NMR IN BIOMEDICINE NMR Biomed. 2004;17:28–32 Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/nbm.861
MRI detects early hindlimb muscle atrophy in Gly93Ala superoxide dismutase-1 (G93A SOD1) transgenic mice, an animal model of familial amyotrophic lateral sclerosis Keith J. Brooks, Mark D. W. Hill, Paul D. Hockingsy and David G. Reid* GlaxoSmithKline, The Frythe, Welwyn, Herts AL6 9AR, UK Received 26 September 2003; Revised 21 November 2003; Accepted 24 November 2003
ABSTRACT: MRI has been used to measure hindlimb muscle volume in female and male transgenic mice overexpressing the Gly93Ala (G93A) mutant human superoxide dismutase 1 (SOD1), a widely used model of familial amyotrophic lateral sclerosis (FALS), over the first 4 months of life. Significant decreases in the hindlimb muscle volume of the female G93A SOD1 mice were evident from 11 weeks of age, before other overt pathology appeared. By 15 weeks volume had decreased by 37% compared with 7 weeks, from 0.84 0.04 cm3 (mean standard deviation, n ¼ 6) to 0.54 0.07 cm3, ( p < 0.05), despite an increase in body weight of ca. 12% (from 16.2 1.4 to 18.1 0.7 g). Female wild-type volume increased by ca. 30% whilst the body weight increased by 15%. Muscle wasteage was less (0.82 0.1 to 0.65 0.02 cm3, p < 0.05, n ¼ 6) in male G93A SOD1 mice between 8 and 16 weeks of age, against a body weight increase trend from 20.7 0.4 to 21.6 0.5 g, ( p > 0.05). Wild-type male muscle volume did not change significantly over this period, with an increase in body weight of 20%. Longitudinal MRI hindlimb muscle volume measurements may provide a straightforward, rapid, non-invasive and sensitive, way of monitoring outcome of experimental ALS treatments. Copyright # 2004 John Wiley & Sons, Ltd. KEYWORDS: MRI; SOD1; G93A; familial amyotrophic lateral sclerosis, FALS; transgenic; mouse; muscle
INTRODUCTION Mice expressing the Gly93Ala mutant form of superoxide dismutase 1 (G93A SOD1) display many of the characteristics of human familial amyotrophic lateral sclerosis (FALS),1–5 probably mediated by an alteration of function in the copper- and zinc-containing enzyme leading to increased production of reactive free radicals.6–8 MRI is an excellent non-invasive technique for imaging changes in soft tissue anatomy and function in experimental animals and man. Evidence has been presented for brain MRI signal9–13 and functional14 abnormalities in human amyotrophic lateral sclerosis sufferers. Although one might expect analagous brain and central nervous system MRI markers to be observeable in models of FALS like the G93A SOD1 transgenic mouse there are no such literature reports, possibly because of the subtlety of the effects, and the constraints on functional MRI imposed by working with anaesthetised animals. *Correspondence to: D. G. Reid, GlaxoSmithKline, The Frythe, Welwyn, Herts AL6 9AR, UK. E-mail:
[email protected] y Current address: AstraZeneca, Pepparedsleden 1, S-43183 Mo¨lndal, Sweden. Abbreviations used: ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; G93A, glycine-93 to alanine-93; Gly93Ala, glycine-93 to alanine-93; SOD-1, superoxide dismutase-1. Copyright # 2004 John Wiley & Sons, Ltd.
We communicate here some results from an MRI phenotyping exercise carried out on groups of G93A SOD1 transgenic mice of both sexes, showing that hindlimb skeletal muscle wasteage is easily measureable and may serve as an early non-invasive disease marker in animal FALS models.
METHODS Animals All experiments complied with the UK Animals (Scientific Procedures) Act 1986 and the GSK Code of Conduct for Animal Experimentation. Six male and six female mutant mice (Jackson Laboratories, Bar Harbor, ME, USA) containing a transgene carrying a high copy number of a mutant allele human SOD1 containing a Gly93Ala substitute [strain designation B6SJL-TgN (SOD1-G93A) 1Gur] backcrossed onto a B6 background, and age- and gender-matched wild-type littermate controls, were imaged at 7 T (Bruker Biospec 7/20) under isoflurane anaesthesia (1–1.5%, flow rate 0.8–1.0 L/min) controlled on the basis of respiratory parameters; body temperature was maintained using a heated mattress. In pilot experiments it was shown that image quality was not affected by motion of the hindlimbs, or by cardiac and NMR Biomed. 2004;17:28–32
EARLY HINDLIMB MUSCLE CHANGES IN G93A SOD1 MICE
respiratory activity. Transgenic animals were euthanased when they began to show overt signs of gait impairment at about 16 weeks of age.
