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In vivo detection of microstructural spinal cord lesions in dogs with degenerative myelopathy using diffusion tensor imaging

Abstract Background Degenerative myelopathy (DM) in dogs is a progressive neurodegenerative condition that causes white matter spinal cord lesions. These lesions are undetectable on standard magnetic resonance imaging (MRI), limiting diagnosis and monitoring of the disease. Spinal cord lesions cause disruption to the structural integrity of the axons causing water diffusion to become more random and less anisotropic. These changes are detectable by the technique of diffusion tensor imaging (DTI) which is highly sensitive to diffusion alterations secondary to white matter lesion development. Objective Perform spinal DTI on cohorts of dogs with and without DM to identify if lesions caused by DM will cause a detectable alteration in spinal cord diffusivity that correlates with neurological status. Animals Thirteen dogs with DM and 13 aged‐matched controls. Methods All animals underwent MRI with DTI of the entire spine. Diffusivity parameters fractional anisotropy (FA) and mean diffusivity (MD) were measured at each vertebral level and statistically compared between groups. Results Dogs with DM had significant decreases in FA within the regions of the spinal cord that had high expected lesion load. Decreases in FA were most significant in dogs with severe forms of the disease and correlated with neurological grade. Conclusions and Clinical Importance Findings suggest that FA has the potential to be a biomarker for spinal cord lesion development in DM and could play an important role in improving diagnosis and monitoring of this condition.

asymmetric pelvic limb proprioceptive ataxia and progress to lower motor neuron paraplegia and eventually flaccid tetraplegia. This condition has no effective treatment and the resultant neurological dysfunction necessitates euthanasia. 2 Currently, DM is only definitively diagnosed postmortem by histopathological examination of the spinal cord. Antemortem diagnosis is limited because the lesions are not visible on conventional magnetic resonance imaging (MRI) sequences and cerebrospinal fluid changes are not specific for DM. 3 The disease is associated with a genetic mutation in superoxide dismutase 1 (SOD1). Dogs with DM are homozygous for the A allele of a SOD1 missense mutation and develop progressive axonal loss, demyelination and astrocytosis within the white matter of the major spinal cord funiculi, 3 but not all dogs with the mutation develop DM. [4][5][6] Clinical diagnosis of DM is therefore presumptive and made by excluding other causes for the clinical presentation and identifying a homozygous A allele SOD1 mutation. 3 Developing an MRI method sensitive for DM-associated spinal cord lesions would be valuable for in vivo noninvasive diagnosis and provide a biomarker for noninvasive monitoring of lesion development throughout the lifetime of an affected dog.
In cohorts of humans with amyotrophic lateral sclerosis (ALS), a disease analogous to DM in dogs, 7,8 diffusion tensor imaging (DTI) has reliably identified diffusivity alterations within the spinal cord that correlate with neurological dysfunction. [8][9][10] Diffusion tensor imagings is an advanced MRI method that measures and quantifies the type of water diffusion within neurological tissue. [11][12][13] The degree of diffusion anisotropy in a voxel is documented by the parameter fractional anisotropy (FA), and the average diffusion within a voxel is documented by the parameter mean diffusivity (MD). 12,14,15 Spinal cord white matter exhibits a characteristic pattern of water diffusion because the axons constrain water resulting in high FA and moderate MD values. When white matter is compromised, water diffusion becomes more random and less anisotropic. As such, when white matter lesions are present, FA values decrease and MD values are altered. 16 Diffusion tensor imaging has been described and optimized in the normal canine spinal cord, 15,17 and changes in FA have been identified in various spinal cord lesions. [18][19][20] This technique has the potential to sensitively detect white matter lesions within the spinal cord of dogs with DM, improving ability to monitor and diagnose the disease.
In this study we used DTI to evaluate the spinal cord of a cohort of dogs with DM. We aimed to evaluate the pattern of diffusivity alteration present in diseased spinal cords and correlate these changes to histopathology and expected lesion load. We aimed to test the hypothesis that diffusivity parameters will be significantly altered in the spinal cords of dogs with DM and that the pattern of these changes will correspond with expected lesion load within the spinal cord. We further hypothesized that the severity of diffusivity alterations will correlate with the neurological status of dogs with DM.

