Abstract
Purpose:
Astrocytes provide vital support to neurons, and in diseases such as glaucoma they become hypertrophic with increased motility. Astrocytes express the transient receptor potential vanilloid (TRPV1) cation channel, which contributes to stress-induced calcium cascades in glaucoma. Previously, we found that TRPV1 modulates astrocyte migration during injury. Here we show that this modulation is through a TRPV1-mediated influx of calcium.
Methods:
Primary astrocytes were isolated by immuno-magnetic separation of postnatal Sprague-Dawley rat retina using an anti-human α-astrocyte antibody (Leinco). Once confluent, cultures were loaded with the calcium indicator, fura-2, and excited at 340 and 380 nm wavelengths under an inverted Nikon microscope. Cultures were scratched with a 10 μL pipet tip, and images were collected at 3 s intervals. Analysis was done using NIS Elements software (Nikon, Melville, NY).
Results:
Scratch wound injury induced a 20% increase in calcium from basal levels at 50 μm that dissipated with time and distance from the injury site. Addition of 3 μM of the TRPV1 antagonist, 5’-iodoresiniferatoxin (IRTX) reduced the calcium influx of vehicle-treated cells by 55% at 50 μm (p < 0.02). IRTX also increased the time needed to reach maximum levels of calcium by over 3-fold compared to basal (p ≤ 0.007). While addition of 10 μM capsazepine, a second TRPV1 antagonist, did not significantly reduce calcium level compared to vehicle following injury, it did increase the time needed to reach maximum levels by 3-fold (p ≤ 0.03).
Conclusions:
Our results support the hypothesis that scratch injury induces an increase in calcium in retinal astrocytes that is partly dependent on the activation of TRPV1. We are currently determining the events that might occur downstream of this calcium influx, such as rearrangement of the cytoskeleton. Our data suggests that TRPV1 may contribute to astrocyte migration via an influx of calcium in response to stressors such as ocular pressure and mechanical injury.
Keywords: 699 retinal glia •
765 wound healing •
675 receptors: pharmacology/physiology