## 4 Discussion  
This study aimed to compare CVR values measured using SPECT and MRI, and to explore the potential of tcMRI as a non-invasive alternative to SPECT. tcMRI, a technique involving timing correction for the stimulus between CO2 administration and its effect on CBF, calculated CVR values using a method with stimulus timing correction to account for the timing of maximum cerebral vessel dilation. Spatial normalization using the AAL2 template facilitated direct regional comparisons. Our findings demonstrate a strong spa-tial agreement between tcMRI and SPECT in CVR assessment, particularly within the top 30% of CVR regions identified by SPECT, where only a single region showed a discrepancy. In a healthy population, some degree of physiological variability is expected; therefore, this high concordance supports the use of tcMRI as a comparable, yet less invasive, alternative to SPECT for CVR assessment avoiding the use of radioisotopes. [PERSON] et al. established SPECT CO2 image acquisition timing based on 99 mTc-HMPAO injection protocols, demonstrating the validity of this approach in patients with cerebrovascular disease [7]. Our study employed a modified protocol with a doubled 99 mTc-HMPAO dose at the same acquisition time. The observed difference in mean CVR between the O2 and CO2 inhalation groups in our SPECT experiments further validates the vasodilatory effect of CO2 and demonstrates the reproducibility of SPECT CVR measurements. While ACZ typically elicits a more potent vasodilatory response than CO2, we observed lower CVR values in patients with cerebrovascular disease compared to healthy individuals, with near zero CVR in the affected hemisphere and values comparable to the CO2 group in the contralateral hemisphere, resulting in a lower overall average [2,10]. Although ACZ has been widely used as a vasodilatory challenge in SPECT, its use has been associated with safety concerns due to reported side effects and limitations for re-peated evaluations due to its sustained effect. In contrast, CO2 offers a safer profile with minimal side effects and immediate, readily reversible vasodilation, making it particularly well suited for repeated CVR assessments [1,19]. Building upon this, tcMRI, as a non-invasive technique utilizing the CO2 stimulus, presents a distinct advantage for repeated evaluations in clinical practice, especially for follow-up assessments. While SPECT can be valuable for the initial evaluation of CBF changes in neurological disorders, long-term follow-up often relies on MRI to assess anatomical recovery. Integrating tcMRI during these MRI follow-up visits allows for the quantitative assessment of CVR recovery without additional radiation exposure [16]. This is particularly valuable for identifying brain regions where CVR remains impaired despite apparent anatomical recovery on structural MRI, providing crucial information for predicting functional outcomes and guiding personalized treatment strategies [8]. Furthermore, for patients requiring frequent monitoring due to chronic conditions like Moyamoya disease, where cumulative radiation dose from repeated SPECT scans is a significant concern, tcMRI offers a safe and effective alternative for serial CVR evaluation [22]. In clinical practice, tcMRI could be integrated into routine brain MRI protocols during follow-up visits for patients with cerebrovascular risk, allowing for non-invasive functional monitoring without altering existing workflows. Its potential utility extends beyond rare conditions such as Moyamoya disease to broader populations, including patients with small vessel disease or vascular cognitive impairment. As the method relies on CO2 inhalation without contrast agents or radiation, it may be particularly beneficial for elderly or vulnerable patients requiring repeated evaluation. With further validation and standardization, tcMRI could be implemented as a supplementary module in clinical MRI platforms, enabling the assessment of both structural and functional recovery in a single session.