Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes

Abstract

Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25 μM. This was paralleled very effectively in the assays measuring cell death and cell viability with half-maximal effects of 241 ± 20 μM and 194 ± 5 μM for tBHP in the lactate dehydrogenase (LDH) release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays, respectively. Pre-treatment with 100 μM Trolox decreased the sensitivity of ONHAs to tBHP. Half-maximal effects increased to 396 ± 12 μM tBHP in the LDH release assay and to 383 ± 3 μM tBHP in the MTT assay. Vehicle treatment (0.1% v/v ethanol) did not significantly affect cellular responses to tBHP. Antioxidant treatment increases ONHA viability and reduces the deleterious effects of oxidative stress. Our experiments provide important feasibility data for utilizing primary rat ONHAs in plate reader-based assays assessing novel therapeutics for glioprotection of the optic nerve and the optic nerve head in glaucoma and related disorders. Furthermore, our novel, standardized protocols have the potential to be readily adapted to high-throughput and high-content testing stra...

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants

“…Primary cultures of adult rat optic nerve head astrocytes were maintained and validated as we have previously described in detail [5,6,26]. Cultures of passages 10 to 20 were used for experiments.…”

“…To determine the glioprotective effects of antioxidants on reactive astrocytosis, we conducted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate de-hydrogenase (LDH) release assays were performed, as previously described by us in detail [5,6]. In brief, supernatants (50 µL) were collected and LDH assays performed.…”

“…Specifically, astrocytes were treated with 0.005% Mn-TM-2-PyP or PBS vehicle, 50 µM resveratrol or 0.1% DMSO vehicle, 100 µM Trolox or 0.1% ethanol vehicle, or 5 µM xanthohumol or 0.01% DMSO vehicle for 24 h. Concentrations were determined based on previous studies [5,6,19], the published literature [23,29,30] and cytotoxicity studies in optic nerve head astrocytes ( Figure S1). Quantitative immunoblotting was performed to determine NOX4, SOD2, and Nrf2 protein expression.…”

“…Previous studies by us and others have shown that primary optic nerve head astrocytes are vulnerable to the deleterious effects of oxidative stress and that antioxidants can protect against chemically induced oxidative stress [5][6][7][8]. Investigations into the underlying mechanisms, however, have primarily focused on the expression of apoptosis-related genes and proteins, such as caspases, Bcl-2, Bcl-2-associated protein and related signaling pathways [9,10].…”

Ion Channels in Aging and Aging-Related Diseases

“…Although the exact pathophysiology is still not completely understood, there is evidence to support ischemic, mechanical, and inflammatory components. In an effort to protect the integrity of the optic nerve and prevent the loss of retinal ganglion cells, research efforts have recently concentrated on glioprotection approaches targeting ONHAs [62,63]. Glia and neuronal cells alike utilize fine-tuned calcium signaling pathways to control physiological functions.…”