Abstract
Purpose:
White matter (WM) degeneration of the visual pathways in primary open-angle glaucoma (POAG) is well documented, but its exact pathophysiology remains unclear. To date, glaucomatous WM degeneration has been exclusively studied using diffusion tensor imaging (DTI) only. However, DTI measures lack direct biological interpretation, and the approach itself suffers from multiple technical limitations. Fixel-based analysis (FBA) is a novel framework for studying WM degeneration, overcoming DTI's technical limitations and providing biologically meaningful metrics. FBA measures fiber density (FD), representing early microstructural changes, and fiber-bundle cross section (FC), representing late macrostructural changes. In this study, we use FBA to study glaucomatous degeneration of the pregeniculate optic tracts (OTs) and postgeniculate optic radiation (ORs) in POAG.
Methods:
This was a cross-sectional case-control study with 12 POAG patients and 16 controls. Multi-shell diffusion-weighted images were acquired. FBA was used to produce a population template, and probabilistic tractography was used to track the OTs and ORs in template space. Finally, FD and FC of the tracts of interest were compared between the two groups.
Results:
Compared with the controls, the OTs of the patients exhibited a significant (familywise error corrected P < 0.05) decrease in FD and FC, whereas their ORs exhibited a significant decrease in FD but not in FC.
Conclusions:
FBA provides sensitive measures to assess WM changes in glaucoma. Our findings suggest that the OTs of glaucoma patients exhibit signs of more advanced WM degeneration compared with the ORs. This potentially implicates anterograde trans-synaptic propagation as the primary cause of glaucomatous spread along the visual pathways.
Primary open-angle glaucoma (POAG) is one of the leading causes of irreversible blindness worldwide, and its prevalence is only expected to increase as the world's population continues to age.
1 POAG results in the death of retinal ganglion cells (RGCs), leading to thinning of the retinal nerve fiber layer (RNFL) and loss of peripheral vision.
2 Furthermore, glaucomatous degeneration of the visual system beyond the retina has been documented in experimental animal models,
3–7 postmortem human histopathologic studies,
8 and human neuroimaging studies.
9–11 Such studies have found evidence of glaucomatous degeneration in the lateral geniculate nucleus (LGN) and the visual cortex, suggesting the involvement of the brain in glaucomatous disease pathology.
12 However, neither the cause nor the origin of this degeneration is fully understood.
Trans-synaptic spread is one of the proposed explanations of glaucomatous degeneration of the central visual system.
13 The conventional view of POAG as simply a degenerative retinal disease implicates anterograde trans-synaptic degeneration in glaucomatous spread, as degeneration would start at the pregeniculate RGCs and spread across the LGN to reach the postgeniculate pathways. However, a small number of primate studies have found evidence of LGN degeneration preceding RGC loss,
5,14 suggesting a retrograde trans-synaptic spread. Furthermore, some MRI studies of POAG patients have reported degenerative changes outside the visual system.
15–17 This has led to the suggestion that an independent brain component may be present in POAG and potentially implicates retrograde trans-synaptic degeneration as a cause of RGC degeneration.
As the visual pathway is uniquely divided into two major white matter (WM) tracts (pre- and postgeniculate), investigating glaucomatous WM degeneration potentially holds the key to understanding the true nature of glaucomatous neurodegenerative spread across the visual system. To date, the most commonly used method for studying WM degeneration in POAG patients has been diffusion tensor imaging (DTI).
15–36 However, DTI metrics have no direct biological interpretation and are commonly interpreted as estimates of WM “structural integrity.” Furthermore, DTI has technical limitations, most notably its inability to account for crossing-fibers within the same voxel.
37 Therefore, several higher-order diffusion models have been recently developed to overcome these limitations and to produce more biologically meaningful measures.
38–45
Fixel-based analysis (FBA) is a recently proposed framework that uses such a higher-order model for analyzing WM in a fiber population–specific manner.
