How the elastase-induced rabbit aneurysm heals following flow diverter treatment: a histopathological study

In Brief

This study aimed to identify the histopathological changes within the aneurysm sac following flow diverter treatment. Healed aneurysms exhibited myofibroblasts, collagen, and a well-organized fibrin network in the aneurysm neck. In contrast, unhealed aneurysms displayed a poorly organized fibrin network with scattered myofibroblasts. This study suggests that the degree of fibrin accumulation within the aneurysm sac and organization of the fibrin network can affect the outcomes of flow diversion.

Abstract

OBJECTIVE

Fibrin deposition is integral to thrombus formation and wound healing. The role of fibrin deposition and subsequent metabolism following flow diversion for aneurysm treatment remains poorly characterized. This study aimed to evaluate the role of fibrin in early thrombus organization after flow diverter treatment.

METHODS

Thirty-five elastase-induced aneurysms were induced in New Zealand white rabbits and subjected to endoluminal flow diversion treatment. The device-bearing arteries were harvested at 1, 3, and 6 months postimplantation and processed for histopathological examination, including a modified picro-Mallory stain (Carstairs method) to visualize fibrin and platelets, immunohistochemical targeting of smooth muscle actin (SMA), and H&E staining for conventional morphological evaluation. Quantitative analysis of tissue components was carried out using the Orbit Image Analysis software. The samples were also assessed qualitatively to investigate the morphology and location of fibrin and other thrombus components within the intra-aneurysmal thrombi. Statistical analyses were conducted using R software version 4.3.1.

RESULTS

Fibrin constituted 27.9% of the thrombus tissue within the aneurysm sac for aneurysms harvested at 1 month, and this rate was significantly lower in the 3-month group (10.2%, p = 0.018). The proportion of blood cells within the sac was also notably higher in the 1-month group compared with other time points. The primary tissue filling the dome at 1 month (14/15, 93%) was an unorganized thrombus primarily composed of fibrin, platelets, and red blood cells. Conversely, aneurysms harvested at 1 month had the lowest collagen level (25.6%). However, collagen became the dominant tissue component within the aneurysm sac, accounting for 71.8% of tissue in the 3-month group (p = 0.007). There were no differences observed among the examined components between the 3-month and 6-month groups. On qualitative analysis, collagen-producing SMA-positive myofibroblasts were located near or in between fibrin molecules. Healed aneurysms exhibited myofibroblasts, collagen, and a well-organized fibrin network on the aneurysm neck. In contrast, unhealed aneurysms displayed a poorly organized fibrin network with scattered myofibroblasts at the neck area.

CONCLUSIONS

These findings indicate that fibrin plays a foundational role in the gradual occlusion of aneurysms after flow diverter treatment. Endovascular approaches that enhance fibrin accumulation could potentially improve aneurysm occlusion rates. Further research is needed to establish the precise role of fibrin in aneurysm occlusion.

Flow diversion has gained widespread recognition among neurointerventionalists over the past decade. Several clinical trials have established flow diversion’s safety profile and efficacy for intracranial aneurysms.^1–3 However, long-term complete occlusion rates have consistently remained under 80% in these trials. Unsuccessful flow diversion attempts burden the healthcare system and put patients at risk due to long-term antiplatelet treatment and periprocedural complications. As a result, many groups have tried to elucidate the key elements of the histopathological aneurysm occlusion process to improve flow diverter design and lead to next-generation devices.

Fibrin, the key protein component of blood clots, acts as a structural biomaterial with biophysical properties to provide binding sites for hemostasis proteins and cells such as platelets, fibroblasts, and endothelial cells to inhibit fluid flow and maintain hemostasis, and it plays a critical role in wound healing.^4–6 However, despite its crucial role in cell-matrix interactions and wound healing, no study has focused on the role of fibrin in aneurysm occlusion. The current literature shows that fibrin deposition in wound healing is a dynamic process, and both fibrin organization and structure change over time.^4–6 Similarly, aneurysm occlusion occurs gradually after flow diversion, and arrests in fibrin deposition and structural differences in fibrin fibers might be associated with incomplete aneurysm occlusion. Considering that fibrin is an integral part of thrombus, understanding fibrin’s role in aneurysm occlusion can improve endovascular treatment outcomes. In this study, we used the Carstairs stain, a histochemical stain modified by the picro-Mallory method and specifically used to stain fibrin, to elucidate how fibrin was involved in the aneurysm’s healing following device implantation.

