| Objective:1.To investigate the effect of only ultrasound treatment on the vascular permeability of rats hind limbs.2.To investigate the effect of low-frequency ultrasound irradiation combined with microbubble contrast agents(Sono Vue)on the vascular permeability of rats hind limbs.3.To investigate the influence of ultrasound irradiation on the viability of endothelial cells.4.To investigate the influence of ultrasound irradiation on the permeability of the endothelial cell barrier model in vitro and the expression levels and distribution of ZO-1 protein.Methods:1.Seventy-two male Sprague Dawley rats were randomly divided into nine groups,Including the control group,2.5 min group,5 min group,10 min group,20 min group,30 min group,60 min group and 120 min group,and each group had eight rats.These time subgroups belonged to the only ultrasound group,which was divided with the interval from the endpoint of ultrasound exposure to the injection of Evans blue.This study quantitively analyzed the vascular permeability of hind limbs of rats by detecting the content of Evans blue(EB)extravasated from the vessels.Meanwhile,two rats in each group were randomly selected and performed hematoxylin-eosin(HE)staining of tissue samples.2.Eighty male Sprague Dawley rats were randomly divided into eight groups,including the control group,only microbubble group,2.5 min group,5 min group,10 min group,20 min group,30 min group and 60 min group,and each group had ten rats.These time subgroups belonged to the microbubble-ultrasound group,which was classified with the interval from the endpoint of ultrasound exposure to the injection of Evans blue.This study quantitively analyzed the change of vascular permeability of rats hind limbs by detecting the content of Evans blue(EB)extravasated from the vessels.Meanwhile,two rats in each experimental group were randomly selected and performed hematoxylin-eosin(HE)staining and transmission electron microscope analysis.3.MTS assay was used to detect the influence of only ultrasound or microbubble combined with ultrasound irradiation on the viability of HUVECs.4.Both the only ultrasound irradiation experiment and microbubble combined with ultrasound exposure experiment consisted of a series of time groups,including 0.5h group,1h group,2h group,4h group,8h group,and 24 h group.These time subgroups were divided with the interval from the endpoint of ultrasound exposure to the test of experiments.Both MTS assay and LDH cytotoxicity assay were used to evaluate the effect of ultrasound exposure on the cell viability of HUVECs.The transwell assay using FITC-Dextran was implemented to evaluate the effect of ultrasound irradiation on the permeability of the endothelial cell barrier.Western blot and immunofluorescence assay was used to detect the changes in expression levels and distribution of ZO-1 protein within HUVECs after the exposure of ultrasound.Results:1.The low-frequency ultrasound irradiation could change the vascular permeability of hind limbs of rats.The permeability of vessels was significantly enhanced 30 min after the ultrasound irradiation.And it resumed the normal condition 120 min after the ultrasound treatment.The only ultrasound treatment had no damage to the vessels and muscles within the range of ultrasound exposure.2.The exposure of ultrasound combined with microbubble could alter the vascular permeability of hind limbs of rats.The optimal time window for extravasation is within 30 minutes after the ultrasound exposure.And the permeability could return to the normal status 1 hour after ultrasound irradiation.There was no apparent damage on arteries and muscles within the area of ultrasound exposure from the observation of the HE slides.The ultrasound exposure combined with microbubble contrast agents could widen the tight junction between endothelial cells on the arterial wall.3.The low-frequency ultrasound irradiation could slightly inhibit the proliferation of HUVECs.Despite the increase of ultrasound acoustic pressure and exposure time,the survival rate of HUVECs was not significantly affected.However,ultrasound exposure combined with microbubble could significantly inhibit the cytoactive of HUVECs.As the increase of ultrasound exposure time and concentration of microbubble contrast agents,the survival rate of HUVECs was significantly inhibited.But the acoustic pressure had no impact on the HUVECs.The optimal conditions for ultrasound exposure are 20% microbubble,500 k Hz,0.4MPa wave,and 1minute exposure.4.Both the ultrasound treatment and ultrasound exposure combined with microbubble could be safely applied to the in vitro experiment of the ultrasonic study.The two ways of ultrasound irradiation could reversibly alter the permeability of the endothelial cell barrier.The ultrasound irradiation could change the distribution of ZO-1 protein on the cytoplasm membrane.But the expression levels of ZO-1 protein did not occur significantly changes after the exposure of ultrasound.Conclusion:1.The low-frequency ultrasound treatment could lead to the delayed enhancement of the vascular permeability of hind limbs of rats.The changes of the vascular permeability are safely reversible.2.The low-frequency ultrasound irradiation combined with microbubble could reversibly change the vascular permeability of hind limbs of rats without any damage to the tissues.3.The exposure of low-frequency ultrasound had no significantly inhibited effect on the proliferation of HUVECs.The ultrasound exposure combined with microbubble could dramatically inhibit the cytoactive of HUVECs.As the increase of exposure time and microbubble concentration,the survival rates of cells are significantly decreased.The optimal conditions for ultrasound exposure are 20% microbubble,500 k Hz,0.4MPa wave,and 1minute exposure.4.Both the ultrasound treatment and ultrasound exposure combined with microbubble could reversibly alter the permeability of endothelial cell barrier model in vitro,for which the underlying molecular mechanism is relevant with the distribution changes of ZO-1 protein on the cytoplasm membrane. |
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