Testing Pathological Variation of White Matter Tract in Adult Rats after Severe Spinal Cord Injury with MRI

Abstract

The purpose of this study was to assess the pathological variation in white matter tracts in the adult severe thoracic contusion spinal cord injury (SCI) rat models combined with in vivo magnetic resonance imaging (MRI), as well as the effect of spared white matter (WM) quantity on hindlimb motor function recovery. 7.0T MRI was conducted for all experimental animals before SCI and 1, 3, 7, and 14 days after SCI. The variation in the white matter tract in different regions of the spinal cord after SCI was examined by luxol fast blue (LFB) staining, NF200 immunochemistry, and diffusion tensor imaging (DTI) parameters, including fraction anisotropy, mean diffusivity, axial diffusion, and radial diffusivity. Meanwhile, Basso-Beattie-Bresnahan (BBB) open-field scoring was performed to evaluate the behavior of the paraplegic hind limbs. The quantitative analysis showed that spared white matter measures assessed by LFB and MRI had a close correlation (R² = 0.8508). The percentage of spared white matter area was closely correlated with BBB score (R² = 0.8460). After SCI, spared white matter in the spinal cord, especially the ventral column WM, played a critical role in motor function restoration. The results suggest that the first three days provides a key time window for SCI protection and treatment; spared white matter, especially in the ventral column, plays a key role in motor function recovery in rats. Additionally, DTI may be an important noninvasive technique to diagnose acute SCI degree as well as a tool to evaluate functional prognosis. During the transition from nerve protection toward clinical treatment after SCI, in vivo DTI may serve as an emerging noninvasive technique to diagnose acute SCI degree and predict the degree of spontaneous functional recovery after SCI.

Figure 1

Set of different tractography ROI regions. (a) Pathological image. (b) MRI image. (c) Color FA map. DC (dorsal column), LLWM (left lateral white matter), RLWM (right lateral white matter), LVWM (left lateral white matter), and RVWM (right lateral white matter).

Figure 2

LFB and Nissl staining of coronal sections at the lesion core (LC), R5mm, and C5mm segments at each time point after SCI. The variation in the percentage of spared white matter area over time was observed. (a) LFB and Nissl staining of coronal sections at the lesion core, R5mm, and C5mm segments at each time point after SCI. The anatomic structure at the lesion core of the spinal cord was clearly changed, where white matter at the DC was greatly lost, gray matter was transformed severely, and a large cyst formed at the spinal cord center, which was filled with a large amount of denatured and dead neurons and disintegrated axons and myelin debris as well as inflammatory cells. The dotted line indicates spared white matter area. (b) Quantitative analysis of LFB- and Nissl-stained spared white matter area percentage in the R5mm, lesion core, and C5mm region over time. Spared white matter at the lesion core decreased at 1 day after SCI, dropped to the lowest point at 3 days after SCI, and remained stable from 7 to 14 days after SCI. ^∗ Significantly different from the value before SCI. DC: dorsal column; LLWM: left lateral white matter; RLWM: right lateral white matter; LVWM: left ventral white matter; RVWM: right ventral white matter.

Figure 3

Structural image of MRI-T2 coronal sections at the lesion core (LC), R5mm, and C5mm segments at each time point after SCI. The variation of spared white matter area percentage over time was observed. (a) T2 images along the horizontal axis taken from the lesion core (LC), R5mm, and C5mm of each time point after SCI. The control rat has a clear boundary between white matter and gray matter at T7-8, gray matter is in an H-shape in the medium signal, white matter in the medium-low signal, and peripheral cerebrospinal fluid in a hyperintensity signal. At 1 day after SCI, a hypointensity signal was observed at the DC of the spinal cord, indicating acute hemorrhage and a blurred boundary between white matter and gray matter. At 3 days after SCI, the hypointensity signal at the lesion core remained, and a larger circle-like hypointensity signal shadow appeared in the region close to the corticospinal tract in the DC of R5mm and C5mm. At 7 days after SCI, a hyperintensity signal appeared in the lesion core (LC), R5mm and C5mm regions, indicating possible edema in these regions. At 14 days after SCI, the abnormal signal shadows shrank in the R5mm and C5mm regions, accompanied by a mixed signal and a clearer boundary between white matter and gray matter. The dotted line indicates spared white matter area. (b) Quantitative analysis of the variation in percentage of spared white matter area in the R5mm, C5mm, and lesion core (LC) regions over time, which is similar to the statistical findings for the morphological changes. ^∗ Significantly different from the value before SCI: ^∗P < 0.05.

Figure 4

Correlation analysis of spared white matter (LFB) and spared white matter (MRI); the two sets of results were positively correlated.

Figure 5

NF-200 immunofluorescent staining and the color FA maps at the lesion core segment showed the axonal variation in the SCI time course. (a) Before SCI and at 1, 3, and 14 days after SCI, axonal morphology of the DC, LLWM, RLWM, LVWM, and RVWM. The number of axons in the dorsal column (DC) region was lost quickly and severely at 1 and 3 days after SCI, and the axonal loss in the DC column was considerable, with spared axons characterized as disorganized, swollen, and ruptured. The axon loss and degeneration were continuously observed over the 14 days after SCI. (b) Before SCI and at different time points after SCI, axonal numbers in different regions. (c) Corresponding color FA maps before SCI and at different time points after SCI. ^∗ Significantly different from those before SCI: ^∗ P < 0.05; ^∗∗ P < 0.01. Scale bar = 100 μm.

Figure 6

Open-field BBB scoring after severe spinal cord contusion. (a) Two observers blinded to the treatment carried out the BBB test on experimental rats in an open-field to evaluate the degree of recovery of locomotor function after SCI. The figure shows behavioral performance prior to the operation and at different time points after SCI. (b) BBB scoring at different time points after SCI. Hindlimb motor function was significantly reduced at 1 day after SCI and began to recover at 7 days after SCI. ^∗ Significantly different from scores before SCI, ^∗P < 0.05.

Figure 7

Correlation analysis of spared white matter at the lesion core (LC) and BBB scores of hind limb motor function. Except for the data at 1 day after SCI, the spared white matter area percentage at the LC is closely correlated with BBB scores (R² = 0.846).