Methods

All rabbits in the present study were originally used as part of another investigation. The original investigation was entirely unrelated to this project.

Aneurysm Creation, Treatment, and Follow-Up

Thirty-five aneurysms were induced in New Zealand white rabbits. The Institutional Animal Care and Use Committee approved all animal procedures. The detailed aneurysm creation procedure has been described previously.^7–9 Briefly, under anesthesia, the right common carotid artery (CCA) was exposed and ligated distally. A 1- to 2-mm beveled arteriotomy was made on the right CCA, and a 5-Fr AVANTI vascular sheath (Cordis) was advanced retrogradely in the right CCA to approximately 3 cm cephalad to its origin. Fluoroscopy (Advantx, GE HealthCare) was performed after the injection of contrast through the sheath retrogradely in the right CCA to identify the junction between the right CCA and the subclavian and brachiocephalic arteries. A 3-Fr Fogarty balloon (Baxter Healthcare) was advanced through the sheath to the level of the origin of the right CCA with fluoroscopic guidance and was inflated with iodinated contrast material. One milliliter of porcine elastase (approximately 130 U/ml; Worthington Biochemical) was incubated within the lumen of the right CCA above the inflated balloon for 20 minutes. The balloon and sheath were then removed, and the right CCA was ligated below the sheath entry site.

Three weeks after aneurysm creation, a 5-Fr sheath was advanced on the right side of the femoral artery via cutdown, followed by a 5-Fr Envoy guiding catheter with a 0.056-inch inner diameter (Codman & Shurtleff). Digital subtraction angiography (DSA) of the aortic arch was performed to confirm the patency of the aneurysm and the parent arteries. Then, a Marksman microcatheter (ev3) was advanced into the distal end of the parent artery (right subclavian artery) over a 0.014-inch microguidewire (Boston Scientific) through the guide catheter to deploy the Pipeline embolization device (Medtronic). DSA was performed through the guide catheter immediately after deployment and before euthanasia. Aspirin (10 mg/kg) and clopidogrel (10 mg/kg) were given daily 2 days before implantation and continued for 30 days after treatment.

The rabbits were followed for 1 month (n = 15), 3 months (n = 10), and 6 months (n = 10). At the time of euthanasia, animals were deeply anesthetized. DSA of the aortic arch was performed using a femoral approach. Animals were then euthanized with a lethal injection of pentobarbital. The device-bearing parent arteries and aneurysms were harvested and immediately fixed in 10% neutral buffered formalin for further evaluation.

Data Analysis

Angiographic Evaluation

A 3-point grading system was used to assess the aneurysm occlusion⁹ on follow-up angiography: grade 1 indicated complete occlusion; grade 2, near-complete occlusion; and grade 3, incomplete occlusion.

Tissue Processing and Staining

The aneurysms and device-bearing parent arteries were harvested and fixed in 10% neutral buffered formalin for at least 24 hours. They were then processed using a modified histological technique as previously reported⁷ and cut into 4-μm sections. Serial sections of each aneurysm/parent artery underwent H&E staining, Carstairs staining, and immunohistochemical (IHC) targeting of alpha–smooth muscle actin (SMA).

H&E Staining

H&E staining was performed as previously reported;⁷ briefly, two minimal serial sections of each block that contained both the aneurysm dome and neck from each sample were stained with H&E for conventional histopathological evaluation. The slides were de-paraffinized and hydrated in water. They were then stained with hematoxylin for 5 minutes. After differentiation in 1% acid-alcohol and rinsing in water, the slides were then stained with eosin for 1 minute, dehydrated in graded alcohol solutions, cleared in xylene, and finally mounted with EZ Mount.

