Computational protocol: Etanercept, a Widely Used Inhibitor of Tumor Necrosis Factor-α (TNF- α), Prevents Retinal Ganglion Cell Loss in a Rat Model of Glaucoma

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Protocol publication

[…] Surviving RGCs were quantified in retinal whole mount preparations that were double-immunolabeled with the TUJ1 antibody directed against βIII tubulin and anti-NeuN. Within the ganglion cell layer, βIII tubulin is expressed only in RGCs, and thus immunostaining for this antigen can be used to distinguish RGCs from the many amacrine cells that are also in this layer , . TUJ1- and NeuN-immunofluorescent cells in the ganglion cell layer were visualized with a Zeiss LSM 510 META laser scanning confocal microscope using a 20× objective and were counted manually in eight confocal fields in each quadrant of the retina using ImageJ software and the cell counter plugin. We counted four fields in the center near the ONH and four in the periphery, and then averaged the 32 counts for each retina to obtain RGCs per mm2. [...] Results are expressed as mean values ± standard error of mean (SEM). Statistical analysis was performed nonparametrically using Mann-Whitney U-test and Kruskal-Wallis tests (SPSS Statistics 17.0, Chicago, IL). A p-value of less than 0.05 was considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Glaucoma, Hypertension, Neoplasms, Ocular Hypertension, Retinal Diseases, Retinitis