Abstract
Purpose: :
In addition to providing vital metabolic and structural support, astrocytes are likely to respond to stressors associated with the degeneration of retinal ganglion cells (RGCs) and their axons in glaucoma. A critical component of the stress response is calcium homeostasis, which can be modulated by activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel in both RGCs and glia. We have shown the expression of TRPV1 in both retinal and optic nerve astrocytes and the increase in TRPV1 message with pressure in the DBA2J mouse model of glaucoma. Here we further characterize TRPV1 in cultured astrocytes and retinal explants subjected to different forms of injury including pressure and scratch wound.
Methods: :
Retinal explants were harvested from Sprague-Dawley rats and cultured on organotypic inserts overnight before exposure to 70mmHg pressure for 24h. To isolate primary astrocytes, retinas from P1-3 rats were harvested and mechanically dissociated. Astrocytes were identified with α-astrocyte antibody (Leinco) and seeded on Labtek chambers. Upon confluency, the cells were treated with 70mmHg pressure for 24h or scratched with a pipet tip. Cells and explants were fixed in 4% paraformaldehyde for immunocytochemistry with antibodies against TRPV1 (Novus) and GFAP (Abcam), and phalloidin (Invitrogen).
Results: :
The isolated cells averaged 93% purity for astrocytes as identified by GFAP immunolabeling. A 24h exposure to 70mmHg hydrostatic pressure increased astrocytic TRPV1 levels by 168% (p≤0.001) in culture and by 32% (p≤0.001) in retinal explants compared to ambient pressure. At ambient pressure, TRPV1 was diffusely expressed throughout the astrocyte. With elevated pressure, TRPV1 translocated from the processes to the perinuclear area of the soma in astrocyte cultures and to astrocyte end-feet in explants. In a scratch wound assay, rearrangement of the actin cytoskeleton was observed at 8h post scratch with concurrent translocation of TRPV1 from the astrocyte processes to the soma.
Conclusions: :
Upregulation and relocalization of TRPV1 suggests that the channel is sensitive to disease-relevant stressors like pressure and may be involved in mobilizing astrocytes in response to injury in glaucoma. Since TRPV1 mediates calcium levels, movement of the channel may contribute to localized changes in calcium to modulate astrocyte function during injury. Elucidating the mechanism of TRPV1 signaling is important in understanding changes in astrocyte activity in glaucoma and other injuries.
Keywords: astrocyte • cytoskeleton • ion channels