Retina and Optic Nerve Research Laboratory, Dalhousie University, Sir Charles Tupper Medical Building 5850 College Street, B3H 4R2, Halifax, Nova Scotia, Canada; Dalhousie Medical School, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building 5850 College Street, B3H 4R2, Halifax, Nova Scotia, Canada.
Retina and Optic Nerve Research Laboratory, Dalhousie University, Sir Charles Tupper Medical Building 5850 College Street, B3H 4R2, Halifax, Nova Scotia, Canada; Department of Medical Neuroscience, Dalhousie University, Sir Charles Tupper Medical Building 5850 College Street, B3H 4R2, Halifax, Nova Scotia, Canada; Nova Scotia Health Authority, 1276 South Park Street, 2W Victoria, B3H 2Y9, Halifax, Nova Scotia, Canada.
Exp Eye Res. 2020 Jul;196:108044. doi: 10.1016/j.exer.2020.108044. Epub 2020 May 4.
Optic neuropathies, such as glaucoma, lead to retinal ganglion cell (RGC) death. Transgenic mouse strains that express fluorescent proteins under the control of the Thy1 promoter have permitted single RGC imaging. Specifically, in one strain of mice expressing yellow fluorescent protein (Thy1-YFP), fluorescence is expressed in only 0.2% of RGCs. This reduced expression allows visualization of the full dendritic arbour of YFP-expressing RGCs, facilitating the investigation of structural changes. As susceptibility amongst RGCs varies with morphology and subtype, labelling methods should ideally non-discriminately label RGCs to accurately determine the effects of experimental glaucoma. This study therefore sought to determine morphological subtypes of RGCs in the Thy1-YFP mouse strain. Retinas from Thy1-YFP mice were imaged ex vivo with fluorescence microscopy. With Sholl analysis, a technique for quantifying the morphology of individual neurons, the dendritic field (DF), area under the curve (AUC), normalized AUC (N), peak number of intersections (PNI), and skew for single RGCs were computed. The distance of the RGC from the optic nerve head (dONH) was also measured. These morphological parameters were inputted into a multivariate cluster analysis to determine the optimal number of clusters to group all RGCs analyzed, which were then grouped into "Small", "Medium", and "Large" sized cluster groups according to increasing DF size. A total of 178 RGCs from 10 retinas of 8 mice were analyzed from which the cluster analysis identified 13 clusters. Eighty-eight (49%), 77 (43.2%), and 13 (7.3%) RGCs were grouped into small, medium and large clusters, respectively. Clusters 1-6 had small DFs. Clusters 1 and 3 had the lowest AUC and N. Clusters 2, 3, and 5 had asymmetric DFs while Clusters 3, 5, and 6 were distal to the ONH. Clusters 7-11 had medium DFs; of these, Clusters 7 and 10 had the lowest AUC, Clusters 8 and 10 had the highest skew, and Clusters 7 and 11 were closest to the ONH. Clusters 12 and 13 had large DFs. Both had low skew and high AUC. High PNI and dONH distinguished Cluster 12 from Cluster 13. We present the largest study to date examining YFP expression in RGCs of transgenic Thy1-YFP mice. Among the 13 clusters, there was a wide range of morphological features with further variation within size categories. Our findings support the notion that YFP is expressed non-discriminatingly in RGCs of Thy1-YFP transgenic mice and this strain is a valuable tool for studies of experimental optic neuropathies.
视神经病变，如青光眼，会导致视网膜神经节细胞（RGC）死亡。在 Thy1 启动子控制下表达荧光蛋白的转基因小鼠品系允许对单个 RGC 进行成像。具体来说，在一种表达黄色荧光蛋白（Thy1-YFP）的小鼠品系中，荧光仅在 0.2%的 RGC 中表达。这种低表达允许可视化 YFP 表达的 RGC 的完整树突状树突，促进结构变化的研究。由于 RGC 之间的易感性因形态和亚型而异，因此标记方法应理想地非选择性地标记 RGC，以准确确定实验性青光眼的影响。因此，本研究旨在确定 Thy1-YFP 小鼠品系中 RGC 的形态亚型。用荧光显微镜对 Thy1-YFP 小鼠的视网膜进行离体成像。使用 Sholl 分析技术，该技术用于量化单个神经元的形态，计算单个 RGC 的树突场（DF）、曲线下面积（AUC）、归一化 AUC（N）、交点峰值数（PNI）和偏斜。还测量了 RGC 距视神经头（dONH）的距离。这些形态参数被输入到多元聚类分析中，以确定将所有分析的 RGC 分组的最佳聚类数量，然后根据 DF 大小将其分为“小”、“中”和“大”大小的聚类组。总共分析了 8 只小鼠 10 只视网膜的 178 个 RGC，聚类分析确定了 13 个聚类。88%（49%）、77%（43.2%）和 13%（7.3%）的 RGC 分别被分为小、中、大聚类组。聚类 1-6 具有较小的 DF。聚类 1 和 3 的 AUC 和 N 最低。聚类 2、3 和 5 的 DF 不对称，而聚类 3、5 和 6 远离 ONH。聚类 7-11 具有中等 DF；其中，聚类 7 和 10 的 AUC 最低，聚类 8 和 10 的偏斜度最高，聚类 7 和 11 最靠近 ONH。聚类 12 和 13 具有较大的 DF。两者的偏斜度都较低，AUC 都较高。高 PNI 和 dONH 将聚类 12 与聚类 13 区分开来。我们提出了迄今为止对转基因 Thy1-YFP 小鼠中 RGC 的 YFP 表达进行的最大研究。在 13 个聚类中，存在广泛的形态特征，并且在大小类别内也存在进一步的变化。我们的发现支持这样一种观点，即 YFP 在 Thy1-YFP 转基因小鼠的 RGC 中无差别地表达，该品系是研究实验性视神经病变的有价值的工具。
