| BackgroundMalignant tumor is one of the major diseases which threaten human health and life, even as the leading cause of death in some countries. The common strategies of the current treatment include surgery, radiotherapy and chemotherapy which is mainly to remove tumor tissue, kill tumor cells or induce cell apoptosis. However, these methods are all failed in inhibiting the formation of tumor vessels, inhibiting the growth of tumors and preventing metastasis, while the chemotherapy lacks of specific target and has side effects for not only be able to kill tumor cells but also kill a large number of bone marrow cells and proliferation cells at the same time,they are limited in treating the tumors.Mammalian cells require oxygen and nutrients for growth and metastasis and are therefore located within microvessels-the diffusion limit for oxygen. Without blood vessels, tumors cannot grow beyond a critical size or metastasize to another organ. It is important significance if we intervene in some ofthe factors and key steps of angiogenesis to cut off the blood supply for the growth and metastasis. However, these antiangiogenic drugs have some limitations in clinical application because different tumor or the same tumor has different reactions to drugs in the process of the treatment. The clinical benefit is very limited when just use the single anticancer drugs even though combined with the chemotherapy drugs. So it is urgent to look for an impressive therapeutic method which is safe and effective.Recent years, with the development of ultrasonic, ultrasound and contrast agent has become more and more widely and important in clinical application. They not only have important scientific significance in diagnosis but also show attractive prospects in the treatment field.The basic theory of the mechanism in the treatment is the ultrasonic cavitation effect, which is endogenous or exogenous microbubble cavitation nuclei process a series of dynamic process such as oscillation, expansion, contraction, implosion and so on.it is peculiar to the ultrasonic propagation in the liquid. In previous studies it shows that Microbubble destruction during ultrasound exposure caused injury of microvessels (mostly capillaries) and the production of nonviable cells in adjacent tissue. Microbubbles can be destroyed by ultrasound, resulting in a bioeffect that could be used for tumor destruction. The next series of researches show that Ultrasound microbubble destruction can lead to the blood perfusion of the tumor significantly reduced by damaging the blood vessel of the tumor. The histopathologic studies showed that the effects of insonation were predominantly on the vascular structures within the tumor and correlated with the findings in the contrast enhanced power Doppler ultrasonographic images. There was disruption of the walls of the tumor blood vessels with associated hemorrhage, vascularcongestion and subsequent thrombosis; edema was also present.But it is not known how long the the cavitation effect which reduced blood perfusion last? Whether it can inhibit the growth of the tumor? Whether it can improve survival rates of the tumor animals? In our preliminary experiments it shows that there is no thrombosis in histopathologic studies when the tumor received1minute treatment which is the same as other scholars. So The question remains as to what were the underlying histopathological changes leading to the observed loss of contrast enhanced power Doppler signal in the treated neoplasms?We have known that it is obviously different between the normal and tumor blood vessels in structure and function. Normal vessel are organized with arteries, veins and capillaries, but the tumor vessels do not display the recognizable features of arterioles, capillaries or venules.The tumor Blood vessels are leaky, tortuous, dilated, and saccular and have a haphazard pattern of interconnection. The endothelial cells lining these vessels have aberrant morphology, pericytes are loosely attached or absent, and the basement membrane is often abnormal—unusually thick at times, entirely absent at others.Based on the above theory, the tumor blood vessels may be more sensitive to mechanical damage of the cavitation effect. It just damages the tumor angiogenesis not normal blood vessels which suppress tumor growth. This therapy can bring us important clinical value of as it guard the safety and effective effect in the treatment. but it is not reported by now, and it is lack the mechanism explanation to a high degree.Objective(1) How long the the cavitation effect which reduced blood perfusion last? Whether it can inhibit the growth of the tumor? Whether it can improve survival rates of the tumor animals?(2) What histopathological changes lead to the observed loss of contrast enhanced power Doppler signal in the treated neoplasms?(3) To observe whether there are some differences in ultrasound microbubble treatment response between normal and tumor blood vessels, if they are different, what’s the mechanism?Materials and Methods1. MaterialsPrincipal Experimental instruments:(1) small, pulsed, focused ultrasonic cavitation treatment apparatus, offered by ultrasound department of the Third Military Medical University. With a treatment probe frequency of0.94MHz, an emission duty of0.1%, a peak negative pressure of1500kPa, time=lmin, Pulse repetition frequency10HZ.