| DM cohort subject selection
Dogs for the DM cohort were recruited from a client-owned clinical population. The DM group candidates were included if they had neurological signs of progressive, nonpainful T3-L3 localized neurological deterioration consistent with a diagnosis of DM, as determined by a board-certified veterinary neurologist. On genetic testing they were required to be homozygous for the A allele mutation (AA) at the SOD1 locus. In addition, for inclusion, imaging and necropsy results, when available, were required to exclude the presence of concurrent disease processes that could have contributed to the neurological status of each subject. All included subjects were required to have an American Society of Anesthesiologists (ASA) score < 2 after physical examinations and preanesthetic blood analysis.

| Control cohort subject selection
Dogs for the control cohort were recruited from research and clientowned populations. Dogs were required to be >8 years of age, > 5 kg in weight, and be neurologically normal and clinically healthy. All included subjects were required to have an ASA score < 2 after physical examinations and preanesthetic blood analysis. Only dogs with a negative (GG) result on SOD1 genetic testing were included in the control cohort. This study had approval from Cornell University's Institutional Animal Care and Use Committee.

| Neurological grading
All included subjects underwent full neurological examination and were graded according to a 15-point motor scale adapted from a scoring system for monitoring pelvic limb function as a result of thoracolumbar myelopathies (Table 1). 21 Subjects in the DM cohort that had a neurological grade of 1 to 7 were placed in the mild DM group and dogs with a neurological grade of 8 to 14 were placed in the severe DM group.
Subjects in the control cohorts that were not grade 0 were excluded from the study.

| MRI protocol
Magentic resonance imaging was performed using a 3T GE Discovery A nonisometric voxel was used (voxel size 1 × 1 × 5 mm 3 ) to allow for whole spine scanning within a feasible time frame. Other parameters T A B L E 1 Each subject underwent a neurological examination and was neurologically graded according to this 5-stage, 15-  Total scan time ranged from 1 hour 20 minutes to 2 hours 11 minutes depending on subject size.

| Data cleaning
The FA diffusion tensor map then was used for quality control and data cleaning. Slices with obvious distortions, such as artificial geometric warping, or that had diffusivity measures outside of normal physiological means were marked and removed before statistical analysis. After this stringent data cleaning, approximately 30% of cervical slices, 64% of thoracic slices and 41% of lumbar slices had been excluded across participants. The remaining FA maps then were masked to a lower threshold of 0.2 and upper threshold of 0.9 to exclude voxels that contained signal from cerebrospinal fluid and ensure that retained voxels were within the normal physiological range. The created masks were applied to both FA and MD maps.

| Region of interest analysis
The ROI analysis was performed at midvertebral level to minimize the

| Control cohort
Thirteen dogs met the inclusion criteria for the control cohort. The signalment and neurological grade for each included dog is provided in the Supporting Information. This cohort had a median age of 10 years (range, 8-12 years) and a median weight of 21 kg (range, 7-31 kg).

| Descriptive statistics
The mean and standard deviation (SD) FA and MD (mm 2 /s) results for the control, mild DM and severe DM groups are provided in Table 2.
The FA and MD results obtained at each midvertebral level from control, mild DM, and severe DM groups are plotted for the cervical, thoracic, and lumbar regions in Figure 3.

