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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 19, 2004 as doi:10.1096/fj.04-2026fje. |
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,||,1
Departments of
* Neurology,
Immunology,
Biochemistry,
Biomedical Imaging Resource, and
|| NMR Core Facility, Mayo Clinic, Rochester, Minnesota, USA
1Correspondence: Department of Neurology, Mayo Clinic, Rochester, Minnesota 55905, USA. E-mail: rodriguez{at}mayo.edu
SPECIFIC AIMS
Stimulation of remyelination may be a restorative treatment for multiple sclerosis (MS). Our laboratory identified both human and murine monoclonal antibodies that promote remyelination in murine models of MS. The recombinant human monoclonal IgM antibody, rHIgM22, promotes remyelination after Theiler’s virus (TMEV)- induced inflammatory demyelination and also following toxin-induced demyelination of the spinal cord.
The primary aim of our study was to determine whether changes detected by 3-D volume-acquisition T2-weighted MRI would allow us to characterize antibody-mediated remyelination in SJL/J mice after TMEV-induced demyelinating disease. These mice develop a progressive demyelinating disease, with lesions almost exclusively confined to the spinal cord. To assess the extent of antibody-mediated remyelination, volumetric measurements of pretreatment and post-treatment spinal cord white matter lesion loads were conducted using in vivo MRI.
A secondary goal was to determine whether remyelination-promoting antibodies could be visualized by MRI in the spinal cords of mice with demyelinating disease, using particulate MRI contrast material (USPIO) labeling of rHIgM22 antibodies.
PRINCIPAL FINDINGS
1. rHIgM22 antibody reduced total lesion load 5 wk after treatment
Two groups of female SJL/J mice had been infected with TMEV for 6 months. One group (13 animals) received a 5 wk treatment with the remyelination promoting antibody rHIgM22, while a control group (8 animals) was treated with phosphate-buffered saline (PBS). The mean lesion load in the antibody-treated group and control groups was not significantly different before rHIgM22 antibody treatment (P=0.37). However, 5 wk after treatment the mean lesion load in the two groups was statistically different (P<0.0001; Student’s t test). The mean lesion load decreased by 40.6% in the antibody-treated group, whereas it increased by 13.6% in sham-treated animals. Lesion volumes decreased in 13 of 13 mice treated with rHIgM22; lesion volume increased in 7 of 8 mice treated with PBS (P<0.01 by 
2 test).
2. Treatment with rHIgM22 antibody decreased average lesion size
We applied 3-D volumetric techniques to monitor changes in each spinal cord lesion. Altogether, the 13 rHIgM22 antibody-treated animals developed a total of 99 lesions; the 8 sham-treated mice developed a total of 64 lesions. The number of lesions per animal was not significantly different between the groups (P=0.92). Before treatment, the average lesion size in the antibody-treated group was 53.4 ± 46.3 units (1 unit=0.01mm3), and in the PBS-treated group it was 57.6 ± 40.8 units. These were not different statistically (P=0.81). Five wk after remyelinating antibody treatment, the average lesion size in the treated group had decreased by 31.5% to 31.7 ± 23.2, whereas in the control group it had increased by 13.6% to 65.4 ± 44.5 units (P=0.01, Student’s t test).
3. Treatment with rHIgM22 increased the number of retracting lesions
To further stratify the data, we characterized the lesions into expanding (lesions that grew in size), retracting (lesions that decreased in size) and stable lesions. In the remyelinating antibody-treated group, only 6.1% of all lesions were expanding, whereas in the control group, 65.6% of lesions were expanding. In the antibody-treated group, 82.8% of all lesions decreased in size, whereas in the control group only 18.8% decreased in size. The rate of change was identical in both groups. This indicated that the remyelinating antibody treatment did not alter the rate of lesion change (growth or decrease), but it shifted the proportion of lesions so that retracting lesions predominate.
4. Decrease in 3-D MRI volume is concurrent with CNS remyelination
After the second MRI session 5 wk after treatment, animals were killed for histologic processing, myelin staining, and microscopic examination. Figure 1
shows representative spinal cord sections with matching MR images. The areas of demyelination on histology (outlined) were smaller and less pronounced on the "after treatment" images compared with the "before treatment" images taken 5 wk after rHIgM22 antibody treatment (Fig. 1A, B
). The reduction in the high MR signal areas corresponded with remyelination in these animals. Conversely, areas of demyelination grew in size after sham treatment (Fig. 1E, F
).
