Quantifying Retinal Ganglion Cell Survival

One quarter of each retinal explant was carefully separated under microscope for subsequent immunohistochemical staining against the brain-specific homeobox/POU domain protein 3A (Brn3a). Unlike Thy1, which is another RGC-specific antigen, the expression pattern of Brn3a does not change after retinal injury and the levels of Brn3a protein are decreased with time after retinal injury, which is in agreement with the loss of RGC [151 (link)]. Therefore, Brn3a immunodetection is a powerful tool to assess RGC survival in rat injury models [152 (link),153 (link)]. Retinal tissue (n = 6 quarters/group) was fixed and stained as previously described [154 (link)]. Immunofluorescent RGCs were visualized and photographed with a fluorescent microscope (Carl Zeiss, Ltd., Hertfordshire, UK). Images of retinal whole mounts were photographed from 4 different regions of each quadrant of the retina (Figure 1) using a Zeiss fluorescent microscope (Carl Zeiss, Ltd., Hertfordshire, UK). Images were captured at a 20-fold magnification using a fluorescent camera (Carl Zeiss, Ltd., Hertfordshire, UK). Total numbers of Brn3a-positive cells were counted with the assistance of ImageJ (ImageJ Fiji v_1, total = 4 counts/quadrant, and six retinal quadrants per treatment group). The mean number of RGCs per quadrant was calculated from a mean count at each of the 4 different regions.

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Liu H., Perumal N., Manicam C., Mercieca K, & Prokosch V. (2020). Proteomics Reveals the Potential Protective Mechanism of Hydrogen Sulfide on Retinal Ganglion Cells in an Ischemia/Reperfusion Injury Animal Model. Pharmaceuticals, 13(9), 213.

Publication 2020

fluorescent microscope

Antigen Brain Homeobox protein Immunofluorescent Injury Microscope Protein Retinal Tissue

Corresponding Organization : Johannes Gutenberg University Mainz

Other organizations : Manchester Royal Eye Hospital, University of Manchester

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Variable analysis

independent variables

Retinal injury

dependent variables

RGC survival, as measured by the number of Brn3a-positive cells

control variables

Experimental procedures for tissue fixation and immunohistochemical staining (as referenced in [154])

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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