**5. Conclusions**

We presented a comprehensive numerical tool for the generation and analysis of imagebased artificial vascular networks. This novel methodology is based on tissue clearing, two-photons microscopy, image acquisition and treatment and 1D computational modeling. We have analyzed five cortical regions showing that the framework is capable of correctly synthesizing the cortex microvasculature from a morphological and hemodynamical point of view. Furthermore, the tissue clearing-based methodology is flexible and it can be applied to human brains that have bigger sizes. In contrast with previous studies, the methodology includes a physiological connection to the venous drainage and some morphological features such as curvature and tortuosity. The obtained results are in line with the literature so that the presented mathematical model allows studying the healthy cerebral microvasculature for computing the hemodynamics of the BBB. Lastly, the presented methodology is feasible to be applied as well to pathological cerebral microvasculature helping understanding the role of the hemodynamics in neurodegenerative diseases.

**Author Contributions:** Conceptualization, N.R., C.O.-d.-S., A.V. and M.M.; methodology, C.E., A.U., E.R.-d.-M., A.O., I.C.-D., N.R., A.V. and M.M.; software, S.M.; formal analysis, S.M., A.S., A.U., E.R.-d.-M. and A.O.; investigation, S.M., A.S., C.E., A.U., E.R.-d.-M., A.O., I.C.-D., N.R., C.O.-d.-S., A.V. and M.M.; resources, N.R., C.O.-d.-S., A.V. and M.M.; data curation, A.S., C.E., A.U., E.R.- d.-M., A.O., I.C.-D. and A.V.; writing—original draft preparation, S.M., A.S., I.C.-D. and M.M.; writing—review and editing, I.C.-D., N.R., C.O.-d.-S., A.V. and M.M.; visualization, S.M., A.S. and A.V.; supervision, N.R., C.O.-d.-S., A.V. and M.M.; project administration, N.R., C.O.-d.-S. and M.M.; funding acquisition, N.R., C.O.-d.-S. and M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study is financially supported by the Economic Department of the Navarre Government through the research project 0011-1411-2020-000025 (3D3B-AVATAR).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the University of Navarra (076-19, 9 October 2019.

**Data Availability Statement:** Some of the presented data could be available from the corresponding author on reasonable request.

**Acknowledgments:** The support of the Instituto de Salud Carlos III (ISCIII) through the CIBER-BBN initiative is gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.
