Introduction
Material and methods
Animals
Intravitreal injections
Measurements of choroidal thickness in alert chickens
Measurements of dopamine and DOPAC with HPLC-ED
Exposure to 1 Hz, 10 Hz, and 400 Hz flickering light conditions
Tissue preparation and immunohistochemistry
Antigen | Antibody | Origin | Working dilution |
---|---|---|---|
α2A-adrenergic receptor | Anti-α2A-adrenergic receptor rabbit polyclonal, ab 92,650 | Abcam, Cambridge, UK | 1:100 |
Nitric oxide synthase (NOS) | Anti-NOS, rabbit polyclonal, ALX-210–529 | ENZO Life Sciences GmbH, Loerrach, Germany | 1:200 |
Tyrosine hydroxylase(TH) | Anti-TH, mouse monoclonal, 22,941 | Immunostar Hudson WI, USA | 1:1000 |
Vascular endothelial growth factor A (VEGF) | Anti-VEGF, mouse monoclonal, orb191500 | Biorbyt Ltd Cambridge, Cambridgeshire, UK | 1:200 |
Experimental groups and treatments
Chickens | Intravitreal injection | Treatment | |
---|---|---|---|
Experiment 1A | 7 | At 10:00, a single intravitreal atropine injection into one eye, saline vehicle into the other | OCT measurements 1 h before (baseline) and 2, 4, 6, and 7 h after injections; final measurement 24 h after baseline measurements |
12 (also used in Exp 1B) | At 10:00, a single intravitreal combined atropine/spiperone injection, vehicle only into the other | OCT measurements 1 h before (baseline) and 2, 4, 6, and 7 h after injections; final measurement 24 h after baseline measurements | |
Experiment 1B | 7 | At 10:00, a single intravitreal atropine injection into one eye, saline vehicle into the other | After about 2 h, animals were euthanized and prepared for HPLC-ED to determine vitreal, retinal, and choroidal levels of dopamine and DOPAC. At this time point, dopamine release was highest, as found in a previous study [16] (same for all HPLC experiments, see below) |
11 | At 10:00, a single intravitreal combined atropine/spiperone injection, vehicle only into the other | After about 2 h, animals were euthanized and prepared for HPLC-ED to determine vitreal, retinal, and choroidal levels of dopamine and DOPAC | |
Experiment 1C | 12 | At 10:00, a single intravitreal spiperone injection into one eye, vehicle only into the other | OCT measurements 1 h before (baseline) and 2, 3, 5, 7, and 9 h after injection |
Chickens | Flickering light condition | Treatment | |
---|---|---|---|
2A | 8 | Broadband white room light (470 lx) | OCT measurements at 9:00 (baseline) and 2, 3, 5, 7, and 8 h after baseline measurements |
2B | 6 | Broadband white room light (470 lx) | After exposure of about 2 h, animals were euthanized and prepared for HPLC-ED to determine vitreal, retinal, and choroidal levels of dopamine and DOPAC |
2C | 6/6/6 (also used in Exp 2D) | Flickering light (duty cycle 0.2, average luminance 20 lx) 1 Hz, 10 Hz, 400 Hz | OCT measurements at 9:00 (baseline), then transfer to either 1 Hz, 10 Hz, or 400 Hz flickering light, continued OCT measurements 2, 3, 5, 7, and 8 h after baseline measurements |
2D | 6/6/6 | Flickering light (duty cycle 0.2, average luminance 20 lx) 1 Hz/10 Hz/400 Hz | After exposure of about 2 h in either 1 Hz, 10 Hz, or 400 Hz flickering light, animals were euthanized and prepared for HPLC-ED to determine vitreal, retinal, and choroidal levels of dopamine and DOPAC |
Chickens | Lighting | Treatment | |
---|---|---|---|
3 | 6 | Broadband white room light (470 lx) | None, naïve controls Localization of tyrosine hydroxylase (TH) in choroid. Double-labeling of TH with neuronal nitric oxide synthase (nNOS), vascular endothelial growth factor (VEGF), and alpha2A adrenoreceptors (α2A-ADR) |