Abstract
Purpose :
To define molecular profiles of distinct subregions of the ONH in a spontaneous large animal glaucoma model, using laser capture microdissection (LCM).
Methods :
Intraocular pressure (IOP) and optic nerve axon counts were available for all animals. ONH tissues of 13 homozygous LTBP2 mutant cats with feline congenital glaucoma (FCG) and 4 normal age-matched control cats (1 year-old) were trephined and cryoembedded. Eight 10µm thick sections were obtained per eye and mounted on RNase-free PEN membrane slides (Zeiss). Each ONH sub-region: pre-lamina (PL), lamina cribrosa (LC) and retro-lamina (RL), was UV-laser microdissected and catapulted into adhesive caps of collection tubes using the Palm MicroBeam system (Zeiss). Total RNA was extracted by RNeasy Plus Micro Kit (Qiagen). cDNA libraries were constructed using SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio), and sequenced by Illumina NovaSeq 6000 to obtain 150 bp strand-specific paired-end reads at ~35 million read depth per sample. RNAseq analysis followed a standard pipeline using STAR-RSEM-DESeq2 packages. Differentially expressed genes (DEGs) were detected at FDR ≤ 0.05. g:Profiler was used to provide functional context for the DEGs identified. RNAscope and immunolabeling validated RNA-seq results and provided further cellular and spatial context. Comparison between groups was by t-test or ANOVA (P < 0.05 significant).
Results :
Mean and cumulative IOP were higher, and axon count lower in FCG than in controls (P < 0.05). In normal ONH, higher expression of fibroblast enriched genes (COL1A1, DCN and IGFBP6) in the collagenous LC and of oligodendrocyte enriched genes (MBP, PLP1, CLDN11, OLIG1 and SOX10) in the myelinated RL region were identified, consistent with resident cell populations of these regions. In comparing transcriptomic profiles of each ONH sub-region between FCG and normal controls, 1205, 628 and 586 DEGs were identified in PL, LC and RL regions, respectively. Only 93 DEGs were shared by all 3 ONH regions and >50% of DEGs were detected in only one ONH sub-region. Functional analysis of these region-specific DEGs identified immune responses and metabolic pathway in PL; cell migration and gap junction in LC, and cell adhesion and fatty acid metabolism in RL region.
Conclusions :
LCM with RNA-seq identified distinct ONH sub-region-specific molecular alternations in a glaucoma model that has similar microanatomy to humans.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.