bioRxiv preprint doi: https://doi.org/10.1101/781815; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 1 Reproducibility Assessment of Neuromelanin-Sensitive Magnetic 2 Resonance Imaging Protocols for Region-of-Interest and Voxelwise 3 Analyses 4 5 Kenneth Wengler1,2, Xiang He3, Anissa Abi-Dargham3,4*, and Guillermo Horga1* 6 7 1: Department of Psychiatry, Columbia University, New York, NY, USA 8 2: Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY, 9 USA 10 3: Department of Radiology, Stony Brook University, Stony Brook, NY, USA 11 4: Department of Psychiatry, Stony Brook University, Stony Brook, NY, USA 12 *: These authors contributed equally 13 14 15 Corresponding Author: Kenneth Wengler, PhD 16 Department of Psychiatry, Columbia University 17 Division of Translational Imaging, New York State Psychiatric Institute 18 1051 Riverside Dr, Unit 31 19 New York, NY 10032 20 Phone: 646-774-5571 21 Email: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/781815; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 1 Highlights 2 • A detailed NM-MRI volume placement protocol is described. 3 • Guidelines covering acquisition through analysis for NM-MRI are given. 4 • A test-retest study in 10 healthy subjects shows high reproducibility for region-of- 5 interest (ROI) and voxelwise analyses. 6 • ~3 minutes of NM-MRI data is needed for high-quality ROI-analysis. 7 • ~6 minutes of NM-MRI data is needed for high-quality voxelwise-analysis. 8 9 Abstract 10 Neuromelanin-sensitive MRI (NM-MRI) provides a noninvasive measure of the content 11 of neuromelanin (NM), a product of dopamine metabolism that accumulates with age in 12 dopamine neurons of the substantia nigra (SN). NM-MRI has been validated as a 13 measure of both dopamine neuron loss, with applications in neurodegenerative disease, 14 and dopamine function, with applications in psychiatric disease. Furthermore, a 15 voxelwise-analysis approach has been validated to resolve substructures, such as the 16 ventral tegmental area (VTA), within midbrain dopaminergic nuclei thought to have 17 distinct anatomical targets and functional roles. NM-MRI is thus a promising tool that 18 could have diverse research and clinical applications to noninvasively interrogate in vivo 19 the dopamine system in neuropsychiatric illness. Although a test-retest reliability study 20 by Langley et al. using the standard NM-MRI protocol recently reported high reliability, a 21 systematic and comprehensive investigation of the performance of the method for 22 various acquisition parameters and preprocessing methods has not been conducted. In 23 particular, most previous studies used relatively thick MRI slices (~3 mm), compared to 24 the typical in-plane resolution (~0.5 mm) and to the height of the SN (~15 mm), to bioRxiv preprint doi: https://doi.org/10.1101/781815; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 1 overcome technical limitations such as specific absorption rate and signal-to-noise ratio, 2 at the cost of partial-volume effects. Here, we evaluated the effect of various acquisition 3 and preprocessing parameters on the strength and test-retest reliability of the NM-MRI 4 signal to determine optimized protocols for both region-of-interest (including whole 5 SN/VTA-complex and atlas-defined dopaminergic nuclei) and voxelwise measures. 6 Namely, we determined a combination of parameters that optimizes the strength and 7 reliability of the NM-MRI signal, including acquisition time, slice-thickness, spatial- 8 normalization software, and degree of spatial smoothing. Using a newly developed, 9 detailed acquisition protocol, across two scans separated by 13 days on average, we 10 obtained intra-class correlation values indicating excellent reliability and high contrast- 11 to-noise, which could be achieved with a different set of parameters depending on the 12 measures of interest and experimental constraints such as acquisition time. Based on 13 this, we provide detailed guidelines covering acquisition through analysis and 14 recommendations for performing NM-MRI experiments with high quality and 15 reproducibility. This work provides a foundation for the optimization and standardization 16 of NM-MRI, a promising MRI approach with growing applications throughout clinical and 17 basic neuroscience. 