Magnetization transfer and diffusion tensor imaging in dogs with intervertebral disk herniation

Abstract Background Quantitative magnetic resonance imaging (QMRI) techniques of magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI) provide microstructural information about the spinal cord. Objective Compare neurologic grades using the modified Frankel scale with MTR and DTI measurements in dogs with thoracolumbar intervertebral disk herniation (IVDH). Animals Fifty‐one dogs with thoracolumbar IVDH. Methods Prospective cohort study. Quantitative MRI measurements of the spinal cord were obtained at the region of compression. A linear regression generalized estimating equations model was used to compare QMRI measurements between different neurological grades after adjusting for age, weight, duration of clinical signs, and lesion location. Results Grade 5 (.79  ×  10−3 mm2/s [median], .43−.91 [range]) and axial (1.47 × 10−3 mm2/s, .58−1.8) diffusivity were lower compared to grades 2 (1.003, .68−1.36; P = .02 and 1.81 × 10−3 mm2/s, 1.36−2.12; P < .001, respectively) and 3 (1.07 × 10−3 mm2/s, .77−1.5; P = .04 and 1.92 × 10−3 mm2/s, 1.83−2.37;P < .001, respectively). Compared to dogs with acute myelopathy, chronic myelopathy was associated with higher mean (1.02 × 10−3 mm2/s, .77−1.36 vs. .83 × 10−3 mm2/s, .64−1.5; P = .03) and radial diffusivity (.75 × 10−3 mm2/s, .38−1.04 vs. .44 × 10−3 mm2/s, .22−1.01; P = .008) and lower MTR (46.76, 31.8−56.43 vs. 54.4, 45.2−62.27; P = .004) and fractional anisotropy (.58, .4−0.75 vs. .7, .46−.85; P = .02). Fractional anisotropy was lower in dogs with a T2‐weighted intramedullary hyperintensity compared to those without (.7, .45−.85 vs. .54, .4−.8; P = .01). Conclusion and Clinical Relevance Mean diffusivity and AD could serve as surrogates of severity of spinal cord injury and are complementary to the clinical exam in dogs with thoracolumbar IVDH.


| INTRODUCTION
Intervertebral disk herniation (IVDH) is considered to be the most common cause of spinal cord injury (SCI) in dogs and prognosis directly correlates to neurologic grade. 1 After severe SCI and loss of deep pain perception neuronal death can ensue; alternatively injured neurons initially might not generate action potentials but will subsequently recover leading to prognostic uncertainty of dogs with loss of deep pain perception. 2,3 Magnetic resonance imaging (MRI) is the ideal tool to noninvasively evaluate the central nervous system (CNS). 4 Despite a high sensitivity in detection of CNS abnormalities, conventional MRI provides limited assessment of SCI severity in dogs with IVDH. 5,6 In dogs with thoracolumbar IVDH, T2-weighted intramedullary hyperintensity (T2W-IH) correlates negatively with prognosis. 5,6 Morphometric measurements, such as the degree of spinal cord compression, vertebral canal compression ratio, and compressive length of the spinal cord, are not predictive of outcome. 5,7,8 Quantitative MRI (QMRI) techniques of magnetization transfer (MT) and diffusion tensor imaging (DTI) have the ability to noninvasively assess myelination and axonal integrity, and are highly predictive of histopathological changes. 9,10 Calculation of the magnetization transfer ratio (MTR) provides a measure of the rate of magnetization exchange between the hydrogen protons in low protein fluid and those protons bound to macromolecules in tissue. 11,12 Magnetization transfer alterations are thought to be directly related to myelin damage, 9 and have been evaluated in people with myelopathy. 13 Quantitative DTI measurements thought to be of clinical importance in SCI include fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD). Variations of FA and MD represent demyelination and changes to axonal integrity. 14 Axial diffusivity is thought to be a surrogate of axonal integrity; RD a surrogate of myelin disintegration. 14 These quantitative DTI measurements have been evaluated in dogs with experimentally induced SCI, but only FA and MD have been evaluated in dogs with IVDH. 15 The goal of our study was to evaluate MTR and DTI in dogs with thoracolumbar IVDH. We hypothesized that MTR and FA would be higher, while MD, AD, and RD would be lower in dogs with more severe neurologic dysfunction. Within the CNS, MTR increase with acute CNS injury, then decrease with chronic CNS injury. 16 We therefore hypothesized that MTR would be higher in dogs with acute and subacute SCI compared to dogs with chronic SCI. Alteration in FA have been associated with the presence of a T2W-IH 17 and we hypothesized that FA would be higher in dogs with a T2W-IH compared to dogs without a T2W-IH. Dogs were grouped based on the severity of their neurological grade at presentation using a modified Frankel scale (Table 1). [18][19][20] To evaluate deep pain perception, the distal limb or nail bed was crossclamped with hemostats (either using the nose or the handle of the hemostats) and then observing a conscious reaction to the painful stimulus or an increase in heart rate, respiratory rate, or pupil size. 21 All dogs in the study were anesthetized for MRI. Anesthesia protocol was at the discretion of the anesthesiologist. Magnetic resonance imaging was performed using a 1.5T magnet (Philips Intera 1.5T MRI scanner, Cleveland, Ohio Once converted, nii images were processed using SCT to obtain the QMRI measurements of MTR, FA, MD, AD, and RD (Figures 1 and 2).