(2) Doppler ultrasonic machine with ultrasonic contrasting mode (Sequoia, Siemens Medical Systems, Mountain View, Calif). A17L5transducer, probe frequency of7-14MHz, the machine with contrast pulse sequencing technology.Experimental reagents,"perflutren protein-type microbubbles developed by Department of Pharmacology, Nanfang Hospital, Southern Medical University, with perfluoropropane as core gas, at approximately2.0-4.25X109microbubbles/ml. A mean of2.07±1.13um in diameter.2. Method(1) The research on microbubble destruction inhibits growth of tumorMice were randomly divided into treated group (n=36) and control group (n=29) each anaesthetized pentobarbital sodium60mg/kg ip) mouse were imaged before and after tumor insonation and histology was performed in a selected number, a tail vein was catheterized before insonation. In the treated group, an initial B-mode study was followed by the intravenous injection of an ultrasound contrast agent (0.02ml perflutren protein-type microbubbles) and contrast-enhanced power Doppler observations were made. After the contrast agent was no longer detectable in these initial observations, a further0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%;) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off), after the procedure the B-mode and contrast-enhanced power Doppler studies were repeated at Oh,24h and72h post-treatment in the identical anatomical plane. In the control group, a similar study to treated group was performed, however, the physiotherapy transducer was applied to the tumor was not switched on after a further0.1ml microbubbles was injected.Tumor CEU imaging:All experimental mice were performed with CEU respectively by using MBa before treatment with the low mechanic index (MI=0.18), the intravenous injection of0.1ml microbubbles and subsequent post-treatment(Oh,24h and72h) images were acquired at the same mechanical index. To identify the signal from microbubbles retained in the tumor tissue, several post-treatment contrast frames representing the signal from circulating microbubbles were averaged and digitally subtracted from several averaged frames which acquired at pre-treatment, Oh, and24h and72h post-treatment with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va), transmission frequency of7.0MHz and receiving frequency of14.0MHz.B-mode:Ultrasound measurements of the growth of the tumor continued every5-7days with the low mechanic index (MI=0.57). In each of the B-mode images, the length (L), width (W) of the tumor was measured and its volume (mm3) was calculated as:Tumor volume=length×(width)2/2.Contrast-enhanced Ultrasound imaging analysis:To identify the signal from microbubbles retained in the tumor tissue, several post-treatment contrast frames representing the signal from circulating microbubbles were averaged and digitally subtracted from several averaged frames which acquired at pre-treatment,0h, and24h and72h post-treatment with use of software (Yabko MCE2.7; University of Virginia, Charlottesville, Va) and then color coded. The background-subtracted video intensity (VI) was measured in a region of interest fit the boundary of the tumor. CEUS of the muscle was obtained at the same method.(2)The mechanism of ultrasound mediated microbubble disruption inhibits the tumor growth20mice were randomly divided into pre-treatment group (n=5), post-treatment Oh group (n=5), post-treatment24h group (n=5) and72h group (n=5).0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off).Histology and Immunohistochemistry:For Immunohistochemistry and Histology, the tissue of the tumor was taken out at the time of pre-treatment, Oh, and24h and72h post-treatment, to observe the expression of the anti-mouse CD31antibody and anti-MPO antibody.Microvessel density measurement:The area of highest neovascularization was identified; individual microvessels were counted first on a100x field and then on a200×field.Any brown-staining endothelial cell or endothelial-cell cluster that was clearly separate from adjacent microvessels, tumor cells, and other connective-tissue elements were considered a single, countable microvessel. Vessel lumens, although usually present, were not necessary for a structure to be defined as a microvessel.MPO assessment:The tissue sections were scanned entirely to assign the scores.The staining intensity was scored as0(negative),1(weak),2(medium), or3(s trong). The extent of staining was scored as0(0%),1(1-25%),2(26-50%),3(51-75%), or4(76-100%), According to the percentages of positively stained areas in relation to the whole version field. The sum of the staining intensity and extent scores was used as the final staining scores (0-7) for MPO.(3) The mechanism and different action to ultrasound microbubble destruction between normal and tumor blood vessels. Mice were randomly divided into tumor group (n=5), muscle group (n=5),0.1ml microbubbles was injected and the physiotherapy transducer (acoustic pressures=1500KPa; f=0.94MHZ; duty factor=0.1%;) was applied to the tumor for1min (transducer worked at an intermittent mode of2s on and10s off).Histology, Immunohistochemistry and Immunofluorescent:For Histology, Immunohistochemistry and Immunofluorescent, the tissue of the tumor and muscle were taken out at the time of pre-treatment and post-treatment Oh.Assessing the maturity of the vessels:To assess the maturity of the vessels, a double-labeling immun technique was used to simultaneously stain endothelial cells (CD31) and mural cells/vascular Muscle Smooth cell (a-SMA).