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Canine degenerative myelopathy (DM) is a model for a form of ALS associated with mutations in SOD1.
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The earliest histopathological changes in DM occur in muscle and spinal cord nerve tracts.
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No significant histopathology was observed in lower motor neurons.
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Effective early therapeutic interventions will likely need to target the muscles directly.
Abstract
Development of effective treatments for amyotrophic lateral sclerosis (ALS) would be facilitated by identification of early events in the pathological cascade of disease progression. Degenerative myelopathy (DM), a naturally occurring disease in dogs, is quite similar to forms of ALS associated with SOD1 mutations and is likely to be a good model for these forms of the human disease. The sequence of histopathological changes that occur in DM was characterized by analyzing tissue samples obtained from affected dogs euthanized at various stages of disease progression. Cervical spinal cord and the associated spinal nerve roots, ulnar nerve, and the extensor carpi radialis (ECR) muscle were obtained from Pembroke Welsh Corgi dogs (PWCs) with early and late stage DM and from age-matched unaffected PWCs. In early stage disease there was a substantial change in the ratio of the two main muscle fiber types and an increase in mean muscle fiber area in the ECR. DM, even in late stage disease, was not accompanied by changes in the number of motor neuron cell bodies, in the number of axons in the motor or sensory nerve roots, or in the ulnar nerve. In addition, no disease-related denervation of the acetylcholine receptors of the ECR was observed at any stage of the disease. On the other hand, axon densities in both motor and sensory nerve tracts in the cervical cord were reduced in affected dogs. SOD1-immunoreactive aggregates were observed in spinal cord motor neuron cell bodies only in late stage disease. These findings suggest that some of the earliest pathological changes in DM occur in the muscle fibers and upper motor and sensory neuron tracts in the spinal cord. Targeting therapeutic interventions to these early events in the disease are most likely to be effective in slowing disease progression for DM and may translate to therapy of SOD1-related forms of ALS.
Amyotrophic lateral sclerosis (ALS) is a group of human neurodegenerative diseases that in most cases are characterized by a late-adult onset and relatively rapid progressive loss of muscle function. Initial muscle weakness progresses to almost total paralysis. At end-stage disease, loss of muscle functions involved with swallowing and respiration ultimately leads to death. Although no underlying genetic or environmental causes have been identified for the majority of ALS patients, mutations in numerous genes have now been associated with some of forms of ALS [
] (http://alsod.iop.kcl.ac.uk/als/). A mutation in only one of the two SOD1 alleles is sufficient to cause ALS, indicating that the disease results not from a loss of superoxide dismutase function, but rather from toxic effects of the expression of the mutant allele. Among the potential mechanisms proposed to explain this toxicity is that the mutant protein assumes an abnormal conformation leading to its aggregation, particularly in neurons, which in turn causes co-aggregation of other normal proteins. The accumulation of these protein aggregates has been hypothesized to cause dysfunction and death of neurons involved in regulating muscle function and thus resulting in impairment of muscle function.
Degenerative myelopathy (DM) is a disease in dogs that is very similar to the form of ALS caused by SOD1 mutations [
]. Most DM affected dogs will progress to nonambulatory status within 1 year from onset of signs but can live 3 years before respiratory dysfunction either necessitates euthanasia or results in unassisted death [
]. In the United States, most affected dogs that suffer from DM are euthanized before they reach disease end stage when respiratory distress becomes apparent. Clinical signs and progression are relatively uniform among dogs of the same breed and between breeds, with the primary breed difference being in the average age of onset. This uniformity in disease phenotype may be partly explained by the fact that in all dogs, except some Bernese Mountain Dogs, DM is associated with the same SOD1 mutation: SOD1:c.118G➔A, which predicts a p.E40K amino acid substitution [
]. The uniformity in DM disease phenotype in dogs contrasts with substantial variability in the phenotypic onset and clinical progression of ALS which in part may be due to variability in the underlying causes, the majority of which have not yet been identified.
