Quantification of Deficits in Spatial Visual Function of Mouse Models for Glaucoma

Department of Ophthalmology, University of Missouri - Kansas City, Kansas City, Missouri, United States
Investigative ophthalmology & visual science (Impact Factor: 3.4). 02/2011; 52(6):3654-9. DOI: 10.1167/iovs.10-7106
Source: PubMed


DBA/2J mice are a standard preclinical glaucoma model, which spontaneously developed mutations resulting in chronic age-related pigmentary glaucoma. The goals of this study were to identify the degree of visual impairment in DBA/2J mice before and after disease onset by quantifying the optokinetic reflex responses and to compare them to the less-researched strain of DBA/2NHsd mice.
Visual performance was measured in healthy, nonglaucomatous, and glaucomatous male DBA/2NHsd or DBA/2J mice using a visuospatial testing box. The optokinetic reflex resulting in optomotor head tracking was manually detected. Measured threshold levels equate to the maximum contrast or spatial frequency the mouse responds to. Intraocular pressure (IOP) was measured by applanation tonometry.
IOP increased with age in both DBA/2J and DBA/2NHsd mice and was not different between the two substrains. Both visual acuity and ability to detect contrast decreased significantly, and similarly with age in both substrains. However, DBA/2NHsd had poorer visual acuity even at a younger age compared to age-matched DBA/2J mice.
Both DBA/2J and DBA/2NHsd mice show a progressive glaucomatous phenotype of age-related increases in IOP and loss of visual acuity and contrast sensitivity when compared to other inbred or outbred strains. Given the similar increases in IOP and contrast sensitivity threshold and loss of visual acuity between these two DBA/2 substrains, we also conclude that DBA/2NHsd mice are a suitable alternative model for pigmentary glaucoma.

Download full-text


Available from: Simon Kaja, Feb 19, 2015
    • "This is supported by a significant body of clinical evidence on normotensive glaucoma (Mozaffarieh and Flammer, 2013), and on glaucoma models where disease progression occurs without ocular hypertension (Mi et al., 2012). Indeed, it has been shown that neuroprotection can acieve preservation of visual function in the presence of continually elevated IOP as a disease-causing mechanism (Burroughs et al., 2011; Prokai-Tatrai et al., 2013). "
    [Show abstract] [Hide abstract]
    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 strategies. Copyright © 2015. Published by Elsevier Ltd.
    Experimental Eye Research 06/2015; 138. DOI:10.1016/j.exer.2015.05.023 · 2.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The mammalian proprotein convertase subtilisin kexins (PCSKs) previously called proprotein or prohormone convertases (PCs) are a family of Ca(+2)-dependent endoproteases in the subtilisin family. These proteolytic enzymes exert their many crucial physiological and biological functions in vivo via their ability to cleave larger inactive precursor proteins into their biologically active mature forms. This event takes place in a highly efficient and selective manner. Such actions of PCSKs either alone or in combination to cleave specific protein bonds are the hallmark events that not only define the normal functions and metabolism of the body but also may lead to a variety of diseases or disorders with associated conditions. These include among others, diabetes, obesity, cancer, cardiovascular diseases, reproduction abnormalities as well as viral bacterial infections. These conditions were the direct consequences of an enhanced level of enzymatic activity of one or more PCSKs except only PCSK9, whose protease activity in relation to its physiological substrate has yet to be characterized. Owing to this finding, a large number of research studies have been exclusively devoted to develop rapid, efficient and reliable in vitro methods for examining the protease activity of these enzymes. Several assays have been developed to monitor PCSK activity and these are widely used in chemical, biochemical, cellular and animal studies. This review will cover various methodologies and protocols that are currently available in the literature for PCSK activity assays. These include liquid phase methods using fluorogenic, chromogenic and intramolecularly quenched fluorescent substrates as well as a newly developed novel solid phase fluorescence method. This review will also highlight the usefulness of these methodologies and finally a comparative analysis has been made to examine their merits and demerits with some key examples.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 768:127-53. DOI:10.1007/978-1-61779-204-5_6 · 1.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we present a virtual experimental arena to perform behavioral experiments on small vertebrates. It consists of a 360° visual stimulation realized by four LC displays and an automated camera-based head tracking system. Based on these two components, visual stimuli are repositioned depending of the animal's head position in a closed-loop approach. We present two algorithms to automatically perform robust head tracking on mice and compare their precision to manual tracking by a human observer.
Show more