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Karen Ho, Carl Weitlauf, David Calkins; The TRPV1 Cation Channel Contributes to Stress-Induced Retinal Astrocyte Migration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):774. doi: https://doi.org/.
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Astrocytes provide both metabolic and structural support to retinal ganglion cells and mediate a variety of stress-related responses in diseases such as glaucoma. Previously we found that retinal astrocytes express the transient receptor potential vanilloid (TRPV1) cation channel. TRPV1 contributes to stress responses in other neural systems via calcium-dependent cascades and is up-regulated in experimental models of glaucoma. Our objective here was to test whether TRPV1 contributes to the migration of retinal astrocytes in response to mechanical injury.
Primary astrocyte cultures were prepared by immuno-magnetic separation of postnatal Sprague-Dawley rat retina using an anti-human α-astrocyte antibody (Leinco). Once confluent, cultures were scratched with a 1mL pipet tip to create a uniform gap of 1mm diameter to induce migration under conditions that minimized cell proliferation. We used DIC imaging to monitor the rate of wound closure in vitro under varying pharmacological conditions. F-actin and proliferation were examined in fixed astrocytes using phalloidin and Ki-67 immunolabeling.
Application of 100pM of the TRPV1-specific agonist capsaicin increased migration by 36% by 36hrs (p=0.03, n=3) compared to vehicle (ethanol). In contrast, treatment with the TRPV1-specific antagonist 5’-iodoresiniferatoxin (IRTX) reduced astrocyte migration compared to vehicle in a dose-dependent manner, with 3µM decreasing migration by 33% by 48 hrs (p<0.03, n=3). Application of 1µM IRTX also reduced phalloidin intensity by 52% compared to control by 12hrs (p=0.01, n=1). Chelation of extracellular calcium with 1mM EGTA also reduced astrocyte migration by 44% compared to vehicle at 48hrs (p<0.05, n=3). Wound closure was not due to proliferation, as Ki-67 expression remained low (5.5±0.7%).
Our results support the hypothesis that in response to injury, TRPV1 contributes to mobilization of retinal astrocytes. This mobilization is at least partially dependent on the influx of extracellular calcium. We are currently using whole-cell voltage clamp recordings to test whether our cultured retinal astrocytes are capable of producing a transient current characteristic of active calcium channels. Our results suggest that TRPV1 may contribute to the known hypertrophy of astrocytes in response to disease-relevant stressors such as ocular pressure.
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