| MATERIALS AND METHODS
Spinal Cord Toolbox obtains QMRI measurements similar to utilizing region of interest (ROI) but provides a more accurate estimation of the QMRI measurement as it is able to account for partial volume effect using Gaussian mixture modeling. 22 Following processing, images were inspected using the open source software FSLView (version 3.2.0) to confirm SCT segmentation (Figures 1 and 2). All images were reviewed by the primary author. In FSLView, the images appear as larger pixels.
In addition to the modified Frankel grade, the sex, age, body

| RESULTS
A total of 52 MRIs were performed on 51 dogs included in the study. When images were evaluated, the mean pixel value for MTR was 23 and for DTI was 10 (Figures 1 and 2). Bivariable analyses did not identify significant differences between sex and MTR, FA, MD, AD, or RD. Significant correlations were found with age and MD (r = 0.67, Magnetization transfer ratio (P = .004) and FA (P = .02) were significantly lower in dogs with chronic SCI compared to dogs with acute SCI, but not when compared to subacute SCI (Figure 3). Mean diffusivity (P = .03) and RD (P = .008) were significantly higher in dogs with chronic SCI compared to acute SCI, but not when compared to subacute SCI (Figure 3). Axial diffusivity was not significantly different when chronic SCI was compared to acute or subacute SCI. FA was significantly lower in dogs where a T2W-IH was present (P = .01; Table 3) compared to dogs without a T2W-IH. Magnetization transfer ratio, MD, AD, and RD were not significantly different in dogs with or without T2W-IH.
After multivariable analyses, MD and AD were found to be significantly different between neurological grades (Figure 4). Mean diffusivity was significantly lower in grade 5 compared to grades 2 (P = .02) and 3 (P = .04), and in grade 4 compared to grade 2 (P = .03). Axial diffusivity was significantly lower in grade 5 compared to grades 2 (P < .001) and 3 (P < .001), and in grade 4 compared to grade 3 (P = .003).

