Purpose: To assess the relationship between levels of energy metabolites, water T2 values and the degree of fat infiltration in the leg muscles of patients with Duchenne Muscular Dystrophy (DMD) and healthy controls.
Introduction: Quantitative MRI and MRS are increasingly important as non-invasive and objective outcome measures in therapy development for DMD(1-4). While several MR indices such as muscle fat fraction, water T2 relaxation time, phosphocreatine (PCr) levels and phosphodiester (PDE) levels, correlated individually with age and functional measures, much less attention has been given to how these indices relate to each other. Recently, it was shown that the water T2 was decreased at 3 months and 6 months after initiation with corticosteroid therapy, while %fat remained unchanged(1), which highlights the fact that it is important to assess more than just one aspect of muscle damage in DMD. 31P-MRS in DMD is commonly performed using surface coil localization, making it difficult to combine these results with spatially-resolved proton imaging. Here, we present combined quantitative MRI and spatially-resolved 31P-MRS data of the leg muscles in DMD to assess pathophysiological processes in a more systematic manner.
Methods: Phosphorous MRS datasets were acquired in the right lower leg of 18 DMD patients (9.18 ± 3.71 yrs.) and 12 healthy controls (9.7± 2.9 yrs.) using a 7T MR System (Achieva, Philips, Best, the Netherlands) with a custom-built double tuned (31P and 1H) volume coil. The protocol consisted of T1-weighted gradient echo images (15 slices; resolution 0.9x0.9x7.0mm; slice thickness/gap 7/0.5mm; TR/TE 10/3.0ms; flip angle (FA) 30°) and a 31P 2D Chemical Shift Imaging (CSI) dataset (10x10 hamming-weighted acquisitions; TR 2000ms; FA 45°; voxelsize 20x20 or 15x15mm depending on leg size). On the same day, turbo spin echo images (17 echoes; TR/TE/ΔTE 3000/8/8ms; resolution 1.4x1.8x10mm; slice thickness/gap 10/20mm; 5 slices; no fat suppression) and a 3-point Dixon sequence (23 slices; slice thickness/gap 10/5mm; TR/TE/ΔTE 210/4.41/0.76 ms; two signal averages; FA 8°; resolution 1x1x10mm) were acquired on a 3T Ingenia MR system with a 32 element receive coil. (Fig.1)
Data-analysis: Outcome measures were determined for five individual lower leg muscles. MR spectra were analysed using AMARES in the JMRUI software package(5). For each muscle the tissue pH and the levels of PDE, inorganic phosphate (Pi), PCr and adenosine triphosphate (ATP) were determined. Water T2 and fat fraction maps were generated using custom-built fitting routines written in Matlab and presented as mean values over multiple slices aligned with the 2D-CSI dataset(6-7). Group differences were assessed with a general linear model, whereas the relation between energy metabolite levels, %fat and water T2 were assessed with a Pearson correlation. Significance level was set at p<0.05.
Results: Group analysis showed significantly elevated Pi/PCr, PDE/ATP, fat fractions, intracellular tissue pH and water T2 values in all lower leg muscles of DMD patients compared to healthy controls. Positive correlations were detected between Pi/PCr (r=0.35; p<0.0001), Pi/ATP(r=0.51; p<0.0001) and PDE/ATP(r=0.56; p<0.0001) with %fat in DMD subjects. No correlations were observed between water T2 and energy metabolite levels.
Discussion & conclusion: The changes in metabolite ratios, fat fractions and water T2 are in agreement with previous work assessing these pathophysiological processes individually(1-3). The positive correlations of Pi/PCr, Pi/ATP and PDE/ATP with fat infiltration, suggest they are linked to progressive muscle damage. These observations are in line with previous work in the lower arm muscles of DMD patients(7). In contrast, tissue pH was not correlated to %fat, which suggests that the more alkaline pH, associated with a leaky membrane, could be a more constant anomaly already altered from disease onset. The absence of correlations between the energy metabolites and water T2 suggest that they reflect independent muscle degeneration processes. One possible explanation for this could be that elevated water T2 is due to an increase in extracellular fluid whereas energy metabolite levels are thought to be strictly intracellular. In the disease cascade, it is thought that persistent inflammation due to muscle damage eventually results in replacement of muscle tissue with fat and fibrosis(8). Consequently, indices which are sensitive to changes before fat replacement occurs are very valuable as it seems unlikely that fat-replaced muscle tissue would revert back to normal. PDE levels have been hypothesized to reflect phospholipid membrane degradation products which in some situations showed to be reversible(9). Interestingly, PDE levels already appear to be elevated above control values at low fat percentages, suggesting that these might reflect muscle damage prior to the occurrence of fat infiltration. Overall, our results show that multimodal MR measures are important tools to objectively assess muscle degeneration processes and potentially the effect of therapeutic interventions in DMD as well as making it possible to detect muscle degeneration early on. Future work will aim to assess longitudinal multimodal MR measures.