Optic Nerve Head Gene Transcription Sequelae to a Single Elevated IOP Exposure Provides Insights Into Known Responses to Chronically Elevated IOP

Purpose: To clarify the optic nerve head (ONH) gene expression responses associated with a single, axon-damaging exposure to elevated IOP in relation to the composite cellular events previously identified in models of chronically elevated IOP. Methods: Anesthetized rats were exposed unilaterally to an 8-hour pulse-train controlled elevation of IOP (PT-CEI) at 60 mm Hg, while others received normotensive CEI at 20 mm Hg. ONH RNA was harvested at 0 hours and 1, 2, 3, 7, and 10 days after either CEI and from naïve animals. RNA sequencing was performed to analyze ONH gene expression. DAVID Bioinformatics tools were used to identify significant functional annotation clusters. Gene function was compared between PT-CEI and two models of chronic ocular hypertension from the literature. Results: The number of significantly changed genes peaked immediately (n = 1354) after PT-CEI (0 hours). This was followed by a lull (point) at 1 and 2 days after PT-CEI. Gene activity increased again at 3 days (136 genes) and persisted at 7 (78 genes) and 10 (339 genes) days. Significant gene functional categories included an immediate upregulation of Defense Response at 0 hours, followed by upregulation in Cell Cycle, a reduction in Axonal-related genes at 3 to 10 days, and upregulation of Immune Response–related genes at 10 days following PT-CEI. The most commonly upregulated gene expression across our PT-CEI study and two chronic models of ocular hypertension were cell cycle related. Conclusions: The PT-CEI model places in sequence ONH gene expression responses previously reported in models with chronically elevated IOP and may provide insights into their role in optic nerve damage

Astrocyte Structural and Molecular Response to Elevated Intraocular Pressure Occurs Rapidly and Precedes Axonal Tubulin Rearrangement within the Optic Nerve Head in a Rat Model.

Glaucomatous axon injury occurs at the level of the optic nerve head (ONH) in response to uncontrolled intraocular pressure (IOP). The temporal response of ONH astrocytes (glial cells responsible for axonal support) to elevated IOP remains unknown. Here, we evaluate the response of actin-based astrocyte extensions and integrin-based signaling within the ONH to 8 hours of IOP elevation in a rat model. IOP elevation of 60 mm Hg was achieved under isoflurane anesthesia using anterior chamber cannulation connected to a saline reservoir. ONH astrocytic extension orientation was significantly and regionally rearranged immediately after IOP elevation (inferior ONH, 43.2° ± 13.3° with respect to the anterior-posterior axis versus 84.1° ± 1.3° in controls, p<0.05), and re-orientated back to baseline orientation 1 day post IOP normalization. ONH axonal microtubule filament label intensity was significantly reduced 1 and 3 days post IOP normalization, and returned to control levels on day 5. Phosphorylated focal adhesion kinase (FAK) levels steadily decreased after IOP normalization, while levels of phosphorylated paxillin (a downstream target of FAK involved in focal adhesion dynamics) were significantly elevated 5 days post IOP normalization. The levels of phosphorylated cortactin (a downstream target of Src kinase involved in actin polymerization) were significantly elevated 1 and 3 days post IOP normalization and returned to control levels by day 5. No significant axon degeneration was noted by morphologic assessment up to 5 days post IOP normalization. Actin-based astrocyte structure and signaling within the ONH are significantly altered within hours after IOP elevation and prior to axonal cytoskeletal rearrangement, producing some responses that recover rapidly and others that persist for days despite IOP normalization

Active Src kinase levels within the optic nerve head are reduced immediately and transiently after intraocular pressure elevation.

<p>(A-B) Superior and inferior optic nerve head (ONH) sections labeled with antibodies against active Src kinase in control eyes and eyes exposed to 8 hours of intraocular pressure (IOP) elevation. The bottom panels in (A) include a validation of antibody specificity using an active Src specific blocking peptide (bottom left panel), as well as an example of co-localization of active Src with astrocyte specific anti-glial fibrillary acidic protein (GFAP) antibodies (bottom right panels). Note similar orientation and directionality of filaments labeled for active Src and GFAP. (C) Mean active Src fluorescence intensity within the superior and inferior ONH of control eyes and eyes exposed to 8 hours of IOP elevation. Day 0 eyes were immediately fixed after 8 hours of IOP elevation, while day 1–5 indicate the period of time the IOP was normalized post IOP elevation prior to fixation. Error bars indicate standard error of the mean (SEM). * = p<0.05 by 2-way ANOVA and indicates statistically significant difference between control and experimental groups; n = 7, 7, 6, 7, and 5 for control, day 0, day 1, day 3, and day 5 groups, respectively. A-P = anterior-posterior.</p

Cortactin phosphorylation levels within the optic nerve head increase in a reversible manner after intraocular pressure elevation.

