Synopsis
Multiple sclerosis (MS) features demyelination
of the brain and spinal cord, resulting in impaired and eventual loss of neuronal
function. Approximately 65% of MS patients experience cognitive impairment and
memory dysfunction. Postmortem analyses reveal hippocampal demyelination and
glutamate receptor loss in MS patients, suggesting impaired synaptic function
in this brain region critical for memory and learning. Using a mouse model of
reversible demyelination, we demonstrate that MRI can detect the loss and
restoration of myelin and neuronal function in the hippocampus. Our results
suggest that MRI is a powerful pre-clinical tool for testing neuroprotective
and reparative therapies targeting the hippocampus.Purpose
To determine if
MRI can detect the effects of demyelination and remyelination on hippocampal
structure and function.
Methods
Animals:
Eight week-old C57Bl/6J mice were fed 0.3%
cuprizone (C) chow pellets or standard rodent chow and received daily IP
injections of Rapamycin (R) (10mg/Kg) for 12 weeks. The animals were divided
into 4 groups: CR12+0 (n=10) receiving 12w C/R to induce demyelination; R12+0
(n=10) control mice receiving 12w R and normal diet and exhibiting normal
myelin; CR12+6 (n=10) receiving 12w C/R to induce demyelination, followed by 6w
normal diet and exhibiting spontaneous remyelination; R12+6 (n=10) control mice
receiving 12w R and normal diet followed by 6w of normal diet without R.
MRI:
3D MRI (voxel
size ~0.140x0.140x0.140 mm3) were acquired at the Case Center for Imaging
Research on a 9.4T Bruker-Biospec MRI. Imaging included: proton-density-weighted
MRI with a magnetization transfer (MT) pre-pulse (TR/TE=50/3.1ms, flip angle=10º);
T2-weighted (T2w) MRI (TR/TE=1980/40ms, RARE factor=12, SA=2); T1-weighted MRI
(TR/TE=120/4.4ms, flip angle=55º) acquired before (PreMN) and 24 hours after (PostMN)
2 days of daily IP manganese chloride (50 mM MnCl2) injections.
MRI
analysis: Brain and hippocampi were segmented from T2w
MRI. To detect myelin changes, hippocampal MTCSF1 (MTCSFhippo),
the ratio of mean intensity of hippocampal voxels to the mean intensity of
cerebrospinal fluid (CSF) voxels based on MT MRI, was computed. To detect
changes in neuronal function,2 the volume of PostMN enhanced voxels in
the hippocampi (eVolhippo) was quantified by comparison of each
PostMN voxel intensity to the mean intensity of its local neighborhood on the
PreMN MRI. Student’s t-test was used to test for statistically significant
differences between groups CR12+0 and R12+0, CR12+6 and R12+6. (Fig. 1)
Results &
Discussion
1. Differences in hippocampal MTCSF are consistent with demyelination and
remyelination.
Hippocampal MTCSF was 20% higher after 12w of
C/R compared to control mice (C/R12+0: MTCSFhippo mean=0.80,
standard deviation (SD)=0.04, N=10; R12+0: MTCSFhippo mean=0.67,
SD=0.04, N=10; p<0.0001), consistent with the severe hippocampal
demyelination typically observed in these mice.3 After spontaneous
remyelination for 6w, we found no significant difference in hippocampal MTCSF
compared to control mice, which is consistent with the remyelination pattern
that is typically observed. (Fig. 2)
2. Differences in MnCl2 enhanced hippocampal volume are consistent with
the impairment and restoration of hippocampal function. In the
hippocampi of the demyelinated mice, we found that MnCl2 enhanced
hippocampal volume was 54% lower compared to control mice (C/R12+0: eVolhippo
mean=0.79 mm3, SD=0.25 mm3, N=8; R12+0: eVolhippo
mean=1.73 mm3, SD=0.41 mm3, N=10; p<0.00001). This is
consistent with evidence for poor memory (on Morris water maze), reduced
synaptic proteins, impaired long term potentiation (LTP) and altered dendritic
spine morphology in demyelinated mice.3,4 After 6w of spontaneous
remyelination, MnCl2 enhanced hippocampal volume was restored to
control levels, consistent with improved water maze performance and partial
restoration of synaptic proteins, LTP and
dendritic spine morphology.3,4 (Fig. 2)
3. Hippocampal
volume decreases with demyelination and is not significantly restored with
remyelination. We found that hippocampal volume was on average
9% lower in the demyelinated mice compared to control mice, consistent with
tissue injury (C/R12+0: hippocampal volume mean=13.97 mm3, SD=0.99
mm3, N=10; R12+0: hippocampal volume mean=15.31 mm3, SD=0.49
mm3, N=10; p<0.005). After 6w of remyelination, the hippocampi remained
significantly smaller than control mice (C/R12+6: hippocampal volume mean=14.45
mm3, SD=0.63 mm3, N=10; R12+6: hippocampal volume
mean=15.78 mm3, SD=0.61 mm3, N=9; p<0.005) suggesting that
hippocampal volume changes are multi-factorial. (Fig. 2)
Conclusion
These results suggest that this mouse model and
these MRI metrics can be used in pre-clinical trials of drugs to treat memory
and learning dysfunction in MS patients.
Acknowledgements
Grant support: NINDS R01NS080976. B. Erokwu and C.A Flask at the Case Center for
Imaging Research for assisting with MRI protocol implementation. J.G. Sled, J.P
Lerch and R. Allemang-Grand at the Toronto Centre for Phenogenomics for
advising on MnCl2 MRI. Renovo Neural Inc. for supplying mice for the study.
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