Elsevier

Clinical Neurophysiology

Volume 123, Issue 10, October 2012, Pages 2080-2091
Clinical Neurophysiology

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1G93A mice

https://doi.org/10.1016/j.clinph.2012.01.028Get rights and content

Abstract

Objective

To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS.

Methods

We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1G93A mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve.

Results

In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1G93A mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease.

Conclusion

The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1G93A mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction.

Significance

Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients.

Introduction

Amyotrophic Lateral Sclerosis (ALS, motor neuron disease) is a neurodegenerative disorder, clinically characterized by inexorable loss of upper (corticospinal) neurons and lower (cranial/spinal) motor neurons, causing progressive weakness and death within 3–5 years of diagnosis (Rowland and Schneider, 2001, Magnus et al., 2002, Mitchell and Borasio, 2007, Dupuis et al., 2011). The fundamental processes that lead to the loss of corticospinal and motor neurons are not understood (Rothstein, 2009). Theories of disease pathogenesis include effects of abnormal proteins, such as Cu/Zn superoxide dismutase 1 (SOD1) (Orrell, 2009), fused in sarcoma protein (FUS) (Blair et al., 2009), tar-DNA binding protein of molecular weight 43 kDa (TDP43) (Kabashi et al., 2008), optineurin (Maruyama et al., 2010) and C9orf72 (DeJesus-Hernandez et al., 2011, Renton et al., 2011), mitochondrial dysfunction and glutamate excitotoxicity (Rothstein, 2009).

The need to quantify motor unit number in disorders of muscle innervation led to the development of motor unit number estimation (MUNE) techniques that estimate the number of motor units innervating a muscle or group of muscles (McComas et al., 1971, Arasaki et al., 1997, Shefner et al., 2002, Daube, 2006, Bromberg and Brownell, 2008). A number of MUNE methods exist. The Poisson method (Daube, 1995) has been used in clinical studies but there are limitations in applying this method through the course of ALS (Shefner et al., 2007). The incremental method uses gradual increases in strength of low intensity electrical stimulation but is limited by sampling issues to determine motor unit size and alternation (Galea et al., 1991). The multiple point stimulation method, which relies on the sampling of motor units that are activated at low strength of electrical stimulation has been widely applied and shown to be reproducible (Felice, 1995). The multi-point incremental method, that has recently been described, incorporates features of the incremental and multipoint methods and appears widely applicable (Shefner et al., 2011).

We have developed a Bayesian method of MUNE (Ridall et al., 2006, Ridall et al., 2007, Henderson et al., 2007) using Bayesian statistics to analyze stimulus–response curves obtained by graded incremental electrical stimulation (from baseline to the maximum compound muscle action potential, CMAP) of a motor nerve. This method is based on the physiology of motor unit activation and accounts for variability in motor unit threshold, variability in size between and within single motor unit action potentials, and alternation of motor unit firing (McCombe et al., 2009). Our method has the advantages of directly recording all motor units as they are progressively activated, of dealing with motor unit variability by allowing motor unit size to vary as part of the Bayesian model, and of successfully incorporating motor unit alternation by recognizing that the size of the applied stimulus will influence probabilistic motor unit activation. Hence, unlike current statistical methods, our Bayesian statistical model makes assumptions that better reflect the natural physiological mechanisms that are involved in the stimulus and response of motor units.

As there is no “gold-standard” for determining the number of motor units in a muscle, debate remains about which MUNE method gives the most accurate results. Hence rather than comparing Bayesian MUNE with an existing MUNE method, we chose to evaluate our Bayesian MUNE method by a direct comparison of the estimated motor unit number with the number of motor neurons in the region of the spinal cord innervating that muscle. Previously, attempts have been made to compare an incremental MUNE method with histological quantification; however this study produced poor correlations between MUNE and histological nerve fiber counts (Arasaki et al., 1997, David et al., 2010). In the present study, we have followed the loss of motor units over time in transgenic mice over-expressing human superoxide dismutase 1 with a glycine to alanine switch at the 93rd codon (SOD1G93A) mutation (Gurney et al., 1994), which is an animal model of ALS (Scott et al., 2008).

There have been previous MUNE studies in SOD1G93A mice (Hegedus et al., 2007, Hegedus et al., 2009b). The incremental twitch MUNE method detected approximately 60 motor units in the medial gastrocnemius at 40 days of age but required surgically invasive isolation of the gastrocnemius tendon (Hegedus et al., 2007, Hegedus et al., 2009b). A modified incremental MUNE method measured approximately 100 motor units in the distal hindlimb at 90 days of age (Shefner et al., 2006) while comparison of the incremental MUNE method with a multipoint stimulation method in 120 day old SOD1G93A mice found approximately 100 motor units for incremental MUNE compared to 110 motor units for multipoint stimulation MUNE (Shefner et al., 2002).

In this study, we evaluate the Bayesian MUNE method by comparing the numbers of motor units estimated by MUNE with the numbers of motor neurons in the spinal cords of the same wild-type mice and SOD1G93A mice. Groups of these mice were studied before the onset of overt signs of motor dysfunction, at the onset of overt symptoms and at the terminal stages of disease, to follow the course of disease. This study is novel in using both neurophysiological and histological techniques in the same mice to estimate motor unit numbers at different stages of disease progression.

Section snippets

Animals

Wild-type and SOD1G93A mice (B6.Cg-Tg(SOD1-G93A)1Gur/J) were bred at the University of Queensland. B6.Cg-Tg(SOD1-G93A)1Gur/J carry a high copy number of the mutated allele of the human SOD1 gene. The University of Queensland Animal Ethics Committee approved all animal procedures, which were in accordance with national guidelines. SOD1G93A heterozygous males were mated to wild-type females and the offspring were genotyped for the SOD1G93A transgene. Experiments were conducted on age-matched

MUNE studies

To perform MUNE estimation, we record the stimulus response curve, which is a graphical display of the CMAPs evoked by progressive increases in the strength of stimulus delivered to the nerve. The MUNE program plots stimulus–response curves as the stimulus level against the area of the CMAP response. In all wild-type animals the stimulus–response curves were continuous (Fig. 1A, C and E). The stimulus–response curves from pre-symptomatic SOD1G93A mice were comparable to age matched wild-type

Discussion

The SOD1G93A mouse is currently the standard animal model of ALS (Turner and Talbot, 2008, Ludolph et al., 2010). There have been previous studies using MUNE to follow disease progression in SOD1G93A mice, but there have been no previous studies that used histological evaluations and neurophysiology studies in the same animals to determine whether the MUNE methods were accurate (Shefner et al., 2002, Shefner et al., 2006, Hegedus et al., 2009a, Hegedus et al., 2009b, Shefner, 2009). In this

Acknowledgements

We thank C. Pfluger for his technical assistance and Dr. F. Steyn for critical reading of the manuscript. This work was supported by the National Health and Medical Research Council of Australia (NHMRC 569698 to PAM, RDH, ANP, PGR and MCB). The authors have no conflicts of interest.

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