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Original research
Reactive tissue proliferation and damage of elastic lamina caused by hydrogel coated coils in experimental rat aneurysms
  1. Chao Zhang1,2,
  2. Neeraj Chaudhary1,3,
  3. Joseph J Gemmete1,3,
  4. B Gregory Thompson1,3,
  5. Guohua Xi1,
  6. Aditya S Pandey1
  1. 1Department of Neurosurgery, University of Michigan, Ann Arbor, Michigan, USA
  2. 2Department of Neurosurgery, Huashan Hospital, Fudan University, China
  3. 3Radiology, University of Michigan, Ann Arbor, Michigan, USA
  1. Correspondence to Dr A S Pandey, Department of Neurosurgery, University of Michigan Health System, 1500 E Medical Center Drive, Room 3552 TC, Ann Arbor, MI 48109-5338, USA; adityap{at}med.umich.edu

Abstract

Background and objective The HydroCoil Endovascular Aneurysm Occlusion and Packing Study clinical trial, comparing HydroCoil with platinum coils, reported an 8.6% reduction in significant recurrence following cerebral aneurysm coil embolization. We sought to better understand the mechanism of aneurysmal healing following HydroCoil implantation using the rat external carotid artery (ECA) sidewall aneurysm model.

Methods We ligated the proximal ECA, creating a blind pouch in our rat model. HydroCoil or bare platinum coil segments (5 mm) were inserted into aneurysms. Sham operated rats underwent identical procedures without coil insertion. 14 days after coil embolization, animals were sacrificed and the common carotid artery/internal carotid artery/ECA complex removed. Sac and surrounding vasculature underwent microscopic and histopathologic evaluation. Cellular and fibrotic components within the sac were defined as the organized area. Percentage of organized area and residual length of internal elastic lamina were calculated.

Results Organized tissue area in ECA sac 2 weeks following coil embolization was significantly greater in the HydroCoil group than the bare coil (60.42±22.58% vs 15.62±19.24%; p=0.01) and sham (60.42±22.58% vs 4.61±3.86%; p=0.002) groups. Elastic lamina was significantly reduced in the HydroCoil group compared with the sham and bare coil groups (21.67±16.50% vs 100% and 96.06±8.78%; both p<0.001). No significant difference was found between the bare coil and sham groups for organized tissue formation or reduction in elastic lamina. Greater numbers of B cells, T cells, and neutrophils were present within HydroCoil induced organized tissue compared with the platinum group; this difference was not statistically significant.

Conclusions In the rat ECA sidewall aneurysm model, hydrogel coated coils cause more tissue reaction and organization compared with bare platinum coils, possibly attributed to observed elastic lamina damage and vascular smooth muscle cell proliferation.

  • Coil
  • Aneurysm

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Introduction

Intracranial aneurysms occur in 1–5% of the population.1 While these cerebral aneurysms remain asymptomatic in the majority of patients, as many as 35 000 aneurysmal ruptures occur on a yearly basis, leading to the diagnosis of subarachnoid hemorrhage.2 Microsurgery is a proven treatment for ruptured and unruptured aneurysms; however, endovascular techniques allow the treatment of cerebral aneurysms with minimal morbidity as well as minimized recovery time. Endovascular occlusion of cerebral aneurysms has been validated by trials such as the International Subarachnoid Hemorrhage Trial (ISAT) and the Barrow Ruptured Aneurysm Trial (BRAT), which have shown a decreased incidence of death and dependence in the coiling group versus those in the microsurgery group.3 ,4 Safety and immediate effectiveness of cerebral aneurysm coiling has continued to improve, as the HydroCoil Endovascular Aneurysm Occlusion and Packing Study (HELPS) trial investigators reported successful coiling in 98.6% of patients with a mortality of 3.6% at the 3 month follow-up.5 ,6