Carstairs Staining

Carstairs staining, a modified picro-Mallory method, stood out as an especially effective technique for distinguishing fibrin and platelets.¹⁰ Briefly, serial sections from each block containing both the aneurysm dome and neck were de-paraffinized and hydrated to a double-distilled H₂O. Next, they were stained in 5% ferric ammonium sulfate for 5 minutes, followed by a rinse in tap water. Mayer hematoxylin was then applied for a 5-minute staining interval, followed by rinsing in running tap water. The slides were stained in Picric Acid–Orange G Solution for 1 minute, with a brief subsequent rinse in double-distilled H₂O. The slides were then stained in ponceau fuchsin solution for 5 minutes, followed by differentiation using 1% phosphotungstic acid for 7 minutes and another rinse in double-distilled H₂O. Finally, the slides were stained in 1% aniline blue solution for 1 minute, followed by rinsing in distilled water, dehydration with alcohol, clearing in xylene, and covering with a mount medium. The reagents used for the staining protocol were obtained from Electron Microscopy Sciences Inc. (no. 26381 series). Sections of thrombus tissue obtained from a patient with acute ischemic stroke were utilized as a positive control.

In the present Carstairs staining method, fibrin stained a clear, bright red and platelets stained a gray, gray-blue, bluish, and/or violet shade. Red blood cells (RBCs) stained a clear yellow or orange, the collagenous matrix/fibers stained a light blue or brilliant blue, and muscle stained red.

Immunohistochemical Analysis

Serial sections of each block containing both the aneurysm dome and neck from each sample underwent immunostaining using monoclonal mouse antihuman alpha-SMA (clone 1A4, no. M0851, Dako). IHC analysis was carried out on a Leica Bond Max Autostainer using a RedMap kit (Bond Polymer Refine Red Detection, Leica Biosystems) following the methodology previously reported.^11,12 The resulting positive signal was conspicuously stained bright red.

Serial sections of thrombus tissue, which were previously stained as a positive control, as discussed in the Carstairs Stain section, were additionally subjected to IHC staining for the platelet marker CD42b.¹¹ This IHC staining served as a point of reference for identifying platelets when interpreting the Carstairs stain.

Quantification of Targeting Tissue Component

The stained slides were scanned using a Miotic Easy Scan Pro system (Miotic Digital Pathology) at a magnification of ×20. An experienced pathologist conducted the interpretation and quantification of the observations. The identification and quantification of individual colors along with their corresponding tissue elements were performed by an experienced pathologist through training on the Orbit Image Analysis software (www.orbit.bio)¹³ to facilitate determination of the percentage composition of various clot components, including fibrin, platelets, RBCs, and the collagenous matrix/fibers, within the aneurysm dome and neck sections stained with the Carstairs method. Additionally, the SMA-positive immunohistochemical stain was used.

Statistical Analysis

The statistical analyses were conducted using R software version 4.3.1 (R Foundation for Statistical Computing), with statistical significance set at a p value < 0.05. Continuous variables were expressed as mean ± SD. To assess the normality of the data distribution, the Shapiro-Wilk test and Q-Q plots were used, revealing a deviation from normal distribution for all outcomes. Consequently, the nonparametric Mann-Whitney U-test was utilized to compare various outcomes across different time points, ensuring robust statistical evaluations despite the nonnormally distributed data.

Results

Aneurysm Sizes and Angiographic Follow-Up

The sizes of the aneurysms are presented in Table 1. At 1 month, the mean dimensions were as follows: 3.4 ± 1.4 mm (neck), 4.7 ± 1.6 mm (width), and 9.3 ± 2.4 mm (height). These measurements exhibited similar trends at the subsequent time points, with minimal deviations: 3.2 ± 1.0 mm (neck), 4.1 ± 0.8 mm (width), 9.1 ± 2.6 mm (height) at 3 months and 3.3 ± 0.8 mm (neck), 4.1 ± 1.0 mm (width), and 10.0 ± 3.1 mm (height) at 6 months. In the pairwise comparisons conducted at various time points, it is noteworthy that no statistically significant differences were observed for any of the size metrics, including aneurysm neck, width, and height.

TABLE 1.

