Validating Myelin Water Imaging with Electron Microscopy in Rat Spinal Cord

Henry Szu-Meng Chen¹, Nathan Holmes², Wolfram Tetzlaff^2,3, and Piotr Kozlowski^4,5

¹Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada, ²Zoology, University of British Columbia, Vancouver, BC, Canada, ³ICORD, Vancouver, BC, Canada, ⁴UBC MRI Research Centre, Vancouver, BC, Canada, ⁵Radiology, University of British Columbia, Vancouver, BC, Canada

Synopsis

Quantitative T2 based myelin water imaging measures myelin content by probing the properties of the water trapped in myelin and therefore depends on its morphology. We compared MR myelin water fraction (MWF) to electron microscopy derived myelin content using a rat injury model and found that MWF correlates strongly with the amount of myelin lipid bilayers in both intact myelin and myelin debris and that myelin debris appears to consist of areas of either normally spaced myelin or large vacuous spaces. No significant differences were found in myelin spacing among normal, 3 week, and 8 weeks post injury time points.

Target Audience

Researchers interested in using quantitative T2 based myelin water imaging.

Purpose

Quantitative T2 based myelin water imaging indirectly measures myelin content by probing the properties of the water trapped between the myelin lipid bilayers. It is therefore important to understand how morphological changes affect this water environment. To do so we compared MR and transmission electron microscopy (TEM) derived myelin measurements in the fasciculus gracilis at 5 mm cranial to injury using a rat model, where based on previous findings¹, we expect a large amount of myelin debris at 3 weeks post injury that are largely cleared by 8 weeks. The use of TEM also enabled direct measurements of myelin spacing.

Methods

Cervical spinal cord sections were excised from Fischer 322 rats at each of the normal (3 rats), 3 weeks (6 rats), and 8 weeks (5 rats) post C5 dorsal column transection time point after intracardial perfusion with buffered fixative solution of 2% paraformaldehyde (PF) and 2% glutaraldehyde. Each section was divided for TEM and MRI, as shown in Figure 1, after overnight post-fixation in the same fixative solution.

A single slice multi-echo CPMG sequence was used to acquire quantitative T2 data² (256 × 256 matrix, TE/TR = 1500/6.738 ms, 32 echoes, 1.79 cm FOV, 1 mm slice, NA = 12, 70 μm in-plane resolution) using a 5 turn, 13 mm i.d. solenoid coil on a 7T preclinical MRI scanner (Bruker, Germany). CPMG data were processed using a non-negative least square analysis technique³. Myelin water fraction (MWF) maps were generated by dividing the integral from 7.75–20 ms range by the total integral of the T2 distribution for each pixel. Region of interest analysis was used to obtain the average MR MWF from fasciculus gracilis.

The TEM sections were further fixed in 1% osmium and 1.5% tetroxide potassium ferrocyanide solution before being set in resin blocks, from which 70 nm thin sections were cut on ultra-microtome, mounted on copper grids, and stained with 2% uranyl acetate and lead citrate. Three grid windows in the fasciculus gracilis were chosen from each section and 9 images taken per window at 16,000x magnification at a resolution of 2048 x 2048. Myelin area was identified using intensity thresholding after manual segmentation, Wiener filtering, and contrast limited histogram normalization (Figure 2a-c). Myelin content was calculated as the myelin area divided by the total area. Myelin spacing/period was sampled from one randomly selected myelin sheath per TEM image using the method shown in Figure 2d. Differences in myelin spacing among the 3 groups were compared using one-way ANOVA.

Results

Results of TEM myelin content vs MR MWF is shown in Figure 3. The two measures are highly correlated (R = 0.83, p < 0.001) despite the lack of functioning myelin at the injured time points. No significant differences were found in the myelin spacing between normal (M = 10.9 nm, SD = 3.0 nm), 3 weeks, (M = 11.2 nm, SD = 1.9 nm), and 8 weeks (M = 11.0 nm, SD =2.6 nm) post injury as shown in Figure 4 (F(2,399) = 0.80, p = 0.45).

Discussion

The fasciculus gracilis was chosen for this study because there is little intermingling of axons at the cervical level, and it provides a consistent myelin content throughout the excised portion. The MR MWF reported here are higher than previously measured in a similar study in vivo⁴. This is likely due to the TEM fixation requirement. In order to resolve lipid bilayers, the use of glutaraldehyde, in addition to PF, results in higher cell shrinkage due to water removal predominantly from the intra/extra cellular space.

Our results suggest that MR MWF accurately reflects the amount of myelin lipid bilayers, regardless of the state of the myelin. Throughout Wallerian degeneration, collapsed/peeled myelin consists of only areas of either normally spaced myelin (with no ultrastructural changes) or large vacuous spaces (Figure 5). The area of normally spaced bilayer in myelin debris would contain similar amount of water as normal healthy myelin, while the large vacuous spaces would be classified as intra/extra cellular water by quantitative T2.

Curiously, while our result here demonstrate no significant difference in myelin spacing, in vivo data in the same WM track using the same rat model have shown higher MR MWF reading at 3 weeks post injury than normal control⁴. This may suggest that factors other than morphology of myelin debris need to be considered to explain this phenomenon.

Conclusion

MWF correlates strongly with the amount of myelin lipid bilayers in both intact myelin and myelin debris.

Acknowledgements

This study has been supported by the Canadian Institutes of Health Research and The Natural Sciences and Engineering Research Council of Canada. The authors would like to thank Susan Shinn for her help with everything TEM.

References

1. Kozlowski P, Raj D, Liu J, Lam C, Yung AC, Tetzlaff W. Characterizing White Matter Damage in Rat Spinal Cord with Quantitative MRI and Histology. J. Neurotrauma 2008;25:653–676.

2. Poon CS, Henkelman RM. Practical T2 quantitation for clinical applications. J. Magn. Reson. Imaging 1992;2:541–553.

3. Whittall KP, Mackay AL, Graeb DA, Nugent RA, Li DKB, Paty DW. In vivo measurement of T2 distributions and water contents in normal human brain. Magn. Reson. Med. 1997;37:34–43.

4. Kozlowski P, Rosicka P, Liu J, Yung AC, Tetzlaff W. In vivo longitudinal Myelin Water Imaging in rat spinal cord following dorsal column transection injury. Magn. Reson. Imaging 2014;32:250–258.

Figures

Figure 1. Sample division. The excised section of the injured spinal cord, centered at 5 mm cranial to injury, is divided into a 3 mm piece used for MRI and a 1 mm piece used for TEM. TEM and MRI slice positions are kept within 1 mm of each other.

Figure 2. a) 2x crop of a TEM image; b) manual segmentation of myelin areas; c) myelin area after processing; and d) magnified view of area highlighted in a). Myelin period is calculated by dividing the length of the blue line in d) by the number of spaces between major dense lines.

Figure 3. TEM myelin content vs MR MWF. TEM myelin content shows a strong correlation (R = 0.83, p < 0.001) with MR MWF. The decrease in MR MWF with time follows the typical response of this injury model.

Figure 4. Box and whisker plot of the myelin period. There is no significant difference in lipid bilayer spacing between the three groups (F(2,399) = 0.80, p = 0.45).

Figure 5. Example of ultrastructure of myelin debris. This figure demonstrates the two distinct types of area – that of large vacuous space and of normally spaced lipid bilayers. Notice that the axonal space is completely missing.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)

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