Myelin loss or damage is a pathological condition that occurs in a variety of neurological disorders, yet non-invasive MRI techniques for myelin quantification in vivo are underdeveloped. An MRI contrast specifically sensitive to myelin would permit the elucidation of mechanisms of demyelination and remyelination, quantification of neuronal loss, and the development more effective treatments in several neurological diseases. Recently, the relaxation time measured with a novel rotating frame method entitled Relaxation Along a Fictitious Field in the rotating frame of rank n (RAFFn)^1,2 was shown to correlate better with myelin content than, e.g., T₁, T₂ and magnetization transfer (MT) ratio (MTR) in healthy perfused rat brains³ and in assessing dysmyelination in the mouse model of mucopolysaccharidosis I (MPS I)⁴. The aim of this work was to demonstrate the benefits of RAFFn in assessing demyelination using lysophosphatidyl choline (LPC) induced lesions, where injections of LPC create demyelination lesions with very little inflammation and gliosis. Changes in tissue were verified with histology.Demyelination lesions were induced in rats (Sprague-Dawley, Charles River) using lysophosphatidyl choline (LPC) injections to the corpus callosum (CC; n_lesion = 6, n_vehicle = 4) which has well-defined fiber orientation and in the dorsal tegmental tract (DTG; n_lesion = 6, n_vehicle = 4) which has heterogeneous fiber orientations. The vehicles were injected with phosphate-buffered saline. The animals were imaged in vivo three days after injection. RAFF4 pulses were used in the pulse train placed prior to a single slice fast spin echo imaging readout (TR = 4 s, TE_eff = 8.3 ms) with ω¹_max/(2$$$\pi$$$) = 324 Hz. RAFF4 pulse train durations were: 0, 27, 54, 81, 108 ms. For MT, hard pulses (ω¹/(2$$$\pi$$$) = 200 Hz) with incremental durations of 0, 0.3, 0.6, 0.9 and 1.2 s were placed +8 kHz from the carrier frequency. DTI parameters were: 42 directions and b = 1000 s/mm² with EPI readout (TR = 2 s, TE_eff = 30 ms, 6 shots). The resolution for all images was 125 x 125 x 500 μm³. After MRI, the animals were perfused and stained for myelin and Nissl, the latter indicating changes in cell density including cell death and gliosis. The changes in MRI metrics were assessed with a region of interest (ROI) analysis.When comparing lesions induced by LPC in the ipsilateral side versus that of vehicle animals in CC (Fig 1), mean diffusivity (MD) and fractional anisotropy (FA) showed significant differences with the largest contrast (Fig 2) as compared to the other MRI metrics in contrast CTR = [mean higher value – mean lower value]/mean higher value *100%: 36.4% (p < 0.01; MD) and 27.8% (p < 0.01; FA). The CTR with RAFF4 was 20.0% (p < 0.01), while the CTR of MTR and T1sat were 9.2% (p < 0.01) and 7.1% (p < 0.01), respectively. Noticeably, in DTG (Fig 3), DTI metrics did not show significant differences (Fig 4), whereas T_RAFF4 showed a significant 26.2% difference (p < 0.01). MTR and T_1sat showed lower CTR: 15.1% (p < 0.01) and 11.3% (p < 0.01), respectively. Our histological analysis demonstrated that demyelination and gliosis took place in both CC and DTG, with greater extent of gliosis in DTG (Fig 5). RAFF4 allowed the detection of LPC-injected vs. vehicle differences in MRI contrast in both CC and DTG. Although DTI has been shown to detect myelin loss in highly anisotropic structures such as CC⁵, a major limitation of DTI is crossing fibers⁶. Our results demonstrate an advantage of RAFF4 in DTG, where RAFF4 outperforms DTI due to independence of the TRAFFn measures from fiber orientation. We have recently demonstrated that RAFFn is predominantly sensitive to slow / ultra-slow motional regimes² which allows detection of demyelination processes. Noticeably, RAFFn also outperforms MT in detecting myelin loss which we attribute to its ability to probe slower motional regime as compared to MT⁴. Furthermore, this regime of motion most likely corresponds to the conformational exchange of methylene groups, which is in the millisecond time scale of motion.The insensitivity of RAFFn to fiber orientation distribution, excellent sensitivity to myelin content in healthy and pathological tissues, and low SAR¹ show the promise of RAFFn to become a useful technique for visualizing demyelinating lesions. Future work will aim at employing a 3D acquisition with later time points to follow also remyelination of the LPC injected animals.