While endovascular methods are preferred for treating most cerebral aneurysms, the current study by Jartti et al7 reports a 3.6% risk of repeat subarachnoid hemorrhage from a previously coiled cerebral aneurysm. In addition, 15–30% of all coiled aneurysms reoccur based on follow-up imaging; thus, these patients require future invasive monitoring as well as endovascular or microsurgical retreatment.8 To prevent aneurysmal recurrence after coiling and the associated complications, much interest has focused on producing bioactive coils that increase the inflammatory response within the aneurysm, thus increasing the probability of permanent thrombosis. Such coil modifications have included polyglycolic acid (PGA) coating (Matrix Coils, Stryker Corp, Kalamazoo, Michigan, USA; Cerecyte Coils, Johnson and Johnson, New Brunswick, New Jersey, USA; Nexus Coils, Micro Therapeutics Inc, Irvine, California, USA) and hydrogel coated platinum coils (HydroCoil Embolization System, Tustin, California, USA). Other proinflammatory and structural proteins (vascular endothelial growth factor VEGF, transforming growth factor β, collagen, laminin, etc) have also been tried in animal models but such coils are not available on the market.9–11 The HELPS trial reported an 8.6% reduction in significant recurrences in those patients treated with HydroCoil technology versus platinum coils.6 In this study, we sought to understand the mechanism of aneurysmal healing after HydroCoil implantation in a rat external carotid artery (ECA) blind pouch aneurysm model.

Materials and methods

Animal preparation and coil placement

The protocols for animal use were approved by the University of Michigan Committee on the Use and Care of Animals. A total of 15 adult male Sprague–Dawley rats weighing between 350 and 400 g were used. Access to food and water was ad libitum. General anesthesia was induced by intraperitoneal injection of 60 mg/kg sodium pentobarbital (Nembutal; Ovation Pharmaceuticals Inc, Deerfield, Illinois, USA). Body temperature was maintained at 37°C by use of a feedback controlled heating pad. Under sterile conditions, bare platinum coils (Microplex 18; MicroVention, Inc, Tustin, California, USA) and hydrogel coated coils (HydroCoil 14 or 18; MicroVention, Inc) were cut into 5 mm long segments.

Once the rats were effectively anesthetized, they were placed in a supine position and the anterior neck region was prepped and draped under sterile conditions. A paramedian incision was then made, and blunt/sharp dissection was carried through the fascia and muscle until the carotid sheath was identified. The carotid sheath was then opened, leading to dissection of the internal carotid artery (ICA), ECA, and common carotid artery (CCA). A permanent ligature was placed 5–7 mm distal to the origin of the ECA using a 6-0 silk suture. A small arteriotomy was made at the site just proximal to the ligature after proximal blood flow of the ECA was controlled by temporary clipping of the CCA and ICA. A 5 mm coil segment (bare coil or HydroCoil) was then inserted into the ECA lumen until the tip of the coil was positioned at the origin of the ECA. Another ligature was made at the proximal site of the arteriotomy to prevent bleeding and coil migration, and the temporary clips on the CCA and ICA were released to restore blood flow. The operative field was inspected to confirm hemostasis and proper location of the coil in the newly created ECA sac. Vasodilation and pulsation of the ECA sac were recognized on removal of the temporary clips, and the wound was closed with 4-0 nylon sutures. Sham operated control rats underwent an identical procedure except that no coil was inserted into the ECA lumen. The rats were returned to their cages and given access to food and water ad libitum.

Histological examination

Rats were divided into three groups: sham, bare coil, and HydroCoil. On day 14 following coil insertion, animals were euthanized by intraperitoneal injection of 300 mg/kg sodium pentobarbital. The previous incision was reopened and the CCA, ICA, and ECA were exposed. The entire bifurcation, including the ECA aneurysmal sac, was harvested for further evaluation. The inner lumens of the CCA and ICA were washed with 10 U/mL heparin saline solution and 4% paraformaldehyde in 0.1 mol/L phosphate buffered saline (pH 7.4), and then fixed in 4% paraformaldehyde for 72 h at room temperature. After fixation, the inserted coils were removed from the ECA sac under microscopic guidance. The aneurysmal sac was then incised in the center so that the carotid artery bifurcation was separated into the dome side and the orifice side. Specimens were then placed in a 70% ethanol–water mixture, and the dome side of the ECA sac segments were embedded in paraffin and cut into 4 μm thick slices in cross section. Coil segments were removed to allow for fine analysis of the reactive tissue via paraffin embedding and histological analysis.