Summary of aneurysm sizes by group

Group	Neck (mm)	Width (mm)	Height (mm)
1 mo	3.4 ± 1.4	4.7 ± 1.6	9.3 ± 2.4
3 mos	3.2 ± 1.0	4.1 ± 0.8	9.1 ± 2.6
6 mos	3.3 ± 0.8	4.1 ± 1.0	10.0 ± 3.1

Data are given as mean ± SD. There were no significant differences between time points.

The angiographic follow-up scores showed that grade 1, 2, and 3 occlusion rates were noted in 24 (69%), 7 (20%), and 4 (11%) of 35 aneurysms, respectively. At 1 month, grade 1, 2, and 3 occlusion rates were noted in 8, 3, and 4 cases, respectively. Grade 1 and 2 occlusion rates were noted in 7 and 3 cases at 3 months, respectively. At 6 months, grade 1 and 2 occlusion rates were noted in 9 cases and 1 case, respectively.

Qualitative Histopathological Evaluation

Eight aneurysms (53%) displayed histologically closed necks at 1 month, characterized by neointima featuring a collagen matrix with SMA-positive cells across the neck, with or without fibrin deposit (Figs. 1 and 2). Conversely, 7 cases (47%) exhibited a patent aneurysm, with the neck interface mainly covered by unorganized fibrin or fibrin mixed with platelets. SMA-positive cells associated with collagenous matrix were limited and primarily located at the periphery of the aneurysm neck (Fig. 3).

FIG. 1.

Photomicrographs of a healed aneurysm at 1-month follow-up. A–C: Images of a routine H&E stain showing a neointima along with device struts (arrows) sealing the aneurysm neck off from the parent artery lumen. D and E: This neointima primarily consists of a collagen matrix (blue in D) and SMA-positive cells (red, arrows in E). A small amount of residual fibrin is still visible within the collagen matrix at the neck (red stains within the square in D). Fibrin and RBCs constitute the primary tissue components within the dome, as seen in panels A and D. Original magnifications: H&E, ×20 (A), ×40 (B), and ×100 (C); Carstairs (fibrin in red, RBCs in yellow, collagen in blue), ×9.6 (D); IHC targeting of SMA (red indicates positive staining), ×6.0 (E). Figure is available in color online only.

FIG. 2.

Photomicrographs of Carstairs stain illustrating the presence of both fibrin and/or platelets and a neointima covering the neck interface at 1-month follow-up after treatment with a flow diverter. A: Low-magnification image demonstrating an overall view of the aneurysm showing separation of the dome from the parent artery lumen. B: Higher-magnification image of the area in the rectangular outline in panel A showing that the neck is constructed with fibrin and/or platelets (red brackets) and a neointima (blue brackets). C: Higher-magnification image of the area in red brackets in panel B showing the fibrin (bright red) and platelets (gray or bluish, arrows). D: Higher-magnification image of the area in blue brackets in panel B showing the collagenous matrix (blue) within the neointima at the neck. E: Image with IHC targeting of SMA showing that the neointima at the neck also contains SMA-positive cells (red, arrows). F: Higher-magnification image of the dome in panel A showing that fibrin (red), platelets (gray, violet, or bluish), and RBCs (yellow/orange) are the primary tissue components within the dome. Original magnifications: Carstairs, ×1.3 (A), ×4.7 (B), ×5.9 (C), ×5.9 (D), and ×4.5 (F); IHC targeting of anti-SMA, ×5.9 (E). Figure is available in color online only.

FIG. 3.

Photomicrographs of an unhealed aneurysm at 1-month follow-up. A and B: Low-magnification images of H&E (A) and Carstairs (B) staining demonstrating an open aneurysm neck associated with a noticeable neck remnant. C and D: Images with Carstairs staining (C) and IHC targeting of SMA (D) illustrating that fibrin (red, thin arrows in C) and fibrin and/or platelets (gray or bluish, thick arrow in C) are the primary tissue present at the neck interface. Collagen matrix/fibers (stars in C) and smooth muscle cells (red, arrows in D) are limited and primarily located at the periphery and side. E–G: Higher-magnification images of the square area in panel A (E) and circled area in panel B (F) showing the spindle cells embedded in fibrin (Fi), platelets (P), RBCs (R), and collagenous matrix (Co). They are positive for SMA stain (red in G) and are primarily located at the side of the aneurysm neck. Original magnifications: H&E, ×1.1 and ×9.2 (A and E); Carstairs, ×1.2 (B), ×3.2 (C), and ×9.2 (F); IHC targeting of anti-SMA, ×3.4 (D) and ×8.4 (G). Figure is available in color online only.

