786072 research-article2018 MSJ0010.1177/1352458518786072Multiple Sclerosis JournalA Shirani, P Sun MULTIPLE SCLEROSIS JOURNAL MSJ Short Report Histopathological correlation of diffusion basis spectrum imaging metrics of a biopsy-proven inflammatory demyelinating brain lesion: A brief report Afsaneh Shirani, Peng Sun, Robert E Schmidt, Kathryn Trinkaus, Robert T Naismith Sheng-Kwei Song and Anne H Cross Keywords: Multiple sclerosis, diffusion basis spectrum imaging, imaging, biomarker Date received: 6 March 2018; revised: 1 May 2018; accepted: 27 May 2018 journals.sagepub.com/home/msj 1­–5 DOI: 10.1177/ https://doi.org/10.1177/1352458518786072 1352458518786072 https://doi.org/10.1177/1352458518786072 © The Author(s), 2018. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav , Abstract: Diffusion basis spectrum imaging (DBSI) models diffusion-weighted magnetic resonance imaging (MRI) signals as a combination of discrete anisotropic diffusion tensors and a spectrum of isotropic diffusion tensors. Here, we report the histopathological correlates of DBSI in the biopsied brain tissue of a patient with an inflammatory demyelinating lesion typical of multiple sclerosis (MS). Increased radial diffusivity (marker of demyelination), decreased fiber fraction (apparent axonal density), elevated nonrestricted isotropic fraction (marker of vasogenic edema), but unchanged axial diffusivity (marker of integrity of residual axons) seen in the lesion appeared consistent with histopathological findings of inflammatory demyelination with relative axonal sparing. Our report supports the application of DBSI as a biomarker in human studies of MS. Introduction Multiple sclerosis (MS) is a pathologically heterogeneous disease with varying degrees of demyelination, inflammation, and axonal injury or loss.1 An unmet need remains for developing pathologically specific, noninvasive quantitative neuroimaging biomarkers of central nervous system (CNS) pathologies in MS.2 Diffusion basis spectrum imaging (DBSI) models diffusion-weighted magnetic resonance imaging (MRI) signals as a combination of discrete anisotropic diffusion tensors (representing the integrity of axon fibers and myelin while resolving crossing fibers), and a spectrum of isotropic diffusion tensors (reflecting the extra-axonal non-anisotropic environment associated with inflammation and tissue loss).3,4 Details of DBSI and its accuracy in capturing whitematter (WM) pathologies have been described in tissue phantoms, spinal cord, and optic nerves of mice with experimental allergic encephalomyelitis, the corpus callosum of mice with cuprizone-induced demyelination, and autopsied MS spinal cord specimens.4–7 Here, for the first time, we report the histopathological correlates of DBSI in the biopsied brain tissue of a living person with inflammatory demyelinating Multiple Sclerosis Journal lesions consistent with MS. We also compared the DBSI findings with those of diffusion tensor imaging (DTI), an earlier diffusion method used to study WM, which is confounded by inflammation and crossing fibers.8,9 Methods A 53-year-old man presented with seizures. Brain MRI demonstrated several WM T2-weighted MRI abnormalities, including a large right frontal lesion with surrounding edema (Figure 1(a)). Gadolinium was not administered due to renal insufficiency. Because of the atypical presentation and tumefactive right frontal WM lesion, brain biopsy was performed. To visualize the pathological processes, a combination of histologic and immunohistochemical stains was used including Luxol Fast Blue (LFB) (for myelin), and antibodies to neurofilament protein (for axons), CD68 (for macrophages/microglia), and CD3 (for T cells; Figure 2). Correspondence to: AH Cross John L. Trotter Multiple Sclerosis Center and Neuroimmunology Section, Department of Neurology, School of Medicine, Washington University in St. Louis, Campus Box 8111, 660 S. Euclid Ave., St Louis, MO 63110, USA. crossa@wustl.edu Afsaneh Shirani Robert T Naismith Anne H Cross John L. Trotter Multiple Sclerosis Center and Neuroimmunology Section, Department of Neurology, School of Medicine, Washington University in St. Louis, St Louis, MO, USA Peng Sun Sheng-Kwei Song Department of