Actin-Based Astrocyte Extensions in the Optic Nerve Head Are Dynamic and Change Orientation after 8 Hours of Intraocular Pressure Elevation in a Rat Model of Glaucoma

Abstract

 

Purpose

 

Optic nerve head (ONH) astrocytes may react structurally and biochemically to elevated intraocular pressure (IOP). This study was designed to quantify the orientation of actin-rich astrocyte extensions and to identify activators of dynamic actin assembly within the ONH after 8 hours of IOP elevation.

 

Methods

 

Brown Norway rats (n=23) were anesthetized with 2% isoflurane. Unilateral corneas were cannulated and linked to a reservoir filled with BSS for IOP control. After an 8 hour exposure to an IOP of 60 mmHg, animals were sacrificed either immediately (day 0), or on day 1, 3, or 5 post IOP elevation. The contralateral eyes were used as controls. Eyes were perfusion fixed. Longitudinal vertical sections of rat ONH (n≥4 per group) were co-labeled with phalloidin and antibodies against tubulin βIII, phosphorylated cortactin (tyrosine 421), or active Src kinase. Confocal microscopy images of the superior and inferior ONH were analyzed by FIJI software. Prism software was used for statistical analysis.

 

Results

 

Astrocyte extensions were orientated stereotypically with respect to the anterior-posterior (A-P) axis (defined by the longitudinal axis of axonal tubulin) in superior and inferior control ONHs (Fig. 1). Astrocyte extensions became rearranged immediately after IOP elevation (day 0 mean superior ONH orientation = 61.4^o ± 6.2^o, compared to 83.3^o ± 1.2^o in controls, p<0.05 ANOVA). Astrocyte extensions re-orientated back to control levels on day 1 (and remained unchanged on day 3-5) post IOP elevation (Fig. 1). Phosphorylated cortactin labeling (suggestive of dynamic actin assembly) significantly increased in the superior and inferior ONH by 1.2 fold per day post IOP elevation by linear regression analysis (Fig. 2A-B), while active Src kinase (upstream of cortactin) was abundantly detected in control ONHs and persisted in ONH tissue at all time points post IOP elevation (Fig. 2C-D).

 

Conclusions

 

Astrocyte extensions are actin-rich, highly dynamic, and rearrange immediately within the ONH after 8 hours of IOP elevation. Markers of dynamic actin assembly are prevalent within the ONH.  

 

A-P = anterior-posterior; ONH = optic nerve head; SEM = standard error of the mean; * = p<0.05 by ANOVA relative to controls

 

 

A-P = anterior-posterior; ONH = optic nerve head; P-Cortactin = phosphorylated cortactin SEM = standard error of the mean