45 The term fixel refers to a specific fiber population within a voxel. FBA uses constrained spherical deconvolution (CSD)
40,44 to model multiple fiber orientations within the same voxel, allowing for the disentanglement of differently oriented WM fiber populations (or fixels). By doing so, FBA solves the classic crossing-fibers problem encountered with DTI and produces biologically meaningful metrics for studying WM changes in vivo. These metrics are as follows: fiber density (FD), fiber-bundle cross section (FC), and fiber density and bundle cross section (FDC). FD represents intra-axonal volume of separate fiber populations within each voxel.
45 FD can be used to probe WM microstructural changes in a fiber-specific manner, with a decrease in FD indicating a loss of axons.
45,46 FC quantifies gross morphologic (or macrostructural) WM changes by measuring fiber bundle cross-sectional area in a plane perpendicular to fixel orientation.
45,46 Finally, FDC is a combined measure of both FD and FC, providing a more comprehensive measure of the information carrying capacity of fiber tracts.
In this study, we use FBA to investigate WM changes in the pregeniculate optic tracts (OTs) and the postgeniculate optic radiations (ORs) in POAG to better understand the underlying pathophysiology of glaucomatous neurodegeneration. Furthermore, we investigate the correlation between FBA metrics and structural and functional clinical measures of glaucoma. Finally, for comparison, we analyze the same tracts using the conventional voxelwise DTI approach.
All subjects underwent tests for visual acuity, IOP, visual fields, and retinal nerve fiber layer (RNFL) thickness.
Visual acuity was measured using a Snellen chart with optimal correction for the viewing distance. IOP was measured using a Tonoref noncontact tonometer (Nidek, Hiroishi, Japan). Visual fields for the POAG group were assessed using a Humphrey Field Analyzer (HFA; Carl Zeiss Meditec, Jena, Germany) using the 30-2 grid and the Swedish Interactive Threshold Algorithm (SITA), and expressed as visual field mean deviation (VFMD). For the controls, visual fields were screened using frequency doubling technology (FDT; Carl Zeiss Meditec) using the C20-1 screening mode. Controls were not allowed to have any reproducibly abnormal test location at P < 0.01. Finally, the RNFL thickness was measured by means of optical coherence tomography (OCT) using a Canon OCT-HS100 scanner (Canon, Tokyo, Japan). Results were expressed as the mean peripapillary RNFL (pRNFL) thickness.
Diffusion-weighted images were acquired using a Siemens MAGNETOM Prisma 3T MRI scanner (Siemens, Erlangen, Germany) with a 64-channel head coil. The following parameters were used: repetition time (TR) = 5500 ms, echo time (TE) = 85 ms, bandwidth = 2404 Hz, field of view (FoV) = 210 × 210 × 132, voxel size = 2.0 × 2.0 × 2.0 mm, 66 slices, in 64 diffusion gradient directions. Two DWI shells were acquired, b = 1000 s/mm2 and b = 2500 s/mm2, in two phase encoding directions: anteroposterior and posteroanterior. Three images with no diffusion weighting (b = 0 s/mm2) were also acquired in each phase encoding direction.
DWI preprocessing included first denoising the data
47 in MRtrix3 (
www.mrtrix.org) and then correction of EPI distortions,
48 motion, and Eddy-current distortions
49 in FSL v5.011.
50
Fixelwise statistical analysis was applied to the fixels included in the OT and OR fixel masks. A general linear model (GLM) was used to compare FD, FC, and FDC between the POAG group and the controls. Sex and demeaned age were added as nuisance covariates. Connectivity-based fixel enhancement was used to perform tract-specific smoothing and enhancement using the default parameter settings.
55 Following 5000 permutation tests, each fixel was assigned a familywise error (FWE) corrected
P value. Streamline segments corresponding to statistically significant fixels were cropped from the population template whole-brain tractogram and used to visualize significant results.
To further analyze our findings, the average FD, FC, and FDC of each tract was calculated for all participants. The average FBA metrics of each tract of the two groups were then compared using analysis of covariance (ANCOVA), adding age and sex as nuisance covariates. To study the correlation between the FBA metrics and the clinical glaucoma tests, the FD and FC of both sides of the OTs and ORs were averaged for each glaucoma patient. Then, a partial Pearson test was used to determine the correlation between the average pRNFL thickness and VFMD of both eyes and the averaged FBA metrics of the OTs and ORs, while controlling for the effects of age and sex.