Companion dogs that suffer from DM are euthanized at different stages of disease progression at the owners' discretion. The ability to obtain tissues from dogs at all stages of the disease provides an opportunity to study the histopathology of disease progression that is not possible with human autopsy specimens that are for the most part obtained from donors who survived to extreme disease end-stage with the aid of artificial respiratory and nutritional assistance. By studying the central and peripheral nervous system and muscles from dogs at different stages of DM disease progression, it should be possible to gain a better understanding of the natural history of the disease. Such an understanding may allow for the identification of targets for early therapeutic interventions to aid in the treatment of at least some forms of ALS. Therefore, as a follow-up to previous studies [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
Degenerative myelopathy associated with a missense mutation in the superoxide dismutase 1 (SOD1) gene progresses to peripheral neuropathy in Pembroke Welsh corgis and boxers.
], research was undertaken to characterize potential histopathological DM disease-related changes in the cervical spinal cord and associated nerves and muscle at different stages of disease progression.
2. Materials and methods
2.1 Sample collection and clinical classification
Tissues were dissected and preserved from companion PWCs between 2014 and 2016. Samples were acquired from 29 PWCs. There were 5 sexually intact females, 12 spayed females, 3 sexually intact males, and 9 castrated males. Median age of early disease dogs (n = 11) was 13 years (range 10–17 years), late disease dogs (n = 10) 13.3 years (range 11.5–13.3 years), and control dogs (n = 8) 14.5 years (range 7–16 years).
All samples utilized in the study were collected with the informed consent of the owners at the time the owners made the decision to have their pets euthanized. The studies were approved by the University of Missouri Animal Care and Use Committee. Dogs were tentatively diagnosed with presumptive DM at academic, private specialty or general practices based on clinical presentation and progression of upper and lower motor neuron signs (Table 1) [
]. Further details on sample acquisition and on the diagnoses and DM disease grade determinations are included in the Supplemental Materials section. Each affected dog was assigned to one of the disease stages listed in Table 1 based on the neurological signs the dog was exhibiting at the time of euthanasia. Dogs with stages 1 and 2 disease were classified as early stage and dogs with stages 3 and 4 disease were classified as late stage.
Table 1Neurological signs present at time of euthanasia used to grade disease stage among dogs with DM.
For this study dogs with stages 1 and 2 disease were pooled into the “early stage” group and dogs with stages 3 and 4 disease were pooled into the “late stage” group.
Neurological signs
1
Asymmetric, general proprioceptive ataxia and spastic paresis in pelvic limbs
Intact spinal reflexes
2
Non-ambulatory paraparesis, paraplegia
Reduced to absent pelvic limb spinal reflexes
Pelvic limb muscle atrophy
±urinary/fecal incontinence
3
Flaccid paraplegia, thoracic limb paresis
Absent spinal reflexes
Severe pelvic limb muscle atrophy
Urinary/fecal incontinence
4
Flaccid tetraplegia
Absent spinal reflexes
Severe generalized muscle atrophy
Urinary/fecal incontinence
Dysphagia, dystonia, respiratory difficulty
a For this study dogs with stages 1 and 2 disease were pooled into the “early stage” group and dogs with stages 3 and 4 disease were pooled into the “late stage” group.
2.2 Motor neuron cell body numbers and SOD1 aggregate content
Portions of the C8 spinal cord segments were embedded in paraffin and 4 μm-thick sections were immunostained for SOD1 and choline acetyl transferase (ChAT, a motor neuron specific marker) and counterstained with hematoxylin as described previously [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. In most cases, a single section from the sample was subjected to SOD1 immunostaining. To determine motor neuron density, multiple sections of the C8 cord segment were used for quantitative assessment of ChAT-positive neuron cell bodies in the Rexed lamina 9 of the anterior horns as described in detail in the Supplemental materials section.
2.3 Axon numbers in spinal nerve roots and ulnar nerve
Approximately 3 mm long slices of the dorsal (sensory) and ventral (motor) roots were dissected from the spinal nerves of cord segment C7 from each dog in which these nerve roots remained attached to the cord segment during sample collection at necropsy. In addition, a 4 to 5 mm long slice of the fixed ulnar nerve from each dog was obtained. The samples were processed and embedded in epoxy resin as described previously [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. Cross-sections of each nerve were cut at a thickness of approximately 0.4 μm, mounted on glass slides and stained with toluidine blue and the number of axons in each nerve was determined as described in the Supplemental materials section.