Conventional and immunohistochemical staining

Thin cut tissue sections were stained with hematoxylin and eosin (H&E) and elastic van Gieson, according to standard protocols. Immunohistochemical staining was performed to identify vascular smooth muscle cells (VSMCs) using the avidin–biotin complex technique and primary monoclonal mouse antirat α smooth muscle actin antibody (1 : 200 dilution; Abcam, Cambridge, Massachusetts, USA). Staining was also performed for the presence of CD3 positive cells (T cells), CD19 positive cells (B cells), and myeloperoxidase positive cells within the organized tissue. After conventional and immunohistochemical staining, the slides were examined microscopically and photographed using a digital camera. The images were analyzed by a single observer who was blinded to the different groups. Coils that had previously been removed were also examined by scanning electron microscopy to evaluate the surface for the presence of cellular tissue.

Morphometric and statistical analysis

Slides from the dome side of treated aneurysms were examined for the encircled area lined by the internal elastic lamina, which was identified as the vascular lumen. Cellular and fibrotic components in the lumen were defined as organized healing tissue. Using image analysis software (NIH ImageJ, V.1.43u), the blinded single observer calculated the percentage of organized area and residual length of the elastic lamina. Data are presented as mean±SD. The Student's t test was used to compare image analysis data from the different groups. Significance levels were measured at p<0.05.

Results

H&E stained histopathology of the experimental aneurysms with treated or untreated coils is shown in figure 1. In most aneurysms in the sham group (figure 1, top row), the vascular lumen showed minimal thrombus formation within the aneurysm lumen. In the bare platinum coil group, there was more blood clot and organized tissue formation (figure 1, middle row), but much less than that observed in the HydroCoil group (figure 1, bottom row). The area of newly formed organized tissue in the ECA sac is summarized in figure 2. The area of organized tissue in the ECA sac 2 weeks after surgery was significantly greater in the HydroCoil group than in the bare coil group (60.42±22.58% vs 15.62±19.24%, p=0.01) and the sham group (60.42±22.58% vs 4.61±3.86%, p=0.002). The bare platinum group had a larger organized tissue area compared with sham animals but the difference was not statistically significant. Such a statistically significant difference was not found between the sham and bare platinum groups.

Figure 1

Hematoxylin–eosin stained sections from external carotid artery sacs harvested on day 14 following coil insertion. Top row, sham group; middle row, bare platinum coil group; bottom row, HydroCoil group. Original magnification ×10; bar=200 μm.

Figure 2

Percentage of organized area in external carotid artery sacs harvested on day 14 following coil insertion. *p=0.01, HydroCoil group compared with the bare coil group; #p=0.002, HydroCoil group compared with the sham group; n=5.

Elastic van Gieson stained histopathology from the three groups is presented in figure 3. The internal elastic lamina was distinct and intact in the sham group (figure 3, top row), minimally damaged in the bare coil group (figure 3, middle row), and severely damaged within the HydroCoil group (figure 3, bottom row). In some animals in the HydroCoil group, the entire elastic lamina was destroyed without any evidence of its presence. The percentage of residual elastic lamina in the different groups is shown in figure 4. Elastic lamina was significantly reduced in the HydroCoil group compared with the sham group and the bare coil group (21.67±16.50% vs 100% and 96.06±8.78%; both p<0.001). However, such a difference was not found between the bare platinum coil and sham groups.

Figure 3

Elastic van Gieson stained sections from external carotid artery (ECA) sacs harvested on day 14 following coil insertion. Top row, sham group; middle row, bare platinum coil group; bottom row, HydroCoil group. Original magnification ×10; black arrows indicate residual elastic lamina of ECA sacs treated with HydroCoil; bar=200 μm.

Figure 4

Percentage of residual elastic lamina in external carotid artery sacs harvested on day 14 following coil insertion. #Statistical significance when comparing HydroCoil treated aneurysms with the bare coil and sham groups; n=5.

