Mechanisms Of The Increased Blood-tumor Barrier Permeability By Associating Of Low-frequency Ultrasound Irradiation With Low Dose Bradykinin

ObjectiveGlioma is frequent malignant brain tumor in central nervous system. chemotherapy becomes one of primary therapy measure on account of it's faster growth and easy recurrence.BTB confine medicine into tumor even if brain tumor may disrupt the function of BBB locally and non-homogeneously.Conventional chemotherapy are often unsuccessful primarily because the anticancer drugs fail to cross the BTB in sufficient quantities.In a word,the efficient delivery of drugs to tumor cells is crucial to effective chemotherapy of glioma.There are two pathways of drugs being delivered into the tumor cells blood-tumor barrier(BTB):paracellular pathway and transcellular pathway.The pathway chosen depends primarily on the physicochemical properties of each pharmaceutical agent.Hence lipophilic drugs traverse the biological membrane by the transcellular pathway,while hydrophilic ones cross the membrane through the paracellular route.The paracellular route is usually the main route of absorption for hydrophilic drugs,however most drugs are transported transcellularly depending on their physiocochemical properties.In normal BBB and BTB,brain capillary endothelium cell has poor endocytosis and pinocytosis.How to enhance transcellular transport is the key that improve anti-cancer drugs entering tumor tissue and chemotherapy's curative effect.To transport drugs into tumor effectively,at the same time no influence for normal brain tissue,strategy that can selectively open BBB is needed.Studies show that low-frequency Ultrasound uninvadly,recoverely and targetly open BBB under control of a certain extent of sound power and sound pressure, through enhancing endocytosis and pinocytosis,no damaging radiated brain tissue,but identified molecular mechanism is unclear.Studies show that small-dose bradykinin(BK) may selectively open the BTB,and no normal brain is involved.But the identified mechanism of BK to selectively open BTB is unclear.Considerable researches reveal that BK increases permeability through BK-2 receptor expressed mainly in the tumor cells.When combined with the BK-2 receptor,BK causes an extracellular Ca²⁺ influx and increases in endothelial intracellular nitric oxide(NO) and cyclic GMP(cGMP),then transendothelial pinocytotic vesicles form and TJ opening.Consequently,the BTB is opened.Caveolae are microinvaginations of the cell plasma membrane involved in cell transport and metabolism as well as in signal transduction,its primary function is to transport macromolecule substance or internalization micromolecule substance through membrane,these functions depend on the presence of integral proteins named caveolins in the caveolar frame.In the brain,various caveolin subtypes have been detected in vivo by immunocytochemistry:caveolin-1 and -2 were found in rat brain microvessels, caveolin-3 was revealed in astrocytes.caveolin-1 and caveolin-2 express in endotheliocyte mainly,they finish pinocytosis together.This study first determined the parameters of low-frequency ultrasound irradiation which can open BBB without brain damage.Then,rat C6 glioma model,morphology and molecular biology methods were used to estimate the molecular mechanisms of low-frequency ultrasound increasing the pinocytosis to open the BTB selectively for the first time,demonstrated that association of low-frequency ultrasound and bradykinin can increase pinocytosis significantly,which provide the theories about the strategy to improve the transcytosis of drug across brain in clinic.Methods1.Low-frequency ultrasound irradiation:1MHz probe frequency,2.0 cm probe diameter,power can be controlled in some scope(EMS-900 brain ultrasound equipment,Delikai Company,China).Approximately 10s before each LFU irradiation, Optison(0.05ml/kg) was injected via right femoral vein.2.Ultrasound window(to degrade the attenuation of low-frequency ultrasound): healthy Wista male rats(weight:100-200 g,provided by experimental anima center of China Medical University).The animals were anesthetized with 10%chloral hydrate (3.5 ml/kg) by peritoneal injection.The skin over the brain was snipped with shears. Fixed the animal in stereotaxic apparatus and cut off a 2 cm incision in center,strip periost inactively.The craniotomy was performed to remove a bone of cranium(3×3mm) so that the ultrasound pressure amplitude could be accurately estimated in the brain.The skin over the craniotomy was sutured and allowed to heal completely about 5 days before the irradiation.3.Rat C6 glioma cells culture:high glucose DMEM medium with 10%fetal calf serum,37℃,100%humidity,culture under 5%CO₂.4.Establishment of rat C6 brain tumor model:the C6 glioma cells were suspended with high glucose DMEM medium without fetal calf serum in 1×10⁶ /10μl.The animals were anesthetized with 10%chloral hydrate(3.5 ml/kg) by peritoneal injection. 