Diana C. Lozano

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.

A period of controlled elevation of IOP (CEI) produces the specific gene expression responses and focal injury pattern of experimental rat glaucoma

Purpose We determine if several hours of controlled elevation of IOP (CEI) will produce the optic nerve head (ONH) gene expression changes and optic nerve (ON) damage pattern associated with early experimental glaucoma in rats. Methods The anterior chambers of anesthetized rats were cannulated and connected to a reservoir to elevate IOP. Physiologic parameters were monitored. Following CEI at various recovery times, ON cross-sections were graded for axonal injury. Anterior ONHs were collected at 0 hours to 10 days following CEI and RNA extracted for quantitative PCR measurement of selected messages. The functional impact of CEI was assessed by electroretinography (ERG). Results During CEI, mean arterial pressure (99 ± 6 mm Hg) and other physiologic parameters remained stable. An 8-hour CEI at 60 mm Hg produced significant focal axonal degeneration 10 days after exposure, with superior lesions in 83% of ON. Message analysis in CEI ONH demonstrated expression responses previously identified in minimally injured ONH following chronic IOP elevation, as well as their sequential patterns. Anesthesia with cannulation at 20 mm Hg did not alter these message levels. Electroretinographic A- and B-waves, following a significant reduction at 2 days after CEI, were fully recovered at 2 weeks, while peak scotopic threshold response (pSTR) remained mildly but significantly depressed. Conclusions A single CEI reproduces ONH message changes and patterns of ON injury previously observed with chronic IOP elevation. Controlled elevation of IOP can allow detailed determination of ONH cellular and functional responses to an injurious IOP insult and provide a platform for developing future therapeutic interventions.

Comparison of MicroRNA Expression in Aqueous Humor of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: A Pilot Study

Purpose MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that have been detected in human aqueous humor (AH). Prior studies have pooled samples to obtain sufficient quantities for analysis or used next-generation sequencing. Here, we used PCR arrays with preamplification to identify and compare miRNAs from individual AH samples between patients with primary open-angle glaucoma (POAG) and normal controls. Methods AH was collected before cataract surgery from six stable, medically treated POAG patients and eight age-matched controls. Following reverse transcription and preamplification, individual patient samples were profiled on Taqman Low Density MicroRNA Array Cards. Differentially expressed miRNAs were stratified for fold changes larger than ±2 and for significance of P < 0.05. Significant Kyoto Encyclopedia of Genes and Genomes pathways influenced by the differentially expressed miRNAs were identified using the predicted target module of the miRWalk 2.0 database. Results This approach detected 181 discrete miRNAs, which were consistently expressed across all samples of both experimental groups. Significant up-regulation of miR-518d and miR-143, and significant down-regulation of miR-660, was observed in the AH of POAG patients compared with controls. These miRNAs were predicted to reduce cell proliferation and extracellular matrix remodeling, endocytosis, Wnt signaling, ubiquitin-mediated proteolysis, and adherens junction function. Conclusions This pilot study demonstrates that miRNA expression within the AH of POAG patients differs from age-matched controls. AH miRNAs exhibit potential as biomarkers of POAG, which merits further investigation in a larger case-controlled study. This technique provides a cost-effective and sensitive approach to assay miRNAs in individual patient samples without the need for pooling.

The effect of age on the response of retinal capillary filling to changes in intraocular pressure measured by optical coherence tomography angiography

PURPOSE To compare the effect of elevated intraocular pressure (IOP) on retinal capillary filling in elderly vs adult rats using optical coherence tomography angiography (OCTA). METHODS The IOP of elderly (24-month-old, N=12) and adult (6-8month-old, N=10) Brown Norway rats was elevated in 10mmHg increments from 10 to 100mmHg. At each IOP level, 3D OCT data were captured using an optical microangiography (OMAG) scanning protocol and then post-processed to obtain both structural and vascular images. Mean arterial blood pressure (MAP), respiratory rate, pulse and blood oxygen saturation were monitored non-invasively throughout each experiment. Ocular perfusion pressure (OPP) was calculated as the difference between MAP for each animal and IOP at each level. The capillary filling index (CFI), defined as the ratio of area occupied by functional capillary vessels to the total scan area but excluding relatively large vessels of >30μm, was calculated at each IOP level and analyzed using the OCTA angiograms. Relative CFI vs IOP was plotted for the group means. CFI vs OPP was plotted for every animal in each group and data from all animals were combined in a CFI vs OPP scatter plot comparing the two groups. RESULTS The MAP in adult animals was 108±5mmHg (mean±SD), whereas this value in the elderly was 99±5mmHg. All other physiologic parameters for both age groups were uniform and stable. In elderly animals, significant reduction of the CFI was first noted at IOP 40mmHg, as opposed to 60mmHg in adult animals. Individual assessment of CFI as a function of OPP for adult animals revealed a consistent plateau until OPP reached between 40 and 60mmHg. Elderly individuals demonstrated greater variability, with many showing a beginning of gradual deterioration of CFI at an OPP as high as 80mmHg. Overall comparison of CFI vs OPP between the two groups was not statistically significant. CONCLUSIONS Compared to adults, some, but not all, elderly animals demonstrate a more rapid deterioration of CFI vs OPP. This suggests a reduced autoregulatory capacity that may contribute to increased glaucoma susceptibility in some older individuals. This variability must be considered when studying the relationship between IOP, ocular perfusion and glaucoma in elderly animal models.