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plied by slice thickness (1 mm) were added to give a measure of hindlimb muscle volume.
Statistical analysis MRI Single spin echo imaging was performed in a Bruker 35 mm i.d. birdcage design RF coil inside an actively shielded Bruker BGA-9 gradient set, using the following parameters: TE/TR 8/1670 ms, SW 50 kHz, image matrix 128 128 pixels; FOV 40 40 mm axial slice orientation covering the entire hindlimb region with 64 1 mm slices, 16 averages. No fat suppression was used so that contrast between muscle and visceral, epididymal and subcutaneous fat deposits was maximized, and because fat–water chemical shift misregistration artefacts were judged to have an insignificant effect on measured volumes.
Intragroup and intergroup statistical significances were calculated in Microsoft Excel using Student’s paired, and unpaired, two-tailed t-tests, respectively.
RESULTS Figure 1 shows representative axial slices from the multislice datasets from female and male G93A SOD1 transgenic and wild-type mice at the beginning and end of their respective 8 week scanning periods. Figure 2 plots the changes in hindlimb muscle volume and body weights in both the female and male G93A SOD1 transgenic and wild-type mice.
Image analysis Hindlimb volumes Muscle was manually segmented from fat and bone using the ROI tool in the Bruker ParaVision (version 2.1) software, the raw output from which is area in number of pixels; resultant cross sectional surface areas multi-
Females [Fig. 2(a)]. As early as 7 weeks of age the hindlimb muscle MRI volumes of the female G93A SOD1 transgenic mice were significantly smaller than
Figure 1. Representative axial slices through the hindlimbs of G93A SOD1 transgenic (top panels) and wild-type (bottom panels) female (left) and male (right) mice at the first (females, 7 weeks; males, 8 weeks) and last (females, 15 weeks; males, 16 weeks) imaging time points. Pairs of panels depict the same individual at the early and late time points Copyright # 2004 John Wiley & Sons, Ltd.
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Figure 2. Time course of hindlimb muscle volume (a, females; b, males) and body weight (c, females; d, males) changes, in G93A SOD1 transgenic (&) and wild-type (&) mice. Intragroup and intergroup statistical significances (Student’s two-tailed paired and unpaired t-tests, respectively) are indicated as follows: intragroup relative to baseline time point values, #p < 0.05, ##p < 0.01; intergroup, *p < 0.05, **p < 0.01. Intergroup statistical significances were all maintained from baseline, and are not marked on the graphs after this time point. Error bars signify standard deviations of the mean
those of the wild-type mice [0.84 0.04 cm3 (mean standard deviation) vs 0.98 0.1 cm3, p < 0.05, n ¼ 6]. Subsequently the G93A SOD1 transgenic muscle volume declined by 25% (0.63 0.04 cm3, p < 0.05 relative to week 7) by 10 weeks and by 36% (0.54 0.07 cm3, p < 0.01 relative to week 7) by 15 weeks of age. The wild-type female controls’ hindlimb muscle volume increased slightly but not significantly during the same period (0.98 0.09 cm3 at week 7 to 1.02 0.15 cm3 at week 15). Males [Fig. 2(b)]. Similarly, at 8 weeks of age the hindlimb muscle volumes of the male G93A SOD1 transgenic mice were significantly smaller than those of the wild-type (0.82 0.09 vs 1.26 0.1 cm3, p < 0.01, n ¼ 6). Thereafter transgenic muscle volume declined significantly (to 0.75 0.03 cm3, p < 0.05) at 12 weeks of age, and to 0.65 0.02 cm3 at 16 weeks ( p < 0.01), reductions of 9 and 21% relative to 8 weeks respectively. We did not observe a significant change in our male wild Copyright # 2004 John Wiley & Sons, Ltd.
type controls’ hindlimb muscle volumes from the value at 8 weeks of age (1.26 0.1 cm3).
