A Porcine Organ-Culture Glaucoma Model Mimicking Trabecular Meshwork Damage Using Oxidative Stress.

Purpose: Re-cellularization of the trabecular meshwork (TM) using stem cells is a potential novel treatment for ocular hypertension associated with glaucoma. To assess the therapeutic efficacy of this approach, improved in vivo and ex vivo models of TM pathophysiology are needed. Here, we investigate whether oxidative stress, induced by hydrogen peroxide (H2O2), can model glaucomatous ocular hypertension in the readily available porcine anterior segment organ culture model. Methods: The impact of H2O2 on TM cell viability and function was first evaluated in vitro using primary porcine TM cells. Oxidative stress was then induced by H2O2 infusion into perfused porcine anterior segments. Trabecular meshwork function was assessed by tracking matrix metalloproteinase (MMP) activity and the ability of the preparation to maintain intraocular pressure (IOP) homeostasis after a flow challenge (doubled fluid infusion rate). Finally, the TM was evaluated histologically. Results: H2O2 treatment resulted in a titratable reduction in cellularity across multiple primary TM cell donor strains. In organ culture preparations, H2O2-treated eyes showed impaired IOP homeostasis (i.e., IOPs stabilized at higher levels after a flow challenge vs. control eyes). This result was consistent with reduced MMP activity and TM cellularity; however, damage to the TM microstructure was not histologically evident in anterior segments receiving H2O2. Conclusions: Titrated H2O2 infusion resulted in TM cellular dysfunction without destruction of TM structure. Thus, this porcine organ culture model offers a useful platform for assessing trabecular meshwork therapies to treat ocular hypertension associated with glaucoma.

A flexible, robust microbead-based assay for quantification and normalization of target protein concentrations.

Although there are many methods for quantifying the concentration of specific proteins in samples, current techniques are technically challenging or do not easily lend themselves to normalization. Here, we describe a microbead-based assay for quantifying specific protein concentration(s) that is high-throughput, inexpensive, simple-to-use, and intrinsically incorporates normalization against the sample total protein content. This assay, termed the FRANC assay, exploits high affinity biotin-streptavidin binding to couple sample proteins to streptavidin-labelled magnetic microbeads. Proteins are then antibody-probed, followed by labeling of proteins on the microbead with fluorescent dye, and flow cytometry-based analysis. The FRANC assay demonstrates detection limits for target proteins in the femtogram range, with a linear range up to as much as 10 ng. Normalization of target protein concentrations resulted in an 80% reduction in variability as compared to non-normalized measurements. We conclude that the FRANC assay offers attractive advantages over current methods for quantifying specific protein(s) in samples.