Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis

Abstract

Canine degenerative myelopathy (DM) is a progressive, adult-onset, multisystem degenerative disease with many features in common with amyotrophic lateral sclerosis (ALS). As with some forms of ALS, DM is associated with mutations in superoxide dismutase 1 (SOD1). Clinical signs include general proprioceptive ataxia and spastic upper motor neuron paresis in pelvic limbs, which progress to flaccid tetraplegia and dysphagia. The purpose of this study was to characterize DM as a potential disease model for ALS. We previously reported that intercostal muscle atrophy develops in dogs with advanced-stage DM. To determine whether other components of the thoracic motor unit (MU) also demonstrated morphological changes consistent with dysfunction, histopathologic and morphometric analyses were conducted on thoracic spinal motor neurons (MNs) and dorsal root ganglia (DRG) and in motor and sensory nerve root axons from DM-affected boxers and Pembroke Welsh corgis (PWCs). No alterations in MNs or motor root axons were observed in either breed. However, advanced-stage PWCs exhibited significant losses of sensory root axons, and numerous DRG sensory neurons displayed evidence of degeneration. These results indicate that intercostal muscle atrophy in DM is not preceded by physical loss of the motor neurons innervating these muscles, nor of their axons. Axonal loss in thoracic sensory roots and sensory neuron death suggest that sensory involvement may play an important role in DM disease progression. Further analysis of the mechanisms responsible for these morphological findings would aid in the development of therapeutic intervention for DM and some forms of ALS.

Keywords: SOD1; axon; dog; dorsal root ganglion; morphometry.

Figure 1

Procedure for obtaining morphometric measurements in T8 motor root cross-sections. (A) Resin embedded nerves were sectioned and stained with P-phenylene diamine (PPD), imaged using light microscopy, and composite images of the entire nerve cross-sections were constructed. (B) Magnified image of area outlined in (A). (C) Images were sharpened using Photoshop™ tools. (D) Imaris® software was used to invert the image, and remove background noise. (E) Multicolored image produced by the automated count tool in MetaMorph® software. Colors indicate what software distinguishes as individual objects. Note that several adjacent axons are being mistaken for a single object. (F) Manual corrections were made in Photoshop by overlaying, and increasing the transparency of, the multicolored image over the sharpened light micrograph. (G) Multicolored images after manual corrections. (H) Corrected multicolored image of entire nerve root cross-section. (Illustration represents a T8 motor root cross section).

Figure 2

Thoracic motor neuron morphometric measurements. (A, B) T7 spinal cord section from an unaffected 9 year old Boxer stained with ChAT. (B). Higher magnification of highlighted box within Rexed lamina 9 in A. This image is a representation of the normal motor neuron morphology seen in unaffected and affected dogs of both breeds. (C) Morphometric measurements obtained from PWC (top) and Boxers (Bottom). Pooled data indicate no difference in the motor neuron density between unaffected and affected samples of either breed (Affected boxers: one grade 1, three grade 2, one grade 3, and two grade 4 dogs; Affected PWC: two grade 2, two grade 3, and four grade 4 dogs). Likewise no differences were seen in the mean soma cross sectional area (CSA) or size range. In PWC's a significant difference was seen in the CSA standard deviation, suggesting more variability in soma area in affected dogs (p=0.02). One Way ANOVA indicated the difference in standard deviation is attributed to a significant difference between unaffected and late stage (grade 3-4) PWC samples (p= 0.04).

Figure 3

Motor root myelinated axon diameter distribution from PWC's (A,B) and Boxers (C,D). Histograms show a bimodal size distribution with one peak representing small caliber axons, and one peak representing large caliber axons (A, C). For both breeds, no obvious differences could be seen between large caliber axons from control and affected motor roots, while the small caliber peak was more variable. (B, D) For both breeds, no statistical difference was seen in the percentage or average numbers of axons ≤5 μm >. Data represented in this figure were obtained from the same samples depicted in Tables IV and V.

Figure 4

Histological sections of T8 sensory roots (A and B) and dorsal root ganglia (DRG) (C and D) in PWC samples. EM-fixed sensory roots and DRG from control (A,C) and grade 4 (B,D) PWC samples were embedded in resin and stained with PPD and toluidine blue respectively. Compared to controls (A), sensory roots from grade 4 PWC (B) exhibited decreased myelinated axon density. DRG neurons from grade 4 PWC (D) contained numerous cells with condensed cytoplasm (arrowhead) and severely pyknotic nuclei (arrow) that were not present in the DRG neurons from age-matched control dogs (arrowhead and arrow in C denote cytoplasm and nucleus of normal neuron for comparison).

Figure 5

PWC sensory root myelinated axon diameter distribution. (A)Histogram shows a bimodal size distribution with one peak representing small caliber axons, and one peak representing large caliber axons. (B) There was a statistically significant difference in the average number of axons ≤5 μm (p= 0.03) and >5 μm (p=0.03). Data represented in this figure were obtained from the same samples depicted in Table VI.