title: 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.
authors.affiliation: Neurotrauma Research Group, Neurobiology Section, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom; School of Dentistry, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Ben
authors.initials: B
authors.lastname: Mead
authors.affiliation: Neurotrauma Research Group, Neurobiology Section, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Adam
authors.initials: A
authors.lastname: Thompson
authors.affiliation: School of Dentistry, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Ben A
authors.initials: BA
authors.lastname: Scheven
authors.affiliation: Neurotrauma Research Group, Neurobiology Section, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Ann
authors.initials: A
authors.lastname: Logan
authors.affiliation: Neurotrauma Research Group, Neurobiology Section, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Berry
authors.affiliation: Neurotrauma Research Group, Neurobiology Section, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Wendy
authors.initials: W
authors.lastname: Leadbeater
mesh_terms: D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D016923:Cell Death; D005260:Female; D060850:LIM-Homeodomain Proteins / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology*; D013194:Staining and Labeling; D050819:Transcription Factor Brn-3A / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
chemical_list: D060850:LIM-Homeodomain Proteins; D050819:Transcription Factor Brn-3A; D014157:Transcription Factors; C488416:Tubb3 protein, rat; D014404:Tubulin; C063843:insulin gene enhancer binding protein Isl-1