Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Optic Nerve Crush and Experimental Glaucoma

Abstract

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor β pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Keywords: astrocytes; gene expression; glaucoma; mouse; nerve crush; optic nerve; retina; transcriptomics.

Figure 1

Transcriptomic analysis of astrocyte-containing tissues in bilaterally naïve mice. (A) Experimental design of the study. Three tissue regions of bilaterally naïve mice were micro-dissected for comparison: (1) myelinated optic nerve (MON), (2) unmyelinated optic nerve (UON), and (3) retina. Tissue from four mice was collected and the sex replicates for each tissue group were pooled after RNA extraction for library preparation and 150 bp paired-end Illumina sequencing. (B) Principal component analysis (PCA) of bilaterally naïve tissue regions. Each symbol represents a single sample, where symbol colors denote the tissue region and symbol shapes signify sex. (C) FPKM (fragments per kilobase of transcript per million mapped reads) expression of cell type markers characteristic of each tissue region: glial (Gja1, encodes for Connexin-43), oligodendrocyte (Mbp, encodes for myelin basic protein), retinal (Rho, encodes for Rhodopsin), microglial (Cd68, encodes for cluster of differentiation 68), and capillary/endothelial (Flt1, encodes for VEGFR1). Dots represent a single sample and lines represent the median FPKM of the replicate samples. (D) Expression of astrocyte genes in three naïve tissue regions: UON, MON, and retina. Left y-axis and filled bars represent FPKM (from RNA-seq data), while right y-axis and empty bars indicate relative expression via qPCR of independent tissue samples. Error bars indicate standard deviation. For RNA-seq, n = 2 (pooled) samples per tissue type. For qPCR, n = 6 samples from 3 mice per tissue group.

Figure 2

Region-specific gene signatures in the naïve ON. (A) Venn diagrams showing the number of significantly enriched genes in naïve UON in pairwise comparisons to MON and retina (top) and MON in pairwise comparisons to UON and retina (bottom). (B) KEGG analysis of enriched UON (top) and MON (bottom) genes compared to all other tissue regions. (C) Clustered heatmaps of significantly upregulated UON genes within the extracellular matrix (ECM)–receptor interactions (top) and MON-enriched genes in the steroid biosynthesis (bottom) KEGG pathways. (D) Volcano plot showing differential expression analysis comparing naïve UON and MON. Dotted lines indicate threshold cut-off for a significantly changed gene (log2FC ± 1, in addition to adjusted p < 0.05). Genes with log2FC > 1 were considered enriched in UON, and genes with log2FC < −1 signified MON-enriched genes. (E) KEGG pathways enriched in UON and MON genes.

Figure 3

Differential responses to ON crush. (A) Experimental design for studying gene expression responses following ON crush in UON, MON, and retinal tissue. (B) PCA of tissue during the ON crush time course. (C) Venn diagrams showing relationships of differentially expressed genes (DEGs) between UON, MON, and retina three days (left, 3 D) and two weeks (right, 2 W) after crush. (D) KEGG pathway analysis of UON and MON DEGs at early (top) and late (bottom) crush time points. (E) Venn diagrams showing relationships of UON (top) and MON (bottom) responses to ON crush. (F) Number of upregulated and downregulated genes in UON and MON at each crush time point. (G,H) Gene expression changes in UON (G) and MON (H) during the ON crush time course.

Figure 4

Differential responses to glaucoma. (A) Experimental design for RNA-seq experiments in the bead-induced glaucoma model. (B) PCA of control and experimental glaucoma tissue time points. (C) Venn diagrams showing relationships of DEGs between UON, MON, and retina three days, two weeks, and six weeks after IOP elevation. (D) KEGG pathway analysis of UON and MON DEGs at different time points following IOP elevation. (E) Venn diagrams showing relationships of UON (left) and MON (right) responses to bead-induced glaucoma. (F) Number of up/down genes in UON and MON at each glaucoma time point. (G,H) Gene expression changes in UON (G) and MON (H) during the glaucoma time course.

Figure 5

Shared responses to ON injury. (A) Venn diagrams comparing DEGs in ON crush and glaucoma injuries in UON (top) and MON (bottom) tissue regions. DEGs are both upregulated and downregulated in at least one time point. (B,C) Gene expression of select UON (B) and MON (C) DEGs in ON crush and glaucoma injury. (D) Heatmap showing PAN-reactive, A1-specific, and A2-specific astrocyte markers in naïve and injured UON and MON regions. UON tissue did not express a dominant A1 or A2 characteristic phenotype in crush or glaucoma, while MON exhibited slightly more consistent A1/A2-specific gene expression compared to UON tissue.