Radiology, Mallinckrodt Institute of Radiology, School of Medicine, Washington University in St. Louis, St Louis, MO, USA Robert E Schmidt Department of Pathology & Immunology, School of Medicine, Washington University in St. Louis, St Louis, MO, USA Kathryn Trinkaus Siteman Biostatistics Shared Resource, Siteman Cancer Center and School of Medicine, Washington University in St. Louis, St Louis, MO, USA Diffusion-weighted imaging (DWI) was performed 3 weeks after the biopsy, prior to administration of 1 Multiple Sclerosis Journal 00(0) Figure 1. (a) and (b). FLAIR image shows the region of interest (ROI) in the biopsied lesion (excluding the actual biopsy site, and separated by a two-voxel layer to avoid possible artifacts). Other disseminated white-matter hyperintensities are seen in the FLAIR image, too. (c) and (d). A comparable ROI in the right frontal white matter of a healthy control was identified. In panels (e)–(i), the central horizontal line in each box plot shows the medians. The vertical thickness of each box represents the interquartile range (IQR). Numbers below each box show median (1st quartile–3rd quartile). Notches show the 95% confidence intervals (CI) around medians (nonoverlapping CIs represent statistical significance). The lower and upper whiskers show the lowest and the highest values within 1.5 IQR of the lower and higher quartiles, respectively. (e) Both DTI-derived and DBSI-derived radial diffusivity (RD) were increased in the lesion compared to the healthy control (consistent with demyelination). (f) DTI-derived axial diffusivity (AD) was increased in the lesion (confounded by isotropic fraction such as edema); whereas DBSI-derived AD was similar to the healthy control (consistent with preserved integrity of residual axons). (g) Both DTI-derived and DBSI-derived fractional anisotropy were decreased (consistent with impaired white matter integrity). (h) DBSI-derived fiber fraction was modestly decreased in the lesion compared to the healthy control (consistent with relatively reduced axonal content). (i) DBSI nonrestricted isotropic fraction was elevated in the lesion (consistent with edema). 2 journals.sagepub.com/home/msj A Shirani, P Sun et al. Figure 2. (a) Pathology of the biopsied right frontal lesion. This image of a hematoxylin and eosin (H&E, 4× magnification) stained neurosurgical specimen shows patchy pallor of the white matter (between arrows), an edge of normal white matter (arrowheads) and perivascular lymphocytic cuffing. (b). Image of the same area as (a) stained with luxol fast blue-periodic acid-schiff (LFB-PAS, 4× magnification) stain for myelin shows loss of myelin involving the pale area of image (a) compared to adjacent white matter. (c). An adjacent section demonstrates relative sparing of neurofilament stained axons in the area lacking myelin (same magnification). This confirms a demyelinative process, that is, near complete loss of myelin with relative axonal preservation (neurofilament immunohistochemistry (NF IHC). (d). Higher magnification of a demyelinated area of (c) (arrow) shows large numbers of normal axons (NF IHC). (e). The same area as (a), (b), and (c) demonstrates large numbers of perivascular and parenchymal macrophages (CD68 IHC). (f). Higher magnification of the area marked with arrow in (e) shows perivascular and parenchymal macrophages. (g). The same area as (a), (b), (c), and (e) shows numerous CD3+T cells (CD3 IHC). (h). Higher magnification of the area marked with arrow in (g) shows a large number of CD+3T cells. journals.sagepub.com/home/msj 3 Multiple Sclerosis Journal 00(0) corticosteroids or any disease modifying therapy. Diffusion data were collected on a 3 Tesla TIM Trio (Siemens) scanner with a 32-channel head coil at 2 × 2 × 2 mm3 resolution in the axial plane with repetition time/echo time = 10,000/120 ms, and employing a 99-direction diffusion-weighting scheme (maximum b value = 2200 s/mm2) with a total acquisition time of 16 minutes, as previously described.3,4 Voxel-wise DBSI and DTI metrics were calculated from six contiguous slices of the biopsied right frontal lesion. The region of interest (ROI) within the lesion (Figure 1(b)) was compared with an ROI in the same frontal lobe area of