| Background:The growth and metastasis of tumor cells are dependent on the neovascularization. Neovascularization not only provides nutrients and oxygen for the growth of tumor cells but also effectively carried away its metabolites. If there are no neovascularization meeted the nutrient supply of the tumor cells and substance excretion, the growth and metastasis of tumor cells will be severely limited. Antivascular therapy is considered to be an important treatment modality for solid tumor. It mainly includes endovascular intervention and antiangiogenic drugs in two ways. Endovascular intervention is an invasive treatment modality which not only limited in curative effect but also have the higher recurrence rates. Likewise, antiangiogenic agents haven’t achieved the ideal effect in clinical practice as they do not obviously improve survival rates and like other antineoplastic drugs can cause drug resistance and some toxic side effects.Recently, low-intensity ultrasound-microbubble (LIUS-MB) treatment is expected to be a new treatment modality for anti-tumor angiogenesis based on the findings from previous studies which shown that the ultrasound-driven cavitation of microbubbles can causes tumor vessels damage and resultantly decreases the tumor perfusion. However, it reflects different influence on tumor blood flow by microbubble enhanced ultrasound among those studies. Some studies indicated that in the treatment of microbubble enhanced ultrasound the tumor blood flow has been complete and persistent blocking. Other studies show that the tumor blood flow just been transiently cut off and will be recovered gradually. As persistent cut off tumor blood flow is the key to the effect of anti-angiogenesis, it is important to make the potential mechanism clear in clinic practice. We analyzed that the reason causing the different influence on tumor blood flow is the different pathological alteration by the different set of ultrasound parameters in their studies. We know that there is a close relationship between ultrasound parameters and ultrasound-driven cavitation. The difference in ultrasound parameters could lead to different effect on tumor blood vessels. From the previous studies we find that there exist different changes in histopathology under the treatment of microbubble enhanced ultrasound. Some studies indicated that the tumor microvessel has been badly damaged. However, in other tirals it only showed congestion and edema. We speculated that the different set of ultrasound pressure is led to different histopathology changes and resultantly bring different effect on the tumor blood flow. As the existing researches are largely used ultrasonic parameters in a single modality and haven’t yet explored the relationship between the blood perfusion and tissue pathology, the evidence is lacking to prove the speculation. Further experiments are needed to clarify.On the other side, we find that there exsit a different influence on blood flow between the tumor tissue and the tissue around tumor by microbubble enhanced ultrasound. Some studies indicated that the blood flow of tumor tissue has been decreased obviously but the tissue around tumor has not been influenced. Other trials show that although the blood flow of the tumor tissue and the tissue around tumor has been immediately reduced by microbubble enhanced ultrasound. However, the blood flow of the tissue around tumor has been graduatelly recovered. As the structure of tumor microvessel is more immature than the microvessel around tumor, we speculated that the reason that tumor microvessel is more sensitive than microvessel around tumor to acoustic cavitation is the difference in the maturity between them. Due to lack of direct evidence to confirm further studies are needed to verify.As the main mechanism of the destruction of tumor microvessel by microbubble enhanced ultrasound is the cavitation effect, yet ultrasound pressure is one of the most important parameters which reflect the level of the cavitation, we hypothesized that low-intensity ultrasound-microbubble (LIUS-MB) treatment under different ultrasound parameters could attribute to different pathologic change and accordingly generate different influence on the blood flow. Furthermore, we also hypothesized that the different influence on blood flow between the tumor tissue and the tissue around tumor by microbubble enhanced ultrasound is the different maturity of their blood vessels.Objection:We attempted to validate the assumption that low-intensity ultrasound-microbubble (LIUS-MB) treatment under different ultrasound parameters could attribute to different pathologic changes and accordingly generate different influence on the blood flow. Furthermore, we also tried to prove that the different influence of blood flow between the tumor tissue and the tissue around tumor by microbubble enhanced ultrasound is the