| Alzheimer’s disease,Parkinson’s disease,epilepsy and depression are disorders of the brain that are difficult to be cured.It has become an urgent task for the scientific community to explore the pathogenesis and the effective diagnosis and treatment of brain diseases.Neuromodulation technologies are basic means to carry out neuroscience research and explore the diagnosises and treatments of brain functional diseases.The conventional neuromodulation methods include medications,deep brain stimulation(DBS),transcranial direct current stimulation(tDCS),transcranial magnetic stimulation(TMS)and optogenetics.The medication method is easy to be used,but it always have disadvantages of poor targeting ability,severe drug reaction and drug resistance.DBS has a high spatial resolution,but it is an invasive method.Although both tDCS and TMS are noninvasive methods,they have low penetration depth and low spatial resolution.Although the optogenetics has extremely high spatial resolution,it is difficult to achieve clinical transformation by relying on genetic manipulation.The neuromodulation method,which has the advantages of noninvasive,high spatial resolution and wide range of brain control,has become the research interests of the clinical therapy of brain disorders.Ultrasound is mechanical wave without ionizing radiation,and can noninvasive penetrate through soft tissue or even skull,and carry out various phisical effects such as oscillation,heating,cavitation or radiation force on target tissue.Over the past years,some scholars had used ultrasound to stimulation research on peripheral nerve and even brain,and had shown that low frequency(< 0.65 MHz)and low intensity transcranial focused ultrasound could achive neuromodulation.It indicated that ultrasound could satisfy the demands of neuromodulation for noninvasiveness,high spatial and temporal resolution,and whole brain positioning control.The scholars had adopted various evaluation methods,such as micro fluorescence imaging,electrophysiology,dialysis,functional magnetic resonance imaging(fMRI),glucose metabolism imaging and behavioral tests measurement,to estimate the effects of neuromodualtion.Those studies had covered most of the common experimental subjects,including neurons,nerve stem,brain slices,mice,rats,mini pigs,rabbits,sheep,monkey,and people.The previous studies had proved the effectiveness of the ultrasonic neuromodualtion technology,but the mechanism of ultrasonic neuromodualtion is still not clear,the stimulating parameters and experimental paradigm are not yet mature,the treatment protocols of the nervous diseases are not sufficient.In order to promote the development of ultrasonic neuromodualtion from the scientific hypothesis to clinical applications,the experimental research on the ultrasonic neuromodualtion of experimental animals should be widely carried out.During the last ten years,among all of the literatures which were cited by scientific cited index and involved experimental animals as their study objects in the field of neuroscience,the proportions of literatures involving rats and mice as their study objects were 49% and 37%,respectively.Thus it could be seen that rodents were the main animal species used in the neuroscientific research,and the early stage of research on ultrasonic neuromodualtion should be mainly based on rodents,especially mice.The reasons include that the mice have a complete range of disease models(such as genetically modified mice),the research resources about mice are rich,the behavioral tests are convenient to carry out and the mice are easy to breed.However,there are two major technical bottlenecks exist in the current ultrasonic neuromodualtion experiments of mice.First,the volumes of the conventional ultrasonic probes used in the experiments of neuromodualtion are too big,lead to the demand for anesthesia and body fixation of the experimental mice,which seriously affects the accuracy of neuromodualtion outcomes,also limits the applications of experimental paradigms relating to cognition and behavior.Second,the diameter of the focus region of low frequency ultrasound(such as 0.5 MHz,-3dB target diameter would be as large as 8 mm)is too large compared with to the size of the mouse brain(about 10 mm in wide).It is difficult to accurately stimulate special brain nuclei,and even more difficult to simultaneously stimulate multiple targets on a neural network in the ultrasonic neuromodualtion studies.Aiming at the above bottleneck problems,the following three research objectives are established:Objective 1: to design and fabricate a miniature head-mounted ultrasonic stimulation system,which is used to ultrasonic stimulation in freely moving mice;to investigate the behavioral responses modulated by noninvasive ultrasonic stimulation.It provides a vital tool to explore the mechanism of ultrasonic stimulation on normal nervous system and the animal behavior.Objective 2: to reduce the focus size of ultrasonic transducer,improve spatial resolution,and explore the effectiveness of high frequency(5 MHz)ultrasound stimulation of mouse brain,and investigate the relationship between the behavioral responses and different stimulation targets.To realize more precise neuromodulation,and provide data for the study of evaluating the influence of ultrasonic frequency on neuromodulation.Objective 3: to design and set up an array ultrasonic stimulation system to realize imaging-guided dual-target neuromodulation of the mouse brain.To provide a tool for investigation of the network function of neuromodulation,to provide an idea for the design of a new generation of neuromodulation system with integrated functions of stimulation and assessment.Based on the above research objectives,this dissertation described the following three aspects of research work and their significances:Study 1: design and fabricate a miniature head-mounted ultrasonic stimulation device,and verify its effectiveness on neuromodulation in the awake and freely moving mice,and record the electrophysilogical signals and assess the behavioral responses when suffering ultrasonic neuromodulation.The core part of the stimulation system is the ultrasonic stimulator.The main components of the ultrasonic stimulator include a small piezoelectric ceramic disk,a concave epoxy acoustic lenses,a housing and a socket components.The action potentials recorded in situ and head-turning behavior can been induced by ultrasound stimulation in the barrel region of the primary somatosensory cortex(S1BF)of the mouse.Experimental results show that the head-mounted stimultaion method can be realized to achive noninvasive ultrasonic neuromodulation in freely moving mice.This method can release the physical restrictions and anesthetic interference of the experimental mice from the conventional ultrasonic system.It provides methods and tools for the ultrasonic neuromodulation in freely moving animals.Study 2: Ultrasonic brain stimulations were carried out in the same group of mice using 1MHz and 5MHz ultrasound,respectively,and the stimulation effects were evaluated by quantitative comparison of the electromyographic signals recorded from the tail muscle,and by observing the video of motion responses.The results showed that the equivalent diameter(0.29±0.08 mm)of 5 MHz ultrasonic stimulation was significantly less than that of 1 MHz ultrasound(0.83±0.08 mm).The latency of 5 MHz ultrasonic stimulation(45±31 ms)was also less than that of 1 MHz ultrasound(208±111 ms).It can be seen that high-frequency ultrasound(5 MHz)could successfully activate neural circuits in mice and improve the anatomical specificity of neural stimulation.The results demonstrate a method of how to improve the spatial resolution for ultrasonic neuromodulation,add new data for parameters design of ultrasonic neuromodulation,and provide tools to discover small scale and sensitive brain nuclei response to ultrasonic neuromodulation.Study 3: based on the experimental results of "study 2",a new system for imaging-guided dual-target neuromodulation was designed and set up.The system can achieve dual-target brain stimulation in mice by alternately switching the focuses of the 5 MHz array transducer.The locations of the targets can be selected in advance through the B-mode image formed by the same ultrasound transducer.The stimulation effects were evaluated by analyzing the electromyographic signal and behavioral video.This study can provides methods and tools for multi-target neuromodulation investigation based on ultrasonic imaging.In conclusion,in order to solve the bottleneck problems exist in the application of ultrasonic neuromodulation in mice,this study had launched some theory analysises of acoustics and electronics,and designed and fabricated prototypes of head-mounted ultrasonic neuromodulation device for freely moving mice and imaging-guided dual-target ultrasonic stimulation system.Verification experiments in mice were also carried out based on the two prototypes.The results of this study not only provide new data for the exploration of the mechanism of ultrasonic neuromodulation,but also provide new tools for the development of experimental research on ultrasonic neuromodulation. |
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