Magnetic resonance imaging versus histological assessment for estimation of lesion volume after experimental spinal cord injury

Laboratory investigation

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Object

The purpose of this study was to compare measures of lesion volume obtained by means of 1.5-T MR imaging to those obtained by the Cavalieri method, 6 weeks after experimental spinal cord injury.

Methods

Nine male Wistar rats were subjected to spinal cord injury by clip compression (50 g) at the T-4 level. Six weeks postinjury, the rats were sacrificed, and spinal cords were analyzed ex vivo for lesion volume by means of 1.5-T MR imaging and subsequently, by the Cavalieri method. In the latter method, cords were cut longitudinally in 25-μm sections and stained with solochrome cyanin for myelin. The area of the lesion was determined for each serial section, and the distance-weighted sum of all area measures was then calculated to estimate the total lesion volume.

Results

Bland–Altman analysis showed that the 2 methods had an acceptable level of agreement for lesion volume estimation, but the Cavalieri method was prone to an overestimation bias. The MR imaging estimates of lesion volume were greater than the Cavalieri method estimates in 3 spinal cords, but the difference between measures was within 1 standard deviation of perfect agreement in these 3 lesions, and the mean difference between measures was 18.3%. In contrast, in those lesions in which the Cavalieri method yielded larger lesion volumes (5 lesions), the difference between measures was 2 standard deviations away from perfect agreement for 2 animals and the mean difference between measures was 72.4%.

Conclusions

The results illustrate that the overestimation bias of the Cavalieri method is due, in part, to artifacts produced during processing of the spinal cord tissue.

Abbreviations used in this paper: SCI = spinal cord injury; SD = standard deviation.

Article Information

Address correspondence to: David S. Ditor, Ph.D., Department of Physical Education and Kinesiology, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario, Canada L2S 3A1. email: dditor@brocku.ca.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Graph illustrating lesion volume as determined by histological analysis and MR imaging. Animals (9 rats) were injured by 50-g clip-compression; 6 weeks postinjury they were killed and their spinal cords were removed. The volume of the lesion (mm3) for each spinal cord was first analyzed ex vivo by means of 1.5-T MR imaging using a 3D steady-state free precession gradient-echo sequence (open bars). The spinal cords were subsequently sectioned longitudinally in 25-μm slices for histological analysis and lesion volume estimation by the Cavalieri method (closed bars).

  • View in gallery

    Bland–Altman plot of lesion volume estimation showing the difference between the lesion volume values (mm3) as determined by MR imaging and as determined by histological analysis for each of the 9 animals (y axis) and the mean of the lesion volume values as determined by these 2 methods (x axis). The Bland–Altman plot shows an acceptable level of agreement between the 2 measures, as all data points are at or within 2 SDs from perfect agreement, but it also shows an overestimation bias of the Cavalieri method compared with MR imaging lesion volume estimation.

  • View in gallery

    Photomicrographs (i and ii) and MR images (iii) of the lesion epicenter 6 weeks post clip-compression SCI. The photomicrographs show spinal cord sections containing the lesion epicenter following staining with solochrome cyanin to detect myelin (ii) and immunoprocessing for neurofilament 200 to identify axons (i). Row A corresponds to Animal 5 in which there was close agreement between lesion volume values as determined by the Cavalieri method (3.45 mm3) and MR imaging (3.89 mm3). Row B corresponds to Animal 3 in which the lesion volume as determined by the Cavalieri method (5.89 mm3) was substantially larger than that as determined by MR imaging (3.42 mm3). The asterisks denote the true lesion epicenter, which is apparent in all images. The arrows point to an artifact that appears to have resulted from histological analysis, causing the overestimation of lesion volume.

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