Ultrasound Microbubble Combined With BDNF Gene Therapy For Autism Rats

Autism has clinical manifestations such as social interaction disorder,speech and intellectual development disorder,narrow interest range,stereotyped and repetitive behavior,which brings huge economic and mental burden to society and families,and becomes a public health problem that needs urgent attention.Abnormal expression of brain-derived neurotrophic factor(BDNF)has been reported in the serum of autistic patients.During the development of central nervous system,BDNF plays important role in supporting survival,differentiation,growth and synapse formation of neurons,maintaining the normal physiological function of neurons,regulating the release of neurotransmitters,and participating the plasticity process of nerves.However,due to the large molecular weight of BDNF,it is difficult to penetrate the blood-brain barrier(BBB).Thus it remains a great challenge to enhance the delivery of BDNF through BBB in the treatment of autism.Low intensity focused ultrasound combined with microbubbles can effectively and noninvasively open BBB,leading to improved efficiency of macromolecular drugs in delivering to the brain.Based on this,we propose to open the blood-brain barrier of autism rats by utilizing ultrasound combined with microbubbles in order to delivering BDNF into the brain.The therapeutic effect is evaluated by examining the behavior and pathology of autism rats.The research work of this paper mainly includes:1.The BDNF plasmid(m BDNF)coated with lentivirus was constructed.It was verified that the plasmid was transfected into SH-SY5Y cells under the action of promoter.The expression of BDNF and GFP in the plasmid was co-expressed in the cells.The results showed that m BDNF could be successfully transfected into nerve cells.2.The preparation and characterization methods of cationic microbubbles and m BDNF-loading microbubbles were established.The results showed that the mean diameter of the cationic microbubbles was 1.33μm with the concentration 6.79×10~8/m L.The half-life of the microbubbles in animals was 10 min.The adhesion efficiency of m BDNF to microbubbles was68.2%-96.9%.Low intensity ultrasound combined with microbubbles could open BBB in rats without obvious neuron injury and hemorrhage.3.A rat model of autism was established.The results showed that the growth and development of autism rats were retarded.The results of orientation and swimming showed that the development of vestibule was relatively slow.The ability of movement,coordination and sense of direction were poor,which provided an important basis for the follow-up experiment.4.The autism rats were treated with ultrasound combined with m BDNF microbubbles,and the therapeutic effects of each group were evaluated by behavioral analysis and pathological analysis.Exploratory behavior experiments showed that the expoloring ability of the untreated autism rats(VPA group)was significantly reduced.The group undergone m BDNF injection only(m BDNF group)and the group treated by ultrasound with CMB-m BDNF(US+CMB-m BDNF group)had certain improvements in the exploratory ability compared with VPA group.Social behavior experiments showed that compared with normal rats,animals in the VPA group preferred to be alone,lacking communication with others.After treatments of US+CMB-m BDNF,the communication disorders have a certain degree of improvement.The results of histology showed that neuron health of both the m BDNF group and US+CMB-m BDNF group was improved in the prefrontal cortex area.The morphology of the neurons tended to be normal.The originality of this paper is as follows:As a noninvasive and repeatable treatment modality,ultrasound combined with microbubbles can effectively open BBB and deliver macromolecular agents into the brain in treating central nervous system diseases.BDNF has the function of improving neuron damage,but it is impeded by BBB.In this paper,we propose to construct microbubbles loading BDNF plasmids to enhance the delivery of BDNF into the brain by ultrasound and microbubbles in the application of treating animal models of autism.