The aneurysm domes were primarily filled with unorganized thrombus, consisting of fibrin, platelets, and RBCs in 14 of the 15 rabbits. Scattered SMA-positive cells associated with collagenous matrix/fibers were minimally present at the periphery or extending toward the dome center from the periphery. Connective tissue primarily composed of collagenous matrix/fibers occupying the aneurysm dome was observed in 1 rabbit.

Organized connective tissue across the neck at 3 months was seen in 6 rabbits (60%), sealing the aneurysm from the parent artery. This connective tissue was primarily composed of collagenous fibers/matrix. Similarly, these 6 aneurysms had a dome occupied by connective tissue predominantly made up of collagenous fibers. In contrast, the remaining 4 rabbits had a partial distribution of collagenous fibers/matrix across the neck, whereas stacked fibrin or fibrin mixed with platelets presented at the interface between the dome and the neck remnant (Fig. 4). Among the 4 aneurysms with open necks, 2 had connective tissue composed of collagenous fibers/matrix filling the domes, while the other 2 had a poorly organized thrombus, composed of fibrin, platelets, and RBCs occupying most of the dome.

FIG. 4.

A–C: Photomicrographs of an unhealed aneurysm at 3-month follow-up. Low-magnification images of H&E (A) and Carstairs (B and C) staining demonstrating that the aneurysm neck remains open to the parent artery. The dome is primarily occupied by an unorganized thrombus composed of fibrin, platelets, and RBCs. D and E: Higher-magnification images of the neck area in panels B (D) and C (E) displaying fibrin (red, red arrows) and fibrin mixed with platelets (gray or bluish, blue arrows) traversing the neck interface. F: Higher-magnification image of the dome area in panel C showing an unorganized thrombus in the dome, composed of fibrin (red), platelets (gray, bluish, or violet), and RBCs (yellow or orange). Original magnifications: H&E, ×0.9 (A); Carstairs, ×1.3 (B and C), ×5.1 (D), ×2.8 (E), and ×4.0 (F). Figure is available in color online only.

Six months after implantation, 8 of 10 aneurysms (80%) had collagenous tissue along with stent struts covering the neck and sealing the aneurysm from the parent artery. The remaining 2 aneurysms demonstrated collagenous tissue stacked with unorganized fibrin, partially covering the neck and resulting in a partially open aneurysm neck. Eight of 10 aneurysms had connective tissue primarily composed of collagen fibers/matrix occupying the dome, while the other 2 had poorly organized thrombus composed of fibrin, platelets, and RBCs partially filling the dome.

Quantitative Analysis of Carstairs Staining and IHC Analysis

The percentages of various tissue components, including fibrin, platelets, RBCs, collagen, and SMA-positive cells, are summarized in Table 2. At the initial 1-month evaluation, fibrin (27.9% ± 14.2%), platelets (23.8% ± 14.1%), RBCs (21.2% ± 12.4%), and collagen (25.6% ± 23.1%) constituted the major components, with variations indicated by the standard deviations. Subsequently, at the 3-month mark, a substantial shift occurred as collagen became the predominant component (71.8% ± 38.9%), while fibrin (10.2% ± 19.4%), platelets (4.0% ± 7.2%), and RBCs (6.0% ± 11.8%) exhibited marked reductions. By the 6-month interval, this trend continued, with collagen (71.9% ± 41.1%) maintaining dominance, complemented by a minor presence of fibrin and platelets (10.6% ± 16.8% and 8.7% ± 11.3%, respectively).

TABLE 2.

