This work should be of interest to people using CEST imaging in order to identify relevant biomarkers of pathology, particularly in the context of neurodegenerative diseases and ataxia.Friedreich Ataxia (FA) is the most common form of recessive inherited ataxia with a prevalence of 1/40000 in the Caucasian population causing severe neurological disabilities and reduced life expectancy¹. FA is caused by mutations in the FXN gene which encodes a mitochondrial protein, the frataxin. Reduced levels of frataxin result in mitochondrial impairment and cellular dysfunction^2,3. Although FA has been described for several decades, in vivo biomarkers are still lacking. There is an urgent need to find objective biomarkers first to better understand the biological processes and second to measure its progression and to evaluate future treatments efficacy. We have recently established a relevant mouse model of FA. As glutamate has been shown to be a potential biomarker of neurodegenerative diseases, we propose to use Chemical Exchange Saturation Transfer (CEST) imaging of glutamate (gluCEST^4,5) in order to characterize our mouse model and to monitor glutamate alterations in the spinal cord.

Mouse model: The model was established using the Cre/LoxP technology to achieve a neuronal-specific deletion of the FXN gene, mainly in large proprioceptive neurons. Knock-out mice (KO, n=6) were compared to Wild Type mice (WT, n=6). Cohorts were imaged at 7 and 14 weeks of age.

MRI: MRI studies were performed on an 11.7T Bruker magnet using quadrature volume coil. MSME sequence was acquired in sagittal plane (resolution 0.1x0.1x0.5 mm³) for anatomical image and T₂ mapping. 20 echo-times equally distributed between 6 and 120ms were used for T₂ fitting.

GluCEST: GluCEST images were acquired using TSE sequence preceded by a frequency-selective continuous wave saturation pulse of 1s with a B₁ intensity of 5µT applied at frequencies ranging from -4 to 4ppm by 0.5ppm steps. Images were acquired in sagittal plane (resolution 0.2x0.2x2 mm³). B₀ inhomogeneity was corrected using WASSR⁶. GluCEST images were calculated using asymmetric Magnetization Transfer Ratio (MTRasym) at ± 3 ppm.

KO mice already displayed an ataxic phenotype at 3.5 weeks of age and electrophysiology studies revealed a decrease of sensory wave at 4.5 weeks and an almost complete loss at 7.5 weeks of age (data not shown). Moreover, the number of neurons within lumbar dorsal root ganglia was significantly decreased at 17.5 weeks of age in KO compared to WT (-14%, data not shown).

Several hyperintense signals were found on T₂-weighted images in the lumbar spine of KO mice (Fig.1, bottom panel, red arrows). Figure 2a shows example of T₂ maps acquired in WT and KO mice (Fig.2a). At 7 weeks, mean T₂ value of lumbar spine was significantly increased in KO (Fig.2b, blue and red bars, +18%, p=0.0012). At 14 weeks, mean T₂ in KO was even larger whereas it remained stable in WT (Fig.2b, hatched blue and red bars).

GluCEST images revealed decrease of gluCEST contrast in KO compared to WT, especially in the lumbar part (Fig.3a, red arrows). MTRasym spectra acquired in WT and KO groups at 14 weeks of age exhibited different profiles, particularly at 3ppm where gluCEST contrast was measured (Fig.3b, blue and red lines respectively). While mean gluCEST contrast in WT and KO was comparable at 7 weeks, it decreased significantly at 14 weeks in KO (Fig.3c, hatched blue and red bars, -11%, p=0.044).

Our neuronal mouse model recapitulates the main clinical and histological features of FA. Similarly to human FA, the neurological phenotype starts developing in the lumbar part of the spinal cord and progresses toward the cervical part. The increase of T₂ at 7 weeks in KO mice was most likely due to degradation of myelin content. Interestingly, T₂ continued to increase 7 weeks later, probably reflecting a progression of the disease along the spinal cord. Contrary to T₂ results, gluCEST contrast was comparable between both cohorts at 7 weeks of age. As glutamate can be considered as a neuronal biomarker, this could indicate that, even if they were altered, neurons were still present at 7 weeks and probably still functional. However, a few weeks later, gluCEST contrast was decreased, indicating that some neurons were suffering or dead.

To our knowledge, this is the first application of gluCEST for mouse spinal cord imaging. Moreover, these results demonstrate the potential of gluCEST imaging in providing innovative and relevant biomarkers of FA. Finally, they might be very informative in the future to evaluate potential therapy of FA by identifying a time-window to rescue affected neurons.