Introduction

Lower limb muscle-water T₂ (T_2m) has been shown in amyotrophic lateral sclerosis (ALS) to be elevated vs. controls, with suggested associations between T_2m and progression¹. Here we also analysed multi-echo MRI data, obtained as part of a wider study from which a first report described elevated fat fraction and STIR hyperintensity particularly at calf level in ALS². We investigated lower-limb T_2m vs. healthy controls at baseline, and longitudinally, correlating T_2m changes and effective fat fraction (ff_a) with functional assessment and myometry.

Methods

Participants, recruited with informed consent, comprised people with ALS (n=20, age 57.3 ±14.8y) and healthy controls (CTR) (n=16, age 55.4 ±13.5y). Participants were assessed using the ALS Functional Rating Scale–Revised (ALSFRS-R)³. Lower limb myometry was obtained with a microFET®2 handheld dynamometer (Hoggan Scientific; UT, USA) for isometric assessment of knee and ankle extension and flexion. Patients were examined at baseline, 6 and 12 months, controls at baseline and 12 months.
MRI was performed at 3T (Siemens Skyra) with a multi-echo spin-echo sequence (TR= 3630/3500ms, 22 TEs from 10-220ms with 10ms interval, 9 x 6 mm slices, matrix 320x160, in-plane resolution 1.3125x1.3125 mm). A multi-component, slice profile-corrected EPG model [s(TE) = (1 - ff_a) · sEPG(B₁f, T_2m, α, σ_N, TE) + ff_a · [ 0.33 · sEPG(B₁f, T₂=40ms, α, σ_N, TE) + 0.67 · sEPG(B₁f, T₂=198ms, α, σ_N, TE)] was fitted pixel-wise to the data using maximum likelihood estimation in MATLAB, to estimate T_2m and an apparent fat fraction ff_a. The 2-component fat-signal model parameters were estimated a priori from 4 subcutaneous fat ROIs in 8 representative subjects. Muscles were manually segmented on single slices at thigh and calf level. Mean T_2m and ff_a were calculated for all individual muscle ROIs, for the anterior and posterior compartments, and the entire musculature cross section at each level.

Results

All calf muscles showed significant T_2m increases (one way ANOVA, p<0.05 considered significant) in patients vs. controls. At the thigh level T_2m differences were more variable: Table 1 shows thigh-level T_2m and ff_a group differences at baseline and 1 year. Significant differences are shown in green. For most thigh muscles T_2m was significantly elevated in ALS vs. CTL, although the sartorius and gracilis appeared largely spared. Significant differences for ff_a were less consistent.

A tendency to negative correlation was seen between both T_2m and ff_a and ALSFRS-R which reached significance at the calf level (entire cross-section left and right combined) at baseline and 6 months. The correlations were stronger for T_2m compared with ff_a (p values: 0.002 and 0.002 vs 0.046 and 0.047, R values - 0.68 and - 0.58 vs - 0.47 and - 0.50, for baseline and 6 months respectively). Figure 1 shows the correlation of calf T_2m and ALSFRS-R at baseline.
In the patients, anterior and posterior muscle compartment T_2m and ff_a at thigh and calf levels at baseline, 6 months and 1 year were tested for correlation with the respective myometry results. Correlations were significant for a number of these (Table 2), most consistently for T_2m at the calf level, with p<0.05 for 9 out of 12 correlations (4 compartments at the 3 time points).
Myometry changes between baseline and 6 months were significant for all compartments (p < 0.039). Concomitant T_2m changes were significant for all calf compartments (p < 0.036) and for left anterior thighs (p = 0.002); ff_a changes were significant for all left limb compartments (p < 0.014).
Four muscle compartments showed significant correlation between longitudinal T_2m (ΔT_2m) change and myometry changes (ΔMyometry) (Figure 3). Analogous correlations for all other muscle compartments were not significant.

Discussion

Consistent with previous reports, in most muscles at calf and thigh level T_2m was at baseline higher in patients than in controls. Notable exceptions were the sartorius and gracilis for which patient T_2m were close to control values.
T_2m and ff_a also showed significant negative correlation with ALSFRS-R scores at the calf level. This somewhat contrasts with previous results¹ where significant positive correlation was observed at thigh level. Differences in these findings may be due to differences in the specific patient groups, or technical differences in the T_2m estimation method⁴.
Several muscle compartments showed significant negative correlation between T_2m and ff_a, and myometry, most notably T_2m in the calf and longitudinal T_2m change correlated significantly with muscle strength change in several compartments, suggesting T_2m reflects functionally relevant cellular changes.

Conclusion

In addition to baseline differences of T_2m between patients and controls, associations between T_2m and ALFRRS-R and myometry results support the validity of T_2m as a disease biomarker. These data provide some evidence of a stronger association between T_2m and function than was seen for ff_a.
This approach using routinely available CPMG pulse sequences provides T_2m and ff_a estimates from a single acquisition which provide complementary indices of neuromuscular pathology.

Acknowledgements

This work was supported in part by GSK and the UCLH NIHR biomedical research centre

References

1. Paoletti, M., et al., Longitudinal Quantitative MRI Evaluation of Muscle Involvement in Amyotrophic Lateral Sclerosis. Frontiers in Neurology, 2021: p. 1998.

2. Klickovic, U., Zampedri, L., Sinclair, C.D., Wastling, S.J., Trimmel, K., Howard, R.S., Malaspina, A., Sharma, N., Sidle, K., Emira, A. and Shah, S., 2019. Skeletal muscle MRI differentiates SBMA and ALS and correlates with disease severity. Neurology, 93(9), pp.e895-e907.

3. Cedarbaum, J.M., et al., The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of respiratory function. Journal of the neurological sciences, 1999. 169(1-2): p. 13-21.

4. Zafeiropoulos, N. Reducing parameter estimate bias due to B1 field error ambiguity in CPMG MRI T2 relaxometry. in ISMRM. 2020. Virtual Conference.