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C. Santano, M. Pérez de Lara, M. Díaz-Hernández, M. Miras Portugal, J. Lucas, J. Pintor Just; Disturbances of VMAT2 and P2Y2 Expression in the Retina of a Mouse Model of Huntington Disease. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4142.
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To determine the biochemical changes in the dopaminergic amacrine cells and the reduction or depletion of P2Y2 receptors expression in the retina of R6/1 mice, a model of Huntington disease.
The eyeballs of R6/1 and wild-type mice were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. After fixation, the eyes were submitted to a cryoprotective process, embedded in tissue freezing medium and cut in 10 µm transversal sections.For immunohistochemical studies, the primary antibodies used were: goat anti VMAT2 (vesicular monoamino transporter 2) and goat anti P2Y2 receptor diluted 1:50 in blocking solution. As secondary antibody, we used a secondary antibody conjugated with FITC and diluted 1:250. The slices were mounted with mounting medium for fluorescence with DAPI (4, 6-diaminido-2-phenylindole), to counterstain the nuclei. For all immunohistochemical experiments, negative controls were carried out by following in the same way without using the primary antibody. The sections were analysed by confocal microscopy using a Zeiss axiovert 200M microscope equipped with a LSM5 Pascal confocal module.
The VMAT2 staining revealed the label significantly reduced or depletion in the central areas of the R6/1 retina. The immunohistochemical studies using P2Y2 receptor antibody showed depletion label in central retina of R6/1 mice while in wild-type mice, P2Y2 was distributed in photoreceptor segments, outer and inner plexiforme layers and ganglion cells layers in the same area.The changes of VMAT2 and P2Y2 receptor label observed in R6/1 match up with the more severe morphological abnormalities in the outer layer which were described in previously papers.
The reduction or loss VMAT2 and P2Y2 immunoreactivity suggest the impairments in the outer layers of the R6/1 retina not only affects phototransduction process but also other retinal neurotransmission system as dopaminergic and purinergic system.
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