Abstract
Purpose :
One of the prominent risk factors for glaucomatous optic neuropathy is the level of intraocular pressure (IOP). Elevated IOP in rodent models causes optic nerve damage at the unmyelinated optic nerve (UON), the region corresponding to the site of injury in human glaucoma. This study compared gene expression changes in retina, UON, and myelinated optic nerve (MON) of normal mice and in mice after experimental glaucoma or optic nerve crush.
Methods :
RNA-seq was performed on retina, UON, and MON tissue of male and female C57BL/6 mice that underwent IOP by microbead injection (n=12, studied at 3, 14 and 42 days) or nerve crush with forceps (n=8, studied at 3 and 14 days). Tissues from bilateral naïve mice were sequenced as controls. Differential expression analysis was performed with each tissue region in naïve mice and between each time point following injury. The data were validated by qPCR and small sample Western analysis (Wes).
Results :
Differentially expressed genes (DEGs) differed among normal retina, UON, and MON. The predominant KEGG pathways in UON-enriched genes were extracellular matrix—receptor, cell membrane signaling, and Wnt, Hippo, PI3K-Akt, and transforming growth factor β signaling pathways. DEGs were more extensive in MON than UON following both injuries, and greater after crush than IOP increase. Of the DEGs in both glaucoma and crush UON, some were expressed in microglia (Dock2, Fcrls, and Pilra) and 2 typically in astrocytes (Timp1 and Vgf), while many were cell cycle genes. More DEGs were upregulated 14 days after crush in UON and glaucoma in MON than in glaucoma UON, including C3, C1q, Il1, and Tnfα. There were no consistent patterns favoring either so-called A1 or A2 astrocyte genes. Western analysis pointed to increased phosphorylated focal adhesion kinase and dephosphorylated cofilin after IOP increase--indications that integrin-linked signaling in UON astrocytes plays a key role in response to experimental glaucoma.
Conclusions :
The genetic phenotype of mouse UON, likely dominated by astrocytes, is significantly different from MON or retina. Their junctional complexes are confirmed as important in the response to IOP-generated stress.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.