Noninvasive in vivo assessment of muscle impairment in the mdx mouse model – A comparison of two common wire hanging methods with two different results

Abstract

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disorder that arises from a mutation in the gene for the cytoskeletal protein dystrophin, normally expressed in the myofibres. The most widely applied animal model in DMD basic research is the C57BL/10ScSn-mdx/J mouse, commonly referred to as the “mdx mouse”. The potential benefit of novel interventions in this in vivo model is often assessed by functioning tests, as the improvement of muscle impairment is the final goal of all approaches to treat DMD.

In this study we compared two (TWHT) and four limb wire hanging tests (FWHT) for utility in evaluating muscle impairment in the mdx-mouse relative to its C57BL/10 wild-type counterpart. Our objective was to determine an optimal approach to perform wire hanging measurements in this model system such that latency to fall is indicative of the dystrophic phenotype that provides a quantitative measure of its presentation, and can be used to assess functional improvements that result from therapeutic intervention.

Surprisingly the results of the latency times in the TWHT did not allow discrimination between the mdx population and their healthy counterparts, whereas hanging times in the FWHT enabled ready discrimination between the muscle function of mutant and wild-type animals. Furthermore, we analyzed confounding factors that explain the strengths and weaknesses of each wire hanging test configuration.

The results of this study are of relevance for investigators who rely on pre clinical function tests to assess potential therapies in DMD.

Introduction

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disorder that arises from a mutation in the gene for the cytoskeletal protein dystrophin, normally expressed in the myofibres (Guglieri and Bushby, 2010, Hoffman et al., 1987). The disease is evidenced by a progressing degeneration of the skeletal muscle and attenuation of muscle function that ultimately leads to a loss of ambulation and death, usually in early adult age. For more than 30 years, DMD has been of subject of much research interest, and the most widely applied animal model in these studies is the C57BL/10ScSn-mdx/J mouse, commonly referred to as the “mdx mouse” (Grounds et al., 2008, Vetrone et al., 2009, Bulfield et al., 1984, Hoffman et al., 1987, Ryder-Cook et al., 1988). The mdx mouse genome contains a stop codon in exon 23 of the dystrophin gene, resulting in the termination of this gene sequence (Willmann et al., 2009). Although there is no detectable dystrophin protein in mdx mice, the dystrophic phenotype manifest is relatively mild in contrast to that evidenced in humans (Partridge, 1991, Sacco et al., 2010), and further, the severity of muscle weakness can vary markedly among individual mice.

To be sure, the ultimate aim of any potential therapeutic approach targeted at treating DMD is improvement of muscle function. The benefit of potential therapies developed in animal models must necessarily be demonstrated through functional testing (Grounds et al., 2008, Lu et al., 2003, Koppanati et al., 2010, Vetrone et al., 2009).

Ideally, functional tests of mdx mice need to be both sensitive and selective such that differences between muscle performance in treated and untreated mutant mice and wild-type controls can be reliably measured. A variety of invasive and non-invasive functioning tests have been suggested in the literature for this purpose (Grounds et al., 2008, Lu et al., 2003, Koppanati et al., 2010). Many of these entail specialized apparatus or other complex arrangements, and hence, the inter-examiner reliability suffers as it is challenging to precisely reproduce the testing configuration between different laboratories. While easier to implement, other, more simplistic methods can suffer the disadvantage of being difficult to administer objectively (Carlson et al., 2010). In our own studies of muscle regeneration in mdx mice, we found apparent skeletal muscle strength and coordination was dependent upon the specific method used for assessment, and that in the context of the subtle presentation of the myotrophic phenotype in this model, artifacts related to the measurement approach often confounded evaluation of our experimental data. Here we report what we have found to be an efficient, easy to implement, and robust method to quantitatively assess in vivo muscle function in the mdx mouse.

We set about defining a protocol for functional testing that meet the following objectives: (a) the test should not depend on cognitive and/or behavioral tendencies of individual animals, and muscle strength and tone should be quantified without being influenced by other abilities, such as innate intelligence or strategies to avoid or solve certain situations; (b) the possibility for bias or subjective interpretation of the test data should be minimized; and (c) the testing apparatus should be uncomplicated and such that other researchers could replicate it and perform the test easily, thereby enabling different approaches to treat DMD to be universally and systematically compared in terms of functional outcome.

There is frequent reporting in the literature of muscle functional evaluation in mice by means of “wire hanging” behavioral tests wherein latency to fall after being suspended from a substrate capable of being gripped is quantified. Although ostensibly straightforward, the term “wire hanging test” has been used to describe implementations as varied as: a single suspended wire, rod or cotton string; an array of wires in the form of a grid, mesh or cage lid; both horizontally and vertically oriented wires and grids; apparatus that is stationary, inclined, shaken, and/or inverted; and placement of the front, hind or all four paws on the wire or grid to commence the test (Vetrone et al., 2009, van Putten et al., 2010, Golumbek et al., 2007, Vieira et al., 2008, Hashemi et al., 2007, Carlson et al., 2010).

This broad usage is ambiguous, and several of these various approaches wire hanging test have been applied to the functional evaluation of the mdx mouse. In our view, the most critical aspect of the various wire hanging evaluations that should be delineated is between the two-limb type of test, where the mouse is placed with its front paws on a rod or wire that is elevated between a pair of vertical posts (Fig. 1A), and the four-limb type, where the mouse is forced to hang with all four paws upside down from a wire-grid (Fig. 1B). Some degree of standardization in the nomenclature does appear to exist as these are frequently referred to respectively as “wire hanging” and “grid hanging” tests. (Vieira et al., 2008, Carlson et al., 2010) Though, these designations are not necessarily, universally used.

Nevertheless, such hanging tests broadly meet the previously outlined criteria. Compliance is usual, and further, these tests draw upon the animals’ instinctive behavior to avoid falling, thus triggering an innate response to hold on as long as muscle endurance lasts. Provided that the test is implemented in a manner so that learned behaviors that influence hanging time, such as coordinated hanging using the whole limb (compared to gripping with the paws alone) or sitting, are restricted, bias and uncertainty in interpreting the results can be mitigated, and a good correlation between latency to fall and muscle strength can be achieved.

We compared two and four limb wire hanging tests for utility in evaluating muscle strength and tone the mdx-mouse relative to its C57BL/10 wild-type counterparts. Our objective was to determine an optimal approach to perform wire hanging measurements in this model system such that latency to fall is indicative of the dystrophic phenotype that provides a quantitative measure of its presentation, and that can be used to assess functional improvements that result from therapeutic intervention.