INTRODUCTION

Multiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system, predominantly affecting young adults^1,2. Remyelination has been introduced as one of the strategies for neuroprotection, nevertheless, the prevention of disability has yet to be achieved³. Sensitive imaging approach is desired to detect myelin loss and regeneration for early diagnosis and treatment. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a non-invasive molecular imaging approach, which is capable of mapping endogenous molecules at low concentrations by detecting the exchangeable protons between solutes and bulk water^4,5. Amide CEST^6,7 and and relayed nuclear Overhauser effect (rNOE) CEST^8,9 have been reported in detecting proteins and lipids, which are major components of myelin. Amide proton transfer-weighted imaging, which is typically analyzed using MTR asymmetry, contains the contributions from both amide and rNOE. It is reported for myelin detection¹⁰. Recently, we also demonstrated the identification of MS patients using rNOE¹¹. Here, we used cuprizone model, a well-established mouse model with myelin pathology in corpus callosum (CC)^2,12,13, to study CEST MRI (rNOE and amide) in detecting demyelination and remyelination in brain at 3T. Significant CEST signal changes within CC were observed and could indicate the corresponding changes in myelin, which were validated by immunohistochemistry.

METHODS

Nineteen C57BL/6 mice (male, 8 weeks old) used in this study were randomly divided into control (CTL, n=9) and cuprizone (CPZ, n=10) groups. CTL mice were fed with control diet for the whole course, while CPZ mice were fed with diet containing 0.2% (w/w) cuprizone for 10 weeks (demyelination) followed by 4 weeks with control diet (remyelination), as shown in Fig. 1A. MRI scans were performed on a 3T Bruker BioSpec system (Bruker, Germany). The 2 mm-thick image slice containing CC was positioned on a collected sagittal image (Fig. 1C). CEST data was acquired using pulsed-CEST (VDMP) module followed by a RARE readout sequence¹⁴. The saturation parameters were as following¹¹: saturation power=0.8 μT, pulsed width=40 ms, mixing time=60 ms and pulse number=30. Frequency offsets were set to -13~13 ppm for Z-spectra and 200 ppm for M₀ image (normalization). Other parameters were set as following: TR=5000 ms, TE=3.69 ms, FOV=16×16 mm², matrix=96×96, scan time=24 min. T1 maps of mouse brains were measured using RAREVTR sequence. CEST data was firstly corrected with B₀ inhomogeneity. Then, rNOE (-3.5 ppm) and amide (3.5ppm) contrasts were extracted using Lorentzian-difference method⁹ and corrected by T1 map (AREX)^15-20. Regions of brain and CC were drawn for analysis (Fig. 1D). Immunohistochemistry was performed and results at week 8 were shown here to validate MRI findings.

RESULTS AND DISCUSSION

We summarized the experimental scheme and slice placement in Fig. 1. Mouse weight was recorded and showed no differences between groups during the study (Fig. 1B). In CC, both rNOE and amide CEST contrasts were comparable initially at week 0 between CTL and CPZ groups. A decrease in rNOE and amide CEST were observable in maps at -3.5 and at 3.5 ppm, respectively, at week 8 post cuprizone diet (Fig. 2A and 3A). rNOE was significantly lower in CPZ group when compared to CTL group (P<0.01, Fig. 2B). Similarly, amide CEST contrast was significantly lower in CPZ group (P<0.05, Fig. 3B). After week 10, cuprizone diet of CPZ group was replaced by control diet for remyelination. Interestingly, at week 14, rNOE contrast in CPZ group recovered to a similar level as that in CTL group (P>0.05, Fig. 2B), indicating both the cuprizone-induced demyelination and remyelination in CC can be sensitively detected by rNOE contrast. This slight increase in rNOE from week 8 to 14 in CPZ during remyelination, leading to a comparable level to CTL, was not observed in amide CEST. The amide contrast remained lower in CPZ than that in CTL at week 14 (Fig. 3B). For whole brain, a slight decrease at week 8 but a slight increase at week 14 in rNOE contrast (P>0.05) were observed (Fig. 2C), while amide contrast slightly decreased (P>0.05) during the course of our study (Fig. 3C). Immunohistochemistry was used to validate the MRI observations. An obviously lower fluorescent myelin signal of CC relative to cortex was found in CPZ than in CTL (0.9 vs. 1.2, Fig. 4), which supported the observations of CEST MRI at week 8.
The distinctive observations at week 14, i.e. a slight increase in rNOE vs a decrease in amide CEST contrast could indicate that rNOE contrast has a high sensitivity towards lipid as myelin contains over 70% of lipids. Moreover, we also demonstrated that rNOE could sensitively detect demyelination in MS patients¹¹.

CONCLUSION

Significant decrease in rNOE and amide CEST contrasts were observed in cuprizone-induced demyelination mouse model at week 8, as validated by immunohistochemistry. Interestingly, we observed a slight increase in rNOE contrast of CPZ, which has a similar contrast as CTL at week 14 during remyelination. This was not observed in amide CEST. These results indicate that both rNOE and amide contrasts can detect demyelination, while rNOE contrast showed a potential sensitivity in detecting remyelination. Overall, our findings indicated that CEST MRI is capable of monitoring demyelination/remyelination and has great potential in identification of myelin-related neuropathology, such as MS at 3T¹¹.

References

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