Ultrasound Combined With Microbubbles Deliver Drug Into Brain For Amyotrophic Lateral Sclerosis Mice Therapy

Amyotrophic lateral sclerosis(ALS)is a fatal,rapidly progressive neurodegenerative disease.To date,there are only two drugs approved by the USA Food and Drug Administration(FDA)for ALS therapy,and edaravone(EDA)is one of them.EDA was found to be effective in halting ALS progression during early stages but not later stages.Most studies only evaluated the therapeutic effect on the spinal cord in preclinical assays in the past,and did not incorporated the motor cortex,which is important in pathology of ALS.Recently the motor cortex has been a therapeutic target in the drug discovery of ALS.However,the blood-brain barrier(BBB)in the central nervous system limits the transfer of therapeutics from the intravascular compartment,which is the key limitation for the development of therapeutics.Here we investigated the effect of edaravone delivery enhanced by low-intensity focused ultrasound combined with microbubbles(FUS/MB),and its therapeutic effect in ALS mouse model SOD1-G93 A mice in order to provide a viable experimental evidence for the ALS therapy.A 0.97-MHz focused ultrasound transducer(peak negative pressure: 0.50 MPa,pulse repetition frequency: 1 Hz,duty cycle: 10%,duration: 1 min)was used to sonicate the bilateral reflexes of limbs of bilateral motor cortex,combined with microbubbles(0.20 μL/g)to open the blood-brain barrier in mice.EDA was co-injected with microbubbles through the tail vein(5 mg/kg),and The content of EDA in the motor cortex and plasma was measured by Liquid Chromatography/mass spectrometry.Then SOD1-G93 A Transgenic mice at 13 weeks were treated with ultrasound combined with microbubbles to deliver EDA(15 mg/kg)into the brain twice weekly for 6 weeks.Before and after treatments,the motor ability of SOD1-G93 A transgenic mice was evaluated by grip strength,hanging test,pole test,open field test and rotation test.After 6 weeks’ treatment,the prefrontal cortex and spinal cord of mice were taken for histological examination.The main experimental results are as fllowing:1.The pharmacokinetic results of EDA in mice plasma were measured.EDA was rapidly metabolized in plasma with a half-life of 2.71 min.The EDA concentration of motor cortex was obtained after administration for 3 minutes: 52.56± 16.48ng/m L in the EDA group and 111.7 ± 27.33 ng/m L in the FUS/MB+EDA group,n=5.We show the promising result of the effect of edaravone delivery enhanced by FUS/MB: Compared with EDA group,the EDA concentration of cortex concentration increased to 2.13 folds(p < 0.01)in the FUS/MB+EDA group.2.The methods of breeding and identification of ALS transgenic mouse model SOD1-G93 A were established.Compared with WT group(the wild type mice),ALS transgenic SOD1-G93 A mice began to decline in motor capacity from 12 weeks of age(p <0.05).At 19 weeks of age,their cortical motor neurons and spinal neurons were under degeneration with loss in number,severe shrinkage and spongiosis.Their gastrocnemius muscles of the hind limbs were significantly atrophic.3.SOD1-G93 A mice were observed significant motor decline delay after treatments by using ultrasound combined with microbubbles to enhance edaravone delivery into the brain.Compared with traditional intravenous administation,the effect is more significant and lasting.After 2 weeks of treatments,EDA treatment only can delay the decline of motor ability to a certain extent,but FUS/MB in conjunction with EDA exhibited a more significant effect.After 4 and 6 weeks of treatments,there were no significant differences in all the behavioral test between EDA group and ALS group,while the gripping force and suspension time of FUS/MB+EDA group were still significantly improved compared with the ALS group(p <0.05).4.Histological results showed that compared with ALS group,EDA group and FUS/MB+EDA group were observed inhabitation of the motor neurons loss not only in the anterior horn of the spinal cord but also in the motor cortex,and inhabitation of the SOD1 protein expression in the lumbar spinal cord.FUS/MB+EDA group exhibited a more significant effect,in which the number of spinal motor neurons increased by 66.67%(p <0.01).To sum up,EDA was effective in halting ALS progression during early stages but not later stages,but FUS/MB enhanced EDA delivery into brain,delay the decline of motor ability,inhabitate the degeneration of the motor neurons in the motor cortex and spinal cord,inhabitate the SOD1 protein expression,exhibiting a more significant and lasting effect compared with traditional intravenous administration.The innovations of this study are as fllowing:Focused ultrasound in conjunction with microbubbles has been proven to be an effective approach to increase the BBB permeability noninvasively,reversibly and temporarily,potential to challenge the bottleneck of the development of therapeutics for central nervous system diseases.But there have been no reports on cooperation use of this method and therapeutic to treat ALS disease up to date.Here is the first study to use this method to deliver clinical drug edaravone into the brain to treat ALS transgenic animals,to explore its delivery efficiency and therapeutic effect.The results show that compared with traditional intravenous administration,ultrasound combined with microbubbles enhance delivery of edaravone into the motor cortex and delay the disease progression of ALS transgenic mice more effectively,which are positive and promising.