18 19 Keywords 20 Neuromelanin; Test-retest; Substantia nigra; Ventral tegmental area; NM-MRI; 21 Voxelwise-analysis bioRxiv preprint doi: https://doi.org/10.1101/781815; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 1 1. Introduction 2 Neuromelanin (NM) is an insoluble dark pigment that consists of melanin, 3 proteins, lipids, and metal ions (Zecca et al., 2008). Neurons containing NM are present 4 in specific brain regions of the human central nervous system with particularly high 5 concentrations found in the dopaminergic neurons of the substantia nigra (SN) and 6 noradrenergic neurons of the locus coeruleus (LC) (Zecca et al., 1996; Zucca et al., 7 2014). NM is synthesized by iron-dependent oxidation of dopamine, norepinephrine, 8 and other catecholamines in the cytosol, to semi-quinones and quinones (Sulzer and 9 Zecca, 1999). While initially present in the cytosol, NM accumulates within cytoplasmic 10 organelles via macroautophagy that results in the undegradable material being taken 11 into autophagic vacuoles (Sulzer et al., 2000). These vacuoles then fuse with 12 lysosomes and other autophagic vacuoles containing lipid and protein components to 13 form the final NM-containing organelles (Zucca et al., 2014). These organelles contain 14 NM pigment along with metals, abundant lipid bodies, and protein matrix (Zecca et al., 15 2000; Zucca et al., 2018). This process was shown to be driven by excess cytosolic 16 catecholamines, such as that resulting from L-DOPA exposure, that are not 17 accumulated in synaptic vesicles and can be inhibited by treatment with the iron 18 chelator desferrioxamine (Cebrián et al., 2014; Sulzer et al., 2000). NM-containing 19 organelles first appear in humans between 2 and 3 years of age (Cowen, 1986) and 20 gradually accumulate with age (Zecca et al., 2008; Zucca et al., 2018). 21 The paramagnetic nature of the NM-iron complexes within the NM-containing 22 organelles (Zecca et al., 1996; Zecca et al., 2004) enables them to be noninvasively bioRxiv preprint doi: https://doi.org/10.1101/781815; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 5 1 imaged using magnetic resonance imaging (MRI) (Cassidy et al., 2019; Sasaki et al., 2 2006; Sulzer et al., 2018; Trujillo et al., 2017). NM-sensitive MRI (NM-MRI) produces 3 hyperintense signals in neuromelanin-containing regions such as the SN and LC due to 4 the short longitudinal relaxation time (T1) of the NM-complexes and saturation of the 5 surrounding white matter (WM) by either direct magnetization transfer (MT) pulses 6 (Chen et al., 2014) or indirect MT effects (Sasaki et al., 2006) (see Trujillo et al. for a 7 detailed investigation of NM-MRI contrast mechanisms (Trujillo et al., 2017)). While 8 most previous NM-MRI studies have used indirect MT effects, images with direct MT 9 pulses achieve greater sensitivity (Langley et al., 2015; Schwarz et al., 2013) and were 10 recently shown to be directly related to NM concentration (Cassidy et al., 2019). NM- 11 MRI has also been validated as a measure of dopaminergic neuron loss in the SN 12 (Kitao et al., 2013) and several studies have shown that this method can capture the 13 well-known loss of NM-containing neurons in the SN of individuals with Parkinson’s 14 disease (Sulzer et al., 2018). More recently, NM-MRI was validated as a marker of 15 dopamine function, with the NM-MRI signal in the SN demonstrating a significant 16 relationship to Positron emission tomography (PET) measures of dopamine release 17 capacity in the striatum (Cassidy et al., 2019). Furthermore, a voxelwise-analysis 18 approach was validated to resolve substructures within dopaminergic nuclei thought to 19 have distinct anatomical targets and functional roles (Cassidy et al., 2019; Haber et al., 20 1995; Roeper, 2013; Weinstein et al., 2017). This voxelwise approach may thus allow 21 for a more anatomically precise interrogation of specific midbrain circuits encompassing 22 subregions within the SN or small nuclei such as the ventral tegmental area (VTA), bioRxiv preprint doi: https://doi.org/10.1101/781815; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 6 1 which may in turn increase the accuracy of NM-MRI markers for clinical or mechanistic 2 research. For example, voxelwise NM-MRI may facilitate investigations into the specific 3 subregions within the SN/VTA-complex projecting to the head of the caudate, which are 4 of particular relevance in the study of psychosis (Weinstein et al., 2017), or help capture 5 the known topography of SN neuronal loss in Parkinson’s disease (Cassidy et al., 2019; 6 Damier et al., 1999; Fearnley and Lees, 1991). An additional benefit of the voxelwise- 7 analysis is avoiding the circularity that can incur when defining ROIs based on the NM- 8 MRI images that are then used to read out the signal in those same regions.