In Vivo Function and Metabolism Evaluation of Glaucomatous RGCs by Two-Photon Scanning Laser Ophthalmology

项目摘要 青光眼是世界范围内最常见的不可逆性失明原因，其特征在于进行性青光眼。 视网膜神经节细胞（RGC）的功能障碍和死亡。我们最近采用了共焦扫描激光 用表达jGCaMP7s的小鼠RGCs成功地获得体内Ca2+成像， 基因编码的钙指示剂。数以千计的ON、OFF和ON-OFF RGC，具有特性响应 通过这种非侵入性的体内成像平台，可以很容易地在活体动物中检测到光刺激。这里 我们寻求开发一种更先进的，首创的具有模式刺激的双光子（2P）-SLO平台 和多个检测通道。通过与体内光学专业知识的多学科合作， 成像，RGC病理生理学，视网膜神经回路和视觉处理，我们将使用互补的 成像技术和最先进的分析方案，以真实的时间了解幼稚RGC生理学。我们 最近扩展了我们最初的小鼠硅油诱导的瞳孔阻滞和高眼压（SOHU） 模型来概括两种形式的青光眼的表型：中度IOP升高的慢性模型和 轻度RGC神经变性;以及具有显著升高的IOP和严重神经变性的急性模型。 重要的是，SO去除几乎立即将IOP降低至正常，允许更好地探索 IOP降低治疗和联合治疗与神经保护策略。我将决定。 在临床相关模型下， 在IOP正常化和/或神经保护治疗之前和之后。这些数据将加深我们对 青光眼的病理生理学，寻找寻找更好的生物标志物来预测进展， 为开发治疗创造更多相关终点，以恢复体内RGC生理学。

PROJECT SUMMARY Glaucoma, the most common worldwide cause of irreversible blindness, is characterized by progressive dysfunction and death of retinal ganglion cells (RGCs). We recently employed confocal scanning laser ophthalmoscopy (cSLO) to successfully obtain in vivo Ca2+ imaging with mouse RGCs expressing jGCaMP7s, a genetically encoded calcium indicator. Thousands of ON, OFF, and ON-OFF RGCs with characteristic responses to light stimulation are readily detected in living animals through this non-invasive in vivo imaging platform. Here we seek to develop a more advanced, first-of-its-kind two-photon (2P)-SLO platform with patterned stimulation and multiple detection channels. Through a multidisciplinary collaboration with expertise in in vivo optical imaging, RGC pathophysiology, and retinal neural circuitry and visual processing, we will use complementary imaging techniques and state-of-the-art analysis protocols to understand naïve RGC physiology in real time. We recently extended our original mouse silicone oil-induced pupillary blocking and ocular hypertension (SOHU) model to recapitulate phenotypes of two forms of glaucoma: a chronic model with moderate IOP elevation and mild RGC neurodegeneration; and an acute model with greatly elevated IOP and severe neurodegeneration. Importantly, SO removal reduces IOP to normal almost immediately, allowing better exploration of the effects of IOP lowering treatment and combined treatment with neuroprotection strategies. Thus, we will determine the longitudinal functional and metabolic changes of glaucomatous RGCs, under clinically relevant models, both before and after IOP normalization and/or neuroprotective treatments. These data will deepen our understanding of the pathophysiology of glaucoma, towards finding much-sought biomarkers to better predict progression, and create more relevant endpoints for developing treatment to restore RGC physiology in vivo.