Thrombus organization and healing in the swine experimental aneurysm model. Part I. A histological and molecular analysis

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Object

The authors describe the process of thrombus organization in the swine surgical aneurysm model.

Methods

Lateral carotid artery aneurysms with immediately induced thrombosis were created in 31 swine for a time-course study. Aneurysms were evaluated at 1, 3, 7, 14, 30, and 90 days after they were created. Histological analyses included quantitative immunohistochemical studies and evaluation of collagen deposition. Complementary DNA microarray analysis was performed for gene expression profiling. The lists of up- and downregulated genes were cross-matched with lists of genes known to be associated with cytokines or the extracellular matrix. The expression of selected genes was quantified using real-time polymerase chain reaction. Functional clustering was performed with the Expression Analysis Systematic Explorer (EASE) bioinformatics package.

Results

Histological analysis demonstrated leukocyte and macrophage infiltration in the thrombus at Day 3, myofibroblast infiltration at Days 7 to 14, and progressive collagen deposition and contraction thereafter. Tissue organization occurred in a centripetal fashion. A previously undescribed reticular network of connective tissue was observed at the periphery of the aneurysm at Day 3. Macrophages appeared critical to this thrombus organization. A total of 1109 genes were significantly changed from reference time zero during the time course: CXCL14, which produces a monocyte-specific chemokine, was upregulated over 100-fold throughout the time course; IGF1 was upregulated fourfold at Day 7, whereas IGFBP2 was downregulated approximately 50% at Days 7 and 14. Osteopontin (SPP1) upregulation increased from 30-fold at Day 30 to 45-fold at Day 14. The EASE analysis yielded eight functional classes of gene expression.

Conclusions

This investigation provides a detailed histological and molecular analysis of thrombus organization in the swine aneurysm model. The companion study will describe the effect of embolic bioabsorbable polymers on this process.

Abbreviations used in this paper:α-SMA = α–smooth muscle actin; CCA = common carotid artery; EASE = Expression Analysis Systematic Explorer; ECM = extracellular matrix; IGF = insulin-like growth factor; IGFBP = IGF binding protein; PCR = polymerase chain reaction; TAMRA = 6-carboxytetramethylrhodamine; TGF = transforming growth factor; UCLA = University of California, Los Angeles; VEGF = vascular endothelial growth factor.

Article Information

Address reprint requests to: Daniel Lee, M.D., Division of Interventional Neuroradiology, UCLA Medical Center and David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, California 90095-1721. email: daniell@mednet.ucla.edu.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Schematic of DNA microarray analysis. Gene expression is measured as the ratio of transcript-related signal in experimental samples to that in control samples.

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    Primer and probe sequences for real-time PCR.

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    Representative images of gross aneurysm specimens obtained at reference time zero (A) and 1 (B), 3 (C), 7 (D), 14 (E), 30 (F), and 90 (G) days after creation.

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    Photomicrographs showing Masson trichrome staining of the aneurysm periphery. A: The reticular connective tissue digitations are evident extending centrally (arrowhead). Original magnification × 100. B: The novel reticular tissue under higher magnification. Original magnification × 200.

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    Photomicrograph showing organized thrombus at the center of a representative Day 14 aneurysm. Note significant collagen deposition, stained blue. Masson trichrome.

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    Photomicrographs of sections stained to demonstrate macrophage presence. A and C: Day 3 aneurysm. Macrophages (arrow) lining the inner wall (W) in A. Near the center of the Day 3 aneurysm (C), almost no expression was observed. B and D: Day 7 aneurysm. Macrophages may be seen infiltrating the thrombus (arrowhead) at its periphery in B. Macrophages (open arrow) were observed near the center of the Day 7 aneurysm (D). Original magnification × 100.

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    Graphs illustrating percentages of mean cross-sectional immunoreactivity as quantified by a modified Photoshop technique. A: Expression of α-SMA at the centers of experimentally thrombosed aneurysms. A significant increase was found to occur between Days 3 and 7 and to stabilize by Day 30. B: Deposition of collagen at centers of experimentally thrombosed aneurysms. A significant increase occurred between Days 7 and 14, stabilizing thereafter. C: Expression of TGF-β2 at the center of experimentally thrombosed aneurysms. There was no significant change during the time course of healing. D: Expression of VEGF at the centers of experimentally thrombosed aneurysms. There was a significant increase only at Days 14 and 30 compared with Day 3 and earlier. *p < 0.05; **p < 0.01; ***p < 0.001.

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    Linear plot of significantly altered gene expression. Described as the ratio of expression in the experimental sample to reference time zero. The expression trend was bimodal. Most genes were either upregulated throughout the time course (red) or down-regulated (blue).

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    Graphical results of functional clustering. Each color denotes a specific functional class of genes. Note that some genes may be related to multiple classes.

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    Graphical representation of the results of real-time PCR. Note that CXCL14 upregulation, at greater than 180- to 300-fold, extends beyond the scale of the chart.

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