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White Matter Microstructural Changes in an Absence Epilepsy Mouse Model
Gustavo Chau Loo Kung1, Juliet Knowles2, Erpeng Dai3, John Huguenard2, Michelle Monje2, and Jennifer McNab3
1Bioengineering Department, Stanford University, Stanford, CA, United States, 2Neurology Department, Stanford University, Stanford, CA, United States, 3Radiology Department, Stanford University, Stanford, CA, United States

Synopsis

A previously unexplored possibility is that maladaptive myelination may contribute to both the predisposition to seizures and cognitive impairment in diseases such as absence epilepsy. This work presents MRI white matter microstructural measurements in ex vivo mouse brains from the Scn8amed+/- model of absence epilepsy. We obtained estimates of fractional anisotropy, myelin volume fraction and g-ratio. Our results are suggestive of thicker myelin sheaths, which is consistent with g-ratio data obtained by electron microscopy. We look to expand the current analysis to perform longitudinal measurements of the evolution of this particular type of epilepsy and its interplay with aberrant myelination.

Introduction

Absence epilepsy is a common form of pediatric genetic generalized epilepsy which causes seizures that entail brief lapses in awareness1. Absence epilepsy is also associated with significant impairment of attention and learning in 30-40% of affected children. Maladaptive structural and/or functional changes in neural networks may underlie both the predisposition to seizures and cognitive impairment; however, this process remains incompletely understood. The recent discovery of activity-dependent myelin plasticity, which is required for neural network function2 suggests that aberrant neuronal activity in the form of seizures might also alter myelin. Previous works have shown that seizures promote maladaptive myelination, based on immunohistochemical quantification of oligodendrocyte precursor cells (OPCs) and electron microscopy (EM) measurements of myelin sheath thickness in rodents2. To better understand the potential interplay between myelination and seizure activity, we aim to develop a non-invasive MRI scan that enables longitudinal, whole brain in vivo assessment of myelination for a more detailed view of absence epilepsy disease progression. Towards this goal, we present here ex vivo brain MRI white matter evaluation in a murine model of absence epilepsy (Scn8amed+/-)3, and we relate these results to EM measurements in the same mouse model and also a closely related rat model (Wag/Rij)4.

Methods

We acquired data on nine excised mouse brains: four wildtype (WT) and five Scn8amed+/- (denote HET) of 87 day old animals using 7T Burker small bore animal scanner (BioSpec 70/30 USR, Gradient amplitude: 440 mT/m, Slew rate: 3440 T/m/s). For all sequences, we obtained 0.7 mm sagittal slices through the corpus callosum with a field of view of 15mm x 16mm and in-plane resolution of 125um x 125um. We used the following protocols:
  • Magnetization Transfer (MT): 2D FLASH images with MT pulses with equivalent flip angles of 1000° and 4000° were used with seven frequency offsets of 1000, 2000, 4000, 6000, 10000, 22000 and 30000 Hz.
  • T1 images: RARE sequence with six different repetition times of 600, 800, 1000, 1500, 2200 and 4000 ms.
  • Diffusion: diffusion weighted EPI images encompassed into two shells with b-values of 3000 and 6000 were acquired. Each shell consisted of 36 encoding directions with 4 additional b=0 images.
We used the pipeline described in West et al.5,6 to process the data. Quantitative T1 maps were obtained by fitting the saturation recovery model to the T1 images7. The T1 maps and the MT images were fitted to the Sled-Pike model8 using qMRlab9 to obtain Myelin Volume Fraction (MVF)5. NODDI10,11 was used to obtain the intra-neurite compartment fraction to estimate the Axon Volume Fraction (AVF)6. Finally, MVF and AVF were used to estimate the g-ratio (ratio of the inner diameter of the axon to the outer diameter encompassing the myelin sheath). For comparisons, fractional anisotropy (FA) was also computed using FSL12. FLIRT12 was used to co-register diffusion, T1 and MT images. NiftyReg13 was used to improve the alignment of the DWI volumes. For comparisons, we defined three ROIs in the genu, body and splenium of the corpus callosum (Fig. 1a). EM measurements were performed on a cohort of three wildtype and three seizure (SZ) animals of the closely related Wag/Rij rat model4. Samples were obtained from the same three aforementioned regions of the corpus callosum. Likewise, samples of the body of a separate cohort of six Scn8amed+/- and six wildtype mice were obtained.

Results and Discussion

Figure 1b-1d show example FA, MVF and g-ratio maps for one of the samples. The corpus callosum can be clearly observed as it is known to show higher anisotropy and myelination. Figures 2 and 3 show the values for MVF and g-ratio between the different brains. Although we are not appropriately powered yet, the results suggest a decrease in estimated g-ratio in the genu (WT:0.75±0.04, HET:0.66±0.04) but not in the body (WT:0.79±0.03, HET:0.77±0.03) or splenium (WT:0.72±0.03, HET:0.71±0.03). EM measurements Wag/Rij rats display a decrease of g-ratio in the genu and body (WT:0.70±0.02, SZ:0.66±0.01 and WT:0.67±0.01, SZ:0.62±0.01, respectively), but not in the splenium (Figure 5). The EM data also showed that such an increase was specifically due to increased myelination, and the small increase in MVF observed in Figure 3 for the genu (WT:0.14±0.01, HET:0.16±0.01) is in agreement with these findings. EM measurements in the body of Scn8amed+/- mice also show a decrease in g-ratio (WT:0.75±0.01, HET:0.72±0.02). The estimated MVF, AVF and g-ratio measures have not yet undergone the slope calibrations recommended in previous works5,6,14. While FA also shows a decrease in the genu (WT:0.24±0.05, HET:0.19±0.05), it suffers from high variability (Fig. 4) and is not specific to myelination15. Future experiments will perform both the MRI and EM in the same brain tissue to better understand how our MRI estimates relate to EM measurements. Ultimately, the aim is to enable longitudinal, whole brain in vivo studies of myelination evolution that capture a more detailed view of absence epilepsy disease progression.

Conclusion

We have presented MRI-derived microstructural metrics in control and absence epilepsy mouse models. Further directions include (i) validating the metrics against EM and (ii) translating the protocol and analysis to in vivo mice to probe longitudinal changes in myelination. These imaging tools will be used to determine network-level seizure-induced myelin abnormalities, a novel disease mechanism.

Acknowledgements

This work has been funded by NIH: R01 NS095985, R01 MH111444 grants, the Charles A. Dana Foundation, NIMH R21 MH116484 grant, and Wu Tsai Neuroscience Institute at Stanford University.


References

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Figures

Figure 1. (a) Example ROIs used in the the analysis. (b) Fractional anisotropy map (c) Myelin Volume Fraction map and (d) g-ratio maps for one of the heterozygote mice.

Figure 2. Estimated Myelin Volume Fraction for the different ROIs and subjects. Blue bars are the wild type mice and red ones represent the Scn8amed+/- mice.

Figure 3. Estimated g-ratio for the different ROIs and subjects. Blue bars are the wild type mice and red ones represent the Scn8amed+/- mice.

Figure 4. Fractional anisotropy obtained for the different ROIs and subjects. Blue bars are the wild type mice and red ones represent the Scn8amed+/- mice.

Figure 5. Electron Microscopy g-ratio comparisons in rats (a) and mice(b). Black dots represent the wild type animals and the red ones represent the seizure animals.

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)
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