different maturity of blood vessels.Methods:1. Materials1.1 Experimental animals:healthy Kunming mice 45, male,6-8 weeks old, weighing 23-30g, purchased from the Laboratory Animal Centre of Southern Medical University.1.2 Principal Experimental instruments:(a) small, pulsed, focused ultrasonic cavitation treatment apparatus, offered by ultrasound department of the Third Military Medical University. With a treatment probe frequency of 0.94MHz, an emission duty of 0.1%, time=lmin, Pulse repetition frequency 10HZ. (b) Doppler ultrasonic machine with ultrasonic contrasting mode (Sequoia, Siemens Medical Systems, Mountain View, Calif). a17L5 transducer, centerline frequency of 7 MHz and a mechanic index of 0.17 the machine with contrast pulse sequencing technology.,1.3 Experimental reagents:perflutren protein-type microbubbles with perfluoropropane as core gas, a mean of 2.07±1.13um in diameter, approximately 2.0-4.2 X 109 microbubbles/ml, developed by Department of Pharmacology, Nanfang Hospital, Southern Medical University.2. Method:(1) To explore the different influence on tumor blood vessels with different ultrasound pressures2.1 Tumor model:Mouse sarcoma S-180 cells were obtained from the cell bank of Sun Yat-sen University (Guangzhou, China). Cells (2×106 cells) were then injected subcutaneously in the left hind limbs of male Kunming mice. LIUS-MB treatment was performed 7 days after tumor implantation, when the tumor reached a size of approximately 10 mm in diameter.2.2 Group of experiments:30 tumor-bearing mice were randomly divided into three groups:a sham group,1.5 MPa, and 3.0 MPa different ultrasound pressure groups (n=10 per group). The sham treatment was performed with the therapeutic ultrasound transducer in the off setting. Both the sham and LIUS-MB treatment were performed in the presence of microbubbles.2.3 LIUS-MB treatment with low-intensity ultrasound:To maintain a distance of 5 mm between the transducer and the skin overlying the tumor, the transducer was fixed on a steel stand with scale followed by applying ultrasound coupling gel to the skin. After intravenous injection of a bolus of 0.1 ml microbubbles, each mouse’s tumor area is treated by low-intensity ultrasound with the random divided ultrasound pressure. The sham treatment was performed with the therapeutic ultrasound transducer in the off setting.The transducer was operated at a frequency of 0.94 MHz with a pulse repetition frequency of 10 Hz and a duty cycle of 0.19%.2.4 B-mode and CEU perfusion imaging:After seletive 5 mice from 1.5, and 3.0 MPa different ultrasound pressure groups, serial B-mode and contrast-enhanced ultrasonic (CEU) perfusion imaging of the tumor was performed on three occasions: before treatment, immediately after treatment (0 h), and 24 h after treatment. Mice were anesthetized and placed in ventral recumbency. An initial B-mode imaging was performed at a mechanic index of 0.27. After intravenous injection of a bolus of 0.02ml microbubbles, CEU perfusion imaging was performed with contrast pulse sequencing at a centerline frequency of 7 MHz and a mechanic index of 0.17. To determine signal from circulating microbubbles alone, post-contrast frames were digitally subtracted by a pre-contrast frame with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va) and then color coded.2.5 Haematoxylin-eosin staining and immunohistochemistry:5 mice were sacrificed at 24 h, whereas the other 5 mice in each group were sacrificed at 0 h after treatment. Tumors were then collected for haematoxylin-eosin and immunohistochemical staining. For evaluation of MVD, sections were incubated with anti-PECAM-1 (anti-mouse CD31 antibody, Santa Cruz Biotechnology) to stain for endothelial cells. Density counts of microvessels were also performed independently by two investigators.(2) To explore the different effect on tumor and para-carcinoma tissue vessels and its potential machanisms2.6 Group of experiments:15 tumor-bearing mouse’s tumor area and para-carcinoma tissue are treated by microbubble enhanced ultrasound. The set of the ultrasonic parameter is 3.0 MPa. The studies were divided into tumor group and para-carcinoma tissue group2.7 B-mode and CEU perfusion imaging:Serial B-mode and contrast-enhanced ultrasonic (CEU) perfusion imaging of the tumor and para-carcinoma tissue were performed on three occasions:before treatment, immediately after treatment (0 h), and 24 h after treatment.2.8 Haematoxylin-eosin staining and immunohistochemistry:15 mice were sacrificed at pre-treatment, immediately after treatment and 24 h after treatment respectively. Tumors and para-carcinoma tissues were collected for haematoxylin-eosin and immunohistochemical staining.2.9 Confocal immunofluorescence:immunofluorescent double-labeling method was carried out in the tumors and para-carcinoma tissues before, and at 24 h after treatment. Anti-CD31 (Abcam) and anti-a-smooth