Body weights Females [Fig. 2(c)]. Body weights of the G93A SOD1 transgenic mice were significantly lower at week 7 than those of the wild-type controls (16.2 1.4 vs 18.6 0.2 g, p < 0.05). In contrast to hindlimb muscle volumes, however, body weights of both the G93A SOD1 transgenic mice and the wild-type controls increased steadily (although not significantly in the transgenic group) over the study period (to 18.1 0.7 and 21.4 0.4 g, respectively, at week 15), although the former were always significantly lighter than the latter. Males [Fig. 2(d)]. Similarly body weights of the male G93A SOD1 transgenic mice were significantly lower at week 8 than those of the male wild-type controls NMR Biomed. 2004;17:28–32
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(20.7 0.4 vs 22.9 0.6 g, p < 0.05). Weights of both groups were increased (to 21.6 0.5 g, p < 0.05, and 28.0 1.44 g, p < 0.05) at week 16.
DISCUSSION Non-invasive tests of potential ALS therapies in mutant SOD1 transgenic mice have involved behavioural observations, measurement of skeletal muscle strength, hindlimb function, and survival time.15–21 Thus, for instance, Klivenyi et al.15 reported ameliorative effects of creatine administration on motor performance and survival, as well as enhanced preservation of motor and substantia nigra neurons. An antagonist of the AMPA subtype of glutamate receptors also improves survival and grip strength.16 Azzouz et al.17 observed decline in body weight (after ca 111 days) and survival correlating with deteriorating motor neuron electrophysiological parameters as animals aged. Genetically engineered overexpression of neurofilament proteins extends survival times.18 Functional muscle strength tests have been used to complement microscopic measurements of motor neuron degeneration in defining stages of pathology progression.19 Survival is also extended by treatment with reactive oxygen scavengers (iron porphyrins)20 and copper chelators.21 Generally, deterioration in function, and sharp decline in number of animals in a given cohort surviving, becomes significant from about 120 days (ca 17 weeks) of age in untreated G93A SOD1 transgenic mice. Although formal behavioural testing was not carried out in the study reported here, the animals were regularly observed for the onset of clinical signs, especially impairment of gait, which only became obvious immediately before the decision to euthenase (15–16 weeks of age). Although our G93A SOD1 transgenic mice were consistently lighter than gender- and age-matched controls, body weight generally increased with time (although not markedly in the transgenic males) until euthanasia; only after about 16 weeks have body weights of such transgenic animals been reported to begin to decline.17 The present study was iniated as an exercise in the use of whole body mouse anatomical MRI to detect morphological markers of disease in a transgenic model in vivo without any preconceptions as to what these markers may be. It soon became obvious that MRI sensitively indicated a significant reduction in G93A SOD1 transgenic mouse hindlimb muscle volume between even the two early (week 7 or 8) and intermediate (week 11 or 12) imaging time points, especially in our group of females. This reduction occurred against the tendency for total body weight itself to increase, and preceded any overt behavioural changes such as gait impairment. Numerous studies have identified biochemical and histopathological abnormalities in G93A SOD1 transgenic mice towards the end stages of the disease when Copyright # 2004 John Wiley & Sons, Ltd.
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muscle weakness has manifested. Kong and Xu have shown that motor neuron mitochondrial histopathological19,22,23 and biochemical22,24 abnormalities do actually precede the onset of the muscle weakness, which becomes measureable after ages of about 120 days, and before massive dendritic mitochondrial vacuolation appears. We did not complement our imaging with these approaches, mainly because they are terminal and preclude following the progression of disease in the same animals (of which only limited numbers were available). Thus, while it is possible that histopathological or biochemical abnormalities in our animals coincided with the onset of hindlimb muscle atrophy, we do not have data to this effect. In conclusion, it is conceivable that MRI will provide a sensitive, straightforward, non-invasive, serial, method of monitoring skeletal muscle atrophy or wasteage in animal models of ALS or FALS, which may be a useful complement to behavioural, survival and histopathological methods of monitoring response to experimental therapies.
Acknowledgements Judy Latcham, Kim Prescott and Julia Reddy are thanked for animal breeding and husbandry, and Steve Skaper for his interest in this work.
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