2.4 Muscle fiber size analyses and histology
Cross-sections of portions of the glutaraldehyde-fixed ECR muscle were dissected, processed and embedded in epoxy resin as described previously [
]. Sections of the embedded tissue were cut perpendicular to the long axes of the muscle fibers at a thickness of approximately 0.4 μm using a Reichart Ultracut ultramicrotome. The sections were stained with toluidine blue and a high resolution composite stitched image of a representative area of each muscle section was obtained with the Leica imaging system. Images were imported as lif files into Leica Application Suite X™ and the cross sectional areas of at least 100 contiguous fibers were determined for each dog using the program's image analysis capabilities. The imaging and analyses were performed in a masked manner. Separate sections from the same blocks were stained using Masson trichrome to assess the muscles for evidence of fibrosis.
2.5 Muscle fiber typing
Cross sections of the ECR muscles that had been preserved in cacodylate buffer were embedded in paraffin. Sections of the tissue were cut perpendicular to the long axes of the muscle fibers. After deparaffinization, the sections were immuno-stained with an anti-myosin heavy chain type 1 (MHC-1) antibody using previously described procedures [
]. Images of the immunostained sections were obtained with a Zeiss™ Axiophot microscope. The number of stained and unstained muscle fibers in each image was counted and the proportion of immuno-positive fibers was determined for each sample. At least 100 muscle fibers per sample were evaluated. The image analyses were performed in a masked manner.
2.6 Analysis of axons in vestibulospinal tracts and fasciculus cuneatus
Approximately 2 mm thick slices of the vestibulospinal (VS) tract and fasciculus cuneatus (FC) were dissected from the glutaraldehyde-fixed C7 cord segments from each dog. The samples were processed and embedded in epoxy resin as described previously [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
] and sections of each were cut at a thickness of approximately 0.4 μm in a plane perpendicular to the long axis of the spinal cord. The sections then were mounted in slides and stained with toluidine blue. Images of the sections were obtained in a with a Zeiss™ Axiophot microscope. The area of tissue included in each VS tract image was 11,200 μm2, and in each CF image was 13,100 μm2. The images were coded and the number of axons in each image was determined as described above for the axon number determinations for the spinal and ulnar nerve cross-sections. The cross sectional areas of 50 randomly selected contiguous axons from each spinal cord tract were measured as described previously [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
Analyses were performed to determine whether the ECR muscles from dogs with DM or age-matched control dogs had acetylcholine receptor complexes that were not contacted by nerve terminals. Segments of the ECR muscles that had been preserved in cacodylate buffer were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA) as described previously [
]. Sections of the muscle were cut at a thickness of 40 μm on a cryostat and mounted on Superfrost Excell microscope slides (Fisher Scientific, #22-034-985). The sections were then incubated with Alexa Fluor 555-conjugated alpha-bungarotoxin (red emission) (Invitrogen, B35451) and anti-neurofilament light antibody (Millipore AB9568) followed by incubation with an Alexa Fluor 488-conugated secondary antibody (Invitrogen A-11034) (green emission) [
]. Confocal images of the stained sections were acquired with a Leica TCP SP8 scanning confocal microscope (Leica Microsystems) equipped with a 40× oil immersion objective. Stacks of multichannel confocal images were recorded in a sequential mode using the following settings: excitation 488 nm and emission bandpass of 505–560 nm (Alexa 488), and excitation 552 nm and emission bandpass of 560–620 nm (α-bungarotoxin- Alexa Fluor 555 conjugate). The size of the pixels was 51 × 51 nm and the distance between images in a stack was set to 200 nm. Stacks of images were deconvolved with the Huygens Essential software program (v. 16.1, Scientific Volume Imaging B.V.) and presented as maximum projections.
2.8 Statistical analyses
For all quantitative data, comparisons among the three groups of dogs were performed using one-way analysis of variance (ANOVA). If the ANOVA indicated a significant difference among the groups, pairwise comparisons between each of the groups were performed using the Holm-Sidak test. The ECR muscle fiber cross-sectional area were not normally distributed when the data were separated into 246 μm2 size increments as displayed graphically in Fig. 8. Therefore, for statistical analysis, the muscle fiber sizes for each dog were classified as either larger or smaller than 988 μm2. Using this size classification, the muscle fiber size data were normally distributed for all three groups of dogs and were analyzed using one way ANOVA and the Holm-Sidak test as were the rest of the quantitative data. All statistical analyses were performed using the SigmaPlot program (Systat Software, Inc., San Jose, CA).