Summary of tissue component by group

Group	Fibrin (%)	Platelets (%)	RBCs (%)	Collagen (%)	SMA (%)
1 mo	27.9 ± 14.2*†	23.8 ± 14.1*†	21.2 ± 124*†	25.6 ± 23.1*†	9.9 ± 8.1
3 mos	10.2 ± 19.4*	4.0 ± 7.2*	6.0 ± 11.8*	71.8 ± 38.9*	11.5 ± 9.1
6 mos	10.6 ± 16.8†	8.7 ± 11.3†	3.4 ± 4.6†	71.9 ± 41.1†	13.8 ± 11.1

Data are given as mean ± SD.

^*Significant difference in percentages of fibrin, platelets, RBCs, and collagen between the 1-month and 3-month groups (p < 0.05).

^†Significant difference in percentages of fibrin, platelets, RBCs, and collagen between the 1-month and 6-month groups (p < 0.05).

In the pairwise comparisons examining the variations in different thrombus components across various time points, noteworthy trends emerged. Fibrin, RBCs, and platelets exhibited statistically significant reductions (p < 0.05) from the initial 1-month assessment to both the 3-month and 6-month intervals. In stark contrast, collagen demonstrated a substantial and statistically significant increase from 1 month to 3 months (p = 0.007) and from 1 month to 6 months (p = 0.001), indicative of a pronounced shift toward a more collagen-rich thrombus organization. Interestingly, no components exhibited significant changes between the 3-month and 6-month time points. Notably, the SMA component displayed no significant variations when all time points were compared, suggesting a consistent presence of this element throughout the observed period (Table 2).

Discussion

In the present study, we conducted an in-depth examination of unhealed aneurysms following flow diversion treatment in an elastase-induced rabbit aneurysm model. Our investigation revealed that fibrin, either alone or in association with platelets, emerged as the predominant tissue element in the neck region of these aneurysms. Furthermore, an unorganized thrombus, primarily comprising fibrin, platelets, and RBCs, initially occupied the domes during the early stages of the healing process and was gradually replaced by collagenous connective tissue over time.

Our study yielded several key findings. First, it became evident that the distribution of fibrin and other thrombus components within healing aneurysms was not uniform, persisting up to 6 months within the dome or at the neck interface. Second, by using a modified picro-Mallory staining method, we successfully identified fibrin and fibrin combined with platelets as the primary constituents at the neck interface in unhealed aneurysms. These elements appeared to serve as attachment points for myofibroblasts and matrix proteins, mirroring processes akin to wound healing. These findings not only underscore the pivotal role of fibrin in aneurysm occlusion but also suggest that treatment strategies aimed at enhancing fibrin or platelet-rich fibrin accumulation may hold the potential to improve endovascular treatment outcomes.

Historically, studies on the histology of intracranial aneurysms have predominantly used the H&E staining method.^14,15 However, H&E staining can pose challenges in distinguishing fibrin from other components like RBCs, myocytes, and platelets, as many thrombus components appear eosinophilic. In contrast, the picro-Mallory/Carstairs staining method stands out as an exceptional histochemical staining technique for identifying fibrin and platelet molecules.¹⁰ Notably, it not only distinguishes fibrin from other thrombus components but also differentiates fibrin from platelets. While a few studies have utilized the picro-Mallory method to explore the wall characteristics of ruptured aneurysms,^16,17 our study is the first to use this staining technique to investigate the occlusion process in aneurysms treated with endovascular techniques. Our results indicate that fibrin and fibrin combined with platelets constitute the dominant components at the neck interface in unhealed aneurysms, as well as within the domes during the early stages of the healing process. These observations suggest that aneurysm occlusion unfolds in a layered organization, with the accumulation of fibrin or platelet molecules playing a pivotal role by providing attachment points for pluripotent stem cells, including myofibroblasts and myoendothelial cells, thereby promoting and driving the occlusion process.