| DISCUSSION
Our study prospectively evaluated the use of QMRI in dogs with thoracolumbar IVDH. Mean diffusivity, AD, and RD had a positive correlation with age and weight. Fractional anisotropy had a negative correlation with age. We also observed that MTR, FA, MD, and RD values in dogs with acute SCI were significantly different from those with chronic SCI caused by IVDH, and that MTR and MD were significantly associated with the affected spinal cord segment. Fractional anisotropy was found to be significantly lower in dogs where a T2W-IH was present compared to dogs without at T2W-IH. On multivariable analyses, MD and RD were significantly correlated with neurological grade, being lower in dogs with grade 5 neurologic dysfunction compared to grades 2 and 3.
Diffusion tensor imaging utilizes MRI to noninvasively measure the molecular diffusion of water in vivo using bipolar magnetic field gradient pulses. 26 As water molecules are able to diffuse 3-dimensionally within a body system, DTI is able to detect not only the magnitude but also the anisotropy of water molecules in the x, y, and z directions. 26 Diffusion tensor imaging therefore provides information about the microstructure and physiologic state of body systems, and changes in the magnitude or anisotropy of water molecules are the result of obstacles limiting the movement of water molecules (change in magnitude), or disarrangement of the body system (change of anisotropy). 26,27 Indices of DTI are difficult to display as images, therefore DTI measurements of water molecules are commonly calculated by diffusion ellipsoids (λ) which represent the average 3-dimensional diffusion over a given distance over time 28 where λ 1 characterizes x, λ 2 characterizes y, and λ 3 characterizes z. 26 Fractional anisotropy represents scalar values, from 0 to 1, and provides directionality information of the anisotropic diffusion of water molecules at each voxel. 29 An FA value of 0 would represent complete isotropy or unrestricted diffusion of water. Within the CNS, this would be expected in areas such as the ventricles. 17 An FA value closer to 1 would represent anisotropy where diffusion is greater in 1 direction, λ 1 , compared to the other directions, λ 2 and λ 3 . 17 In our study, the quantitative DTI parameter FA was not significantly different in dogs with more severe neurologic dysfunction. Interestingly, a recent study on SCI in dogs found FA to be higher in dogs with a worse outcome. 30 For our study, we did find a trend for FA to increase in dogs with a higher neurologic grade when a T2W-IH was absent, and the opposite when a T2W-IH was present. Comparing our findings to Wang-Leandro et al, a possible explanation for FA being higher in dogs with a worse outcome could be related to the effects of T2W-IH on FA. 30 In our study, FA was significantly lower in dogs with a T2W-IH when compared to dogs without at T2W-IH. It has been shown that a T2W-IH is associated with a worse outcome in dogs with IVDH. 6 Fractional anisotropy has been shown to change if a T2W-IH is present. 31 In rats, FA will increase with vasogenic edema then return to normal values once the edema resolves. 17 Therefore, the FA being decreased in our population of dogs could be related to other causes of T2W-IH other than edema including: gliosis, inflammation, hemorrhage, or malacia. Fractional anisotropy was also found to be lower in dogs with chronic SCI compared to acute SCI. This correlates to a previous study evaluating experimental SCI in dogs. 32 Axonal injury is known to decrease FA values. 33 A possible explanation for the difference in FA values between chronic and acute SCI is the effects of vasogenic edema on FA compared to the effects of chronic axonal injury. However, more information on FA is needed to confirm these conclusions.
Directional magnitude of water molecules can be measured by MD, AD, and RD. The most extensively studied metric of directional magnitude in dogs is the apparent diffusion coefficient, for which DTI is similar to MD, although AD and RD have also been evaluated in dogs with SCI. 33-36 A significant increase in MD has been correlated to severe axonal degeneration in dogs with SCI 34 thought to be associated with demyelination, inflammation, and edema. 37 A decrease in MD represents an increase in resistance to diffusion rates. 37 In our study, the DTI measurements MD and AD were significantly lower in dogs with more severe neurologic dysfunction after accounting for the effects that age, body weight, duration of SCI, and longitudinal location of IVDH were demonstrated to have on these metrics.
Studies on SCI suggest that acute injury compared to chronic injury may exhibit different cellular response which can alter DTI measurements. 15 With severe acute injury, MD and RD will generally decrease and with chronic injury it will tend to increase. 15 Axial diffusivity should not to be affected by the chronicity of disease. 34 In our population of dogs, MD and RD were significantly lower in dogs with been previously published. 15 Axial diffusivity was not significantly different in dogs with acute compared to dogs with chronic IVDH.
Chronic SCI is generally associated with demyelination and axonal loss, along with gliosis and syrinx formation. 15 Therefore, with chronic SCI diffusion will increase but anisotropy will decrease, leading to the increase in MD and RD seen with chronic IVDH, and AD being unaffected. 15 Acute SCI is often associated with ischemia, contusion, and inflammation leading to lower diffusion values. 15 A positive correlation was found with age, body weight, and MD, AD and RD. In people, MD, AD, and RD have been shown to increase with age and body mass index, which suggests there is a decline in white matter composition and integrity related to age and weight. 38,39 This leads to a decrease in anisotropy and increase in isotropy as there is loss in the white matter volume. In this population of dogs, a negative correlation was seen between FA and age which is also present in human patients likely associated to a decline in white matter composition and integrity similar to MD, AD, and RD. 38 Our study also prospectively evaluated the use of MTR in dogs with IVDH. Magnetization transfer ratio was not significantly different between neurologic grades but was found to be significantly lower in dogs with chronic compared to acute injury. To the author's knowledge, this is the first study to evaluate MTR in dogs with SCI. Magnetization transfer ratio measures the ability of low protein fluid to exchange magnetization, and alterations to MTR correlate to myelination state. 9,12 Values of MTR vary depending on the time frame of imaging with MTR increasing within the first 2 weeks of CNS injury, and between 16 and 28 days this will reverse to a reduction in MTR. 16 Although the reason for the rise in MTR is not well understood, it is likely related to the stages of Wallerian degeneration and acute secondary injury, and is not related to edema. 16 Instead, there is an increase in available proton exchange sites which can occur with axonal membranes collapsing into ellipsoid bodies and a physiochemical change in the myelin lipid bilayer altering the availability of cholesterol for relaxation exchange. 16 Our study did not find a significant difference in MTR when com-  22 It is an open source software which utilizes an atlas-based system to properly capture the spinal cord shape, including gray matter and white matter. 40 Spinal Cord Toolbox has been utilized in cats and therefore shows promise in dogs. 41 We were able to analyze all QMRI measurements on each dog with SCT. Motion correction methods for DTI are utilized by the SCT software. All images can be evaluated with FSLview after processing to inspect that the software is working correctly.
One limitation of the study is that MTR was not performed on any normal dogs, and therefore we cannot make a conclusion of how important the change of MTR was compared to normal dogs. It was also found that MTR and DTI measurements varied by body weight, age and spinal cord segment. The significance of this is unknown but stresses the importance of age and body weight matched controls in future studies, along with normal values for each spinal cord segment.
In our study, a b-value of 800 was used for DTI. Although we were able to perform quantitative DTI measurements on all dogs, only 1 dog had a cranial thoracic lesion. We were still able to perform DTI on this dog without the use of gating; however, we cannot make a

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

This study was approved by the Department of Small Animal Clinical
Sciences at the Virginia-Maryland College of Veterinary Medicine.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.