an age-matched healthy control (Figure 1(c) and (d)). The boundary of the site of needle insertion (dark area in Figure 1(a)) was separated from the ROI by a two-voxel layer to avoid possible artifacts from blood products, or possible damage to the immediately surrounding area by the biopsy needle placement. DTI-derived singletensor metrics included radial diffusivity (RD), axial diffusivity (AD), and fractional anisotropy (FA). DBSI-derived multi-tensor metrics included RD, AD, FA, fiber fraction (FF), nonrestricted isotropic fraction, and restricted isotropic fraction.3 Whereas the DTI-derived metrics were based on single-tensor analysis, the DBSI-derived RD, AD, FA, FF, nonrestricted isotropic fraction, and restricted isotropic fraction were derived from multitensor modeling to resolve underlying intravoxel structure and pathology components.3 As previously reported,3,4 in the settings of acute inflammation and tissue loss commonly seen in MS, DTI-derived axon/myelin injury metrics are confounded by infiltrating cells and extracellular water. Results were displayed using notched box plots representing median and 95% confidence intervals of image voxels from the defined ROI for each diffusion metric. Nonoverlapping notches between boxes indicate that the corresponding medians differ. Formal hypothesis testing was not performed, given the single sample. The Institutional Review Board at Washington University in St Louis approved the study. Results Figure 1(e)–(i) show the DBSI- and DTI-derived metrics in the biopsied lesion (Figure 1(a) and (b)) and in normal WM of the corresponding region in a healthy subject (Figure 1(c) and (d)). Compared with the healthy subject, both DTI- and DBSI-derived RD were significantly increased (consistent with demyelination) in the biopsied lesion (Figure 1(e)). DBSIderived AD in the biopsied lesion did not appear 4 different compared to the healthy control (Figure 1(f)), however, DTI-derived AD in the lesion was higher than that of the healthy control. The DBSI finding was consistent with preserved integrity of residual axons, whereas the DTI result likely reflected confounding effects of increased water content in the setting of inflammation. DBSI-derived nonrestricted isotropic fraction was increased (Figure 1(i)) a finding consistent with edema. Both DTI-derived and DBSI-derived FA were significantly decreased in the biopsied lesion compared to the healthy control (Figure 1(g)). DBSI-derived FF (apparent axonal density) was modestly decreased in the biopsied lesion compared to the healthy control (Figure 1(h)), suggesting relatively reduced axonal content in the lesion. Microscopic images of the biopsied lesion (Figure 2) showed regions of reduced LFB stain (demyelination) with relatively less reduction in neurofilament stain (relative axonal preservation). Increased cellular content was also present (Figure 2(a)). Only infrequent signs of axonal injury (spheroids) were observed (Figure 2(d)). Overall, the DBSI metrics of increased radial diffusivity, unchanged axial diffusivity, mildly decreased fiber fraction, and increased nonrestricted isotropic fraction (Figure 1 (e)–(i)) were consistent with the histopathology findings of an inflammatory demyelinating lesion with relative axonal sparing (Figure 2). Discussion Based on animal data and human autopsy results,4,5,7 increased RD and decreased AD are indicators of demyelination and axonal injury, respectively, and an increased proportion of nonrestricted isotropic diffusion is a marker of vasogenic edema or loss of axonal fibers. Compared to healthy control tissue, DBSIderived metrics in this biopsied lesion demonstrated increased RD, decreased FF, and elevated nonrestricted isotropic fraction, but with normal AD. This pattern was consistent with inflammatory demyelination with relative axonal sparing that was observed by histopathology. The mildly decreased FF in the lesion was in accord with the relative preservation of axons seen histologically. In this study, DBSI was more consistent with the underlying WM pathologies found in the biopsy than DTI. DTI metrics, both RD and AD, are known to be impacted by the presence of inflammation and vasogenic edema.10 This confounding effect