Abstract
Purpose: :
To develop a DBA/2J mouse line with CFP (cyan fluorescent protein) fluorescent ganglion cells for evaluating ganglion cells during the development of progressive glaucoma.
Methods: :
A thy1–CFP/DBA/2J mouse line was created using congenic breeding. The DBA/2J line, which develops progressive pigmentary glaucoma, and the thy1–CFP/C57/Bl line (JAX Labs), which expresses CFP in ganglion cells, were bred for 5 generations. Genetic analysis was performed using microsatellite markers for the isa and ipd loci, located on chromosomes 4 and 6, respectively. Specific mutations within these loci lead to the production of dysfunctional melanosomal protein genes. RT–PCR with primers to the thy1–CFP gene construct was used to determine CFP in retinal extracts. Immunohistochemistry with antibodies to neurofilament light (NF–L), protein gene product (PGP 9.5) and the POU family homeodomain protein Brn 3a, were used to confirm the presence of CFP in ganglion cells.
Results: :
Microsatellite marker analysis of thy1–CFP/DBA/2J progeny verified the genetic inclusion of the two loci responsible for the DBA/2J glaucomatous phenotype. Analysis of the ipd locus yielded a 119 bp PCR product for DBA/2J mice and a 125 bp product for C57BL/6 animals, and the isa locus analysis produced 160 bp and 204 bp fragments for DBA/2J and C57BL/6 mice, respectively. RT–PCR analysis confirmed the inclusion of the thy1–CFP transgene. Strongly CFP–fluorescent ganglion cell bodies ranging from 10 to 30 µm in diameter were in all retinal regions and CFP–fluorescent axons were in the fiber layer and optic nerve head. Weakly–CFP–fluorescent displaced amacrine cells were also in the ganglion cell layer. CFP–containing dendrites were in all laminae of the inner plexiform layer. NF–L, PGP 9.5 and Brn 3a immunoreactivity was observed in medium and large CFP–expressing ganglion cells. NF–L immunostaining was strong in ganglion cell bodies, dendrites, and axons. PGP 9.5 immunostaining was mainly in large ganglion cell bodies and Brn3a immunostaining was in some small, medium and large ganglion cell bodies.
Conclusions: :
A new thy1–CFP/DBA/2J line has been developed. This line has 1) the genetic loci that lead to the development of pigmentary glaucoma and 2) CFP–expressing ganglion cells in all retinal regions. This new line will be of great value for experimental and pharmacological studies requiring the evaluation of ganglion cell survival during the progressive development of glaucoma.
Keywords: ganglion cells • neuroprotection • retina: proximal (bipolar, amacrine, and ganglion cells)