Magnetic resonance imaging (MRI) is extensively used in clinical and basic biomedical research. However, MRI detection of pH changes still poses a technical challenge. Chemical exchange saturation transfer (CEST) imaging is a possible solution to this problem. Using saturation transfer, alterations in the exchange rates between the solute and water protons because of small pH changes can be detected with greater sensitivity. In this study, we examined a fatigued skeletal muscle model in electrically stimulated mice. The measured CEST signal ratio was between 1.96 ppm and 2.6 ppm in the z-spectrum, and this was associated with pH values based on the ratio between the creatine (Cr) and the phosphocreatine (PCr). The CEST results demonstrated a significant contrast change at the electrical stimulation site. Moreover, the pH value was observed to decrease from 7.23 to 7.15 within 20 h after electrical stimulation. This pH decrease was verified by ³¹P magnetic resonance spectroscopy and behavioral tests, which showed a consistent variation over time.

The chemical exchange saturation transfer (CEST) signal at −1.6 ppm is attributed to the choline methyl on phosphatidylcholines and results from the relayed nuclear Overhauser effect (rNOE), that is, rNOE(−1.6). The formation of rNOE(−1.6) involving the cholesterol hydroxyl is shown in liposome models. We aimed to confirm the correlation between cholesterol content and rNOE(−1.6) in cell cultures, tissues, and animals. C57BL/6 mice (N = 9) bearing the C6 glioma tumor were imaged in a 7 T MRI scanner, and their rNOE(−1.6) images were cross-validated through cholesterol staining with filipin. Cholesterol quantification was obtained using an 18.8-T NMR spectrometer from the lipid extracts of the brain tissues from another group of mice (N = 3). The cholesterol content in the cultured cells was manipulated using methyl-β-cyclodextrin and a complex of cholesterol and methyl-β-cyclodextrin. The rNOE(−1.6) of the cell homogenates and their cholesterol levels were measured using a 9.4-T NMR spectrometer. The rNOE(−1.6) signal is hypointense in the C6 tumors of mice, which matches the filipin staining results, suggesting that their tumor region is cholesterol deficient. The tissue extracts also indicate less cholesterol and phosphatidylcholine contents in tumors than in normal brain tissues. The amplitude of rNOE(−1.6) is positively correlated with the cholesterol concentration in the cholesterol-manipulated cell cultures. Our results indicate that the cholesterol dependence of rNOE(−1.6) occurs in cell cultures and solid tumors of C6 glioma. Furthermore, when the concentration of phosphatidylcholine is carefully considered, rNOE(−1.6) can be developed as a cholesterol-weighted imaging technique.