led to falsely increased DTI-derived AD. In contrast, DBSI-derived AD was not increased. DBSI results were more in accordance with histological neurofilament staining journals.sagepub.com/home/msj A Shirani, P Sun et al. (Figure 2(c)). In addition, DTI-derived FA was underestimated due to averaging effects of increased isotropic diffusion from vasogenic edema. In contrast, DBSI models multiple intravoxel pathologies and structures to estimate the extent of vasogenic edema and tissue loss, represented by the nonrestricted isotropic diffusion tensor component.3,4 This study provides evidence supporting the feasibility of DBSI to differentiate the coexisting pathologies in acute MS lesions in human brain in vivo. DBSI metrics better reflected the actual histological findings than DTI, because DBSI models multiply intravoxel structural and pathological components using multiple anisotropic and isotropic tensors,3,4 whereas DTI parameters represent “averaged” data within imaging voxels. Several advanced diffusion MR methods have been developed to address the limitations of DTI. Much can be learned from the comparison of such advanced methods. Our study suggests that DBSI is a promising noninvasive biomarker of MS neuropathology. Declaration of Conflicting Interests The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S. is funded through a clinician scientist development award from the National Multiple Sclerosis Society (USA), and a clinical research training scholarship from the American Academy of Neurology. P.S., R.E.S., and K.T. report no conflicts of interest. R.T.N. has consulted for Alkermes, Acorda, Bayer, Biogen, EMD Serono, Genentech, Genzyme, Novartis, and Teva. S.K.S is currently funded by NIH U01EY025500, R01NS047592, P01NS059560, and NMSS RG 5258-A-5. He is a co-founder of DxGPS and may financially benefit if the company is successful in marketing its product(s) that is/are related to this research. A.H.C has been a paid consultant for Biogen, EMD-Serono, Genzyme/Sanofi, Genentech/Roche, and Novartis. A.H.C. was funded in part by the Manny & Rosalyn Rosenthal—Dr. John L Trotter MS Center Chair in Neuroimmunology of Barnes-Jewish Hospital Foundation. Washington University may receive royalty income based on a technology licensed by Washington University to DxGPS LLC. That technology is evaluated in this research. journals.sagepub.com/home/msj Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by a grant from the U.S. National Institutes of Health (PO1 NS059560, PI: A.H.C.). ORCID iD Robert T Naismith org/0000-0003-0520-4283 https://orcid. References 1. Compston A and Coles A. Multiple sclerosis. Lancet 2008; 372: 1502–1517. 2. Fox RJ, Beall E, Bhattacharyya P, et al. Advanced MRI in multiple sclerosis: Current status and future challenges. Neurol Clin 2011; 29: 357–380. 3. Cross AH and Song SK. A new imaging modality to non-invasively assess multiple sclerosis pathology. J Neuroimmunol 2017; 304: 81–85. 4. Wang Y, Sun P, Wang Q, et al. Differentiation and quantification of inflammation, demyelination and axon injury or loss in multiple sclerosis. Brain 2015; 138: 1223–1238. 5. Lin TH, Chiang CW, Perez-Torres CJ, et al. Diffusion MRI quantifies early axonal loss in the presence of nerve swelling. J Neuroinflammation 2017; 14: 78. 6. Wang Y, Wang Q, Haldar JP, et al. Quantification of increased cellularity during inflammatory demyelination. Brain 2011; 134: 3587–3598. 7. Wang X, Cusick MF, Wang Y, et al. Diffusion basis spectrum imaging detects and distinguishes coexisting subclinical inflammation, demyelination and axonal injury in experimental autoimmune encephalomyelitis mice. NMR Biomed 2014; 27: 843–852. 8. Song SK, Yoshino J, Le TQ, et al. Demyelination increases radial diffusivity in corpus callosum of mouse brain. Neuroimage 2005; 26: 132–140. 9. Naismith RT, Xu J, Tutlam NT, et al. Radial diffusivity in remote optic neuritis discriminates visual outcomes. Neurology 2010; 74: 1702–1710. 10. Chiang CW, Wang Y, Sun P, et al. Quantifying white matter tract diffusion parameters in the presence of increased extra-fiber cellularity and vasogenic edema. Neuroimage 2014; 101: 310–319. Visit SAGE journals online journals.sagepub.com/ home/msj SAGE journals 5