Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.
OBJECTIVE
To characterize Brn3a expression in adult albino rat retinal ganglion cells (RGCs) in naïve animals and in animals subjected to complete intraorbital optic nerve transection (IONT) or crush (IONC).


METHODS
Rats were divided into three groups, naïve, IONT, and IONC. Two-, 5-, 9-, or 14-day postlesion (dpl) retinas were examined for immunoreactivity for Brn3a. Before the injury, the RGCs were labeled with Fluorogold (FG; Fluorochrome, Corp. Denver, CO). Brn3a retinal expression was also determined by Western blot analysis. The proportion of RGCs double labeled with Brn3a and FG was determined in radial sections. The temporal course of reduction in Brn3a(+) RGCs and FG(+) RGCs induced by IONC or IONT was assessed by quantifying, in the same wholemounts, the number of surviving FG-labeled RGCs and Brn3a(+)RGCs at the mentioned time points. The total number of FG(+)RGCs was automatically counted in naïve and injured retinas (2 and 5 dpl) or estimated by manual quantification in retinas processed at 9 and 14 dpl. All Brn3a immunopositive RGCs were counted using an automatic routine specifically developed for this purpose. This protocol allowed, as well, the investigation of the spatial distribution of these neurons.


RESULTS
Brn3a(+) cells were only present in the ganglion cell layer and showed a spatial distribution comparable to that of FG(+) cells. In the naïve retinal wholemounts the mean (mean +/- SEM; n = 14) total number of FG(+)RGCs and Brn3a(+)RGCs was 80,251 +/- 2,210 and 83,449 +/- 4,541, respectively. Whereas in the radial sections, 92.2% of the FG(+)RGCs were also Brn3a(+), 4.4% of the RGCs were Brn3a(+)FG(-) and 3.4% were FG(+)Brn3a(-). Brn3a expression pattern was maintained in injured RGCs. The temporal course of Brn3a(+)RGC and FG(+)RGC loss induced by IONC or IONT followed a similar trend, but Brn3a(+)RGCs loss was detected earlier than that of FG(+)RGCs. Independent of the marker used to detect the RGCs, it was observed that their loss was quicker and more severe after IONT than after IONC.


CONCLUSIONS
Brn3a can be used as a reliable, efficient ex vivo marker to identify and quantify RGCs in control and optic nerve-injured retinas.
Nadal-Nicolás
Francisco M
FM
Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain.
Jiménez-López
Manuel
M
Sobrado-Calvo
Paloma
P
Nieto-López
Leticia
L
Cánovas-Martínez
Isabel
I
Salinas-Navarro
Manuel
M
Vidal-Sanz
Manuel
M
Agudo
Marta
M
D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D002452:Cell Count; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009409:Nerve Crush; D020221:Optic Nerve Injuries / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D013266:Stilbamidines / Q000378:metabolism; D013997:Time Factors; D050819:Transcription Factor Brn-3A / Q000378:metabolism*
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D015415:Biomarkers; D005456:Fluorescent Dyes; D013266:Stilbamidines; D050819:Transcription Factor Brn-3A