<p>(A-B) Superior and inferior optic nerve head (ONH) sections labeled with anti-phosphorylated cortactin (p-cortactin) antibodies in control eyes and eyes exposed to 8 hours of intraocular pressure (IOP) elevation. The bottom panels in (A) include a validation of antibody specificity using a p-cortactin specific blocking peptide (bottom left panel), as well as an example of co-labeling with p-cortactin and axon specific anti-tubulin antibodies to demonstrate the non-axonal source of the majority of p-cortactin label (bottom right panels). (C) Mean p-cortactin fluorescence intensity within the superior and inferior ONH of control eyes and eyes exposed to 8 hours of IOP elevation. Day 0 eyes were immediately fixed after 8 hours of IOP elevation, while day 1–5 indicate the period of time the IOP was normalized post IOP elevation prior to fixation. Error bars indicate standard error of the mean (SEM). * = p<0.05 by 2-way ANOVA and indicates statistically significant difference between control and experimental groups; n = 7, 6, 7, 8, and 7 for control, day 0, day 1, day 3, and day 5 groups, respectively. A-P = anterior-posterior.</p

Paxillin phosphorylation levels within the optic nerve head increase and remain elevated after intraocular pressure elevation.

<p>(A-B) Superior and inferior optic nerve head (ONH) sections labeled with anti-phosphorylated paxillin (p-paxillin) antibodies in control eyes and eyes exposed to 8 hours of intraocular pressure (IOP) elevation. The bottom panels in (A) include a validation of antibody specificity using a p-paxillin specific blocking peptide (bottom left panel), as well as an example of co-labeling with p-paxillin and axon specific anti-tubulin antibodies to demonstrate the non-axonal source of the majority of p-paxillin label (bottom right panels). (C) Mean p-paxillin fluorescence intensity within the superior and inferior ONH of control eyes and eyes exposed to 8 hours of IOP elevation. Day 0 eyes were immediately fixed after 8 hours of IOP elevation, while day 1–5 indicate the period of time the IOP was normalized post IOP elevation prior to fixation. Error bars indicate standard error of the mean (SEM). * = p<0.05 by 2-way ANOVA and indicates statistically significant difference between control and experimental groups; n = 7, 7, 5, 7, and 7 for control, day 0, day 1, day 3, and day 5 groups, respectively. A-P = anterior-posterior.</p

Actin-based astrocyte extensions change orientation within hours after intraocular pressure elevation.

<p>(A) Low magnification images of control optic nerve head (ONH) sections labeled for actin (TRITC-phalloidin), tubulin (Tuj1 anti-tubulin βIII antibody), and nuclei (DAPI). The left and right boxes in the merged image indicate the anterior superior and inferior regions of the ONH, respectively. (B, C) High magnification images of the superior and inferior regions of the ONH, as indicated by boxes in panel (A). Representative superior (D) and inferior (E) ONH regions from control eyes and eyes exposed to 8 hours of intraocular pressure (IOP) elevation, labeled for actin, tubulin, and nuclei. Day 0 eyes were immediately fixed after 8 hours of IOP elevation, while day 1–5 indicate the period of time the IOP was normalized post IOP elevation prior to fixation. A-P = anterior-posterior, B = Bruch’s membrane, I = inferior, R = retina, and S = superior.</p

Model of optic nerve head astrocyte signaling downstream of integrin activation in response intraocular pressure fluctuation.

<p>Physiologic and experimental intraocular pressure (IOP) fluctuation within the optic nerve head (ONH) results in tissue strain and extracellular matrix (ECM) stretch. Interaction between the ECM and integrin receptors on astrocyte cell membranes, leads to integrin activity, which phosphorylate focal adhesion kinase (p-FAK). Active p-FAK can target paxillin for phosphorylation, which is important for focal contact/adhesion formation. FAK phosphorylation also allows for cortactin phosphorylation through active FAK and Src kinase. Phosphorylated cortactin enhances actin polymerization. Increased focal contact formation and actin polymerization are required for dynamic astrocyte extension formation and maintenance within the ONH.</p

Mean IOP Measurements During 8 Hours of IOP Elevation.

<p>Mean IOP Measurements During 8 Hours of IOP Elevation.</p

Quantitative analysis of actin-based astrocyte extension and axonal microtubule reorganization in response to elevated intraocular pressure.

<p>(A) Mean astrocytic actin bundle orientation relative to the anterior-posterior axis within the superior and inferior optic nerve head (ONH) of control eyes and eyes exposed to 8 hours of intraocular pressure (IOP) elevation. (B) An example of the superior ONH with actin and nuclear co-labeling, before and after application of a grid to allow for filament length measurement analysis. Note, in this example, only nuclear-adjacent actin bundles are identified and measured. (C) Mean length of actin bundles (with or without contact with a nucleus) within the superior and inferior ONH of control eyes and eyes exposed to 8 hours of IOP elevation. (D) Mean length of actin bundles in contact with a nucleus within the superior and inferior ONH of control eyes and eyes exposed to 8 hours of IOP elevation. (E) Mean axonal tubulin fluorescence intensity within the superior and inferior ONH of control eyes and eyes exposed to 8 hours of IOP elevation. Day 0 eyes were immediately fixed after 8 hours of IOP elevation, while day 1–5 indicate the period of time the IOP was normalized post IOP elevation prior to fixation. Error bars indicate standard error of the mean (SEM); * = p<0.05 by 2-way ANOVA and indicates statistically significant difference between control and experimental groups; n = 7, 7, 7, 8, and 6 for control, day 0, day 1, day 3, and day 5 groups, respectively. A-P = anterior-posterior.</p