Association Between Magnetic Resonance Imaging-Based Spinal Morphometry and Sensorimotor Behavior in a Hemicontusion Model of Incomplete Cervical Spinal Cord Injury in Rats

Abstract

Aim: Structural connectivity in the reorganizing spinal cord after injury dictates functional connectivity and hence the neurological outcome. As magnetic resonance imaging (MRI)-based structural parameters are mostly accessible across spinal cord injury (SCI) patients, we studied MRI-based spinal morphological changes and their relationship to neurological outcome in the rat model of cervical SCI. Introduction: Functional connectivity assessments on patients with SCI rely heavily on MRI-based approaches to investigate the complete neural axis (both spinal cord and brain). Hence, underlying MRI-based structural and morphometric changes in the reorganizing spinal cord and their relationship to neurological outcomes is crucial for meaningful interpretation of functional connectivity changes across the neural axis. Methods: Young adult rats, aged 1.5 months, underwent a precise mechanical impact hemicontusion incomplete cervical SCI at the C4/C5 level, after which sensorimotor behavioral assessments were tracked during the reorganization period of 1-6 weeks, followed by MRI of the cervical spinal cord at 8 weeks after SCI. Results: A significant ipsilesional forelimb motor debilitation was observed from 1 to 6 weeks after injury. Heat sensitivity testing (Hargreaves) showed ipsilesional forelimb hypersensitivity at 5 and 6 weeks after SCI. MRI of the cervical spine showed ipsilateral T1- and T2-weighted lesions across all SCI rats compared with no significant lesions in sham rats. Morphometric assessments of the lesional and nonlesional changes showed the diverse nature of their interindividual variability in the SCI receiving rats. While the various T1 and T2 MRI lesional volumes associated weakly or moderately with neurological outcome, the nonlesional spinal morphometric changes associated much more strongly. The results have important implications for interpreting functional MRI-based functional connectivity after SCI by providing vital underlying structural changes and their relative neurological impact. Impact statement Functional connectivity assessments on patients with SCI relies heavily upon MRI based approaches. Hence, underlying MRI based structural and morphometric changes in the reorganizing spinal cord and its relationship to neurological outcomes is vital for meaningful interpretation of functional connectivity changes across the complete neural axis (both spinal cord and the brain).

Keywords: MRI; T1; T2; behavior; morphometry; rat cervical spine; spinal cord injury.

FIG. 1.

(A) Schematic of the rat brain and cervical spinal cord showing the hemicontusion injury impact site lateralized to the left side of the spinal cord at the C4 vertebral level. (B) Typical MRI T1-RARE images in the axial plane (1 mm slice thickness; in plane resolution 110 × 110 μm) covering from the brainstem rostral to C1 all the way to the T1 vertebral level. (C) Lateral sagittal plane image showing CSF, spinal parenchyma, and spinal roots (appearing bright) with the hypointense vertebral bodies. Masked and linearly registered T1- and T2-weighted images from C1 to C7 from typical (D) sham and (E) SCI animal, respectively. The laminar level of each coronal slice position was determined from the unique vertebral bodies, spinal roots, and the cross-sectional spinal parenchymal topography at each laminar level, as shown in (B, C), respectively. The contusion injury epicenter at C4 vertebral level and the periinfarct zones are evident on adjacent slices rostral and caudal to C4 from the T1- and T1-weighted images of the SCI animal. (E) All SCI animals (n = 11) showed similar pattern of lesions with no detectable lesions across all sham animals (n = 7), as shown in Supplementary Figure S1. CSF, cerebrospinal fluid; MRI, magnetic resonance imaging; RARE, rapid acquisition relaxation-enhanced; SCI, spinal cord injury.

FIG. 2.

Percentage forelimb use behavior in sham (n = 5) and SCI (n = 7) animals. (A) SCI animals showed a steep decrease in the simultaneous use of both forelimbs at 1 week after injury, followed by a gradual increase until 6 weeks after injury. Significantly different ***p < 0.0001; df = 1; F = 315.13 two-way ANOVA with repeated measures. (B) No ipsilateral forelimb use was observed in the SCI animals, whereas sham animals showed ∼15% use. Significantly different **p < 0.001; df = 1; F = 71.52 two-way ANOVA with repeated measures. (C) One hundred percent contralateral forelimb use was observed across SCI at 1 week, followed by gradual decrease up to 6 weeks. Significantly different ***p < 0.0001; df = 1; F = 516.19 two-way ANOVA with repeated measures. (D–G) Assessment of heat sensitivity (Hargreaves test) in sham (n = 5) and SCI (n = 5) animals. (D) Ipsilateral forelimb showed significantly lower paw withdrawal latency to heat stimulus in SCI. Significantly different ***p < 0.0003; df = 1; F = 35.7 two-way ANOVA with repeated measures. (E–G) No significant differences in paw withdrawal latencies were observed across contralateral forelimb or both hindlimbs between sham and SCI. ANOVA, analysis of variance.

FIG. 3.

Typical T1- and T2-weighted MRIs along the C3–C5 vertebral columns with the injury epicenter at C4. (A) CSF and gray matter (butterfly shaped) appear brighter with relatively less intense white matter regions in the T1-weighted images. Lesions from the mechanical impact contusion injury show both hypo- and hyperintense regions within the ipsilateral spinal parenchyma. (B) In the T2-weighted images, CSF is the brightest followed by less intense gray matter (butterfly shaped) and hypointense white matter. Lesions from the contusion injury appear hypo- or hyperintense. (C) Parenchymal horizontal cross-sectional length (central axis) at the injury epicenter slice; i-ipsilateral length and c-contralateral length. Box and whisker plots of the various spinal MRI morphometric parameters. (D) Lesion volumes determined by T1 hypointensity, T1 hyperintensity, T2 hypointensity, and T2 hyperintensity, respectively, across the spinal parenchyma in sham (S; n = 7) and SCI (n = 11) animals. (E) T2 parenchymal volume across the contralateral and ipsilateral spinal cord and the contralateral–ipsilateral volume difference across sham (n = 7) and SCI (n = 11) animals. Significant difference between sham and SCI was observed across all MRI variables. Ipsilateral parenchymal volume and the contra–ipsi volume difference also significantly differed between sham and SCI. Two tailed t-test with a p < 0.05 required for significance. Color images are available online.

FIG. 4.

Relationship between various T1- and T2-weighted morphometric parameters across the SCI animals (n = 11). (A) Moderate correlation between T1 hyperintensity versus T1 hypointensity (R = 0.51), and (B) strong correlation between T2 hypointensity versus the i/c ratio at the epicenter (R = 0.65). (C) Table showing the association between all other combinations of the MRI variables.

FIG. 5.

Relationship between the i/c ratio, contralateral–ipsilateral volume difference, and behavioral outcomes across seven SCI animal subjects. (A–D) Motor behavior at both the subacute (1 and 2 weeks) and chronic (4 and 6 weeks) after injury correlated strongly with the i/c ratio. (E–H) A similar relationship was observed between the acute/subacute and chronic behavioral outcomes and the contralateral–ipsilateral volume difference.

FIG. 6.

Relationship between MRI parameters and heat sensitivity behavior (Hargreaves) across five SCI animal subjects. (A, B) Five weeks after SCI and (C, D) 6 weeks after SCI. Only the i/c ratio at the epicenter correlated moderately with heat sensitivity at both 5 and 6 weeks after SCI. T1 hyperintensity and T1 hypointensity also showed a moderate correlation with heat sensitivity behavior at 6 weeks after SCI.