Abstract
Purpose :
Lipoxin formation has been documented in rat, mouse, and human eyes, and in the retina of rat and mouse. However, it is unclear whether neuronal tissues more generally, regulate the lipoxin pathway across multiple species. In this study, we investigate whether lipoxin formation is functionally present in the retina, optic nerve and brain, across a range of mammalian species.
Methods :
Cell-specific gene expression of Alox5, Alox15, and lipoxin receptors (Fpr2 and Fpr3), were assessed by RNAscope in-situ hybridization in the retina and optic nerve of C57BL6 mice (n=4). Gene expression of the lipoxin pathway in retinal astrocyte culture and optic nerves of Aldh1l1 mice was established by qPCR (n=3). Lipoxin formation in macaque optic nerve (n=2) was analyzed by RNA-seq. LC/MS/MS-based lipidomic analysis was performed in macaque optic nerve and primary human brain astrocyte cultures (n=3) to quantify lipoxin formation.
Results :
RNAscope identified Alox5, Alox15, Fpr2, and Fpr3 expression in the ganglion cell layer (GCL), along with the inner nuclear layer (INL) and outer NL (ONL) and expression in the myelinated optic nerve region. qPCR data from cultured mouse retinal astrocytes established Alox5, Alox15, and Fpr2 gene expression with average Ct values of 28.2±0.2, 29.0±0.2, and 28.5±0.2 respectively. Notably, high expression was observed in optic nerves of mice with average Ct values of 27.7±0.1 (Alox5), 24.6±0.5 (Alox15), and 25.9±0.7 (Fpr2). Macaque optic nerve RNA-seq data showed very high expression of Alox15; moderate expression of Alox5 and Alox5AP; and lower expression of Alox12, Alox15B, Fpr2, and Fpr3 genes. Lipidomic analysis of human brain astrocyte culture media and macaque optic nerve established formation of lipoxin pathway intermediates (5-HETE, 12-HETE, 15-HETE), and PUFAs. More importantly, LXA4 is formed in macaque optic nerve, and both LXA4 and LXB4 in human brain astrocytes.
Conclusions :
These data demonstrate that neuroprotective lipoxins are formed by astrocytes in the retina, optic nerve, and brain, and are present in murine and primate species, including humans. Therefore, the regulation and function of the neuroprotective lipoxin pathway is not just of interest in the retina but also in the brain and optic nerve as potential therapeutic targets for neurodegenerative diseases, including glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.