Numerous studies have documented the changes in metabolite concentrations and water properties during ischemia [1, 2]. Less attention was put on the metabolite diffusion, especially for lipid. The intramyocellular lipid (IMCL), is an important dynamic organelle and it provides mitochondria with fatty acids for the energy generation [3]. IMCL turnover has been proved to be fast and sensitive to the metabolism changes [4]. In previous studies we showed that the IMCL apparent diffusion coefficient (ADC) can reflect the lipid droplet size alterations [5]. In this study, we measured the ADCs of IMCL and creatine in the rat muscle ischemia model, in order to show that IMCL and creatine ADCs had drastic increases during muscle ischemia and IMCL ADC is sensitive to the lipid metabolism changes.

Animal Model: Twelve Sprague-Dawley rats (~330 g) were evenly separated into two groups. Hindlimb ischemia was created on one group of rats (ischemia group, N=6) by ligation of the right common iliac artery and right femoral artery [6, 7]. Sham surgeries were performed on the other group (sham control group, N=6). MR exams were performed 18 hours after surgery. During the MR exam, the rats were anesthetized, mechanically ventilated and paralyzed as previously described [5].

MR protocol: All experiments were performed under 7T with a 25-mm-diameter volume coil. A STEAM based single-voxel diffusion weighted MRS (DW-MRS) sequence was implemented. The diffusion gradient was added perpendicular to the muscles fiber direction. Twelve b-values (0 to 1.5×10⁶ s/mm²) were used for IMCL ADC measurement (TR/TE=1500/100 ms, Δ/δ=80/40 ms, NEX=64). Five b-values (0 to 3000 s/mm²) were used for creatine ADC measurement (TR/TE=1500/40 ms, Δ/δ=80/10 ms, NEX=32). Both IMCL and creatine ADCs were measured from the same voxel (8×8×8 mm³).

Data Analysis: The MR signals from IMCL (1.28 ppm) and creatine (3.02 ppm) were quantified by AMARES algorithm using JMRUI software. The IMCL and creatine ADCs were calculated from the exponential fitting of the b-value dependent DW signals. Their relative concentrations were measured as the peak area to spectral noise ratio without diffusion weighting (TR/TE=1500/40 ms). The results in the sham control and ischemia groups were compared (Two-tailed unpaired t-test, *p<0.05; **p<0.01; ***p<0.001).

Typical T1 weighted images acquired from sham control and ischemia groups were shown in Figure 1. Severe edema induced by ischemia was found in the regions of low signal intensity (pointed by arrows). The representative DW spectra and the corresponding exponential fittings of the DW signals of sham control and ischemia groups for IMCL ADC measurement (Fig. 2a) and for creatine ADC measurement (Fig. 2b) were presented. In Figure 3, the ADCs of IMCL and creatine in both sham control and ischemia groups were summarized. The IMCL ADC in ischemic muscles was about two time higher than the sham control (Fig. 3a). The creatine ADC was also significantly increased in the ischemia group (Fig. 3b). Figures 4a and 4b summarized the relative IMCL and creatine concentrations in the two groups. Both IMCL and creatine concentrations were lower in the ischemia group but the differences were not statistically significant. No obvious difference of IMCL/creatine ratio between the two groups was found (Fig. 4c).In this study, we found that IMCL ADC increase was more dramatic than creatine during ischemia (Fig. 3). Unlike creatine, the IMCL diffusion is assumed to be isotropic and restricted by the lipid droplet boundary but not the intracellular barriers [5, 8]. Thus the drastic IMCL ADC increase in the ischemic muscles should be due to the size change of lipid droplets. Muscle ischemia is a complex process and the primary events are the prohibited supply of oxygen and the impaired fatty acid metabolism [3, 6, 9]. The increase of the lipid droplets sizes might be the direct consequence of the discontinuous fatty acid consumption in the mitochondria and the persisting synthesis and storage of lipids in the short term. In addition, the creatine ADC was also higher during ischemia. One possible reason was the hydrolysis of the phosphocreatine [10]. On the other hand, creatine diffusion in skeletal muscles might be hindered by intracellular structures as previous studies implied [11, 12]. During ischemia, the edema might cause the expansion of muscle fibers and decrease of the density of intracellular hindrances, thus resulted in the increase of creatine ADC.IMCL and creatine diffusivities, suggested by our data, are more sensitive to muscle ischemia than metabolite concentrations. The IMCL ADC, measured by DW-MRS, can reveal the changes in lipid droplet size and probe the alterations of lipid metabolism in skeletal muscles during ischemia.