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Histologic evaluation by myelin staining was available for 7 antibody-treated animals and 4 PBS-treated animals, revealing a 1.9-fold increase in spinal cord quadrants showing remyelination after rHIgM22 treatment compared with controls, which corresponded to a 4.2-fold decrease in mean lesion volume by MRI.
5. USPIO-labeled rHIgM22 antibodies were detected by MRI in the spinal cord of SJL/J mice with demyelination
We tested whether rHIgM22 could enter the demyelinated lesions in the spinal cords of SJL/J mice at 4 wk after TMEV infection. The spinal cords of SJL/J mice were imaged 4 wk after TMEV infection. Mice were given intravenous injection of biotinylated rHIgM22 antibodies, followed 4 h later by streptavidin-USPIO complexes.
After the injection of the contrast material complex, high T1 signal areas were identified in comparison to matching images of the same animals before treatment (Fig. 2
). In noninfected control mice, postcontrast enhancement was not seen after the injection of the same contrast material complex (Fig. 2A, D
). Infected SJL/J mice were injected with labeled isotype control sHIgM39 antibody, which does not bind to CNS cells. In these mice, enhancement was not seen (Fig. 2E, F
). The injection of avidin-USPIO in mice without prior antibody injection also did not result in any noticeable enhancement (Fig. 2G, H
). We conclude that rHIgM22 antibody enters the spinal cord of animals with demyelinating lesions.
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CONCLUSIONS AND SIGNIFICANCE
Previous histology-based studies demonstrated that CNS-type remyelination occurred in 59.7% of spinal cord quadrants in animals treated with rHIgM22 antibody compared with 15.8% in animals treated with PBS. In the present MRI study we found that 82.8% of lesions in animals treated with rHIgM22 showed retraction of varying degrees, presumably the effect of remyelination. Volumetric MRI may provide a more sensitive measure compared with two-dimensional random-sampling using histopathology methods. However, it is possible that some of the decrease in 3-D volumes could be related to postprocessing errors due to the applied windowed sync interpolation methods during coregistration.
Our observation that the rate of lesion growth or lesion retraction is not different between individually identified lesions in the antibody-treated group compared with the PBS-treated group, is a novel finding. This indicates that the processes of demyelination and remyelination have the same chronological characteristics, but the treatment with rHIgM22 shifts the dynamics toward lesion retraction (i.e., remyelination).
At present, there is no universally accepted marker for remyelination using standard MRI techniques in MS or MS models. In some models, the resolution of T1 hypointensities has correlated with remyelination whereas, in others, increasing magnetization transfer ratio was seen. T2-weighted sequences are especially sensitive to structural alterations of CNS tissue; they can represent edema, inflammation, demyelination, axonal loss, and gliosis. Our results indicate that T2-weighted 3-D volume acquisition MRI is capable of identifying antibody-induced remyelination after chronic demyelination in mice.
The recovery of T2-weighted hyperintensities does not necessarily follow the formation of remyelinated shadow plaques in human MS. In our model, the volume of T2 hyperintensity clearly decreased in the rHIgM22-treated animals and correlated closely with the appearance of remyelination. An alternative explanation is the appearance of T2 hypointense areas at the lesion edges. Such areas could "mask" parts of the hyperintense lesions, resulting in an apparent decrease. Such hypointensities have been observed in MS and are related to susceptibility artifacts caused by the presence of strong paramagnetic materials (iron, free radicals, etc.). Such materials may be byproducts of enhanced immune activity. Considering that CNS-type remyelination does require the presence of antibody and immune cells in the plaques, it is possible that immune activity is responsible in part for the observed MRI phenomenon.
The direct binding of rHIgM22 to demyelinated lesions using the biotin-streptavidin-USPIO-based MRI contrast material is consistent with the hypothesis that these antibodies work directly on CNS glia to induce remyelination.
MRI appears to be a potentially powerful tool as an outcome measure for future studies of remyelination in the TMEV model of MS, and possibly in the future human trials aimed at using antibodies to promote remyelination and functional improvement in MS.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2026fje;
This article has been cited by other articles:
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  I. Pirko, T. K. Nolan, S. K. Holland, and A. J. Johnson Multiple Sclerosis: Pathogenesis and MR Imaging Features of T1 Hypointensities in Murine Model Radiology, March 1, 2008; 246(3): 790 - 795. [Abstract] [Full Text] [PDF]
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 D. R. Ure, T. E. Lane, M. T. Liu, and M. Rodriguez Neutralization of chemokines RANTES and MIG increases virus antigen expression and spinal cord pathology during Theiler's virus infection Int. Immunol., May 1, 2005; 17(5): 569 - 579. [Abstract] [Full Text] [PDF]
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