Aortic dissection detection and thrombus structure quantification using volumetric ultrasound, histology, and scanning electron microscopy

Abstract

Aortic dissection occurs when a weakened portion of the intima tears, and a separation of layers propagates along the aortic wall to form a false lumen filled with active blood flow or intramural thrombus. The unpredictable nature of aortic dissection formation and need for immediate intervention leaves limited serial human image data to study the formation and morphological changes that follow dissection. We used volumetric ultrasound examination, histology, and scanning electron microscopy (SEM) to examine intramural thrombi at well-defined timepoints after dissection occurs in apolipoprotein E-deficient mice infused with angiotensin II (n = 71). Stratification of red blood cell (RBC) morphologies (biconcave, intermediate biconcave, intermediate polyhedrocyte, and polyhedrocyte) in the thrombi with scanning electron microscopy (n = 5) was used to determine degree of thrombus deposition/contraction. Very few biconcave RBCs (1.2 ± 0.6%) were in the thrombi, and greater amounts of intermediate biconcave RBCs (25.8 ± 6.7%) were located in the descending thoracic portion of the dissection while more polyhedrocytes (14.6 ± 5.1%) and fibrin (42.3 ± 4.5%; P < .05) were found in the distal suprarenal aorta. Thrombus deposition likely plays some role in patient outcomes, and this multimodality technique can help investigate thrombus deposition and characteristics in experimental animal models and human tissue samples.

Keywords: Aortic dissection; Intramural thrombus formation; Scanning electron microscopy.

Fig 1

Daily ultrasound scans detect dissections less than 24 to 32 hours after they develop. Using this method, we found young mice develop dissections earlier after pump implant compared to older mice. These data can also provide serial diameter/volumetric data both before and after dissection. (A) Three-dimensional ultrasound (3DUS) scans provide volumetric data of dissecting aortic aneurysms in vivo. (B) Diameter measurements at three locations along the aorta using the 3DUS scans. (A, B) Yellow, true lumen; green, false lumen. (C) Diameter change from baseline to 3 days after pump implant showed no difference between young (9.7-21.7 weeks of age) and old (35.0-88.1 weeks of age) mice. (D-F) We observed a significant difference in the timing of dissections post-implant between young and old mice, with young mice developing dissections earlier after implant (∗∗∗∗P < .0001). Scale bars, 1 mm. Ao, aorta; SMA, superior mesenteric artery; VC, vena cava.

Fig 2

Daily ultrasound scans help detect when dissections occurred after pump implantation (8.0 ± 0.6 days on average) and segmentation revealed regions of thrombosed and patent false lumen. Histology and scanning electron microscopy (SEM) analysis showed high percentages of fibrin and red blood cells (RBCs) in the intramural thrombus that formed, a compressed true lumen, and collagen surrounding the adventitia. (A) Daily ultrasound scans collected hemodynamic and morphological data in vivo.(B) Short-axis schematic of abdominal aorta with surrounding organs and tissue. (C) Aorta was removed and sliced into 12 sections where every other section was saved for histology or SEM. (D-F) Ultrasound images, H&E stained histology, and SEM cross-sections can be aligned anatomically. Histology and SEM sections help with the comparison of structural differences observed in ultrasound images, helping to identify composition and structure of the resulting intramural thrombus in the false lumen. Yellow, true lumen; cyan, patent false lumen; green, thrombosed false lumen. Scale bars, 500 μm. (A-C created in Biorender.com.)

Fig 3

Histology showed a significant number of red blood cells (RBCs) (P < .001) and fibrin (P < .001) compared with all other structures, but no statistically significant differences between proximal, middle, and distal regions. (A, B) Histology sections stained with hematoxylin and eosin (top) and Movat’s Pentachrome (bottom). (C, D) RBCs and fibrin comprised a large portion of the intramural thrombus. Thrombus composition differs slightly in the proximal, middle, and distal sections of the aorta. (Created in Biorender.com.) (E, F) A semiquantitative analysis identified the presence of significantly more intramural immune cells in middle and distal regions compared to proximal sections (P < .05) and slightly increased intramural thrombus and thrombus organization in middle sections. Scale bars, 50μm.

Fig 4

We found that 98.3% of red blood cells (RBCs) in the intramural thrombus had deformed morphologies and regional analysis revealed 25.8% of proximal intramural thrombus was intermediate biconcave RBCs, whereas 14.6% of distal thrombus was polyhedral RBCs. Fibrin made up 29.7% of all intramural thrombus in the sections analyzed. (A) Low magnification SEM images are used to identify different thrombus structures (1-4) in the false lumen to be scanned at higher magnification. Scale bar, 500μm. (B) A grid was placed on high magnification (2000×) scanning electron microscopy (SEM) images and FIJI ImageJ cell counter was used to identify RBC types, fibrin, and other structures. Scale bar, 25 μm. (C) Black and white arrows indicate borders of different thrombi in a proximal section of thrombus. Scale bar, 500μm. (D) Intermediate biconcave RBCs were located primarily in proximal sections of thrombus. Black and white arrows point to intermediate biconcave RBCs. Scale bar, 10 μm. (E) Black and white arrows indicate circumferential fibrin and thrombi organization in a distal section. (F) Black and white arrows point to polyhedral RBCs found mostly in distal portions of the thrombus. (G, H) Each dissection has a unique composition of RBCs with varying morphology and fibrin. Composition as well as clot contraction was different in proximal and distal sections of the dissection, which can likely be attributed to local hemodynamics.

