Supplementary Material for: Microvascular Plasticity After Experimental Stroke: A Molecular and MRI Study

Background: Microvasculature plays a key role in stroke pathophysiology both during initial damage and extended neural repair. Moreover, angiogenesis processes seem to be a promising target for future neurorestorative therapies. However, dynamic changes of microvessels after stroke still remain unclear, and MRI follow-up could be interesting as an in vivo biomarker of these. Methods: The aim of this study is to characterize the microvascular plasticity 25 days after ischemic stroke using both in vivo microvascular 7T-MRI (vascular permeability, cerebral blood volume (CBV), vessel size index (VSI), vascular density) and quantification of angiogenic factor expressions by RT-qPCR in a transient middle cerebral artery occlusion rat model. CBV and VSI (perfused vessel caliber) imaging was performed using a steady-state approach with a multi gradient-echo spin-echo sequence before and 2 min after intravenous (IV) injection of ultrasmall superparamagnetic iron particles. Vascular density (per mm2) was derived from the ratio [ΔR2/(ΔR2*)2/3]. Blood brain barrier leakage was assessed using T1W images before and after IV injection of Gd-DOTA. Additionally, microvessel immunohistology was done. Results: 3 successive stages were observed: 1) ‘Acute stage' from day 1 to day 3 post-stroke (D1-D3) characterized by high levels of angiopoietin-2 (Ang2), vascular endothelial growth factor receptor-2 (VEGFR-2) and endothelial NO synthase (eNOS) that may be associated with deleterious vascular permeability and vasodilation; 2) ‘Transition stage' (D3-D7) that involves transforming the growth factors β1 (TGFβ1), Ang1, and tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains 1 (Tie1), stromal-derived factor-1 (SDF-1), chemokine receptor type 4 (CXCR-4); and 3) ‘Subacute stage' (D7-D25) with high levels of Ang1, Ang2, VEGF, VEGFR-1 and TGFβ1 leading to favorable stabilization and maturation of microvessels. In vivo MRI appeared in line with the angiogenic factors changes with a delay of at least 1 day. All MRI parameters varied over time, revealing the different aspects of the post-stroke microvascular plasticity. At D25, despite a normal CBV, MRI revealed a limited microvessel density, which is insufficient to support a good neural repair. Conclusions: Microvasculature MRI can provide imaging of different states of functional (perfused) microvessels after stroke. These results highlight that multiparametric MRI is useful to assess post-stroke angiogenesis, and could be used as a biomarker notably for neurorestorative therapy studies. Additionally, we identified that endogenous vessel maturation and stabilization occur during the ‘subacute stage'. Thus, pro-angiogenic treatments, such as cell-based therapy, would be relevant during this subacute phase of stroke.