Comparative Evaluation of Methods for Estimating Retinal Ganglion Cell Loss in Retinal Sections and Wholemounts

Abstract

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a+, βIII-tubulin+ and Islet-1+ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG+ and Brn3a+ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin+ RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.

A Mouse Model of Chronic Ocular Hypertension Induced by Circumlimbal Suture

“…Brn3a is commonly used as a biomarker of RGCs, and it must be acknowledged that it can detect only 80% to 90% of RGCs and furthermore is likely to be down-regulated following the ocular hypertension insult. 30,31 The protocol for Brn3a-labeled RGC counting was adopted as previously described. 32 Two equal-sized areas (450 3 320 lm, 203 magnification, 850 lm from the center of the optic nerve head) within each quadrant of the retina were imaged, and hence, eight areas were included for each retina (~6.2% of retinal area).…”

siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms

“…Survival of RGCs was analyzed as previously described. 55 Briefly, RGCs were identified and counted in a standard 250-lm linear strip of the ganglion cell layer (GCL) in radial sections on either side of the ON head (four radial sections/retina, n ¼ 5 eyes/treatment group), using the RGC phenotypic antibody marker Brn3a, 56 and results were expressed as the mean number of RGCs per 250-lm GCL. Survival analysis of RGCs in the initial in vivo studies used bIIItubulin as the RGC phenotypic marker (Supplementary Table S1), which was superseded with the more specific Brn3a for the studies reported here.…”

“…Retinal analysis is therefore simplified compared with wholemount preparations and enables both RGC survival and axon regeneration analyses from the same animals, thereby reducing animal usage and associated costs. 56 Quantification of axon regeneration in longitudinal ON sections was undertaken using a method described previously. 24 Briefly, composite images were constructed from individual 3200 magnification ON sections in Photoshop CS3 (Adobe Systems, Inc., San Jose, CA, USA).…”

Evaluating retinal ganglion cell loss and dysfunction

“…The ganglion cell layer (GCL) is occupied not only by RGC but also astrocytes and displaced amacrine cells, potentially leaving RGC at only 40-50% of the total GCL population(Bunt and Lund, 1974, Perry, 1981, Schlamp et al, 2013, Nadal-Nicolas et al, 2015b). Previously used markers such as βIII tubulin(Chou et al, 2013, Leibinger et al, 2013, Jiang et al, 2015) and islet-1(Johnson et al, 2014) stain RGC and amacrine cells(Sharma and Netland, 2007, Mead et al, 2014), calling into question their accuracy at reporting RGC numbers. Despite this, βIII-tubulin is still suggested to be a reliable marker(Jiang et al, 2015).…”