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.3. Results(1)The research on microbubble destruction inhibits growth of tumorResults for CEU imaging:Significant ultrasound imaging was observed both in the tumor treatment group and control group. While there was no significant changes of the CEU imaging after injecting the microbubble in the control group, CEU signal reduced obviously in the treated group after treatment. The signal was a little recovery at the time of post-treatment24h and72h, but there is significant difference compare to pre-treatment.Results for CEU-derived VI:CEU image were acquired at the same of pre-treatment, Oh, and24h and72h post-treatment with the MCE software and then color coded. There was significant reduced of VI at the time of post-treatment Oh (16.677±3.297),24h (26.524±2.193) and72h (33.225±19.952) compared with pre-treatment (66.012±4.129)(P<0.001).Tumor volume:the growth of the tumor in the treated group is significantly inhibited, while the growth was not in the control group. at7days after implantation, the tumor volume were0.315±0.080cm3and0.364±0.135cm3between the control and treated group respectively, which is no difference (p=0.098).At11,18and25days after implantation, the mean tumor volume were1.112±0.340cm3,2.526±0.483cm3and5.718±1.078cm3in the control group which is significantly larger than the treat group that the volume were0.662±0.186cm3,1.856±0.349cm3and3.290±0.772cm3respectively(p<0.001)Survival of the tumor mouse:The mean survival time of tumor mice was50.931±8.426days in the control group, while the survival time is significantly extend in the treated group that is72.722±10.870days (p<0.001).(2)The mechanism of ultrasound mediated microbubble disruption inhibits the tumor growthImmunohistochemistry result:The expression of the anti-mouse CD31antibody which represents Endothelium, the structure of the vessels is integrated,the endothelial cells lines very closed before the treatment.but immediately after treatment, the structure of the vessel is Severe and diffused disruption, the endothelial cells lines very loose, some parts of which has missed. at the time of post-treatment24h and72h, the structure of the vessels have not recovered, in the edge of the tumor, a small amount of vessels formed.Microvascular density measurement:Before the treatment, there are a number of vessels in the tumor filed, the number of which was decreased significantly at the time of post-treatment Oh,24h and72h(p<0.001).Expression of MPO which represents enhanced neutrophil infiltration: pre-treatment and immediately post-treatment, the expression of myeloperoxidase is little, the expression is increased significantly at post-treatment24h(p<0.001), and it is declined at post-treatment72h, but which is also significantly increased compared to pre-treatment(p<0.001). Scores of MPO:There was no significant increase the scores of MPO at neither pre-treatment (1.400±0.548) nor immediately post-treatment (1.600±0.548)(p=0.572). As compared to pre-treatment, the scores are significant increased at post-treatment24h (6.600±0.548) and post-treatment72h (3.600±0.548)(p<0.001).Results for examination of pathology:Before treatment, the tumor cell lines compact solidlike pattern with a tight intercellular space, the neoplastic cells varied considerably in size and shape with relatively large, deep-blue dyed nuclei. There are abundant of vessels in the tumor, the structure of the vessel is intact, and the endothelial cells lines very closed. There was no remarkable hemorrhage. At immediately post-treatment, the structure of the tumor is Severe and diffused disruption, the continuity of the endothelial cell is interrupted, hemorrhage was present in large part of the tumor, there are a lot of red blood cells. At the time of post-treatment24h, the red blood cells have been absorbed, massive tumor cell necrosis was observed in the tumor, and the area was increased at post-treatment72h.(3) The mechanism and different action to ultrasound microbubble destruction between normal and tumor blood vesselsResults for CEU imaging:Significant ultrasound imaging was observed in the tumor group before treatment the signal was reduced obviously after treatment(p<0.001); Significant ultrasound imaging was also observed in the muscle group before treatment, but the signal was not significant reduced after treatment (p=0.134).Immunohistochemistry result:The expression of the anti-mouse CD31antibody which represents Endothelium, the structure of the vessels is integrated, the endothelial cells lines very closed before the treatment. But immediately after treatment, the structure of the vessel is Severe and diffused disruption, the endothelial cells lines very loose, some parts of which has missed. In the muscle group, the structure of the vessels is also integrated, the endothelial cells lines very closed before the treatment, but there is not much change of the structure of the vessels.Microvascular density measurement:Before the treatment, there are a number of vessels in the tumor filed (66.600±2.408), the number of which was decreased significantly after treatment (12.800±1.923)(p<0.001), in the muscle group, the microvascular density is34.400±3.847before treatment, the number of the vessel was also decreased (27.000±3.162), but compare to decrease of the tumor, the degree is slight.Immunofluorescence result:It shows that mature blood vessels take up a small part of proportion which is31.907±3.911%in the tumor group, the proportion is87.869±3.061%in the muscle group, it is significant differences (p<0.001) before treatment. After treatment, the percent of the mature blood vessels is significantly improved which is87.851±1.755%(p<0.001) in the tumor group, the proportion is also improved which is90.444±4.298%, but there is no significant statistical difference (p=0.307).4. Conclusions(1) Ultrasound microbubble by directly destroy the tumor microvascular blood perfusion decreased significantly, leading to tumor cells in the ischemic necrosis of large area, in this process, the activation of neutrophils play an important role.(2) Ultrasound microbubble destruction leading to the blood perfusion of the tumor significantly reduced by disruption the blood vessels directly, which lead to massive tumor necrosis. The activation of neutrophils play an important role in this process.(3) The tumor blood vessels are far more serious damage to ultrasound microbubble destruction than the normal blood vessels which guard the safety and effective effect in the treatment because the tumor vessels are immature than the normal. |
PDF Full Text Request