This work characterized an in vitro 3-D neural co-culture model electrophysiologically via multi electrode arrays (MEAs), and morphologically via immunocytochemistry. Since MEA surface insulation SU-8 2000 can be used in neural micro- and multi- electrode arrays, this investigation first developed techniques to make SU-8 2000 cytocompatible. The in vitro 3-D neural co-culture model was then used to study viability and electrophysiological responses to physical injury as well as drugs known to affect network signaling. 1) SU-8 2000 cytotoxicity to neuronal cultures was linked to both poor adhesive properties and toxic components, such as solvents and photo acid generator elements. Surface treatments of oxygen plasma or parylene coating following optimal combinations of heat and isopropanol sonication showed improvement in SU-8 2000 cytocompatibility. 2) The 3-D neural networks within the 3-D co-cultures maintained considerable process outgrowth and complex 3-D structure. The cultures were viable up to three weeks in vitro with functional synaptic connections and spontaneous electrophysiological activity that was responsive to chemical modulation. This electrophysiological activity was modulated by synaptic inhibition. 3) Injury experiments demonstrated that both shear and compression loading significantly increased acute membrane permeability of cells in a strain rate dependent manner. Cell death correlated with higher membrane permeability, and shear resulted in more death than compression in these 3-D cultures. While techniques were developed for making a major micro-fabrication material cytocompatible, engineering the 3-D neural co-culture resulted in a more physiologically-representative neural tissue platform, allowing an increased understanding of structure-function relationships. Overall, this research established and characterized a neural culture system for the mechanistic study of cell growth, cell-cell and cell-matrix interactions, as well as the responses to chemical or mechanical perturbations. This is the first investigation of the network-level electrophysiological activity of 3-D dissociated cultures. This system can be used to model various pathological states in vitro, testing various reparative drugs; cell-, and tissue-engineering based strategies; as well as for pre-animal and pre-clinical testing of neural implants.

It would be of great value to have a precise and non-damaging neuromodulation technique in the field of basic neuroscience research and for clinical treatment of neurological diseases. Infrared neural modulation (INM) is a new modulation modality developed in the last decade, which uses pulsed or continues infrared (IR) light with a wavelength of 1200 to 2200 nm to directly alter neural signals. INM includes both infrared neural stimulation (INS) and infrared neural inhibition (INI). INM is widely investigated for use on peripheral nerves, cochlear nerve fibers, cardiac cells, and the central nervous system. This technique holds the advantages of contact-free and high spatiotemporal precision compared to the traditional electrical stimulation. It does not depend on genetic modification and exogenous absorbers as other optical techniques, such as the optogenetic technique and the enhanced near-infrared neural stimulation (e-NIR). These advantages make INM a viable technique for research and clinical applications. The primary mechanism of the INM is believed to be a photothermal effect, where the IR laser energy absorbed by water leads to a rapid local temperature change. However, so far the details of the mechanism of action potential (AP) generation and inhibition remain elusive. Another issueis that the cells may be endangeredbythe heat exposure, consequently triggering a physiologicalmalfunction or even permanent damage.These concernshave hindered the transfer of the INM technique to the clinical therapy.Therefore, the general aim of this study was to improve the understanding of the details of how INM affects the cells. Laser parameters for safe and efficient stimulation were investigated on the basis of being useful for clinical applications. A tailored heating model and in vitro INM experiments on cortex neurons were used to reach this goal.The first paper was a feasibility study. A 1550nm laser with a beam spot diameter of around 6 mm was used to irradiate the rat cortex neurons, which were seeded on multi-electrode arrays (MEA) and formed well-connected networks. A heating model based on an estimated laser beam (standard Gaussian distribution) was used to simulate temperaturechanges. The damage signal ratio (DSR),based on the temperature,was calculated to predict the heat damage. The average spike rate of all the working electrodes from two MEAs was used to evaluate the degree of theinhibition of the neural networks. Results IVshowed that it is possible to use the 1550 nm laser to safely inhibit the neural network activity and that the degree of the INI is dependent on the power of the laser.The second paper wasan application and mechanism study. The aim of this study was to investigate the safety, efficiency, and cellular mechanism of INI. The same laser as in paper Iwas used in this study. A 20 X objective was used to decrease the beam spot diameteraround 240 μm. The measured laser profile (high order Gaussian beam) was used in the heating model to predict the temperature. The model was verified by local temperature measurements viamicropipette. The action potential rates, measured by the MEA electrodes, were quantified for different temperatures. Bicuculline was added to the cortex neuron cultures to induce hyperexcitation of the neural network. The results showed that the INI is temperature dependent and that the temperature needs to be less than 46 °C at 30 s laser irradiation for safe inhibition. The IR laser couldalso be used to inhibit the hyperexcitedactivity. The degree of inhibition, for the assessed subpopulation of neurons, was better correlated with the action potential amplitude than the width of it and INIcan be accomplished without inhibitory synapses / <p>QC 20180920</p><p></p>