Abstract
Purpose: :
To study the degeneration of rods and cones induced by an MNU injection in a cone-rich rodent model: Arvicanthis ansorgei.
Methods: :
Male Arvicanthis ansorgei, diurnal rodents whose retinas contain approximately 33% cones, were injected intra-peritoneally with a 150 mg/kg dose of MNU (N-methyl-nitrosurea) dissolved in 0.9% NaCl and sacrificed 3, 5, 8, 11, 15, 20 and 25 days after injection. Control animals were injected with a solution of 0.9% NaCl and 0.05% acetic acid (as contained in the commercial MNU) and sacrificed 25 days after injection. For each animal, one retina was fixed for paraffin sectioning, and the second was frozen for biochemistry. Hematoxylin/eosin staining was performed on histological sections and measurements of Outer Nuclear Layer (ONL) width were made for each time point, every 275 µm, on sagittal sections cut through the head of the optic nerve. In order to assess whether apoptosis could be involved in ONL changes, terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining was performed. To determine whether the apoptotic cells were either cones or rods, immunohistochemistry with specific antibodies produced against cone or rod opsins was performed. The kinetics of rod and cone degeneration were quantified by Western-Blot analysis. Electroretinograms (ERGs) were recorded for light responses to determine retinal function two months after MNU injection .
Results: :
Hematoxylin/eosin stained sections showed that after 8 days, the ONL thickness of MNU injected animals was significantly reduced. The topography of the degeneration showed that, as in most retinal degenerations, the superior hemisphere was more damaged than the inferior. TUNEL staining confirmed that ONL decrease was due to apoptosis. Most of the TUNEL-stained cells were localised in the central region of the ONL, corresponding to rod cells. Immunohistochemistry revealed that 20 days after injection, rhodopsin staining could not be seen whereas anti-cone opsin antibody labelling was still present. Dual labelling with TUNEL and anti-rhodopsin antibody confirmed that the first cells to disappear were rods.
Conclusions: :
Arvicanthis ansorgei injected with MNU represents a good model to study the degeneration of both photoreceptor types; rods and cones. The model may serve to test some putative molecules that could slow down or stop the degenerative process.
Keywords: retinal degenerations: cell biology • photoreceptors • drug toxicity/drug effects