Abstract
Purpose: :
To demonstrate the presence of alpha 2 adrenergic agonist receptor available in chick retina.
Methods: :
We performed experiments on post-hatched 7 days old chick retina (n= 4). Immediately after decapitation the eyes were removed, the retina were isolated and fixed in 4% Paraformaldehyde solution in Phosphate Buffer (PB) 0,16M for 2 hours. Then, the retinas were washed in PB Saline solution (PBS) 3 times for 10 minutes each time. After this, they were placed in 15% sacarose PBS solution for 1 hour, in order to crioprotection. Followed, the retinas were stored in a 30% sacarose PBS. The retinas were cut in a criostat device in 14µm sections and collected in slides. Bovine Serum Albumine (BSA) solution (3%) was placed over the retina section for 40min, and after that some slides were submitted to 1:100 alpha-2A antibody and 1:500 alpha-2A antibody (ABCAM) for 24h. The tissue were then washed and submitted to the secondary antibody (Donkey anti-rabbit) with the fluorescent marker (Alexa Fluor 555, Molecular Probes) in 1:800 dilution for 4 hours. Finally, the retinas were analyzed in APOTOME Zeiss Microscope, and the images were acquired. We also performed immunohistochemistry of a chick specific marker of glial cells (Müller cells), the antibody against 2M6. The immunohistochemistry process were repeated for alpha-2C antibody.
Results: :
Alpha 2 receptors were only detected in retinas with alpha-2A antibody in 1:100 dilutions. The observation of the immunolabel was topology compatible with Müller cells. The location in Müller cells of the receptors were confirmed with the use of glial cell markers. The immunohisitochemistry process using alpha-2C antibody was negative in all dilutions.
Conclusions: :
The experiment confirms the presence of alpha-2 adrenergic agonist receptor, subtype 2A, in newborn chicken retina. This is the first step to study the presence of these receptors in humans, that will permit further studies of the existence of neuroprotective effect of brimonidine, an alpha-2 agonist drug.
Keywords: retina • retinal glia • microscopy: light/fluorescence/immunohistochemistry