3. Results
3.1 SOD aggregates in cervical cord motor neurons
In previous studies we demonstrated that motor neurons in the thoracic segments of the spinal cords of dogs with degenerative myelopathy accumulated aggregates that immunostained with antibodies directed against the SOD1 protein [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. Sections of cervical spinal cord segment C8 were examined to determine whether these aggregates were also present in motor neurons in this region of the cord. SOD1-positive aggregates were not observed in the cervical spinal cord motor neurons in either control dogs or dogs with early stage DM (Fig. 1 A and B). These aggregates were present in the motor neurons of 7 of 10 PWCs with late stage DM (Fig. 1 C).
Fig. 1Sections of the ventral horn of cervical spinal cord segment C8 immunostained to reveal SOD1 from a control PWC (A), a PWC with early stage DM (B), and a PWC with late stage DM (D). Ventral horn neurons indicated with arrows. Dark brown staining inclusions were present in only in motor neurons from PWCs with late stage disease. Bar in (A) indicates magnification of all 3 micrographs.
3.2 Cervical spinal cord motor neurons and motor root axon numbers
To determine whether DM was accompanied by a loss of motor neuron cell bodies from the cervical cord ventral horn, the number of ChAT-positive cells per 4 μm thick section was determined in PWCs with early-stage and late-stage DM and in unaffected age-matched normal PWCs. No significant differences in ChAT-expressing motor neurons were detected between these three groups of dogs (Fig. 2).
Fig. 2(A) Numbers of ChAT immunolabeled cells per section in the ventral horns of the C8 segment of spinal cords from PWCs with early-stage and late-stage DM and from unaffected age-matched PWCs. There were no significant differences between the groups of dogs. (B) Micrograph showing an area of a section of a C8 spinal cord segment from a late-stage DM PWC immunolabeled with an anti-ChAT antibody. Arrows indicate some of the ChAT-positive motor neurons. The images used for quantitative analyses included both ventral horns from at least 10 non-adjacent sections from each dog.
While the motor neuron cell bodies may remain intact in the cervical spinal cord of dogs with DM, it is possible that the axons of these cells degenerated. We therefore determined the total axon numbers in the C7 motor roots from affected and age-matched unaffected PWCs. No significant differences in motor root axon numbers were detected between these three groups of dogs (Fig. 3).
Fig. 3(A) Numbers of axons in the C7 motor roots of PWCs with early-stage and late-stage DM and from unaffected age-matched PWCs. There were no significant differences between the groups of dogs. (B) Micrograph showing cross-section of a C7 motor root from a PWC with late-stage DM.
Some of the signs of DM could be attributable to peripheral sensory loss. Therefore, the numbers of axons on the C7 cord sensory roots were determined in affected and age-matched unaffected PWCs. No significant differences in sensory root axon numbers were detected between these three groups of dogs (Fig. 4).
Fig. 4(A) Numbers of axons in the C7 sensory roots of PWCs with early-stage and late-stage DM and from unaffected age-matched PWCs. There were no significant differences between the groups of dogs. (B) Micrograph showing cross-section of a C7 sensory root from a PWC with late-stage DM.
Some of the signs of DM could be attributable to degeneration of the distal portions of motor and sensory axons. Therefore, the numbers of axons in the ulnar nerves were determined in affected and age-matched unaffected PWCs. No significant differences in ulnar nerve axon numbers were detected between these three groups of dogs (Fig. 5).
Fig. 5(A) Numbers of axons in the ulnar nerves of PWCs with early-stage and late-stage DM and from unaffected age-matched PWCs. There were no significant differences between the groups of dogs. (B) Micrograph showing cross-section of an ulnar nerve from a PWC with late-stage DM.
