Introduction

Cerebral perfusion is critical for early detection of neurological diseases and for effectively monitoring disease progression and treatment responses. A BOLD dynamic susceptibility contrast (DSC) MRI with transient hypoxia was successfully adopted to noninvasively measure cerebral perfusion in mouse with high spatiotemporal resolution¹. Given the importance of reliability and sensitivity in routine perfusion studies, particularly considering the challenges faced by other perfusion techniques like DSC with Gadolinium injection or ASL^2,3, this study aims to assess these aspects of BOLD-DSC measurements within a single session and across multiple sessions.

Methods

Set up and Hypoxic Gas Stimulation
The experimental setup for transient anoxic stimulus under Isoflurane anesthesia was depicted in Fig. 1. Gas stimulus paradigm was delivered using a block design paradigm of 60s rest (40% O₂/ 60% N₂) and 5s stimulation (100% N₂) alternatively repeated five times (Figure 2.A).
BOLD acquisitions
BOLD MRI studies were acquired on a 9.4T system using GE-EPI sequence with TR/TE =1000/11ms, FA=50°, 156x156x500 μm³ , 20 slices. To assess the reliability of technique, we conducted BOLD-DSC measurements with 10 C57BL/6 mice over four weekly sessions spanning a month. The identical procedure was repeated every week. Hypoxic stimulus was administered under 1.5% Isoflurane. Each subject underwent a total of 3 GE-EPI block-design runs.
Data analysis
The reproducibility of trial-wise BOLD-DSC measurements within each session was assessed for each animal during week 1, while the reproducibility of weekly BOLD-DSC measurements was evaluated across all animals from week 1 to week 4. To quantify perfusion values from dynamic hypoxia-induced BOLD responses, we adopted the DSC theory¹.

Results

Reproducibility of trial-wise hypoxia-induced BOLD signal change within single session
The reproducibility of hypoxia-induced signal changes was examined across 15 trials in each animal. The voxel-wise absolute signal change induced by the hypoxic stimulation (∆S) was obtained from an average of 3-s data points around the peak for each trial. ∆S maps of several representative trials are shown for a single selected slice in one mouse (Fig. 2C). Next, voxel-wise ∆S values of all possible pairwise trials among 15 trials were compared (Fig. 2D). The ∆S values of the first trial were highly correlated with those of three randomly selected trials 5, 10, and 15 with Pearson’s r value of 0.859, 0.848, and 0.837, respectively (Fig. 2D). All r values of all paired trials were computed and presented as color maps (Fig. 2E, one animal (left) and group-average of 10 animals (right)). Overall, strong correlations (r values of >0.80) were consistently found, suggesting that the hypoxia-induced ∆S was highly reproducible across repeated trials.
Sensitivity of hypoxic-induced BOLD response within session
The contrast to noise ratio (CNR), an index of detectability, was examined from an average of 15 repeated trials within each animal, as proportional to ∆S/N (Fig. 3A). CNR maps of 6 exemplary slices in one representative animal (Fig. 3B) clearly differentiate the gray matter and white matter as well as the area containing large vessels. The distributions of voxel-wise CNRs in the white matter corpus callosum (1021 voxels, dark gray violins) and in the dorsal cortical ROI (16071 voxels, red violins) were plotted across 10 mice (Fig. 3C). The mean voxel-wise CNR of the dorsal cortex and CC region was 7.85± 1.15 and 2.76 ± 0.73 (Fig. 3D), respectively, and a ratio of mean CNR values between gray and white matter was 2.03 ± 0.15.
Reproducibility of hypoxic-induced BOLD-DSC measurements across four weekly sessions
We observed almost identical hypoxia-induced BOLD responses in the primary somatosensory area (SSp) from week 1 to week 4 in all animals (Fig. 4B, data from four representative animals). Notably, there were slight variations in the peak amplitudes of BOLD responses in SSp among the 10 animals over four weeks (Fig. 4C). Hypoxia-induced BOLD responses were converted into absolute CBV and CBF values. We observed consistency in CBV and CBF measurements across four sessions. Importantly, no significant differences in quantitative regional CBV and CBF metrics were observed across the four repeated weekly measurements in the CC, SSp, and thalamus area (Figs. 4D-E). In summary, our BOLD-DSC measurements have high reproducibility and consistency over multiple scan sessions.

Discussion and Conclusions

We demonstrated excellent reproducibility and sensitivity of hypoxic-induced signal changes in BOLD data at the voxel level both within a single session and multiple sessions. The high reproducibility and sensitivity of our technique indicate its potential for routine perfusion measurements with high repeatability, which is particularly promising when considering the challenges faced by other techniques such as DSC with Gd injection and ASL^2,3.

Acknowledgements

This research was supported by the Institute of Basic Science (IBS-R015-D1).