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Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.
Invest Ophthalmol Vis Sci. 2009 Aug; 50(8):3860-8.IO

Abstract

PURPOSE

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.

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Authors+Show Affiliations

Nadal-Nicolás FM
Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain.
Jiménez-López M
No affiliation info available
Sobrado-Calvo P
No affiliation info available
Nieto-López L
No affiliation info available
Cánovas-Martínez I
No affiliation info available
Salinas-Navarro M
No affiliation info available
Vidal-Sanz M
No affiliation info available
Agudo M
No affiliation info available

MeSH

AnimalsBiomarkersBlotting, WesternCell CountFemaleFluorescent Antibody Technique, IndirectFluorescent DyesMicroscopy, FluorescenceNerve CrushOptic Nerve InjuriesRatsRats, Sprague-DawleyRetinaRetinal Ganglion CellsStilbamidinesTime FactorsTranscription Factor Brn-3A

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19264888

Citation

Nadal-Nicolás, Francisco M., et al. "Brn3a as a Marker of Retinal Ganglion Cells: Qualitative and Quantitative Time Course Studies in Naive and Optic Nerve-injured Retinas." Investigative Ophthalmology & Visual Science, vol. 50, no. 8, 2009, pp. 3860-8.
Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, et al. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci. 2009;50(8):3860-8.
Nadal-Nicolás, F. M., Jiménez-López, M., Sobrado-Calvo, P., Nieto-López, L., Cánovas-Martínez, I., Salinas-Navarro, M., Vidal-Sanz, M., & Agudo, M. (2009). Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Investigative Ophthalmology & Visual Science, 50(8), 3860-8. https://doi.org/10.1167/iovs.08-3267
Nadal-Nicolás FM, et al. Brn3a as a Marker of Retinal Ganglion Cells: Qualitative and Quantitative Time Course Studies in Naive and Optic Nerve-injured Retinas. Invest Ophthalmol Vis Sci. 2009;50(8):3860-8. PubMed PMID: 19264888.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. AU - Nadal-Nicolás,Francisco M, AU - Jiménez-López,Manuel, AU - Sobrado-Calvo,Paloma, AU - Nieto-López,Leticia, AU - Cánovas-Martínez,Isabel, AU - Salinas-Navarro,Manuel, AU - Vidal-Sanz,Manuel, AU - Agudo,Marta, Y1 - 2009/03/05/ PY - 2009/3/7/entrez PY - 2009/3/7/pubmed PY - 2009/8/12/medline SP - 3860 EP - 8 JF - Investigative ophthalmology & visual science JO - Invest Ophthalmol Vis Sci VL - 50 IS - 8 N2 - PURPOSE: 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. SN - 1552-5783 UR - https://www.unboundmedicine.com/medline/citation/19264888/Brn3a_as_a_marker_of_retinal_ganglion_cells:_qualitative_and_quantitative_time_course_studies_in_naive_and_optic_nerve_injured_retinas_ L2 - https://iovs.arvojournals.org/article.aspx?doi=10.1167/iovs.08-3267 DB - PRIME DP - Unbound Medicine ER -