A Novel High-Content Flow Cytometric Method for Assessing the Viability and Damage of Rat Retinal Ganglion Cells

PURPOSE: The 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/RESULTS: Retinal 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. CONCLUSION: The 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

Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury

Pannexin1 (Panx1) forms large nonselective membrane channel that is implicated in paracrine and inflammatory signaling. In vitro experiments suggested that Panx1 could play a key role in ischemic death of hippocampal neurons. Since retinal ganglion cells (RGCs) express high levels of Panx1 and are susceptible to ischemic induced injury, we hypothesized that Panx1 contributes to rapid and selective loss of these neurons in ischemia. To test this hypothesis, we induced experimental retinal ischemia followed by reperfusion in live animals with the Panx1 channel genetically ablated either in the entire mouse (Panx1 KO), or only in neurons using the conditional knockout (Panx1 CKO) technology. Here we report that two distinct neurotoxic processes are induced in RGCs by ischemia in the wild type mice but are inactivated in Panx1KO and Panx1 CKO animals. First, the post-ischemic permeation of RGC plasma membranes is suppressed, as assessed by dye transfer and calcium imaging assays ex vivo and in vitro. Second, the inflammasome-mediated activation of caspase-1 and the production of interleukin-1β in the Panx1 KO retinas are inhibited. Our findings indicate that post-ischemic neurotoxicity in the retina is mediated by previously uncharacterized pathways, which involve neuronal Panx1 and are intrinsic to RGCs. Thus, our work presents the in vivo evidence for neurotoxicity elicited by neuronal Panx1, and identifies this channel as a new therapeutic target in ischemic pathologies

Iris ischaemic changes and visual outcome after acute primary angle closure

10.1111/j.1442-9071.2005.01064.xClinical and Experimental Ophthalmology335473-47