Global changes in optic nerve head gene expression after exposure to elevated intraocular pressure in a rat glaucoma model

Abstract

Purpose: In glaucoma, the optic nerve head (ONH) is the likely site of initial injury and elevated intraocular pressure (IOP) is the best-known risk factor. This study determines global gene expression changes in the pressure-injured ONH.

Methods: Unilateral sustained IOP elevation (glaucoma, n = 46) or optic nerve transection (n = 10) was produced in rats. ONHs were removed, and the retrobulbar optic nerves were graded for degeneration. Gene expression in the glaucomatous ONH with extensive injury was compared with that in the fellow ONH (n = 6/group), by using cDNA microarrays. Data from 12 arrays were normalized, significant differences in gene expression determined, and significantly affected gene classes identified. For the remaining ONH, grouped by experimental condition and degree of injury, quantitative reverse transcriptase-PCR (qPCR) and ANOVA were used to compare selected message levels.

Results: Microarray analysis identified more than 2000 significantly regulated genes. For 225 of these genes, the changes were greater than twofold. The most significantly affected gene classes were cell proliferation, immune response, lysosome, cytoskeleton, extracellular matrix, and ribosome. A 2.7-fold increase in ONH cellularity confirmed glaucoma model cell proliferation. By qPCR, increases in levels of periostin, collagen VI, and transforming growth factor beta1 were linearly correlated to the degree of IOP-induced injury. For cyclinD1, fibulin 2, tenascin C, TIMP1, and aquaporin-4, correlations were significantly nonlinear, displaying maximum change with focal injury.

Conclusions: In the ONH, pressure-induced injury results in cell proliferation and dramatically altered gene expression. For specific genes, expression levels were most altered by focal injury, suggesting that further array studies may identify initial, and potentially injurious, altered processes.

Figure 1

(A) Example of the dissected ONH used in this study. Each 1-mm-long ONH consisted of the optic disc and the unmyelinated and the initial myelinated portions of the nerve. A representative optic nerve cross-section from a normal, fellow eye (control) is shown in (B). All fellow nerves were graded 1.0. For the microarray study, glaucoma model nerve cross-sections exhibited extensive axonal degeneration (C). This nerve was graded 4.7. In the microarray study, glaucoma model ONH had nerves with grades between 4.6 and 4.95, indicating ongoing and extensive axonal degeneration affecting approximately 50% of the axons. ONH with grade-5 optic nerves that demonstrated more extensive gliosis (D) were excluded from the microarray study. The experimental design of the microarray study is illustrated in (E). For each gene, RNA from each of six fellow and six glaucomatous model eyes were independently compared by using 12 separate arrays with an ONH RNA reference standard.

Figure 2

Elevated IOP results in ONH cellular proliferation. mRNA levels of cyclin D, the key regulator for cell-cycle progression, are shown in (A) for ONH with graded optic nerve injury due to elevated IOP. ONHs were grouped according to injury grade (■; n = 7–11/group). ( ) For comparison, ONH from eyes with transected optic nerves (n = 7) are shown. Each bar represents the mean ± SEM for that group relative to the mean for untreated fellow eyes (n = 14). *Significant increases compared with fellow eye levels (ANOVA with the Dunnett posttest, P < 0.01). (B) In a pressure-injured ONH, the relationship of mean IOP to optic nerve injury grade is shown over the 5-week experimental period, by group. Mean IOP differences between groups are significant for all injury groups (*P < 0.05), except that between fellow and injury grade <1.5. For two separate groups of fellow and grade-5 ONH, total DNA measurements are shown in (C), illustrating the approximately threefold increase in cellularity in ONH with grade-5 optic nerve damage due to elevated IOP (*P < 0.000002).

Figure 3

Microglial activation in response to elevated IOP. In the same nerve injury groups as described in Figure 2, mRNA levels for the microglial marker, IBA1, were measured (A). *Significantly different from fellow eye (P < 0.05). IBA1 mRNA levels were significantly elevated in ONHs damaged by elevated IOP (■) with optic nerve injury grades greater than 3.5, as well as in ONHs at 2 weeks after optic nerve transection ( ). Immunohistochemical labeling indicated activation and potential proliferation of ONH microglia. Representative fellow ONH (B) and glaucoma model ONH with an optic nerve injury grade of 3.6 (C). (D, E) Higher magnifications of the boxed regions in (B) and (C), respectively. Arrowheads: labeled cells; arrows: Bruch’s membrane. Brown: chromogen labeling; pale blue: counterstained nuclei. Scale bars: (B, C) 10 μm; (D, E) 3 μm.

Figure 4

Two response patterns for altered ONH ECM message expression accompanying pressure-induced injury. For the ECM components, periostin, collagen IV, and collagen VI, increasing optic nerve injury was accompanied by linear increases in ONH message level (A). For periostin and collagen VI, ONH groups with injury grades greater than 3.5 demonstrated significant increases compared with the fellow eye group (*P < 0.05). Note that the magnitude of increase for periostin is much greater, as indicated by the fivefold difference in y-axis scale labels. Collagen IV demonstrated a similar pattern, although the increase was only statistically significant in the transection group. In contrast, message levels for fibulin2, tenascin c, and TIMP-1 were highest in ONHs from eyes with focal optic nerve injury (B). *Significant elevation relative to untreated, fellow ONH (P < 0.05). In addition, for fibulin 2, the grade-3.5 to less than grade-5 group, had significantly greater responses than all other groups. For TIMP-1, the grade-1.5 to less than grade-3.5 group, had significantly greater values than all groups, except the grade-3.5 to less than grade-5 group. ANOVA with the Tukey multiple comparison posttest.

Figure 5

TGFβ isoform mRNAs were differentially regulated in pressure-injured ONH. For TGFβ1, there was a significant increase in mRNA level that correlated with the degree of optic nerve injury, so that levels were significantly elevated in the group of pressure-injured ONHs with grade 5 (*P < 0.01). In contrast, expression levels for TGFβ2 demonstrated a significantly nonlinear correlation to optic nerve injury grade, with lowest levels in ONH with focal optic nerve injury. This resulted in a significant (approximately twofold) difference in the ratio of TGFβ1 to TGFβ2 in all ONHs with pressure-induced optic nerve injury grades greater than 1.5.

Figure 6

ONH Aquaporin-4 mRNA levels decreased in a pattern similar to that of TGFβ2. For all injury groups, except that with injury grades <1.5, there was a significant decrease in aquaporin-4 mRNA expression (*P < 0.05) compared with levels in the fellow eye. Correlation of expression levels with optic nerve injury grade demonstrated a significant, nonlinear fit, with the lowest expression levels in ONH from eyes with focal optic nerve injury (F test; P < 0.01).

Figure 7

Expression levels of two other proteins associated with differentiated astrocytes, GFAP and connexin43, were not altered in the ONH by either elevated IOP or transection. Although the pattern of expression in the various optic nerve injury groups may appear similar to that seen for TGFβ2 and aquaporin-4, levels in the optic nerve injury groups were not significantly different from fellow eye levels. Regression analysis also indicated no significant correlation.