Effects Of Ultrasound On Blood - Brain Barrier Permeability In Rats

Objective:The purpose of this paper was to explore the feasibility of opening rat blood-brain barrier by the commonly used ultrasonic equipment. Firstly, we chose ultrasound device, and screened the optimal ultrasonic parameters from frequency, intensity, irradiation time, type and dosage of the micro bubbles; secondly, we explored the change of permeability of blood brain barrier in rats under different ultrasonic conditions. Whether the opening of the blood-brain barrier was reversible and whether the ultrasonic conditions were safe were also investigated. We explored if the ultrasound can promote the methotrexate, an expected drug to be used for brain tumor, through the blood-brain barrier.Methods:1 Selecting the ultrasonic equipmentThe rats was weighted, and fixed after anesthesia. Their head was shaved, and coated with ultrasonic coupling agent, their tails was put the remaining needle with heparin cap. The ultrasonic probe was fixed on the retort stand and contacted with the head skin closely. In the meanwhile, micro bubbles was injected as a dose of 2 mL/kg, then normal saline (NS) was injected as a dose of 1 ml and following 2% EB,2.5 mL/kg.4 hours later, rats’chest was opened, and its’hearts was poured into normal saline. Then cut down its’head and removed the brain tissue. Evans blue was extracted in formamide and quantitative analysis by enzyme.2 Optimizing the experiment parameterThe existing frequencies and maximum strength of pulsed ultrasound and fetus-voice meter were used to radiate the rats. Frequency 3.5 MHz and 7.5 MHz probe cooperating with ultrasonic power in 5、10、15 level of doppler ultrasound devices were chose to radiate the rats. Optimal radiation parameters were used in the following experiments. And to select the best radiating time, the rats were exposed to ultrasound for 5min,20min,40min, and 60min, respectively. SonoVue, the ultrasound contrast agents commonly used in clinical, need to be mixed into the concentration gradient at the concention of 0.5,1.0,2.0 mg/mL according to the body surface area ratio between the human and rat. Mixing the micro bubbles compounded by PLGA, approved by FDA(Food and Drug Administration), into the concentration gradient of 2.5,5.0,10.0 mg/mL to explore the impact of different types and doses of the micro bubbles’effects on the permeability to blood-brain barrier.3 The blood-brain barrier closing timeAfter ultrasound irradiation, injecting EB to the tail vein after 0 h,0.5 h,1.0 h,2.0 h, 3.0 h, and 4.0 h, respectively, then extracted the EB from cerebral cortex by formamide. The reversibility of the BBB was measured by the content of the EB.4 The quantitative analysis of ultrasonic promoting drug through the BBBThe Rats preparation method was the same as before, the optimal parameters of ultrasound were used. The content of methotrexate in brain tissue was measured by high performance liquid chromatography (HPLC).5 The safety of the ultrasonicBrain tissue pathological slices were dyed by HE to observe whether there were any tissue damage. Ultrathin slices for the conventional uranium lead staining, ultrastructure were observed to make sure if there was a change of the ultrastructure.6 The data analysisMicrosoft Excel, GraphPad Prism 5 and other statistical software were used for analysis. Data was measured by x±s. Multiple comparison were using one-way ANOVA.Results:1 Primary equipment screeningThe DUS-3.5 group and DUS-7.5 group had significant difference with the control group (P<0.01), and there were statistically difference when compared with other groups (P<0.05). Other groups have no difference when compared with each other or control group.2 Doppler ultrasound diagnostic parameter optimization3.5 MHz ultrasonic power had a significant difference when set to 5 and 10(P<0.05). 3.5-10 Group and 3.5-5 Group also had a significant difference when compared to each other (P<0.01).3.5-10 Group and 10-15 Group had little significant difference when compared to each other. This showed that when it is 3.5 MHz, the ultrasonic set to 10,the influence of ultrasonic intensity on the blood-brain barrier had a significant improvement.7.5 MHz group, there was a significant difference compared to only when the power was set to 15 and power 5(P<0.01), while between 7.5-15 group and 7.5-10 group, P<0.05. This showed that, at the frequency of 7.5 MHz, the influence of ultrasonic power to BBB had a significant upgrade when it was set to 15.The results of the analysis about the time length of the radiation showed that 20 min statistically difference compared with 5 min.40 min has no difference with 60 min.3 Reversibility of BBB openingWithin two hours, there were significant differences between the BBB permeability and control group (P<0.01).3 hours late, there was no difference, which indicated that BBB permeability returned to normal levels.4 SafetyNo tissue damage and abnormal bleeding cells was observed in HE staining pathological section. Ultrastructure showed that brain microvascular endothelial cells and surrounding tissue structure were normal and there was no significant damage or edema. 5 There was a significant difference between MTX group and the control group.Conclusion:1 Only doppler ultrasound device successfully changed the blood-brain barrier permeability and successfully increased the amount of Evans blue dye through the blood-brain barrier in rats.2 Without craniotomy, the optimal parameters for doppler ultrasound equipment to enhance the permeability of the blood-brain barrier in rats is that:mechanical index set to maximum, the frequency was 7.5 MHz, micro bubbles used PLGA, with a dose of 10 mg/ kg in rats, irradiation 20 min. And the security of these parameters of the ultrasound was good.3 Ultrasound open blood-brain barrier was reversible, about 3 h later the permeability returned to normal levels.4 Ultrasound could promote anticancer methotrexate through the blood brain barrier in rats.