Investigation of MicroRNA Expression in Experimental Glaucoma

MicroRNAs are small, endogenous noncoding RNAs that modulate post-transcriptional gene expression. Recent evidence suggests that they may have a potential role in the regulation of the complex biological responses that develop in response to elevated intraocular pressure. However, contemporary microRNA assay techniques (e.g., microarrays and next-generation sequencing) typically require large amounts of RNA template that are often times difficult to obtain from glaucomatous tissue. We describe in detail an experimental protocol utilizing targeted pre-amplification and low-density polymerase chain reaction arrays to circumvent this hurdle. This approach optimizes the simultaneous high-throughput screening of small tissue samples, such as the rodent optic nerve head, for up to 754 microRNA probes while also providing an opportunity for subsequent confirmatory reactions of technical or biological replicates.

Author Response: Comparison of MicroRNA Expression in Aqueous Humour of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: A Pilot Study

We thank Li and Wang for their interest in our work and thoughtful comments about our recent pilot study on the differential expression of microRNA (miRNA) within the aqueous humor (AH) of patients with primary open angle glaucoma (POAG) and normal controls. Until recently, the study of miRNA expression within biofluids was hindered by small sample volumes, which led to pooling of samples. However, this can now be overcome either through the use of targeted sample preamplification with low-density polymerase chain reaction (PCR) arrays, as we demonstrated, or through next-generation sequencing with adapter-driven amplification. Analyzing individual AH samples paves the way for future studies that can benefit from increased statistical power and precision. This will further our understanding of the mechanistic role of miRNAs upon the aqueous outflow facility and advance the pursuit of molecular biomarkers of outflow function. Li and Wang highlight two pertinent issues. First, they point out that comorbidity of dry age-related macular degeneration (AMD) may be a confounding factor in the analysis of AH miRNA expression. Confounding factors, from known and unknown sources, always are a concern in medical research. Our pilot study was not of sufficient size to examine formally whether the presence of AMD may or may not be a confounding factor, since AMD was present only in three of 14 samples analyzed. However, when considering whether a factor is likely to be a confounder, it is important to consider whether its presence provides a reasonable explanation for the observed outcome. The predicted biological impact of altered miRNA expression in these patients was to reduce cell proliferation, extracellular matrix remodeling, endocytosis, Wnt signaling, ubiquitin-mediated proteolysis, and adherens junction function. These biological processes have been described frequently in relation to trabecular meshwork (TM) dysfunction, but are not consistent with contemporary thinking regarding the pathogenesis of AMD. Thus, it is unlikely that the presence of AMD is a confounder for these TM-specific processes. Furthermore, our study specifically identified upregulation of miR-143 in glaucomatous AH. Since the publication of our report, targeted deletion of miR-143 in mice increased outflow facility and reduced IOP, while knockdown of miR-143 in human TM cells regulated actin dynamics. Thus, their results are consistent with ours and independently validate the important role of miR-143 in outflow regulation. Nonetheless, future studies with larger study populations can address this question more thoroughly through the stratification of miRNA expression not only by AMD status, but also by other ocular abnormalities to generate adjusted stratum-specific measures of relative miRNA expression. Secondly, Li and Wang argue that miRNA expression within plasma is equally important to that of AH. Only approximately 1% of plasma-derived proteins (and presumably miRNAs) are present in AH due to the blood–aqueous barrier. Thus, it is not surprising that we detected six miRNAs that were common to AH and plasma. However, it remains unclear whether the majority of plasma-derived miRNAs affect TM function and outflow regulation. Thus, we would propose, for the study of outflow regulation and IOP, that it would be more prudent to place the emphasis upon identifying AH miRNAs. However, if the intention is to discover novel systemic mechanisms that may underpin the highly complex pathogenesis of POAG, then the study of circulating miRNAs within plasma may be preferable, to include miRNAs known to affect retinal cell function.