In SOD1 mutant mouse models of ALS it has been reported that there is loss of contact between nerve terminals and muscle acetylcholine receptor complexes. To determine whether this is the case in DM, sections of the ECR were labeled with an anti-neurofilament antibody that labels nerve processes with a green-emitting fluorophore and Alexfluor 555-tagged alpha-bungarotoxin that labels the muscle receptor complexes with a red-emitting fluorophore. Sections were then examined for the presence of receptor complexes that were not contacted by nerve terminals. Over 1000 receptor complexes from DM-affected PWCs at early and late stage disease were examined and results showed no disease-related loss of contact with a nerve terminals (Fig. 6).
Fig. 6Confocal microscopy images of ECR muscles from age-matched normal PWCs that were euthanized without having exhibited clinical signs of DM (A) or at late stage DM (B and C). The muscle acetylcholine receptor complexes are labeled red and the neurofilaments of terminal nerve processes are labeled green. In a high magnification image, the nerve terminals were seen to branch at the site of contact with the muscle receptor complexes (C).
Skeletal muscles are composed of two main fiber types that can be distinguished by the predominance of either heavy chain or light chain myosin. Sections of the ECR were immunolabeled with an antimyosin heavy chain type 1 (MHC-1) antibodies [
], and the percent of fibers that contained primarily heavy chain myosin was determined for PWCs with DM and age-matched normal dogs. In age-matched normal dogs, there were few heavy chain type fibers. The proportion of this fiber type increased significantly in early-stage DM and continued to increase with the progression of disease severity (Fig. 7).
Fig. 7(A) Percent of myosin heavy chain type fibers in the ECR of PWCs with early and late stage DM and from age-matched unaffected dogs. There was a significant increase in the heavy chain myosin fiber type in early stage DM (p < 0.05) that was more pronounced in dogs with late stage disease. (B) Micrograph of a section of ECR from an unaffected 14.5 year old PWC immunolabeled with an anti myosin heavy chain antibody. (C) Micrograph of a section of ECR from a 15.1 year old PWC with late stage DM immunolabeled with the same anti myosin heavy chain antibody. Bar in (B) indicates magnification of micrographs in both (B) and (C).
3.7 Extensor carpi radialis muscle fiber size distribution
In a previous study we found that DM resulted in an alteration in muscle fiber size distribution in the intercostal muscles of PWCs. Analyses were performed to determine whether similar alterations were observed in the ECR muscles in DM-affected PWCs (Fig. 8). Among the early and late stage affected dogs, there were significant increases in the proportions of muscle fibers with cross-sectional areas > 988 μm2 (control:39%; early stage:68%; late stage:52%). Using this size cut-off, the average cross-sectional areas of the muscle fibers from early stage and late stage affected dogs were significantly greater than those of the control dogs (P < 0.01). The early stage dogs (Fig. 8) had significantly higher proportions of larger muscle fibers than either the control or late-stage affected dogs for all muscle fiber size classes with cross-sectional areas > 1720 μm2. Among early stage affected dogs, a mean of 24% of the muscle fibers were in this size class compared to a mean of only 5.4% of the muscle fibers from the late stage affected dogs and 9% of the fibers from the unaffected control dogs.
Fig. 8ECR muscle fiber cross-sectional area distribution from PWCs with early and late stage DM and from age-matched unaffected dogs. The number of control, early stage and late stage dogs represented in this graph are 5, 11, and 12 respectively. Data in each bar represents the mean value for that group.
3.8 Cervical spinal cord vestibulospinal tract axon density and size
The mean number of axons in a 11,200 μm2 area of the C7 spinal cord VS tract was 50% lower in PWCs with early stage DM than in age-matched controls (p < 0.01) (Fig. 9). No further decrease in axon density was observed between dogs with early stage and late stage disease. There was no significant difference in mean axon size between the 3 groups of dogs. There was also no significant difference in axon size frequency distribution between the 3 groups when axon sizes were divided into size increments of approximately 5 μm2 each. For all 3 groups the largest numbers of axons were <10 μm2 in cross-sectional area (46% of the axons in control dogs, 48% of the axons in dogs with early stage DM, and 59% in dogs with late state DM). The remainder of the axons in all 3 groups were distributed equally among size classes ranging from 10 to >60 μm2.
