Laser Diode for Ion Channel Stimulation

第二阶段SBIR项目的目标是开发和验证激光二极管刺激器，以评估 用于体外和体内研究的热敏瞬时受体电位（TRP）离子通道，使用 膜片钳记录、钙显像和复合动作电位记录。 这种激光器对于新药的发现和评估将非常有用，因为目前还没有 具有类似功能的商用工具。 TRP家族蛋白质的评估可用于描述假定的镇痛药的潜在效用。 毒品选择性（通过温度阈值）、可靠和可重复地激活TRP的能力 还可以在理解拮抗剂作用（结合）的机制方面提供独特的效用， 提供关于细胞中不同TRP蛋白相互作用机制的额外信息， 神经组织也有一个潜在的效用，能够有区别地评估疼痛介导的 TRPV 1或TRPV 2蛋白在诊断和监测疼痛患者进展中的应用。照经上所 我们的总体目标是建立用于体外和体内激光刺激器的刺激方案， 激活热门控离子通道。激光刺激用于选择性激活C和A 人类和大鼠中的δ纤维（在我们的平行项目中），允许可重复的协议来测试 从细胞培养到人类的假定镇痛药。 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， 符合FDA对激光器械的要求。为了做到这一点，我们将开发一个额外的电子 电路，允许通过显微镜的激光发射的封锁，保护眼睛免受任何 损害我们还将提交激光器及其组件进行电气和磁性认证 干扰标准，并进行任何必要的设备更改，以满足要求。 此外，我们还将开发一种直接测量激光诱导温度的方案， 温度和修改设备，以允许可调的加热速率。我们还将确保 激光与钙成像的兼容性，并将继续使用TRPV 1 DRG测试激光 和HEK 293细胞，以研究致敏/脱敏过程。为此，我们将应用 短时间和长时间的激光脉冲和辣椒碱。我们将测试TRP相关的概念 通过神经纤维的直接激光激活来激活C和A δ纤维以及该方法的应用 TRPV 1和TRPV 2拮抗剂辣椒平和钌红。

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.