Supplementary Fig 1

Color Doppler imaging displayed the presence or lack of active flow in the false lumen and pulsed-wave Doppler measured velocity measurements showed increases in true lumen velocity after intramural thrombus constricts/applies force to the true lumen. (A) LAX color Doppler shows blood flow in the true lumen (red) traveling from proximal to distal while we observed recirculating flow in the false lumen (blue). (B) SAX color Doppler shows similar flow in the true and false lumens. Yellow, true lumen; cyan, patent false lumen; green, thrombosed false lumen. (C) We observed increases in true lumen flow and velocity after dissections occurred owing to constriction of the true lumen by the false lumen. Scale bars, 1 mm. (D) Old mice had significantly higher mean flow rates at baseline compared with young mice. (E) There were no significant differences in the mean flow at baseline between the dissection and no dissection group. (F) There were significant increases in mean velocity once a dissection occurred as the true lumen is compressed. ∗P < .05; ∗∗P < .01.

Supplementary Fig 2

After pump implantation, all groups underwent a significant increase in aortic diameter from baseline to maximum diameter before dissection. There were no significant differences in diameter growth percentage between the young and old or dissection and no dissection groups. (A, B) All mice experienced aortic diameter growth from baseline measurements to 3 days after pump implantation with the young mice and ones that developed dissections increasing slightly more than old and nondissecting mice, respectively. (C, D) Mice experienced significant aortic dilation from baseline to maximum diameter, but significant diameter growth was not deterministic in the development of dissections. ∗∗∗P < .001; ∗∗∗∗P < .0001.

Supplementary Fig 3

Hematoxylin and eosin (H&E)-stained aortic slices display extracellular matrix, red blood cells (RBCs), and nuclei present in the true and false lumen of the aortic dissections (ADs). H&E samples were examined for intramural thrombus presence and organization, immune cell infiltration, intramural hemorrhage, atherosclerosis, and tertiary lymphoid organs and differences in proximal, middle, and distal sections were observed. (A-D) Four ApoE^–/– mice displayed in columns A-D, where the top sections were taken from the descending thoracic aorta down to the bottom section taken just proximal to the renal arteries. Scale bars, 500 μm.

Supplementary Fig 4

Movat’s pentachrome stained slides identified RBCs (purple), fibrin (red-pink), collagen (yellow-brown), elastin (black), and proteoglycans (blue). Fiji ImageJ color segmentation revealed large numbers of red blood cells (RBCs) followed closely by fibrin in the false lumen. Each section is heterogeneous with some containing more fibrin than RBCs and varying amounts of collagen, elastin, and proteoglycans. Collagen consistently remained localized to the outer adventitia. (A-D) Four ApoE^–/– mice displayed in columns A-D with top to bottom being proximal to distal. Scale bars, 500 μm.

Supplementary Fig 5

Low magnification scanning electron microscopy (SEM) images were used to identify different thrombi structures in the false lumen of the AD. (A-D) Four ApoE^–/– mice displayed in columns A-D with top to bottom being proximal to distal. Scale bars, 500 μm.

Supplementary Fig 6

Structures identified in thrombi from aortic dissection (AD) murine model. (A) Biconcave red blood cells (RBCs), black arrows. (B) Intermediate biconcave RBCs, black arrows. (C) Polyhedral shaped RBCs, black arrows. (D) Intermediate polyhedral RBCs, black arrows. (E) Fibrin, black arrows. Other structures include: (F) collagen, (G) neutrophils, (H) fibroblasts in black arrows. Scale bar, 10 μm.

Supplementary Fig 7

We used FIJI ImageJ color segmentation and cell counter plugins to quantify cellular structures in histology and scanning electron microscopy (SEM) images. Histology provides compositional information for the entire aorta while SEM allows for the stratification of red blood cell (RBC) morphology and the presence of fibrin strands in the intramural thrombus. (A) Movat’s pentachrome stains for RBCs (dark red/maroon), fibrin (light red/magenta), proteoglycans (blue), elastin (black), and collagen (yellow/brown). Scale bar, 500 μm. (B) Color segmentation plugin in ImageJ was used to sample colors for each cellular structure and background (white). The hidden Markov model algorithm was applied to calculate the percentages of each color present. (C) After loading a high magnification (2000×) SEM image, set the scale of the image in pixels. (D) Apply a grid size that roughly encompasses one RBC per square. Grid size = 15 μm². (E) Rename each counter to correspond with the RBC type, fibrin, or other/space. (F) Designate each square as a RBC type, fibrin, or other/space depending on what structure the majority of the square is occupied by.