Fig. 9(A) There was a significantly lower VS tract axon density in dogs affected with early or late stage DM compared to unaffected dogs of similar age (p < 0.02), but no difference between early and late stage affected dogs. Micrographs of representative areas of the VS tract from unaffected (B) and early stage (C) and late stage affected (D) dogs. The number of axons were counted in an 11,200 μm2 area of the VS tract from each dog.
3.9 Cervical cord fasciculus cuneatus axon density and size
The mean number of axons in a 13,100 μm2 area of the C7 spinal cord FC tract did not differ significantly between control PWCs and those with early stage DM. However, the axon density in this tract was 42% lower in PWCs with late stage DM than in age-matched controls (p < 0.01) (Fig. 10). There was no significant difference in mean axon size between the 3 groups of dogs nor was there a significant difference in axon size frequency distribution between the 3 groups when axon sizes were divided into size increments of approximately 5 μm2 each. However, as can be seen in Fig. 10C, in some of the dogs in only the latest stage of disease progression (stage 4), there were many axons with cross-sectional areas that significantly larger than any of the axons seen in normal or early-stage affected dogs.
Fig. 10Micrographs of representative sections of the fasciculus cuneatus (FC) from an unaffected dog (A), dog with early stage DM (stage 2) (B) and a dog with late stage DM (stage 4) (C). Quantitative analysis (D) indicated that the density of axons in the FC did not differ between unaffected dogs and dogs with early stage DM (A, B, and D). However, in dogs with late stage disease there was a significant decline in axon density in the FC compared to normal dogs and dogs with early stage DM (p < 0.01). The numbers of axons were counted on in a 13,100 μm2 area of the FC from each dog.
]. The underlying disease mechanism for DM is likely to be the same as that for SOD1-related ALS. Therefore DM associated with SOD1 mutations in dogs is likely to be a good model for the form of ALS associated with mutations in the human ortholog of this gene.
Any treatment for ALS is likely to be most effective at the earliest stages of the cascade pathology that characterizes the disease. Characterization of this cascade of pathological events is likely result in a better understanding of disease mechanisms. Therefore, it is important that the earliest pathological changes associated with the disease be identified as well as the order in which these changes occur relative to clinical disease progression. In this study we were able to distinguish between pathology that developed at different stages of disease progression and thus to identify targets that would be appropriate for therapeutic intervention at each disease stage.
In a previous study we examined pelvic limb muscles and nerves from DM-affected dogs and found many pathological changes that have been reported in end-stage ALS [
Degenerative myelopathy associated with a missense mutation in the superoxide dismutase 1 (SOD1) gene progresses to peripheral neuropathy in Pembroke Welsh corgis and boxers.
]. However, because the pelvic limbs are affected early in DM, most of the pathology reported probably did not reflect the earliest disease-related pathology. Clinically, impairment of thoracic motor function occurs very late in DM disease progression. Therefore, to identify earlier indicators of disease pathology we subsequently examined the thoracic spinal cord, thoracic spinal nerves and the muscles they innervate in dogs with DM [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. Although these studies were informative with respect to identifying the early pathological changes in nerve and muscle, the entire spectrum of disease pathology progression could not be characterized since all of the dogs available to us were euthanized prior to the stage at which they would have exhibited respiratory distress. With regard to clinical disease progression, the thoracic limb muscles that are innervated by cervical spinal neurons exhibit clinical signs after the pelvic limbs, but become severely affected prior to the onset of respiratory signs (Table 1) which only become apparent if affected dogs are allowed to progress to exhibiting respiratory distress that shortly precedes natural death from the disease [
]. Therefore, in order to examine the widest range of DM disease pathological progression, thoracic limb muscle, nerve, and the cervical spinal cord were examined from PWCs euthanized at all stages of disease progression with the exception of extreme disease end stage in which they would have been completely paralyzed and in a state of respiratory failure.
