Reduction of lower motor neuron degeneration in wobbler mice by N-acetyl-L-cysteine

Abstract

The murine mutant wobbler is a model of lower motoneuron degeneration with associated skeletal muscle atrophy. This mutation most closely resembles Werdnig-Hofmann disease in humans and shares some of the clinical features of amyotrophic lateral sclerosis (ALS). It has been suggested that reactive oxygen species (ROS) may play a role in the pathogenesis of disorders such as ALS. To examine the relationship between ROS and neural degeneration, we have studied the effects of agents such as N-acetyl-L-cysteine (NAC), which reduce free radical damage. Litters of wobbler mice were given a 1% solution of the glutathione precursor NAC in their drinking water for a period of 9 weeks. Functional and neuroanatomical examination of these animals revealed that wobbler mice treated with NAC exhibited (1) a significant reduction in motor neuron loss and elevated glutathione peroxidase levels within the cervical spinal cord, (2) increased axon caliber in the medial facial nerve, (3) increased muscle mass and muscle fiber area in the triceps and flexor carpi ulnaris muscles, and (4) increased functional efficiency of the forelimbs, as compared with untreated wobbler littermates. These data suggest that reactive oxygen species may be involved in the degeneration of motor neurons in wobbler mice and demonstrate that oral administration of NAC effectively reduces the degree of motor degeneration in wobbler mice. This treatment thus may be applicable in the treatment of other lower motor neuropathies.

Fig. 1.

NAC reduces axonal loss in wobblerfacial nerve. A, Analysis of the superior medial branch of the facial nerve. B, Plot of the total cross-sectional area of the medial facial nerve for each group. Values represent the mean ± SEM; Asterisk signifies a significant difference (p < 0.05), as compared with the wr/wr group. Control-treatedwr/wr mice, n = 17; NAC-treatedwr/wr mice, n = 11; NW mice,n = 11; NW(N) mice, n = 10.wr/wr versus wr/wr(N),p < 0.004; wr/wr versus NW,p < 0.005; wr/wr versus NW(N),p < 0.006. C, Number of axons in nerve cross sections. For all groups n = 10.wr/wr versus wr/wr(N),p <0.027; wr/wr versus NW,p < 0.030.

Fig. 2.

NAC prevents losses in axon caliber inwobbler mice. Shown is the distribution of axon areas in medial branch of the facial nerve. A, Axon distributions of NW and NW(N) mice,n = 7 per group. B, Axon distribution of wr/wr mice, n = 6.C, Axon distribution of NAC-treated wr/wrmice, n = 6. D, Comparison of axon distributions in each treatment group. Values represent the mean ± SEM; the asterisk signifies a significant difference (p < 0.05) between wr/wr(N)and wr/wr groups at the indicated axon area; (+) signifies a significant difference between NW andwr/wr groups at the indicated axon area. Within a given group, individual percentage values did not vary by more than ±2% of the mean.

Fig. 3.

NAC reduces the loss of ChAT-positive neurons in the cervical spinal cord of wobbler mice. Total numbers of choline acetyltransferase-positive neurons are indicated for each group (n = 4). Numbers represent counts from every fifth section in a serial series of 10 μ transverse sections through the entire c4–c7 region. Values represent the mean ± SEM; asterisk signifies a significant difference (p < 0.05), as compared with thewr/wr group.

Fig. 4.

NAC reduces the atrophy of forelimb muscles inwobbler mice. A, Plot of triceps muscle mass for each treatment group (n = 12). Values represent the mean ± SEM; (*) signifies a significant difference (p < 0.05), as compared with the wr/wr group. For wr/wrversus wr/wr(N), p < 0.003.B, Plot of flexor carpi ulnaris muscle mass for each treatment group. n ≥ 12 animals per group; values represent the mean ± SEM; asterisk signifies a significant difference (p < 0.05), as compared with the wr/wr group. For wr/wrversus wr/wr(N), p < 0.021.C, Percentage of distribution of muscle fiber areas. For each treatment group, mean cross-sectional areas were determined for 70 muscle fibers in each of four separate individuals. Muscle cross sections were taken at the midpoint of the main body of the flexor carpi ulnaris. Then areas were determined for the medial fibers within each cross section. All fiber areas were determined in a blind manner from coded 1 μ thin sections. For each pair-wise comparison of the treatment groups [wr/wr:wr/wr(N),wr/wr:NW,wr/wr(N):NW],p < 0.001.

Fig. 5.

NAC reduces functional losses in the forelimb ofwobbler mice. Seven-week-old mice from each of the treatment groups were examined by an observer blinded with respect to the animals’ treatment status. Functional classes were divided as follows: 1, no apparent atrophy, limbs outstretched when suspended, continuous grasping at proximal surface, normal inclination of the phalanges; 2, visible atrophy of limbs, limbs outstretched when suspended, continuous grasping at proximal surface, normal inclination of phalanges; 3, significant atrophy of limbs, limbs not outstretched when suspended, tentative grasping at proximal surface, abnormal (≤60°) inclination of phalanges;4, severe limb atrophy, limbs held closely to body when suspended, no grasping at proximal surface, abnormal (≥90°) inclination of phalanges. Forelimbs were scored separately for each animal. For the NW and NW(N) groups,n = 6, with all animals scoring “1.” Forwr/wr, wr/wr(N) groups,n = 8 animals per group. For each pair-wise comparison of the treatment groups [wr/wr vswr/wr(N), wr/wr vs NW orNW(N), wr/wr(N) vs NW orNW(N)], p < 0.001.

Fig. 6.

NAC treatment augments glutathione peroxidase (GPx) levels in the cervical spinal cord of wobblermice. Segments of the cervical spinal cord (c3–c7) were dissected and examined for GPx activity in 9-week-oldwobbler mice. GPx values are plotted as a percentage of the NW group. The NW(N+) group represents a positive control consisting of NW animals (glucose supplement) given daily intraperitoneal injections ofN-acetyl-l-cysteine (1 mg · gm⁻¹ · d⁻¹) for 3 d before killing. For NW and NW(N) groups,n = 4; for wr/wr andwr/wr(N) groups, n = 6. Values represent the mean ± SEM; asterisk signifiesp < 0.05 for a given group versus thewr/wr group. wr/wr versuswr/wr(N), p < 0.003;wr/wr versus NW or NW(N),p < 0.01.