Fig. 2

Histological and immunofluorescence analysis of vessel morphological changes in the mouse IA samples. a Schematic representation of experimental design. b Modified Verhoeff-Van Gieson staining on cryosections from the mouse brain, continuously infused with Ang II for 14 days. Samples were obtained at Day 15. Elastic laminae are visualized by the dark purple lines in the media of the artery. Collagen appears red/purple, nuclei in black and other structures in yellow. IA vessel shows visible degradation of internal elastic lamina, collagen turnover in red, and cell accumulation (nuclei in black). Closer look at the aneurysm (20× magnification, on the right). Representative section of IA in greater detail revealed multi-lobed nucleus characteristic for neutrophils—found at the lumen of the IA vessel. Scale bar: 100 µm. c Representation of how the measurements were carried out (Scale bar: 50 μm). d Intima media thickness and wall-to-lumen ratio. Based on the histological images, the widest vessel and vessel lumen diameters of both IAs and the contra arteries were measured. Vessel wall diameter was defined as the difference in intima-media thickness. Both intima-media thickness and the wall-to-lumen ratio were significantly increased in the aneurysms compared to the contra arteries. (****P < 0.0001, N = 11 mice, n = 60 aneurysms/n = 41 contra arteries, Mann–Whitney U test). e Representative images of Verhoeff–Van Gieson staining with IAs assigned different stages of the progression according to the manifested morphological changes. Inflammatory cell infiltration in the IA walls and at the luminal surface are indicated with the arrow. Scale bar: 50 µm f Vessel wall thickness—aneurysm vessel wall shows substantially increased diameter in compared to the healthy, contralateral artery. g Weak and dispersed laminin signal in the IAs as a sign of internal laminae degradation. Visible difference in laminin expression between the healthy contralateral artery and the IA. h Confirmation of Col IV turnover (characteristic for IAs) with Col IV positive signal in the aneurysm vessel. i SMC proliferation and migration. j Some IAs presented a complete degradation of SMCs. k Damage of the endothelium characteristic for IAs. CD31 (platelet endothelial cell adhesion molecule; PECAM-1) marker reveals discontinuous signal in the IA vessel wall with no cell–cell junctions were clearly visible. l Fibrinogen accumulation on the luminal side of the IA. m, n Immunofluorescence images with visible P-selectin signal (in turquoise blue, m) and VCAM-1 (magenta pink, n) signal, characteristic of increased endothelial activation found within the vessel lumen. Scale bar: 100 µm. Statistical analysis was performed using Mann–Whitney U test; **P < 0.01, ***P < 0.001; NS, not significant. Further detailed information on statistics is provided in the Methods and Materials