With respect to both the spinal nerve root axons and a peripheral nerve of the thoracic limb our findings were very similar to those we previously reported for spinal cord root axons in the thoracic region of DM-affected PWCs [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. There was no significant loss of axons in the cervical motor and sensory roots or in the ulnar nerve of affected dogs and relative age-matched controls, even in late stage disease. This indicates that axons are not dying back from their nerve terminals toward the cell bodies of the lower motor neurons or lower sensory neurons. Consistent with this finding, we found that the acetylcholine receptor complexes on the ECR muscle retained contact with motor nerve terminals (Fig. 6), similar to what we had observed in the thoracic intercostal muscles in a previous study [
]. These data indicate that impairment of forelimb motor function is not secondary to denervation by lower motor neurons. Consistent with the absence of disease-related decreases in motor root axon numbers, there was no disease-related decline in the density of ChAT-positive motor neurons in the ventral horn of the cervical cord. Unlike our previous findings in the thoracic cord motor neurons, we did not observe SOD1-immunlabeled aggregates in the cervical cord lower motor neurons until disease late stage, suggesting that accumulation of significant amounts of these aggregates occurs relatively late in the pathological process and correlates with the timing of the appearance of clinical signs in the muscles innervated by cervical cord lower motor neurons.
In a previous study, we found that there was a significant decrease in thoracic cord sensory root axon numbers in PWCs with late-stage DM when compared to age-matched unaffected PWCs [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. A similar trend of decreasing sensory root axon numbers in the cervical cord with disease progression was observed in this study, although the disease effect was not statistically significant. Our data do not allow us to rule out that cervical cord sensory root axon loss occurs in DM because of our small sample size. We did observe a significant disease-related decline in axon density in the C7 FC, which consists of sensory axons. The extent of sensory axon loss in DM warrants further investigation, particularly in light of reports of functional sensory deficits in some forms of ALS [
In contrast to the lack of significant early changes in the neurons of the cervical region, there were pronounced alterations in the ECR muscle that included a shift toward type 1 muscle fibers early in the disease process accompanied by a disease-related increase in mean muscle fiber area (hypertrophy). The increased numbers of type 1 muscle fibers were relatively uniformly distributed throughout the muscle, suggesting that this change was not secondary to denervation followed by re-innervation which typically results in clustering of fibers of the same type [
Degenerative myelopathy associated with a missense mutation in the superoxide dismutase 1 (SOD1) gene progresses to peripheral neuropathy in Pembroke Welsh corgis and boxers.
]. These findings suggest that pathology associated with DM may originate at least in part in the muscles. This conclusion is consistent with our previously reported characterization of the thoracic muscles and nerves of DM affected PWCs in which muscle pathology was significant in the absence of any evidence of denervation [
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
]. In addition, it has been reported that gene expression profile of muscle samples from an SOD1 mutant mouse ALS model collected before the mice were symptomatic is substantially different from that of muscles that were denervated as a result of axotomy [
], again suggesting that muscle pathology can be primary in this form of ALS and is not necessarily secondary to neurological pathology or alterations in muscle innervation. Thus, data from both canine DM and from SOD1 mutant mouse models support the idea that the muscle should be considered as a primary target for therapeutic intervention in at least some forms of ALS. Unfortunately, as for the nerve pathology associated with both DM and ALS, it is not yet apparent by what mechanism the SOD1 mutation would lead to the disease-related changes in the ECR. However, the mouse data showing that an SOD1 mutation is associated with changes in expression of numerous muscle genes suggests that mutant SOD1 protein may directly alter gene expression profiles in such a manner as to cause the observed phenotypic changes in the ECR in early stage DM. One possible mechanism by which this could occur would be via direct or indirect interactions between muscle-specific transcription factors and the mutant form of the SOD1 protein. A number of studies have demonstrated altered transcription factor expression in SOD1 mutant mouse models of ALS and human ALS patient tissues [
S[+] Apomorphine is a CNS penetrating activator of the Nrf2-ARE pathway with activity in mouse and patient fibroblast models of amyotrophic lateral sclerosis.
]. In particular, significant alterations in mRNA expression of the transcription factor PGC-1alpha were observed in skeletal muscle from an SOD1 mutant mouse model and from human ALS patients [
The potential that muscle pathology can be primary in some forms of ALS is supported by findings that mutations in genes associated with primary myopathies are also associated with some cases of ALS. For example, mutations in VCP, CHCHD10 and MATR3 are associated with both primary myopathy and ALS [
]. These findings are consistent with the possibility that SOD1 mutations can be associated with primary myopathy as suggested by our data. One way to test this would be to create an animal model in which the mutant form of SOD1 was expressed exclusively in muscle and determine whether this results in the same type of alterations that were observed in the ECR in early stage DM in this study.