For voxel-based DTI analysis, threshold-free cluster enhancement and permutation testing was performed in MRTrix3 using the provided default parameters.
59 FA and MD of the OTs and ORs were tested in a voxelwise manner using the same design matrix created for the FBA.
To compare participants' demographics and clinical characteristics, an independent-samples t-test was used for parametric continuous variables of equal variance, the Mann–Whitney U test was used for nonparametric continuous variables, Welch's t-test was used for continuous variables of unequal variance, and the χ2 test was used for categorical variables.
Statistical significance for FBA and DTI analysis was reported at an FWE-corrected P < 0.05. For other tests, statistical significance was reported at P < 0.05.
To summarize our results, FBA of the OTs revealed a significant loss of FD, FC, and FDC in the POAG group compared with controls, whereas the ORs showed a significant decrease in FD and FDC only. For glaucoma patients, FD of the OTs showed a significant correlation with the pRNFL thickness and VFMD, whereas FD of the ORs showed a significant correlation with pRNFL thickness only. FC measures of both OTs and ORs did not show any significant correlation with pRNFL thickness or VFMD. Using conventional DTI, the POAG group showed a significant decrease of FA in both OTs and the left OR, whereas a significant increase in MD was found in the OTs only. These results are described in more detail below.
The main findings of this study are that, in POAG patients, OTs exhibit both micro- and macrostructural degeneration, whereas ORs show evidence of microstructural degeneration only. To the best of our knowledge, this is the first study to use FBA to investigate WM degeneration in POAG. We find that FBA is more sensitive to glaucomatous degeneration in the ORs compared with conventional DTI, highlighting the importance of adopting higher-order diffusion models for studying glaucomatous WM changes. We describe these conclusions in more detail.
The pRNFL thickness showed a stronger correlation with the FD of both the OTs and ORs than did the VFMD. This is expected, as both FD and pRNFL thickness are measures of structural degeneration, whereas VFMD is a measure of functional loss. Additionally, the FD of the OTs showed a stronger correlation with both clinical measures compared with the FD of the ORs. This could be attributed to the fact that the clinical tests examine the structure and function of the optic nerves, which are essentially formed of the same RGCs axons as the OTs. Furthermore, the FC of both tracts was not correlated with either of the clinical measures. A possible explanation for this lack of correlation is that FC changes, unlike FD and structural and functional optic nerve changes, are a delayed secondary outcome of axonal loss and not a direct response to it. This further strengthens our assertions regarding the time course of micro- and macrostructural WM changes in POAG.
Surprisingly, the average VFMD maps (
Supplementary Fig. S1) failed to explain the difference in spatial pervasiveness of FD loss between the left and right ORs. A similar (unexplained) difference has been previously reported in a meta-analysis of DTI studies of glaucoma,
62 although the meta-analysis found a greater decrease of FA on the right side.
The main limitation of this study is that our interpretations regarding the nature of glaucomatous spread along the visual pathways are based on cross-sectional data. The OTs and ORs differ considerably in size and shape, which means they might exhibit different patterns of FBA changes independent of the effect of glaucomatous degeneration. Although FBA metrics describe distinct aspects of WM degeneration, which we used as potential surrogate biomarkers for glaucomatous degenerative advancement, evidence of actual disease progression over time is still needed to confirm our findings.
Another study limitation is the relatively moderate sample size of our POAG group. However, finding statistically significant changes despite this sample size and while following rigorous correction for multiple comparisons is a testament to how sensitive FBA is to glaucomatous WM changes.
In this study, we set a precedent for the use of FBA in investigating POAG, and potentially other diseases that may affect the integrity of the visual pathways. Future studies with larger sample sizes and a longitudinal nature are required to verify our present suggestion that visual pathway WM changes are primarily caused by anterograde trans-synaptic degeneration originating from the eye.
Supported by the Graduate School of Medical Sciences (GSMS), University of Groningen, Groningen, The Netherlands (SH). This project has received funding from the European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie Grant 675033 (Egret-plus). The funding organizations had no role in the design, conduct, analysis, or publication of this research.
Disclosure: S. Haykal, None; B. Ćurčić-Blake, None; N.M. Jansonius, None; F.W. Cornelissen, None