradiation; the sham group was subjected to sham ultrasonic treatment. For each group,8 rabbits were irradiated across the abdominal wall for blood perfusion assessment, and the other 3 rabbits were directly irradiated after laparotomy for mechanism analysis.2.Experimental time points:Ultrasonic contrasting was carried out in each group before and after treatment, and the time points for analysis included pre(contrasting),0 min,15 min,30 min,45 min,60 min,24h (a total of 7 time points). The changes in the grayscale value of peak ultrasonic contrast perfusion were recorded, and the peak perfusion images were scored visually. The duration of and recovery from blockage of blood flow to the normal rabbit liver parenchyma were analyzed.3. Protocol of ultrasonic cavitation:The rabbit was anesthetized and an intravenous access established through the ear rim vein. Zhifuxian solution was injected in bolus at 0.01 ml/kg for ultrasonic contrasting (for the simple ultrasonication group and the sham group, ultrasonic contrasting was performed one day before treatment and the images were stored). After the liver was located, ultrasonic irradiation was applied to the liver according the predetermined time and direction. In the ultrasonic microbubble group, microbubbles (0.1 ml/kg, diluted with normal saline to 5 ml) were injected slowly and simultaneously the liver was irradiated for 5m using the treatment probe across the abdominal wall. In the simple ultrasonication group,5 ml normal saline was used to replace microbubbles and, meanwhile, the liver was irradiated for 5min vertically using the treatment probe. The sham group was sham-irradiated using the treatment probe for 5min, and 5ml normal saline was used to replace microbubbles. For laparotomic irradiation, a midline incision was made below the xiploid process, and the liver was exposed and the left and middle liver lobes were pulled out of the abdominal cavity for treatment. The protocol of ultrasonic cavitation treatment was the same as above. Specimens of the irradiation target area were harvested immediately after treatment for pathological study.4. Analyses:(1) Visual density analysis:The dynamic ultrasonic contrasting images of the target area were recorded at various time points in each group. The digital images of points of interest were imported in the JPG format, and then were subjected to quantitative grayscale analysis using the histogram feature of Adobe Photoshop CS3 software. The cross-section of large or medium vessels should be avoided during sampling. The mean grayscale value in the sampling window was calculated automatically by the software.(2) Visual score analysis:For blood perfusion analysis, dynamic ultrasonic contrasting images of the target liver area were recorded at various time points. Topical liver blood perfusion was visually scored as 0-3 grades. The grayscale value changes of peak contrast perfusion were visually scored at each time point. Ⅲ. Experiment on the blockage of VX2 tumor microvessels by microbubbles enhanced ultrasonic cavitation1. Experimental groups and methods:24 VX2 tumor-bearing rabbits were grouped and treated as described previously. The time points for tumor treatment included:Oh (starting treatment),48h,96h (a total of three time points). The time points for observation included pre (treatment), post (treatment),30min,24h,48h,96h, 10d (a total of 7 time points). The grayscale value changes in the peak tumor contrast perfusion were observed at various time points (pre, post,30min, and 24h). The tumor volume was measured, and tumor specimens were subjected to apoptosis detection upon completion of the experiment.2. Analytical methods:The grayscale value changes in the peak tumor contrast perfusion were analyzed by visual density method (see 4(1)). The tumor volume was calculated by the ellipsoid formula, and the tumor growth curve was plotted after treatment. Tumor necrosis was assessed pathologically and the apoptotic index was calculated by TUNEL.Results:1. Preliminary study on blockage of normal liver blood perfusion by microbubbles enhanced ultrasonic cavitation(1) Visual density:Before treatment, the mean grayscale value and ROI contrast mean grayscale value (88.4-89.1) differed insignificantly between the groups at various time points. At Omin after treatment, in the US+MB group, the mean ROI grayscale value reduced significantly to 2.7±2.7, and then restored to 92.8±13.9 gradually in 24h. The mean contrast grayscale value was significantly higher before treatment than at Omin,15min and 30min after treatment, but the value before treatment differed insignificantly from the value at 45min,60min and 24h after treatment. In the US group and the Sham group, the mean grayscale value changes were within 88.2-89.7 at all time points (P>0.05). In the experimental group (US+MB), the mean peak contrast grayscale value changed in a "U"-shaped manner; that is, the value decreased steeply virtually to 0 after treatment, and then restored gradually to the normal level before contrasting.