Abstract
PurposeThe aim of the study was to develop a high-content flow cytometric method for assessing the viability and damage of small, medium, and large retinal ganglion cells (RGCs) in N-methyl-D-aspartic acid (NMDA)-injury model.Methods/ResultsRetinal toxicity was induced in rats by intravitreal injection of NMDA and RGCs were retrogradely labeled with Fluoro-Gold (FG). Seven days post-NMDA injection, flatmount and flow cytometric methods were used to evaluate RGCs. In addition, the RGC area diameter (D(a)) obtained from retinal flatmount imaging were plotted versus apparent volume diameter (D(v)) obtained from flow cytometry for the same cumulative cell number (sequentially from small to large RGCs) percentile (Q) to establish their relationship for accurately determining RGC sizes. Good correlation (r = 0.9718) was found between D(a) and apparent D(v). Both flatmount and flow cytometric analyses of RGCs showed that 40 mM NMDA significantly reduced the numbers of small and medium RGCs but not large RGCs. Additionally, flow cytometry showed that the geometric means of FG and thy-1 intensities in three types of RGCs decreased to 90.96±2.24% (P<0.05) and 91.78±1.89% (P>0.05) for small, 69.62±2.11% (P<0.01) and 69.07±2.98% (P<0.01) for medium, and 69.68±6.48% (P<0.05) and 69.91±6.23% (P<0.05) for large as compared with the normal RGCs.ConclusionThe established flow cytometric method provides high-content analysis for differential evaluation of RGC number and status and should be useful for the evaluation of various models of optic nerve injury and the effects of potential neuroprotective agents.
Highlights
Retinal ganglion cells (RGCs) are neurons that receive visual information from photoreceptors via intermediate neurons and transmit messages to the brain
The goal of this study was to develop a flow cytometric method associated with biomarkers and neuronal tracers for assessing the viability and damage of small, medium, and large RGCs in an N-methyl-D-aspartic acid (NMDA)-induced rat retinal damage model
The density of RGCs in the control group obtained from two methods were similar
Summary
Retinal ganglion cells (RGCs) are neurons that receive visual information from photoreceptors via intermediate neurons and transmit messages to the brain. A combination of retrograde labeling and retinal flatmount is frequently applied to quantify RGCs in intervention-induced RGC toxicity. Several neuronal tracers, such as fluoro-Gold (FG) [2], di-I (1, 1-dioctadecyl-3, 3, 39, 39-tetramethyl-indocarbocyanine perchlorate), and fast blue have been used to label RGCs [3]. After injecting the FG tracer into superior colliculi, the tracer is transported in a retrograde way through the optic nerve to obtain FG-labeled RGCs up to 85% [4]. The FG-tracer method provides a reliable measurement to determine the number of RGCs, but no further information regarding the function or damage of RGCs is obtained. Pattern electroretinography can be used for determining the function of RGCs in vivo, but the methodology is limited only qualitatively measuring the overall RGC function [7]
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