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Jeffrey T. Henderson, Mohammed Javaheri, Susan Kopko and John C. Roder
Samuel Lunenfeld Research Institute, Mount Sinai
Program in Molecular Biology and Cancer, 600 University Ave., Toronto, Ontario M5G-1X5
Treatment groups and conditions.
Wobbler mice were maintained by taking litters derived from wr/+ x wr/+ crosses, and mating these pups to mice previously confirmed to be heterozygous for the wobbler gene. This method was used since wr/wr mice are infertile, and the genetic basis of the wobbler mutation is unknown. In addition, there are no genetic loci which can definitively be used to determine the genotype of wr animals. Following the identification of sufficient numbers of wr/+ mice, three wr/+ x wr/+ breeding pairs were set up from our sibling stock. Pups subsequently born to these breeding pairs were used for analysis.
Each of the breeding pairs received normal acidified (pH 3.5) drinking water supplemented with either 1% N-acetyl L-cysteine (BDH, B24001-30), L-alanine (Sigma, A5824), or D-(+) glucose (Sigma, G7528). Alanine and glucose solutions were adjusted to the same pH and molarity as NAC-treated animals (61.2 mM) and served as control agents. Thus for a given litter, all animals were exposed to the same agent. Breeding pairs and the mice derived from them were treated continuously with a given factor, which was replenished every 48 hours, to ensure that pups would ingest the agent at the earliest possible time point. Four week old NAC-treated mice drank an average of 3.4+0.6mls. per 24 hour period, and had an average weight of 14.6+0.8 grams (n=20). The mean quantity of NAC ingested was approximately 2.3 mg/g body weight. For 9 week old wr/wr mice, the amount of water consumed was 4.5+0.9 mls. per 24 hours, and mice had an average weight of 21+1.3 grams (n=16, wr/wr weights were approximately 73% of NW controls). The mean quantity of NAC received was 2.1 mg/g body weight. Mice born from each breeding pair were treated for a period of nine weeks, at which time they were sacrificed for analysis. At this time, mice within each treatment group were designated as either wobbler (wr/wr) or "non-wobbler" (NW) and the tissues coded for analysis. Even within the NAC treated group the distinction between wr/wr and NW mice was apparent at this time point. The "NW" groups are thus composed of both wr/+ and +/+ genotypes. For all of the analyses presented below, no significant differences in any experimental parameter were observed between "NW" animals from either the L-alanine or D-(+) glucose groups. Thus data for these animals are all presented under the "NW" groups. Similarly, no significant differences were observed between wr/wr animals from either the L-alanine or D-(+) glucose treatment groups. Data for these animals is presented as the wr/wr group.
The litter size and numbers of wr/wr mice observed per litter did not differ significantly from that expected by Mendelian segregation (22%), suggesting that wr/wr mice do not exhibit elevated mortality rates compared to pups from wr/+ x +/+ breeding pairs from 0-9 weeks. This also suggests that the ratio of wr/+ to +/+ in the non-wobbler group is 2:1.
Two distinct populations of lower motor neurons were examined. These were, cervical spinal motor neurons, and motor neurons of the facial nerve. Both of these populations have been shown to undergo progressive degenerative changes in wr/wr animal. These degenerative changes result in overt muscular weakness which is first apparent at 3 weeks of age (Bird et al., 1971). For this study, changes in axon, nerve, and muscle caliber were determined as a function of cross-sectional area rather than diameter, due to the intrinsically higher reliability of this measure.
Analysis of the facial nerve.
In order to asses changes occurring within the facial nerve of wobbler mice, the major medial branch of the facial nerve was examined in cross section at a point 2 millimeters distal to the stylomastoid foramen; as shown in figure 1a . Measurement of the cross-sectional nerve area of this branch of the facial nerve for wr/wr, NW and NAC-treated wr/wr and NW mice is shown in figure 1b . These data demonstrate that wr/wr mice undergo a substantial reduction in nerve caliber compared to NW controls by 9 weeks of age. In NAC-treated wr/wr mice [wr/wr(N)], this reduction was eliminated; and mice exhibited nerve areas which were not significantly different from NW controls. In order to further define the nature of the reduction in nerve caliber, complete axon counts through the entire nerve segment were collected for animals of each treatment group; as shown in figure 1c . These data indicate that wr/wr mice exhibit a small, but significant, reduction in the number of axons which project through this branch of the facial nerve compared to NW controls. Wobbler animals treated with NAC do not show this loss of motor axons, instead giving values which are similar to the NW group.
The relatively small reduction in the number of axons in wr/wr mice compared to NW controls indicates that only a portion of facial motor axons have undergone complete degeneration by nine weeks of age. The comparatively larger magnitude of the reduction in gross nerve caliber suggests that the predominant cause of this reduction is one of axonal atrophy, rather than axonal loss. To examine this possibility, the area of each axon within the superior median branch of the facial nerve was determined for each treatment group. Axon areas, rather than axon diameters were determined due to their inherently greater accuracy. Measured axon areas represent the intra-luminal area of each axon and do not include components of the myelin sheath. Following their collection, axon areas were rounded to the nearest whole integer (um2) and the resulting distribution plotted as a percent of the total axons in a given nerve; as shown in figure 2 . Displaying the data in this format eliminates the compounding influence of a change in total axon number within a given group. Thus, if a given treatment group were to lose a subset of axons, but the axons which remained were of normal caliber (in normal proportions) with respect to the control group, no difference in the axon distribution between the two groups would be observed. We have observed that the frequency and size distributions of axon areas (when measured for an entire nerve cross-section) is a highly reproducible and sensitive measure of the changes which occur within a nerve fiber.