muscle actin (SMA; Abcam) antibodies for detection of endotheliocytes and pericytes, respectively. Immature vessels are CD31-positive vessels but lacking a-SMA-positive periendothelial cells. Mature vessels are CD31-positive vessels and a-SMA-positive periendothelial cells.Statistical analysis:Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by a Bonferroni correction. Data were expressed as mean± standard deviation and p<0.05 was considered to indicate statistical significance. Statistical analyses were performed using SPSS 17.0 (IBM SPSS Inc., Chicago, IL, USA).Result:Effects of LIUS-MB treatment at varying ultrasound pressuresResults for CEU imaging:Significant ultrasound imaging was observed in the tumor group after injecting the microbubble. The tumor perfusion was substantially decreased immediately after treatment (P< 0.05) without significant recovery for 24 h in the 3.0MPa groups, while it was partially reduced immediately after treatment (P< 0.05) and gradually recovered (VIpost-0 h vs. VIpost-24 h, P< 0.05) in 1.5 MPa groups.Results for Hematoxylin-eosin staining:It showed that tumor microvessels were severely disrupted with their tubular architectures invisible and diffuse haemorrhage immediately after treatment in the 3.0 MPa groups, while there was vasodilation and congestion as well as haemorrhage in the 1.5 MPa groups. At 24 h after treatment, the number of extravasated RBCs was reduced in all the treatment groups. Meanwhile, massive necrosis of tumor cells was observed in the 3.0 MPa groups, whereas the necrosis area was much smaller in the 1.5 MPa groups.Result for immunohistochemistry:Immunohistochemistry results further revealed that immediately after treatment, the structures of tumor microvessels were incomplete with vessel fragments scattered in the region in the 3.0MPa groups, while the diameters of tumor microvessels appeared to increase in the 1.5 MPa groups compared with the sham group. At 24 h after treatment, there was a significant reduction in the MVD of tumor in the 1.5 and 3.0 MPa groups compared with the sham group. The MVD of tumor was reduced by 28% and 83% in the 1.5 and 3.0 MPa groups, respectively (P<0.05).The different effect on tumor and para-carcinoma tissue vessels and its potential machanismsResults for CEU imaging:Significant ultrasound imaging was observed both in the tumor group and the para-carcinoma tissues group after injecting the microbubble. The tumor perfusion was substantially decreased immediately after treatment and in some area without recovery for 24 h, while it was slightly reduced after treatment and gradually recovered in the para-carcinoma tissues groups.Results for Hematoxylin-eosin staining:It showed that the structure of tumor microvessels was intact before treatment and were severely disrupted with their tubular architectures invisible and diffuse haemorrhage immediately after treatment, while there was vasodilation and congestion as well as a little haemorrhage in the para-carcinoma tissues group. At 24 h after treatment, the number of extravasated RBCs was reduced in all the treatment groups. Meanwhile, massive necrosis of tumor cells was observed in the tumor group, whereas the necrosis area was much smaller in the para-carcinoma tissues groups.Result for immunohistochemistry:Immunohistochemistry results further revealed that the structure of tumor blood vessel was intact and immediately after treatment, the structures of tumor microvessels were incomplete with vessel fragments scattered in the region, while the diameters of tumor microvessels appeared to increase in the para-carcinoma tissues groups. By contrast, immediately after treatment, microvessels in the para-carcinoma tissues were mostly circular and clearly visible. At 24 h after treatment, there was a significant reduction in the MVD of tumor compared with the pretreatment group (P< 0.05) and also a slightly reduction in the MVD of para-carcinoma tissues.Result for Confocal immunofluorescence staining:Confocal immunofluorescence staining showed that a majority of tumor microvessels were absent of pericytes (89±6%), which is denoted as immature, while microvessels in the peritumor tissue were immature 20±5%. At 24 h after treatment, the number of immature microvessels was significantly reduced in these tissues, especially in the tumor (reduced by 90%, P<0.05), while there was no significant reduction in the number of mature vessels.Conclusion:In conclusion, Low-intensity ultrasound-microbubble (LIUS-MB) treatment under different ultrasound parameters could attribute to different pathologic change and accordingly generate different influence on the blood flow. Furthermore, the different influence of blood flow between the tumor tissue and the tissue around tumor by microbubble enhanced ultrasound is the different maturity of blood vessel. |
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