10μl C6 glioma cells suspension were infuse into the right caudate nucleus(1 mm before anterior fontanel;3 mm right beside biparietal suture;4.5 mm deep).To prepare for 10-14 days after C6 glioma transplantion before use.5.In C6 brain glioma rats,the BTB permeability was determined by Evans blue (EB) assay;the quantity of pinocytotic vesicles were determined by transmission electron microscopy(TEM) before and after low-frequency ultrasound irradiation.6.EB assay was used to detect the permeability of BTB after association of low-frequency ultrasound irradiation and low dose bradykinin.7.Transmission electron microscopy was used to detect the change of the number of pinocytotic vesicle in BMECs in BTB after association of low-frequency ultrasound irradiation and low dose bradykinin.8.After low-frequency ultrasound irradiation and combining with low dose bradykinin,immunohistochemistry and immunofluorescence assays were used to determine the distribution and expression of caveolae structure proteins caveolin-1 and caveolin-2.9.RT-PCR and Western-blot assessments were used to detect the mRNA and protein expression levels of caveolin-1 and caveolin-2 in C6 brain glioma rats after low-frequency ultrasound irradiation and combining with low dose bradykinin.10.In BTB model in vitro,immunofluorescence assays were used to determine the distribution and expression of caveolae structure proteins caveolin-1 and caveolin-2 in BMECs after low-frequency ultrasound irradiation.Results1.Demonstrated the acoustic parameters of low-frequency ultrasound which were used to open the BBB:1MHz probe frequency,12mW power,20 s exposure time. These parameters can enhance endocytosis in BBB to induce its opening without brain injury in exposure location.2.In C6 glioma rats,EB exudation and the number of pinocytotic vesicles were increased after irradiation by low-frequency ultrasound.The peak of EB exudation is observed at 1.5 h after the irradiation and also the number of the pinocytosis in BBB as well.These were all recovered after 12 h.3.After low-frequency ultrasound irradiation,the expressing levels of protein and mRNA of caveolin-1 and caveolin-2 in BMECs in BTB rised up and reached to the peak at 1.5 h.Then they retum to the normal level after 12 h.4.In BTB model in vitro,the protein expressing levels of caveolin-1 and caveolin-2 increased significantly after 1.5 h exposure of low-frequency ultrasound, which were recovered after 12 h.The distribution of caveolin-1 and caveolin-2 was demonstrated to change from cellular membrane to cytoplasm.5.Association of low-frequency ultrasound irradiation with low dose bradykinin increased EB exudation and the number of pinocytotic vesicles significantly,compared with the separate utility,and also increased the expressing levels of mRNA and protein of caveolin-1 and caveolin-2.Conclusions1.Low-frequency ultrasound irradiation(1MHz probe frequency,12mW power,20 s exposure time) can open BBB locally without brain injury in exposure location.2.Low-frequency ultrasound irradiation increased EB exudation and the number of pinocytotic vesicle,which revealed that low-frequency ultrasound irradiation may increase the blood-tumor permeability through transcellular pathway.3.Low-frequency ultrasound irradiation may increase the expressing levels of mRNA and protein of caveolin-1 and caveolin-2.4.Association of low-frequency ultrasound irradiation with low dose bradykinin increased EB exudation and the number of pinocytotic vesicle significantly,compared with the separate utility.5.Association of low-frequency ultrasound irradiation with low dose bradykinin increased the expressing levels of mRNA and protein of caveolin-1 and caveolin-2 significantly,compared with the separate utility..6.The up-regulation of the expressing levels of caveolin-1 and caveolin-2 may be mechanisms of increasing pinocytotic vesicle in BMECs in BTB.