Scientist or clinicians who are interested in tissue fibrosis, muscular dystrophy, and CEST MRIMuscular dystrophy (MD) is a chronic process that causes weakness and wasting away of muscle tissue.¹ The progression of muscular damage leads to muscle fiber necrosis, inflammation, and replacement by myofibroblasts, ultimately leading to death.¹ Currently MRI techniques used to evaluate tissue fibrosis include dynamic contrast enhancement (DCE),² T_1ρ,³ and MR elastography.⁴ These methods measure the changes in structure and tissue property, including water density, membrane leakage, fat deposition, vasculature, and elasticity. Given that metabolic and bioenergetic changes typically occurred in early stage, MRI techniques that are sensitive to these changes will have significant clinical impacts. As such technique, chemical exchange saturation transfer (CEST) MRI derives its contrast from in-vivo metabolites that have exchangeable protons with tissue water.⁵ Furthermore, it has been used to image muscular creatine under stress.⁶ In this study, we aim to evaluate creatine changes in the fibrotic muscle of MD mice by using creatine CEST (CrCEST) MRI.MD mice with mdx/Sgcg deficiency (n=6) ⁷ were studied and compared with results from wild-type mice (n=5) (Age of 5.9±0.4 mo). In-vivo MRI of lower limbs was performed at a Varian 9.4-T horizontal small-animal MRI scanner under an approved IACUC protocol. Structural T₂-weighted MRI was acquired and used to delineate muscular fibrosis based on hyper-intensity signal. CrCEST MRI was acquired according to previous published methods.^6,8-11 Briefly, CEST Z-spectra were collected using a custom sequence with a frequency selective rectangle saturation pulse (B₁=150 Hz, 1s) followed by a Fast Low-Angle Shot (FLASH) readout.¹² The acquired Z-spectrum contained 49 saturation offsets at -5 to 5 ppm with steps of 0.25 ppm, and ±10, ±20, ±50, ±100 ppm. B₀ and B₁ maps were acquired for correction of B₀ and B₁ field inhomogeneities.¹² CrCEST contrast was then derived from the signal difference at ±2 ppm normalized by the signal without saturation. CrCEST mapping was repeated with various saturation amplitudes (B₁) from 50 to 350 Hz to assess the optimized saturation amplitude that best differentiates fibrotic from normal tissue. Figure 1 shows the representative CEST Z-spectra obtained from fibrotic and normal tissue of one MD mouse. The CrCEST contrast in the fibrotic region was significantly higher than the normal region in MD mice shown in Figure 2 (4.26±0.56% vs. 1.70±0.15%, p<0.05). The CEST contrast of the normal muscle in MD mice was not significantly different than that in the wild-type mice (1.70±0.15% vs. 2.06±0.08%, p=0.14). The maximal contrast between fibrotic and normal tissues from the CEST maps was expressed with saturation amplitude around 50 Hz (Figure 3). The regions of fibrotic tissue observed from CEST contrast map mostly corresponded to hyper-intensity regions in the T₂-weighted images (Figure 4). In contrast, there was no obvious hyper-intensive signal in the muscle of the wild-type mice (Figure 4). A representative histology section of muscle fibrosis from one of MD mice is shown in Figure 5. Further comparison with histological studies will help us to validate our findings. Tissue fibrosis is known to be associated with decreased glycolysis and oxidative phosphorylation, or reduced metabolic rate. Therefore we expect to observe an increased creatine concentration and thus CrCEST contrast in the fibrotic tissue, corresponding to the hypometabolic states or reduced metabolism. In this study, we demonstrated that CrCEST contrast in the fibrostic muscle is higher than that of the normal muscle in MD and wild-type mice. Findings are confirmed by the conventional structural T₂-weighted images. In addition, CEST map may detect fibrotic tissue that is not observable on T₂-weighted images. Our results indicate that the CrCEST MRI can be useful for detecting tissue fibrosis and serving as a quantitative imaging biomarker for tissue fibrosis.