| Differences in diffusivity parameters between control and DM cohorts
Two-tailed Student's t-tests comparing control and DM cohorts for each metric, identified statistically significant differences in FA in the caudal  F I G U R E 6 Violin plots documenting the fractional anisotropy (FA) and mean diffusivity (MD) (mm 2 /s) found at sites graded on histopathology as mild, moderate, and severe. Although there is overlap between the groups, the mean FA for the mild sites is higher than that of the moderate and severe sites imaging to histopathological evaluation and histopathological grade are presented in Table 3. All spinal cords had the most marked lesion loads in the thoracic and cranial lumbar regions. When diffusivity values were compared to histopathological grade, measured mean FA was higher for sites with mild lesions (0.46 ± 0.09) when compared to sites with moderate (0.39 ± 0.07) or severe (0.4 ± 0.06) lesions, and mean MD values were similar for all grades: mild (0.00069 ± 0.00022), moderate (0.00069 ± 0.00021) and severe (0.00067 ± 0.00025). These data are plotted for each grade (mild, moderate, and severe) in Figure 6.

| DISCUSSION
These data support the tested hypotheses and identified that at spinal cord sites where lesion load was expected to be the highest, FA was significantly decreased in the DM cohort. These changes were most notable in dogs with severe forms of DM and were negatively correlated with neurological grade, suggesting that this metric has potential to be used as a clinical biomarker for the disease. Identifying the relationship of DTI metrics to neurological outcome measures helps explore the utility of in vivo imaging for monitoring lesion load, as shown with our findings in a DM canine population and provides support for the use of DTI metrics as potential clinical biomarkers in DM and other conditions. 10,36 In human disease, FA is a promising biomarker for disability in numerous spinal cord pathologies. 10  A limitation of spinal cord DTI is that it often is impacted by low signal and artifacts. 43 We designed our protocols using previouslypublished descriptions of spinal DTI in dogs 15,17 and applied a small field-ofview technique to minimize the impact of motion and magnetic field inhomogeneity. 44,45 Despite these sequence optimization and postprocessing measures, data cleaning resulted in substantial loss of data at the cervicothoracic and thoracolumbar junction regions. At the cervicothoracic level, the loss of data was mostly a result of low signal and high levels of noise, likely caused by increased distance of the spinal cord from the surface coil used. 43 This effect may have been decreased by using with a different coil combination or with altered coil placement. At the thoracolumbar junction level, data loss occurred secondary to substantial cardiovascular and respiratory motion artifact, magnetic field inhomogeneity and wrap-around. These effects are likely caused by the close proximity of the pulsating aorta and lung-bone interface to the thoracolumbar spinal cord, causing offresonance artifacts, susceptibility signal drop-out and anatomic distortions. 43 These artifacts potentially may have been minimized if cardiac or respiratory gating techniques had been used, but with our system, gating would not allow for DTI acquisition within a reasonable time frame. With continual improvement in DTI and MRI technology, the impact of artifacts and noise on DTI data is likely to be decreased. Our sequence protocol also utilized a nonisometric voxel size (1 × 1 × 5 mm).
This allowed us to have a high in-plane resolution without low signal, and is a common method used in spinal DTI. 15,17 However, this method comes with limitations because nonisometric voxels introduce bias in the quantitative assessment of fiber orientation and anisotropy. 46,47 Future research is needed to optimize DTI protocols for the canine population and for challenges specific to spinal cord imaging.
In our study the disease cohort was relatively small and included dogs of variable breeds and sizes. Increasing subject numbers would improve statistical power and potentially provide more information on the diffusivity alterations observed in dogs at different stages of the condition. In addition, focused breed-specific studies would help limit breed-related variability and assist in identifying differences across breeds in DM.
We evaluated the diffusivity parameters FA and MD, but DTI data can provide other measures of diffusivity, including radial diffusivity (RD) and axial diffusivity (AD). 48 These measures provide information on different aspects of diffusion, with RD relating to myelination, axon diameters and density and AD relating to axonal injury and disease. 14,49 Although these parameters have been shown to be altered with neuropathology, including ALS lesions, 8

ACKNOWLEDGMENTS
Funding provided by Morris Animal Foundation, grant D18CA-310. We thank the Vaika Foundation for providing subjects for the control cohort.
We acknowledge Dr Mathew Brunke for assisting with recruitment.