MRI assessment of white matter lesions is the primary diagnostic surrogate and clinical trial imaging outcome measure in multiple sclerosis (MS). Nevertheless, the processes leading to the formation of lesions and underlying their subsequent evolution on conventional imaging are poorly understood. Proton MR spectroscopy (¹H-MRS) can inform about these processes; however, the technique’s inherent low spatial resolution poses challenges for monitoring all but the largest lesions. Here we implement ¹H-MRS imaging (¹H-MRSI) with stringent partial volume corrections and absolute quantification to enable the study of transient and persistent lesions over their full evolution: from pre-lesional state, through formation, to either resolved or chronic status.Subjects: 10 relapsing-remitting MS patients within 6 years from diagnosis, scanned semi-annually for 3 years. Data acquisition: pre- and post-contrast T1-weighted MRI (MP-RAGE), T2-weighted MRI (FLAIR), B₀ shimming, 10×8×4.5 cm (AP×LR×IS)=360 cm^{3 1}H-MRS VOI (PRESS TE/TR=35/1800 ms), encoded to 480 voxels, each 1.0×1.0×0.75 cm³ (Fig. 1). Segmentation: Lesion masks were obtained from FLAIR¹ and gray, white matter (GM, WM) and CSF masks were segmented from MP-RAGE². Only lesions >0.3 cm³ (>40% of ¹H-MRSI voxel) were retained. Co-registration: FLAIR was co-registered to the MP-RAGE and the transformation matrix was applied to the lesion mask co-registering it to the MP-RAGE space. All masks, now in MP-RAGE space, were co-registered with the ¹H-MRSI, yielding their volume in every ¹H-MRSI voxel. To delineate pre-lesional tissue, FLAIR from the timepoint at which the lesion first appeared were co-registered to the FLAIR of the preceding timepoint(s), and the transformation matrix was applied to the lesion mask, creating a “ghost” mask of the forthcoming lesion (Fig. 2). Lesion characterization: Based on their MP-RAGE contrast, lesions were quantitatively defined as isointense or hypointense. A T1-contrast ratio was calculated for each lesion as a continuous measure of T1-hypointensity³. The rubric followed for lesion definitions is presented in Fig. 3. Pre-lesional tissue was defined as a region in which a lesion satisfying the size inclusion criterion (>0.3 cm³) appeared at a subsequent time point. Metabolic quantification: Phantom replacement with MS tissue- and lesion-specific T1 and T2 relaxation times^4,5. ¹H-MRSI quality control and partial volume considerations: Voxel shifting⁶ was performed for each lesion and pre-lesional tissue mask assuring that only voxels with >40% mask, <30% CSF, <30% GM; metabolite Cramer Rao lower bounds<20% and 4<linewidths<13 Hz are retained. Metabolite concentrations were corrected for partial CSF volume. Statistics: ANCOVA, random coefficients regression and Pearson correlations with 2-sided p values. Analyses controlled for variable lesion/pre-lesion volume in the ¹H-MRSI voxel.

Six lesions were classified as acute; all were T1-hypointense, rendering them “acute black holes”^7,8 (Fig. 3). They were distributed amongst four patients, as shown in Table 1, which also demonstrates contrast enhancement status and lesion evolution.

Metabolite levels in pre-lesional tissue giving rise to resolving black holes (n=5) were not different from those in pre-lesional tissue giving rise to chronic black holes (n=3) (all p>0.08).

After the appearance of a resolving acute black hole, there was significant increase in NAA (0.1 mM/month, p=0.01) and T1-contrast ratio (0.004/month, p=0.01) (Fig. 4).

After the appearance of a persisting acute black hole, there was significant decrease in Cr (-0.1 mM/month, p=0.01) and T1-contrast ratio (-0.002/month, p=0.01).

The key finding was that NAA levels showed a statistically significant recovery in the months following the appearance of an acute lesion which concurrently resolved on T2-weighted imaging. This correlated with decreasing T1-hypointensity, indicating that in transient black holes, retaining isointensity is associated with a normalization of axonal function. Previous ¹H-MRS studies support the notion of post-acute NAA recovery^9,10,11, but in this study we test it statistically, and distinguish between transient and persistent black holes. The results here have important implications for trials utilizing lesion MRI outcomes, as they substantiate the notion that transient new lesions represent a return to pre-lesional axonal state, and are therefore the preferred outcome after new lesion formation also from a metabolic point of view.

The decreasing Cr levels in persisting black holes were driven by a spike at the acute stage, with a return to pre-lesional levels at the next follow-up. Without concurrent changes in the other glial markers or NAA, this is best interpreted as inflammation-related energy imbalance without a post-acute manifestation.

NAA declines during the formation of all acute lesions, but recovery to pre-lesional levels occurs in only in transient lesions. This implies that transient lesions do not cause permanent axonal damage and are therefore a positive outcome after lesion formation.