Diffusion fMRI (dfMRI) is a presumably non-BOLD technique sensitive to transient microstructural changes underlying neural activity. Previous task-fMRI studies have assessed the characteristics of the dfMRI signal and potential BOLD contamination, with conflicting results. Here we acquired resting-state fMRI data with five protocols with incrementally reduced BOLD contributions and analyzed the characteristics of resting-state networks and functional connectivity using model-free approaches. We report dfMRI data does not contain fundamentally different information to BOLD-fMRI, with the exception of a few regions that switch from anti- to positively-correlated. Future work will focus on removing any remaining BOLD contribution from the dfMRI acquisition.
All experiments were approved by the local Service for Veterinary Affairs and performed on a 14T Varian system using a home-built surface quadrature transceiver. Sprague-Dawley rats (N=3) were anesthetized using isoflurane for initial setup and promptly switched to medetomidine sedation (bolus: 0.1mg/kg, perfusion: 0.1mg/kg/h), which preserves neural activity and vascular response better than isoflurane. An anatomical whole-brain scan was acquired for atlas-registration (0.15x0.15x0.50mm3 resolution). Five fMRI protocols, designed to contain gradually reduced sources of BOLD contrast, were compared (Fig. 1): gradient-echo EPI (GE-EPI, #1), spin-echo EPI (SE-EPI, #2), diffusion-weighted SE-EPI (DW SE-EPI, #3), ADC derived from alternating b=0 and b=1ms/μm2 images (ADC b=0/1, #4), and ADC derived from alternating b=0.4 and b=1ms/μm2 images (ADC b=0.4/1, #5). Each block of five protocols was acquired twice on each rat.
Data pre-processing included denoising6, slice-timing correction and spatial smoothing7 (Gaussian kernel: 0.36x0.36x1mm3). For the protocols alternating two b-value acquisitions, ADC timecourse was calculated as $$$ADC=\ln\left(S_{2}/S_{1}\right)/\left(b_{1}-b_{2}\right)$$$. Each signal timecourse (protocols #1-3) and ADC timecourse (protocols #4-5) was analyzed using FSL’s MELODIC8 with high-pass temporal filtering (f>0.01Hz) and 30 independent components (IC’s). Group-ICA was further performed for each protocol, using time-concatenation of all runs from all rats (N=6). Anatomically-relevant resting-state networks (RSNs) were compared between protocols. In parallel, noise-related IC’s were identified and cleaned from each individual fMRI run9 and the “cleaned” fMRI data were used to compute individual functional connectivity (fc) matrices between 18 atlas-defined ROIs, using global signal regression. Mean fc matrix for each protocol was calculated.
We identified at least three RSN’s based on ICA analysis of resting-state dfmri data, whose spatial extent and temporal characteristics were highly comparable to GE-EPI or SE-EPI BOLD rs-fmri. In the low b-value regime, the overall ADC can be expressed as a weighted sum of local ADC’s, which justifies the use of ICA analysis on quantitative ADC timecourses. Functional connectivity matrices were also very similar between all spin-echo based protocols (with or without diffusion weighting).
Protocol #5 (ADC b=0.4/1) should have limited contributions to functional contrast from: T2-weighting (both images are weighted similarly) and perfusion (fast-flowing spins should be suppressed at b≥0.4ms/μm2) and should be closest to potential non-BOLD dfmri signal. It is noteworthy that the significant differences in fc matrices between SE-EPI and ADC b=0.4/1 were driven not by the magnitude of the correlation, but by the change in sign – with anti-correlated ROIs in SE-EPI being positively correlated in terms of ADC changes. This could suggest a genuinely different source of functional contrast in dfmri.
The similarity to SE-EPI in terms of RSN’s and fc matrix raises nonetheless the question of residual BOLD contrast. BOLD could stem from variations in T2 between the acquisition of successive b=0.4 and b=1 images – which could be minimized using an INDI scheme10 – or cross-terms between venous susceptibility gradients and diffusion gradients, potentially significant at ultra-high field but which could be reduced using a bipolar gradient design. Future work will include minimization of aforementioned confounds and suppression of vascular response using nitroprusside2.
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