It is possible that the alterations seen in the ECR in early stage DM are, at least in part, secondary to increased stress on the thoracic limbs due to disease-related loss of function in the pelvic limbs. Muscle fiber remodeling may occur in disease states secondary to changes in muscle utilization [
]; so it also is conceivable that the increased ECR muscle fiber size and type distribution seen in early stage DM was associated with an increase in reliance on the thoracic limbs for locomotion. Dogs normally have 60% of their weight bearing in the thoracic limbs compared to 40% in the pelvic limbs [
], and DM-related impairment of pelvic limb function could increase the proportion of weight born by the thoracic limbs. This could contribute to the ECR muscle changes observed in early stage DM. Consistent with this possibility, endurance training consisting of repetitive, low intensity contractions can induce changes in muscle fiber types. In the triceps brachii muscle of dogs running on a treadmill for over a year, there was a shift from type II to type I muscle fibers, which was also observed in the thoracic and cervical spinal muscles [
In order to functionally determine whether stress placed on the thoracic limbs is altered in DM, it would be necessary to document changes in weight distribution to the thoracic limbs in DM affected dogs as well as disease-related changes in thoracic limb utilization over time. Functional gait analyses using kinetic [
] studies have been established in normal dogs and warrant further investigation in DM affected dogs. Such studies would also need to take into account whether overall locomotor activity is altered during DM progression. It seems likely with the gradual loss of muscle function in DM, affected dogs would become less physically active overall. In this case, there may be no net effect of utilization muscle stress on the ECR.
Based on the animal studies, it would be informative to examine muscles from human patients with early stage SOD1-related ALS for pathological and biochemical changes. Since serial samples for such analyses could be obtained by biopsy, it should be possible to collect and evaluate muscle samples from subjects who are pre-symptomatic [
]. Identification of intrinsic muscle abnormalities at early stages of the disease or even prior to the onset of clinical signs would suggest that such changes could be primary and should therefore be considered as targets for therapeutic interventions [
Voluntary muscle function of course depends not only on the functional and structural integrity of the lower motor neurons and the muscles themselves, but also on interneurons and brain neurons whose axons traverse the ascending and descending tracts of the spinal cord. Therefore, axon densities were examined in the VS tract of the cervical spinal cord in PWCs at various stages of disease progression. We found that there was a significant decline in axon density in the VS tracts of affected dogs in early stages of clinical disease. The concurrent loss of these axons with forelimb muscle pathology does not necessarily indicate that there is a causal link between these changes, so early therapeutic interventions may need to target both the muscles and the upper motor neurons since the integrity of both is necessary for normal forelimb function.
Overall our data indicate that the lower motor neurons remain morphologically intact even in DM affected dogs with relatively advanced clinical signs. In contrast, changes in muscle fiber type and size occurred early in the disease process and appeared to be due to primary alterations in the muscles. These findings suggest that there should be a focus on primary changes in the muscles in developing early therapeutic interventions for some forms of ALS. Based on our findings, upper motor neurons and sensory neurons should also be considered as targets for early therapeutic interventions.
Acknowledgments
The authors thank Lauren Gillespie for assistance with some of the morphometric analyses, the Veterinary Medical Diagnostic Laboratory for assistance with sample preparation, and Alexander Jurkevich of the University of Missouri Molecular Cytology Core Facility for assistance with confocal microscopy and image analyses. Our thanks also to Dr. Dawn Corneison for providing us with alpha-bungarotoxin. We would also like to thank the many dog owners who allowed tissues to be collected from their animals and the veterinarians who collected the samples that were evaluated in this study. Support for this research was provided by the Missouri Spinal Cord Injury Research Program.
Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.
Degenerative myelopathy associated with a missense mutation in the superoxide dismutase 1 (SOD1) gene progresses to peripheral neuropathy in Pembroke Welsh corgis and boxers.
S[+] Apomorphine is a CNS penetrating activator of the Nrf2-ARE pathway with activity in mouse and patient fibroblast models of amyotrophic lateral sclerosis.
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