Abstract
Purpose :
To investigate the role of 5-lipoxygenase (LOX) in regulating microglia function during glaucomatous stress. 5-LOX is a key enzyme in generating neuroprotective lipoxins in the retina and optic nerve. Lipoxins maintain and restore the homeostatic function of microglia during glaucoma pathogenesis.
Methods :
Silicone oil-induced ocular hypertension (OHT) was used to induce glaucomatous injury. Alox5-/- mice were used to study the effect of loss of lipoxins. Intravitreal delivery of adeno-associated virus (AAV) vector (shH10-CMV-h.5LOX-mcherry) was used for gain of 5-LOX function in retinas. Retinas (n=4-13) and myelinated optic nerves (n=5-6) were immunostained for Iba1 (microglia marker) and CD74 (disease-associated microglia marker) and imaged by confocal microscopy. Microglia skeleton graphs were reconstructed using Imaris. MorphOMICs, a tool that captures morphological information, was used to characterize microglia. Monocle3 was used to map pseudotime trajectories.
Results :
Untreated normotensive Alox5-/- mice had distinct microglial phenotypes in retina and optic nerve compared to congenic wild-type (WT) controls. AAV-mediated expression of 5-LOX in retina of Alox5-/- restored microglia phenotype towards the WT homeostatic phenotype in optic nerves. Optic nerves of Alox5-/- mice had an increased population of CD74+ (disease associated) microglia (0.5-fold, p=0.027) compared to WT. During OHT, the optic nerve had two morphological subpopulations of microglia in WT mice, with one distinct from the homeostatic WT phenotype and defined to be reactive. OHT upregulated CD74 expression in optic nerves of WT mice (3-fold, p=0.0017). With OHT, 5-LOX deletion further removed microglial phenotype in optic nerve from the WT based on pseudotime trajectory analysis, and more importantly, gene therapy with retinal 5-LOX overexpression corresponded with elimination of the reactive microglia population in the optic nerve.
Conclusions :
Our results demonstrate that loss of 5-LOX function, i.e., loss of lipoxin pathway, causes dysregulated optic nerve microglia phenotypes and expression of a disease-associated microglia marker in normotensive eyes. Retinal 5-LOX gene therapy regulated myelinated optic nerve microglia phenotype towards homeostasis during OHT. These findings identify a fundamental and protective role of 5-LOX in retina and optic nerve for regulating microglia function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.