Neurologists, neuroradiologists, and scientists with an interest in arterial spin labeling and cerebrovascular diseaseMoyamoya disease is characterized by progressive stenosis of the distal internal carotid arteries and the proximal anterior and middle cerebral arteries1. The patients are usually young and often suffer multiple strokes. The standard therapy consists in an external-internal carotid artery bypass operation. The indication to treat is determined based on the severity of the symptoms, the occurrence of strokes, and surrogate imaging markers, such as cerebrovascular reserve (CVR). We assessed the changes in arterial spin labeling (ASL) cerebral blood flow (CBF) and arterial transit time (ATT) between before and after acetazolamide (a cerebral vasodilatative agent) in Moyamoya patients as function of the severity of vessel stenosis. In addition, we studied whether sub-clinical, “pre-stroke” changes in apparent diffusion coefficient (ADC) could occur under those circumstances.Images were acquired preoperatively in 37 patients (mean age ± standard deviation 38.5±14.8 years old, 25 women) at 3 Tesla on a GE MR750 scanner. A 3D, multi-delay pseudo-continuous ASL2 and a diffusion-weighted sequence were acquired before and 15 minutes after acetazolamide injection (1g i.v.). Imaging parameters for the ASL were: 3D FSE stack-of-spiral read-out with 8 interleaves, TR/TE 6484/23 ms, labeling duration = 2 s, post label delay = 0.7, 1.3, 1.9, 2.5, and 3 s, voxel size = 6x6x5 mm3, NEX = 1, scan time = 3:36 min. Imaging parameters for the diffusion sequence were TR/TE = 5000/87 ms, bandwidth 1953 Hz, voxel size = 1.9x1.9x5 mm3, b-value = 0 and 1000 s/mm2. All images were co-registered to the MNI brain template4 using MINC5. The severity of the occlusion of anterior, middle, and posterior cerebral arteries involvement was graded (0 = normal, 1 = mild/moderate, 2 = severe/occluded) in consensus on time of flight MRA images by two experienced neuroradiologists (Fig. 1A). 20 standardized regions of interests (ROI’s) of two centimeters thickness were defined on the MNI brain template and corresponded roughly to ASPECTS levels6 (Fig. 1B). ADC, CBF, and ATT was measured in these ROI’s and evaluated as function of the severity of vessel stenosis. Paired two-tailed Student t-tests were used to calculate statistical significance. Significance level was set to α < 0.006 (= 0.05/9), using a Bonferroni correction of 9 to account for multiple testing.51% of vessels were normal, 27% mildly/moderately stenosed, and 22% severely stenosed/occluded. After acetazolamide challenge, a significant increase in CBF (mL/min/100g) was observed in territories of normal (50.9 ± 19.0 to 66.8 ± 19.3, p < 0.0001) and mildly/moderately stenosed (52.9 ± 18.8 to 66.2 ± 23.4, p < 0.0001) vessels, but not in the territories of severely stenosed/occluded vessels (57.8 ± 31.7 to 58.1 ± 23.4, p = 0.92). In contrast, a significant decrease in ATT was observed in all territories (Table 1, Fig 2). No significant difference was found in ADC between pre- and post-acetazolamide injection in any of the groups (Table 1). We found a significant difference after acetazolamide challenge for ATT between territories of normal and mildly/moderately stenosed vessels (p = 0.0009), as well as severely stenosed/occluded vessels (p = 0.005), while interestingly, for CBF we found a significant difference between territories of normal and severely stenosed/occluded vessels (p < 0.0001), as well as mildly/moderately stenosed and severely stenosed/occluded vessels (p = 0.0006). Finally, independently of the acetazolamide challenge, we found significant ADC increases in regions with increasing severity of large vessel stenosis (Table 2).This study demonstrates in a large cohort of Moyamoya patients, that their CVR, as measured with multi-delay ASL CBF is impaired primarily in the territories with severe stenoses or occlusions, compared with mild/moderate or no vascular abnormalities. ATT was significantly longer in regions with either mild/moderate and severe stenosis/occlusion. A significant ATT decrease after acetazolamide was seen in all territories, but without any differences based on feeding vessel status. No ADC changes were seen in any vascular group following acetazolamide. This supports the safety of acetazolamide challenge for measuring CVR in patients with cerebrovascular disease. The increase in ADC with vessel stenosis might be explained by an increasing number of chronic ischemic lesions in territories with higher stenosis. Given the growing concerns of chronic gadolinium deposition in the brain7, ASL offers a robust non-contrast method for measuring CBF and ATT to evaluate cerebrovascular reserve in Moyamoya patients.