Laser Diode for Ion Channel Stimulation

描述(申请人提供)：该SBIR二期项目的目标是开发和验证激光二极管刺激器，以分别使用膜片钳记录和钙成像和复合动作电位记录来评估用于体外和体内研究的热敏瞬时受体电位(Trp)离子通道。这种激光器将对新药的发现和评估非常有用，因为目前还没有类似能力的商业工具。色氨酸家族蛋白的评估有助于描述可能的止痛药的潜在效用。选择性地(通过温度阈值)、可靠和可重复地激活TRPs的能力也可能为理解拮抗剂作用(结合)的机制提供独特的有用信息，并提供关于不同Trp蛋白在细胞和神经组织中相互作用的机制的更多信息。此外，能够对TRPV1或TRPV2蛋白介导的疼痛进行差异性评估在疼痛患者的诊断和病情监测中也具有潜在的实用价值。因此，我们的总体目标是建立与激光刺激器一起使用的刺激方案，用于在体外和体内激活热门离子通道。激光刺激被用于选择性地激活人类和大鼠的C和A三角洲纤维(在我们的平行项目中)，允许重复性的方案来测试从细胞培养到人类的推定的止痛药。因此，我们假设激光离子通道刺激器将被证明是安全可靠的，并将满足FDA对激光设备的要求。为了做到这一点，我们将开发一种额外的电子电路，允许通过显微镜封锁激光发射，保护眼睛免受任何损害。我们还将提交激光器及其部件进行电气和磁干扰标准认证，并进行必要的设备更改以满足要求。此外，我们将开发一种直接测量激光感生温度的协议，并对设备进行修改，以允许加热速度可调。我们还将确保激光与钙成像的兼容性，并将继续使用TRPV1 DRG和HEK293细胞测试激光，以研究敏化/脱敏过程。要做到这一点，我们将应用短暂和长时间的激光脉冲和卡萨西平。我们将通过直接激光激活神经纤维以及应用TRPV1和TRPV2拮抗剂卡萨西平和钌红来测试C和A三角纤维色氨酸相关激活的概念。体外和体内无创、可重复、可重复的热门离子通道(Trp)激活方案可为止痛药的发现和开发提供有用的工具。在细胞培养和组织(体内)中可靠地激活TRPV1/TRPV2蛋白的能力也可能为基于色氨酸拮抗剂的疼痛治疗的疗效提供更准确的预测。短暂的加热可以研究热门离子通道激活的分子基础。目前市场上还没有类似功能的商业可用工具。

DESCRIPTION (provided by applicant): The goal of this Phase II SBIR project is to develop and validate a laser diode stimulator to assess heat-sensitive Transient Receptor Potential (TRP) ion channels for in vitro and in vivo studies, using patch clamp recording and calcium imaging and compound action potential recording, respectively. This laser will be very useful for new drug discovery and evaluation, as there is currently no commercially available tool of similar capabilities. Evaluation of TRP family proteins can be useful in describing the potential utility of putative analgesic drugs. The ability to selectively (by temperature thresholds), reliably and reproducibly activate TRPs may also provide unique utility in understanding the mechanisms of antagonist action (binding) and provide additional information about mechanisms of interaction of different TRP proteins in cells and nerve tissue. There is also a potential utility of being able to differentially evaluate pain mediated by TRPV1 or TRPV2 proteins in the diagnosis and monitoring of progression of pain patients. Thus, it is our overall goal to establish stimulation protocols for use with a laser stimulator for in vitro and in vivo activation of heat gated ion channels. Laser stimulation is used for selective activation of C and A delta fibers in humans and rats (in our parallel projects), allowing reproducible protocols to test putative analgesic, from cell culture to humans. Thus, we hypothesize that the laser ion channel stimulator will prove safe and reliable action and will meet the FDA requirements for a laser device. To do this, we will develop an additional electronic circuit that allows the blockade of laser emission through the microscope, protecting eyes from any damage. We will also submit the laser and its components for certification of electrical and magnetic interference standards, and make any device changes that are necessary to meet the requirements. In addition, we will develop a protocol of direct temperature measurement of laser-induced temperature and modify the device to allow a tunable rate of heating. We will also insure the compatibility of the laser with calcium imaging and will continue to test the laser with TRPV1 DRG and HEK293 cells in order to study sensitization/desensitization processes. To do this we will apply brief and prolonged laser pulses and capsazepine. We will test the concept of TRP-associated activation of C and A delta fibers by direct laser activation of nerve fibers and application of the TRPV1 and TRPV2 antagonists capsazepine and ruthenium red. In vitro and in vivo protocols of non-invasive repeatable and reproducible activation of heat gated ion channels (TRP) can be useful tool for discovery and developing of analgesics. The ability to reliably, activate TRPV1/TRPV2 proteins in cell cultures and in tissue (in vivo) may also provide more accurate prediction of the efficacy of pain treatments based on TRP's antagonists. The brief heating allow investigating of molecular bases of activation of heat gated ion channels. There is currently no commercially available tool of similar capabilities on the market.