Abstract
Purpose :
We previously discovered astrocytes ability to synthesize lipoxins in the inner retina and the neuroprotective actions of Lipoxin B4 (LXB4). Muller glia are the major macroglia cell type in the retina. Our aim was to investigate the lipoxin pathway in Muller glia and determine if LXB4 can regulate Muller glia reactivity and homeostatic function.
Methods :
Primary and immortalized Muller glia were validated by immunocytochemistry. Functional activity of the lipoxin pathways was analyzed by LC/MS/MS-based lipidomics. Muller glia were treated with LXB4 in the absence or presence of the TRPV4 agonist GSK101 to induce Muller glia reactivity. LXB4 signal transduction pathways in Muller glia were analyzed by kinase screening assays. Regulation of Muller glia reactivity by OHT and LXB4 treatment was studied in vivo using the silicone-oil model of chronic ocular hypertension (OHT). Reactivity and function in vitro and in vivo were assessed by qPCR and immunohistochemistry.
Results :
Primary and immortalized Muller glia express Muller glia markers (Gs, CD44, and Rlbp1). Lipidomic analysis revealed Muller glia produce LXB4 and lipoxin pathway intermediates (5-HETE and 15-HETE, P<0.05, n=5). qPCR analysis established upregulation of Gfap and Vim, markers of reactivity, following GSK101 treatment. Reactive Muller glia show increased expression of 5-Lipoxygenase (Alox5) and decreased expression of 15-Lipoxygenase (Alox15). Glutamate transporter, Glast was downregulated in reactive Muller glia. Kinase assays identified that LXB4 inhibits phosphorylation of p38, Stat2, Stat5, and Stat3 (p< 0.05, n=4), which are pathways linked to regulation of Muller cell gliosis. LXB4 treatment inhibits GSK101 induced vimentin expression (p < 0.05, n=4). qPCR analysis show downregulation of Gfap, Vim, and Alox5 following LXB4 treatment and upregulation of the homeostatic gene Glast. In vivo data confirmed LXB4 regulation of reactivity by reducing Gfap expression in Muller glia following LXB4 treatment in an OHT model. Additionally, qPCR analysis revealed that LXB4 significantly upregulated Glast expression.
Conclusions :
Muller glia express a functional lipoxin pathway, which is dysregulated during Muller cell gliosis. Moreover, LXB4 downregulated Muller cell reactivity in response to both activation of TRPV4 and OHT and restored expression of a homeostatic pathway, identifying a new neuroprotective macroglia mechanism for LXB4.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.