(2) Visual scores:In all groups, the visual scores of liver blood perfusion were 0 before treatment. After treatment, Kruskal-Wallis test indicated that the statistics in the MB+US, US, and sham groups were 37.34,1.26, and 0, respectively. This suggested that the visual scores differed significantly in the MB+US group between various time points, and that visual scores differed insignificantly in the US and sham groups between various time points. Nemenyi test demonstrated that in the MB+US group, the pretreatment scores were significantly superior to those at Omin and 15min post-treatment (P<0.05). The scores at Omin post-treatment differed significantly from those at pretreatment, and 45min,60min,24h post-treatment (P<0.05); and the scores at 15min post-treatment differed significantly from those at pretreatment, and 60min, and 24h post-treatment (P<0.05). The scores at 30min post-treatment differed insignificantly from those at the other time points (P>0.05). The scores differed significantly between 45min and Omin post-treatment. The scores at 60min and 24h post-treatment differed significantly from those at Omin and 15min post-treatment (P<0.05).(3) Pathological study:The sham group did not exhibit abnormal liver histology. In the simple ultrasonication group, focal, patchy hyperemia and hemorrhage were observed occasionally in the liver tissue. In the ultrasonic microbubble group, the following pathological changes were observed:massive hemorrhage of liver tissue, hepatic sinus congestion, and breakage of vascular wall, strip-like shedding of endothelial cells and thrombosis, and liver cell edema and degeneration around the central vein.2. Experimental results of ultrasonic cavitation blockage of tumor microcirculationIn each group, pretreatment ultrasonic contrasting indicated abundant tumor blood supplies (hyperperfusion) and patchy filling defect in a few large tumors, and the peak tumor contrast GSV(changes between 93.36 and 96.4) differed insignificantly(P>0.05). In the sham group and simple ultrasonication group, tumor hyperperfusion was observed immediately and 30min,24h after treatment, and the grayscale value of tumor tissue contrasting images differed insignificantly from that before treatment (P>0.05).In the ultrasonic microbubble group, ultrasonic contrasting immediately after treatment did not reveal contrast medium perfusion in the tumor and surrounding liver tissues, and the contrasting grayscale value decreased significantly to 13.87±10.04(P<0.05). At 30 min after treatment, contrasting revealed partial recovery of tumor perfusion, but the mean grayscale value (66.09±21.85) was still significantly lower than the value before treatment (P<0.05). At 24h after treatment, tumor hyperperfusion recovered (P>0.05), which differed insignificantly from the pretreatment findings (P>0.05).Tumor volume and apoptosis:Before treatment, the tumor volume (0.37-0.45 cm3) differed insignificantly between the groups (P>0.05). At 10d after treatment, the mean tumor volumes were 1.53 cm3,2.27 cm3 and 2.46cm3 in the treatment group, the simple ultrasonication group and the sham group, respectively. The volumes were significantly smaller in the treatment group than the control group (P<0.05). TUNEL detection indicated an obvious increase in the apoptotic index in the microbubbles ultrasonication group at 10d after treatment, and that the apoptotic index was relatively low in the simple ultrasonication group and the sham group.Conclusions:The cavitation effect resulting from the use of microbubbles and a pulsed ultrasonic cavitation treatment apparatus can block normal liver microcirculation temporarily, and the microcirculation restored gradually within lh. The mechanism mainly involves liver tissue congestion, hemorrhage, hematoma and thrombosis. Microbubbles induced ultrasonic cavitation may lead to microcirculation blockage of VX2 tumor in rabbits. However, blood flow restored apparently after 24h, and the precise mechanism remains unclear.