Synopsis

Keywords: Arterial spin labelling, Animals

Blood-brain barrier (BBB) plays a critical role in brain health and diseases. However, there is a scarcity of tools to assess BBB integrity, particularly in mouse models. Here, we aimed to develop a non-contrast arterial-spin-labeling-based MRI technique to estimate BBB permeability in mice by measuring relative fractions of labeled water in cerebral veins. Systematic optimizations were performed to enhance signal sensitivity with a further study investigating reproducibility. The proposed method revealed significant BBB dysfunctions in a mouse model of Huntington’s disease, which were further validated with histology. Our method may open new avenues for preclinical mechanistic research or therapeutic trails.

INTRODUCTION

Blood-brain barrier (BBB), which plays important roles in blocking toxins and regulating fluid transportations, is a promising biomarker for normal aging and several neurological disorders.^1-3 Dynamic contrast-enhanced (DCE) MRI is a commonly used method for evaluating BBB function.⁴ However, its sensitivity in studying diseases with subtle BBB damages at the early stage, e.g., Alzheimer’s disease, is limited. Alternatively, approaches based on arterial-spin-labeling (ASL) MRI have been proposed to investigate the BBB’s permeability to water^5,6 and were applied in disease studies^2,7. In this work, we aim to develop a non-contrast MRI technique to estimate BBB permeability to water in mice by measuring arterially labeled water in major veins, similar to the principle of water-extraction-with-phase-contrast-arterial-spin (WEPCAST)⁵ MRI. We optimized the sequence in terms of labeling duration and post-labeling delay. Test-retest reproducibility was characterized. An initial application of the proposed technique to a mouse model of Huntington’s disease (HD) was performed to demonstrate the utility of the proposed method in pathological conditions, which was further validated with histology.

METHODS

BBB permeability can be defined as the permeability surface-area product (PS) per unit-mass tissue (Renkin-Crone model^8,9):
$$PS=-ln(1-E)×CBF, (1)$$
where CBF denotes cerebral blood flow (by phase-contrast [PC] MRI) and E denotes water extraction fraction (by ASL signals at vein). Analogous to WEPCAST,⁵ ASL signals at vein are:$${\Delta}M_{vein}(t)=2{\alpha}(1-E)M_{0,blood}e^{-\frac{BAT_{vein}}{T_{1b}}}c(t), (2) $$ where α denotes inversion efficiency, M_0,blood the blood equilibrium magnetization (by M₀ scan at long TR), BAT_vein the bolus arrival time of vein, T_1b the blood T₁, and c(t) the arterial input function (a step function convolved with a Gaussian function to account for bolus dispersion). Therefore, a complete PS measurement requires a PC MRI scan and a venous-ASL scan.
All experiments were approved by local IACUC. A total of 41 mice (17 female 24 male; age: 14-44 weeks; body weight: 21-40g) were included in four sub-studies.
Study 1: Estimation of BAT_vein in mice (N=5). Eq. (2) requires the knowledge of BAT_vein, which has not been elucidated before. Therefore, Study 1 aims to measure BAT_vein. ASL (sagittal slice covering brain midline) was performed with a short labeling duration (500ms) and multiple post-labeling delays (PLDs) (25,100,200,300,400,500,600,700,800,900,1000 ms) to estimate BAT_vein. Other parameters followed previous reports.^10,11
Study 2: Optimization of labeling duration (LD) (N=5). ASL was performed with a fixed PLD=25ms and 11 LDs (50,100,200,300,500,700,900,1100,1300,1600,2000 ms) to examine the dependence of ΔM_vein on LDs.
Study 3: Assessment of BBB permeability in wild-type (WT) mice (N=5). ASL (optimized parameters from Studies 1&2 used) and PC MRI were repeated by three times to test the reproducibility of measurements.
Study 4: BBB permeability in a mouse model of HD (N=26). 17 mice (20-24 weeks; 9 zQ175 HD and 8 WT) were included in MRI sessions to test the sensitivity of our method in detecting BBB dysfunction induced by disease pathology. Another 9 age-matched mice (4 zQ175 and 5 WT) were included for Western blotting analyses focusing on two types of tight-junction proteins (ZO-1 and Claudin-5), which were critical components of the BBB structure.
Linear mixed-effect (LME) model and Student's t-test were used for statistical analyses (significance level at P<0.05).

RESULTS AND DISCUSSION

Study 1: Great vein of Galen (VG) was larger than the superior sagittal sinus (used in human WEPCAST5) and therefore was vein of interest in mice (Fig. 1A). Figures 1B-1D present the control, labeled, and difference ASL images with PLDs. By fitting VG signals at different PLDs (Fig. 1E) into Eq. (2), it was estimated that BAT_vein=691.2±14.5 ms.
Study 2: Figures 2A-2C show the control, labeled, and difference ASL images as functions of LDs. A progressive buildup of ASL signals can be noticed at the VG (Fig. 2C). Fitting of VG signals at different LDs (Fig. 2D) led to the estimation that LD=1200 ms with PLD=100ms provides a trade-off among sensitivity, scan duration, and specific absorption rate.
Study 3: Figure 3 summarizes the test-retest reproducibility results. The BBB permeability index, PS, has a CoV of 6.1±1.2%, suggesting excellent reliability. We also observed a significant increases in CBF (Fig. 3B; P=0.0004) and ΔM/M0 signal (Fig. 3C; P=0.012), a significant decrease in E (Fig. 3D; P=0.009), but no significant change in PS (Fig. 3E; P=0.38), which we attributed to accumulation effect of anesthesia. There was a significant negative correlation between CBF and E (Fig. 3F, y=-0.17x+116.82, R²=0.61, P=0.0006), suggesting that the variations in CBF and E have a physiological origin.
Study 4: HD mice showed a higher E (69.7±2.4%, Fig. 4A, P=0.026) and PS (318.1±17.1 ml/100g/min, Fig. 4B, P=0.040) when compared to WT mice (E=59.9±3.2%, PS=260.9±18.9 ml/100g/min), suggesting a higher BBB permeability. There were no significant differences in brain volume (Fig. 4C, P=0.72), baseline CBF (Fig. 4D, P=0.26), and BAT_vein (Fig. 4E, P=0.43). There were significant differences in ZO-1 (Fig. 5B, P=0.037) and Claudin 5 (Fig. 5C, P<0.001). The reduced ZO-1 and Claudin-5 in HD mice showed agreement with the increased E and PS, supporting the BBB-related measurements with non-invasive imaging.

CONCLUSION

We developed a quantitative MRI method for measuring BBB permeability in mice. Initial application in the mouse model of Huntington's disease supports the sensitivity of this technique in detecting subtle BBB breakdown.

Acknowledgements

Z Wei and H Liu contributed equally to this work.