Efficiency and Safety of Microstimulation Via Different Electrode Materials

微刺激一直是神经科学研究人员推断功能连接的宝贵工具 或者结构和行为之间的因果联系。近年来，治疗 微刺激在恢复视觉、听觉和躯体感觉功能方面也越来越受到关注 作为生物电子医学的新兴应用。当前神经刺激参数和安全限值 需要使用更系统和先进的方法对微电极进行修订。刺激通过 微电极通常需要高电荷注入来有效地调制神经组织，而不超过 伤害组织或电极的阈值。因此，具有高电荷的先进电极材料 非常需要注入能力和稳定性。我们已经开发了几种类型的刺激材料 基于导电聚合物PEDOT和纳米材料复合材料。这些材料呈现不同的电荷 转移和电化学性能以及生物相容性，以及这些性能对 微刺激还有待全面表征。该提案旨在建立新的体外 和体内模型，以检查通过多种电极材料进行刺激的效率和安全性， 从临床批准的Pt和铱氧化物（IrOx）到新兴的PEDOT纳米复合材料。另一 微刺激的挑战在于其对宿主组织反应的敏感性。电极植入原因 电极污染、进行性神经元损失和紧邻植入物周围的炎性神经胶质增生。 附近神经元和轴突的损失导致刺激功效降低，而电极污染和神经胶质增生 增加阻抗。另外，如果高于预定阈值，则刺激本身可进一步加剧宿主组织反应。 安全限值，微电极和新兴电极材料尚未定义。使用体内 在荧光标记的小鼠成像中，我们将检查微刺激对小鼠的急性和慢性影响。 神经元、小胶质细胞和脉管系统，同时监测电极材料和电化学产物。我们 将使用体外多电极阵列（MEA）系统来研究电刺激对材料的影响 和细胞，以查明材料和组织损伤的机制。 第一个目的是评估神经刺激的效率和安全限度，通过不同的方法， 电极材料在体内急性实验。为了进行效率测试，我们将电极植入 GCaMP小鼠的皮质，并使用双光子显微镜对钙信号进行成像，以确定 刺激阈值和每种电极材料最佳刺激参数。的函数 刺激参数不同脉冲宽度、相间周期、偏压的刺激阈值和效率 将确定来自每种电极材料类型的电势和频率。为了安全测试，我们将使用 Syn-RCaMP/Cx 3Cr 1-GFP小鼠，使神经元和小胶质细胞可视化并确定损伤 阈值 第二个目的是检查刺激对电极材料和培养的细胞的影响。 体外细胞使用高通量的体外MEA系统，其中六种微电极材料可以 我们将在安全和不安全的参数（从目标1中确定的体内参数）下刺激长达12周。 我们将评估电极材料的稳定性，并分析刺激介质，以确定电化学和 降解产物刺激培养基的毒性将在神经元、小胶质细胞、 内皮细胞和神经元-小胶质细胞以不同剂量共培养，以确定 这些电池上的电化学和降解产物。最后，我们将直接刺激培养在 MEA和使用定量RNA和蛋白质分析表征细胞行为，神经记录/刺激 和免疫组织 第三个目的是表征体内微刺激的长期安全性和稳定性， 不同的电极材料。每天对微电极阵列施加一小时刺激 在Syn-RCaMP/Cx 3Cr 1-GFP动物中长期植入12周。在每周的成像会议中，我们 将测量体内阻抗、CV、电荷注入限值和刺激阈值。神经元 反应（活动，健康，密度），小胶质细胞（形态，覆盖和运动）和BBB完整性将被 记录，并在材料类型之间的时间点上进行比较，并与非刺激部位进行比较。此外，本发明还提供了一种方法， 我们将通过电化学询问、成像和外植体分析来密切跟踪电极的健康状况。

Microstimulation has been an invaluable tool for neuroscience researchers to infer functional connections between brain structures or causal links between structure and behavior. In recent years, therapeutic microstimulation is gaining interest for the restoration of visual, auditory and somatosensory functions as well as emerging applications in bioelectronic medicine. Current neural stimulation parameters and safety limits need to be revised for microelectrodes using more systematic and advanced methodologies. Stimulations via microelectrodes often require high charge injection for effective modulation of neural tissue without exceeding the threshold to harm the tissue or the electrodes. Therefore, advanced electrode materials with high charge injection capability and stability are highly desired. We have developed several types of stimulation materials based on conducting polymer PEDOT and nanomaterial composites. These materials present different charge transfer and electrochemical properties as well as biocompatibility, and the effects of these properties on microstimulation have yet to be comprehensively characterized. This proposal aims to establish new in vitro and in vivo models to examine the efficiency and safety of stimulation via multiple electrode materials, ranging from the clinically approved Pt and Iridium Oxide (IrOx) to the emerging PEDOT nanocomposites. Another challenge with micro-stimulation is its sensitivity to host tissue responses. Implantation of electrodes causes electrode fouling, progressive neuronal loss and inflammatory gliosis immediately surrounding the implants. Loss of nearby neurons and axons leads to decreased stimulation efficacy, while electrode fouling and gliosis increase impedance. Additionally, stimulation itself may further exacerbate host tissue responses if above the safety limit, which has yet to be defined for microelectrodes and emerging electrode materials. Using in vivo imaging in fluorescently labeled mice, we will examine the acute and chronic effects of microstimulation on neurons, microglia and vasculature, while monitoring the electrode material and electrochemical products. We will use an in vitro multielectrode arrays (MEA) system to study the effects of electrical stimulation on material and cells, in order to pinpoint the mechanisms of material and tissue damage. The first aime is to assess the efficiency and safety limit of neural stimulation via different electrode materials in vivo in acute experiments. For efficiency testing, we will implant the electrodes in the cortices of GCaMP mice and use 2-photon microscopy to image the calcium signal in order to determine stimulation threshold and optimum stimulation parameter for each electrode material. as a function of stimulation parameters. Stimulation threshold and efficiency for different pulse width, interphase period, bias potential and frequency from each electrode material type will be determined. For safety testing, we will use Syn-RCaMP/Cx3Cr1-GFP mice to visualize both neuronal and microglia cells and determine the damage threshold. The second aim is to examine the effects of stimulation on electrode materials and cultured cells in vitro. Using a high-throughput in vitro MEA system in which the six microelectrode materials can be deposited, we will stimulate at safe and unsafe parameters (identified in vivo from Aim 1) for up to 12 weeks. We will assess electrode material stability and analyze the stimulated media to identify electrochemical and degradation products. The toxicity of stimulated media will be tested in cultures of neuron, microglia, endothelial cells and neuron-microglia co-culture at varying doses to determine the detrimental effects of electrochemical and degradation products on these cells. Finally, we will directly stimulate the cells cultured on MEAs and characterize cell behavior using quantitative RNA and protein analysis, neural recording/stimulation and immunohistochemistry. The third aim is to characterize the chronic safety and stability of microstimulation in vivo from different electrode materials. Stimulation will be applied one hour per day to microelectrode arrays chronically implanted in Syn-RCaMP/Cx3Cr1-GFP animals for 12 weeks. In each weekly imaging session, we will measure the in vivo impedance, CV, charge injection limit, and stimulation threshold. The neuronal response (activity, health, density), microglia (morphology, coverage and motility) and BBB integrity will be recorded, and compared over time points between material types, and to the non-stimulated sites. In addition, we will closely track the electrode health with electrochemical interrogation, imaging and explant analysis.