Figure 2a shows the normal distribution of axon areas for the NW control groups. These distributions are similar to that observed for this branch of the facial nerve in other genetic backgrounds at the same location and age (J.T. Henderson, unpublished observations). Figure 2b shows the distribution of axon areas in wr/wr, and NAC-treated wr/wr mice respectively. Comparison of these values, as shown in figure 2c , indicates that wr/wr mice do exhibit a reduction in axon caliber, consistent with the overall reduction in nerve area compared to NW controls. This appears to be a generalized atrophy which affects axons of all caliber, and is marked by a shift in the mean distribution toward smaller axon areas compared to the NW group. In contrast, NAC-treated wobbler mice do not show a marked reduction in axon caliber by nine weeks of age, exhibiting an axon distribution which is not substantially different from NW control animals. If one assumes that the wobbler mutation exerts a generalized effect on all axons of this population equally, the data can be interpreted in terms of an effect on the true distribution. In this case, the wobbler controls are significantly different from both the wr NAC treatment and NW groups (pair-wise comparisons: wr/wr vs. wr/wr(N) p<0.007, wr/wr vs. NW p<0.030, wr/wr vs. NW(N) p<0.028; wr/wr(N) vs. NW p<0.43, two tailed distribution). Even if one takes the more conservative view (as we have in figure 2c ) that one cannot be assured that the wobbler mutation affects all axons of this population equally, and that the data from different groups can only be analyzed in terms of their absolute area, significant differences between these groups still exist at several discrete axon calibers.
The data given above demonstrate that oral treatment of wobbler mice with NAC can significantly reduce the degree of axonal atrophy and loss which normally occurs in this murine mutant.
Analysis of cervical motor neurons.
Atrophy was apparent in the forelimb of wr/wr animals by nine weeks age. In order to examine the effects of NAC on neuromuscular aspects of the forelimb, 10 micron serial frozen sections were taken through the c4-c7 level of the spinal cord. Every 5th section was then stained for choline acetyl-transferase. The number of ChAT positive neurons shown in figure 3 represents the number of ChAT positive neurons counted for every 5th section. As shown in the figure, wr/wr mice exhibit a substantial reduction in the number of ChAT positive neurons by nine weeks of age. Wobbler mice treated with NAC exhibited significantly greater numbers of ChAT positive neurons than wr/wr mice (58 versus 44 percent of control values respectively). However NAC-wobbler mice still exhibited a substantial loss of ChAT positive neurons compared to the NW mice of the same age.
Structure and function of forelimb muscles.
To determine the effects of NAC treatment on the innervation targets of these spinal motor neurons, proximal and distal muscles of the forelimb were examined. As shown in figure 4a and b , animals treated with NAC show a significant increase in the overall mass of the triceps and flexor carpi ulnaris respectively, in comparison animals in the wr/wr group. Thus, treatment with NAC reduces the degree of overt muscular atrophy which is induced by the wobbler mutation. In order to more clearly delineate the effects of NAC on muscle morphology within the distal forelimb, cross-sectional areas of muscle fibers within the flexor carpi ulnaris were determined for each group. The results, shown in figure 4c , indicate that animals treated with NAC show a marked increase in mean fiber area (~603 um2) compared to wobbler controls (~403 um2). As indicated the figure, each group exhibits a distribution of muscle fiber areas which were significantly different (for each comparison wr/wr:wr/wr(N), wr/wr:NW, wr/wr(N):NW, p<0.001).
These data demonstrate that oral administration of NAC can significantly reduce motor neuron loss and axonal atrophy in wobbler mice; as well as retarding muscle atrophy within the forelimbs of these animals. However, it is also important to determine what the functional consequences, if any, are of this treatment. In order to determine this, animals were scored in a blinded manner for several overt properties of forelimb function. As shown in figure 5 , the distribution of forelimb function was significantly different for each of the pair-wise comparisons (wr/wr vs. wr/wr(N), wr/wr vs. NW or NW(N), wr/wr(N) vs. NW or NW(N); p<0.001). These results demonstrate that while wobbler mice receiving NAC do exhibit a significant reduction in forelimb function, they perform better on average than animals from the control wr/wr group. These data suggest that treatment with NAC does result in some reduction in the functional losses which occur within the forelimbs of wobbler mice.
Glutathione peroxidase activity.
Previous work has suggested that NAC acts in part by increasing intracellular supplies of cysteine; the rate limiting step in glutathione synthesis (Ferrari et al., 1995). Glutathione is an important component of the bodys' free-radical scavenging system, since it serves as a substrate for glutathione-dependent enzymes such as glutathione peroxidase (GPx). The importance of GPx in the protection of neural cells from programmed cell death, and its regulation by neurotrophins, has been demonstrated in several studies (Pan and Perez-Pollo, 1993; Sampath et al., 1994; Kroemer et al., 1995; Mattson et al., 1995). In order to determine the ability of NAC to enhance the free-radical scavenging ability of the glutathione peroxidase system, the level of glutathione peroxidase activity was directly assessed in wobbler mice. Segments of c3-c7 spinal cord were dissected from nine week old mice in each of the treatment groups, and assayed in triplicate for GPx activity. As shown in figure 6 , animals which received NAC in their drinking water exhibited a substantial increase glutathione peroxidase activity within the cervical spinal cord. The level of GPx activity was similar to that of NW mice which did not receive NAC supplementation. In order to further assess the ability of NAC to effect GPx activity levels in non-wobbler mice, several NW mice (glucose treatment group) were injected daily with 1 mg/g NAC for three days prior to the analysis. As indicated in the figure, this treatment resulted in a marked increase in GPx activity. These results demonstrate the ability of NAC to enhance antioxidant activity in tissues affected by the wobbler mutation. This effect was not specific to wr/wr mice, but rather reflected a generalized enhancement of glutathione peroxidase activity.
Materials and Methods
Materials and Methods