Research On Photothermal Effects Of Near-Infrared Laser Modulating Neural Activity Of Rat Cortex Neurons

Neural modulation technique is a rapidly developing medical engineering technology to treat neurological diseases.It has been successfully applied in control and treatment of diseases such as epilepsy,depression and Parkinson’s disease.Electrical,magnetic,thermal,optical and other physical factors have been proved to be effective in modulating neural activity,among which electrical stimulation is the most widely used.However,the tissue is easy to be damaged under electrical stimulation due to the contact between electrodes and target neurons,and the current spread beyond the stimulation electrode would lead to poor spatial selectivity.Therefore,researchers continue to explore neural modulation techniques with minimal damage and high spatial resolution.Infrared neural modulation(INM)is a new developing modulation technology which has the potential for clinical application.The primary mechanism of INM has been recognized as photothermal effects,where the near-infrared(NIR)laser energy absorbed by water introduces to a local temperature change altering the neural activity.Currently,limited research focusing on the effects of INM in the central nervous system(CNS)was reported,which makes the temperature conditions of INM in CNS unclear.The limitations of temperature measurement techniques and the complexity of brain network structure and function make it impossible to investigate the temperature conditions of INM in neurons of CNS according to experiments in vivo.INM has made great progress in peripheral nerves,such as sciatic nerve and auditory nerve.However,the optimal temperature changes for modulating the neural activity in PNS are not suitable for modulating the neural activity in CNS due to the structure and function differences between PNS and CNS.Numerous neurological diseases are related to the central nervous system.Thus,it is of great value to understand and master the temperature conditions of infrared neural modulation in CNS for its clinical application.Therefore,we built an optical stimulation and recording platform for neurons in vitro and created the temperature model during infrared laser stimulation,and investigated the temperature parameters for neural inhibition and activation in cortex neurons to establish temperature conditions of neural inhibition and excitation using near-infrared lasers.Which would provide the theoretical evidence for selective neural activity modulation in CNS.Firstly,an optical stimulation and recording experimental setup was built by combing a multi-electrode array(MEA)recording system and a laser system with an inverted microscope system through the reflection mirror device.The laser beam from the laser source can be focused by the reflection mirror device and the microscope system to target neurons on the MEA surface.The results of measurements suggest that,after the laser beam traveled through the 20X objective lens of an inverted microscope,the optical energy transmission efficiency is around 59%,the laser spot diameter in the focus plane is around 240μm and the shape of the laser beam in the air is a 4-order Gaussian distribution.After that,we deduced the energy density distribution equation based on the shape of the laser beam and the Beer-Lambert law.The energy density distribution equation can be used to calculate the energy as the heat source for the temperature model.Secondly,a temperature model was implemented in COMSOL Multiphysics to predict the temperature change behaviors of cells solution under the NIR irradiation based on the classical heat transfer theory and heat source calculated from the energy density distribution equation.Local temperature measurements were performed via the open glass pipette method to validate the temperature model.The results suggest that no matter the temperature changes over time or in space at the same laser power or the maximum temperature rises at different laser powers,model simulations and local temperature measurements are matched very well.Thus,the temperature model built in this thesis is reliable and can be used to predict the temperature spatiotemporal behaviors for the NIR irradiation with the different laser parameters.Thirdly,based on the above experimental platform,a continues wave laser with a wavelength of 1550 nm,an irradiation time of 30 seconds and laser powers varied from2-56 mW was used to irradiate the rat cortex neurons.The model estimated that the temperature increase from 37.2°C to 46°C when the laser power varies from 2-56 mW.Damage signal ratio(DSR)suggests that no damage occurs under the above laser stimulation parameters.Through the investigation of NIR laser on the spontaneous activity of neurons,it was found that(1)the most effective neural inhibition accrued at increasing the temperature by 5 to 9°C with 37°C of initial temperature,resulting in a decreased spike rate of above 40%to around 80%,when the laser irradiation time is 30seconds;(2)The average shape of spikes(the amplitude and half-width of spikes)changes with the temperature increasing.The degree of inhibition was better correlated with the action potential amplitude than the half-width of it;(3)The shape of spikes at increased temperatures shows a prominent increase at the end phase of spikes.Thus,it is speculated that an outward current of K~+is involved in the mechanism of infrared neural inhibition(INI).Afterward,we appliedγ-aminobutyric acid A(GABA_A)receptors antagonists bicuculline(BCC)to cortex neuron cultures to induce epileptiform activity and used the NIR laser to irradiate the abnormal neural activity.Results suggest that NIR laser can be also used to inhibit the epileptiform neural activity.Thus,it is demonstrated that INI can be used to treat some certain neurological disorders in CNS.Finally,a pulsed laser with a wavelength of 1940 nm,a laser power of 600 mW,a frequency of 10 Hz,pulse durations varied from 200μs-1 ms,was used to irradiate the rat cortex neurons.It was found that(1)NIR irradiation with a pulse duration of 800μs and 1 ms could elicit neuronal action potentials,corresponding to temperature rising by18.5°C and 23.9°C respectively.(2)The evoked neuronal responses were frequency locked to the laser pulses.(3)Both local temperature rise(ΔT)and transient temperature rise(dT/dt)could be involved in the mechanism of neural excitation under NIR irradiation.(4)DSR is only 2.41%with the pulse duration of 1 ms,which illustrates that no damage occurs on neurons(the damage threshold is 5%of DSR).Thus,for the first time,we achieved that the NIR laser pulsing directly activated the network activity of cultured neurons on MEAs in this kind of setup.In conclusion,the neural inhibition and excitation using infrared laser were achieved based on the optical stimulation and recording experimental setup we developed.It is demonstrated that it is feasible to modulate the neural activity using NIR laser irradiation.and it also demonstrated that this experimental platform can be used to investigate the efficiency and mechanism of INM for neurons in vitro.A reliable temperature model was created for neuronal stimulation experiments.Through the results of temperature model and electrophysiological recording experiments,it is demonstrated that neurons activity can be inhibited safely and efficiently with temperature rise from 5-9°C with the 30seconds CW NIR irradiation;neurons activity can be elicited safely with the temperature rise 18.5°C and 23.9°C with the pulse duration of 800μs and 1 ms of NIR irradiation.It was also found that the degree of inhibition was correlated with the action potential amplitude;the inhibitory synapses are not necessary for INI to be effective and an outward current of K+is involved in the mechanism of INI.In addition,the slow temperature rise(ΔT)is also involved in the mechanism of infrared neural activation(INA).Until now,in this thesis,it was shown how to modulate the neural activity of cortex neurons using near-infrared laser and explanations of the efficacy,safety,and mechanism of INM were proposed.All these results provide theoretical evidence for the INM technique potentially used to treat some neurological disorders of CNS.