Furthermore, our study highlighted the synergistic relationship between fibrin and myofibroblasts, which potentiate each other’s effects during the wound healing process. Fibrin serves as an initial attachment point for myofibroblasts during the acute phase of wound healing.^4–6 Subsequently, myofibroblasts proliferate, activate, secrete extracellular matrix, and mediate fibrin accumulation at the injury site.^4–6 We observed significant parallels between aneurysm occlusion and wound healing processes. Through the combination of IHC analysis and modified picro-Mallory staining, we were able to identify SMA-positive myofibroblasts located either adjacent to or embedded within the fibrin matrix. Therefore, our findings strongly suggest that fibrin molecules guide myofibroblast migration and proliferation within the aneurysm sac through mechanisms akin to those seen in wound healing.

Considering fibrin’s role in thrombus formation, it has been used in various endovascular applications. For example, fibrin sealants have been utilized to treat endoleaks arising after endovascular treatment of aortic aneurysms and pseudoaneurysms.^18–20 Surprisingly, fibrin’s potential to enhance thrombus formation has not been fully harnessed in the management of intracranial aneurysms. In contrast, several research groups have explored fibrin’s heparin-binding capability to coat flow diverters with heparin, thus reducing the thrombogenicity of these devices.^21,22 In vitro studies by Haworth et al. have demonstrated that the choice of endovascular devices can impact fibrin network characteristics and stiffness.²³ Consequently, the development of next-generation treatment tools aimed at enhancing fibrin accumulation holds promise for improving aneurysm occlusion rates.

The elastase-induced aneurysms in the right CCAs of New Zealand white rabbits have similar hemodynamic, morphological, and histological characteristics to human aneurysms.²⁴ Ding et al. demonstrated that untreated elastase-induced aneurysms remain patent for at least 24 months following creation, indicating that thrombotic changes within the created aneurysms can be attributed to the deployed device.²⁵ Additionally, in a study of 51 elastase-induced right CCA aneurysms, Zeng et al. found that the number of recirculation regions and ranges in values of pressure, wall shear stress, and oscillatory shear index were all similar to the hemodynamic features of human aneurysms.²⁶ Therefore, our findings might provide useful insights into the occlusion process of intracranial human aneurysms following flow diversion.

However, it is important to acknowledge the limitations of our study. The small sample size and the nature of old lesions, characterized by organized thrombus where platelets and fibrin masses have become organized, posed challenges in using the picro-Mallory stain to distinguish fibrin and/or platelets from collagenous/fibrous tissue. In such cases, the picro-Mallory stain offered only marginal advantages over the H&E method.

Conclusions

Our study reaffirms the well-established role of fibrin in thrombus formation and wound healing. We extend these findings to aneurysms treated with flow diverters, demonstrating that fibrin, often in conjunction with platelets, predominates within thrombi and serves as a pivotal element in thrombus organization and aneurysm healing. These insights suggest the potential for enhancing aneurysm occlusion rates through the development and application of next-generation fibrin/fibrinogen-coated flow diverters. This work lays the foundation for further investigations into the precise mechanisms governing fibrin’s role in aneurysm occlusion and the clinical performance of fibrin-coated devices, with the goal of advancing endovascular treatments for intracranial aneurysms.

Acknowledgments

The research reported in this publication was in part supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number R01 NS076491 (to R.K.).

Disclosures

The research reported in this publication was in part supported by Medtronic. Dr. Kallmes reported grants from Medtronic, Stryker, MicroVention, and NeuroGami Medical; and stockholder of Monarch Biosciences outside the submitted work. Dr. Kadirvel reported grants from Piraeus Medical, Bionaut Labs, Neurogami Medical, Monarch Biosciences, MIVI Biosciences, and Stryker Neurovascular; and nonfinancial support from Medtronic outside the submitted work.

Author Contributions

Conception and design: Bilgin, Dai, Mereuta, Kallmes, Kadirvel. Acquisition of data: Dai, Ding, Kallmes. Analysis and interpretation of data: Bilgin, Dai, Ghozy, Kallmes, Kadirvel. Drafting the article: Bilgin, Dai, Ghozy, Kadirvel. Critically revising the article: Bilgin, Dai, Ghozy, Mereuta, Kallmes, Kadirvel. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Bilgin. Statistical analysis: Ghozy. Administrative/technical/material support: Ding, Kallmes. Study supervision: Kallmes, Kadirvel.