We detected α-smooth muscle actin positive cells in organized tissue and vascular walls of all animal groups. These cells are considered to be VSMCs. Such a cellular presence was similar in overall appearance in the sham (figure 5, top row) and bare platinum coil (figure 5, middle row) groups. Within the HydroCoil group (figure 5, bottom row), the entire sac vascular wall consisted of smooth muscle cells and was significantly increased in size compared with the bare platinum coil or sham group. Immunohistochemical staining of the organized tissue was performed and revealed greater numbers of B cells (CD19), T cells (CD3), and neutrophils within the HydroCoil group compared with the bare platinum group but the difference was not statistically significant (figure 6).

Figure 5

α-Smooth muscle actin stained sections from external carotid artery sacs on day 14 following coil insertion. Top row, sham group; middle row, bare platinum coil group; bottom row, HydroCoil group. Original magnification ×10; bar=200 μm.

Figure 6

Immunohistochemical staining for CD3 (T cells), CD19 (B cells), and myeloperoxidase (neutrophils) on organized tissue within the sham, bare platinum, and HydroCoil implanted animals 14 days after coil insertion. Bar graphs illustrate greater numbers of inflammatory cells in the HydroCoil group but the difference was not statistically significant.

Discussion

Endovascular embolization using platinum coils is a safe and effective method for treating intracranial aneurysms.12 The goal of endovascular treatment is to exclude the aneurysm from the vasculature, thus preventing the risk of subarachnoid hemorrhage. Embolization of cerebral aneurysms initiates a string of biological changes within the aneurysmal sac that include stasis of blood flow, thrombus formation, and fibrovascular tissue formation leading to exclusion of the aneurysm from the cerebral circulation. However, not all coiled aneurysms develop the tissue reaction required for permanent cure and, instead, these aneurysms reoccur.

Current limitations of coil embolization of cerebral aneurysms include incomplete initial occlusion, coil compaction, lysis of the initial thrombosis, and de novo aneurysm formation.13 ,14 The mechanisms of aneurysmal recurrence are unknown but likely relate to lysis of the initially formed coil induced thrombus.15 This allows coil compaction, as most aneurysms are coiled to a packing density of 20–35%, thus leaving 65–80% of the sac volume filled with acute thrombus.16 PGA and hydrophilic coating of coils had been thought to reduce aneurysmal recurrence but the Cerecyte and Matrix trials (comparing PGA coated coils with bare platinum coils) have not shown a decreased rate of recurrence in coiled aneurysms.5 ,17

Recurrence of coiled aneurysms leads to the necessity for invasive imaging follow-up as well as potential future surgical and endovascular therapies. The design of the HydroCoil incorporates a layer of hydrogel polymer surrounding a platinum core. The hydrogel polymer expands by absorption of fluid from the blood over a 30 min period, thereby occupying additional space within the aneurysm beyond what is possible with bare platinum coils. This allows for increased packing density within the aneurysm and less space between the interstices of the coils, theoretically decreasing the amount of thrombus formation within the aneurysm and providing a surface for cell adhesion and proliferation. There also appears to be a decreased tendency for aneurysm regrowth secondary to thrombus resorption or reorganization, and it provides a biocompatible surface for cellular proliferation across the neck of the aneurysm.18 Our in vitro analyses show a greater number of human endothelial cells attaching and proliferating on the HydroCoil compared with bare platinum coils (manuscript submitted for publication). Clinical results have shown that HydroCoils can achieve greater aneurysm packing density than bare platinum coils, which may result in better long term results. In the first randomized control trial using HydroCoils, 8.6% fewer patients presented with major recurrences versus those treated with bare platinum coils.6 ,19–21

We used a rat aneurysm model in the ECA to compare the tissue reaction induced by HydroCoils with that induced by bare platinum coils. This model allows for the formation of arterial aneurysms as compared with venous pouch aneurysms in other animal models (swine, canine, etc). In addition, the rat aneurysm model allows evaluation of a larger group of animals for a longer duration at a lower cost. We were also able to evaluate the presence of specific cell types within the treated experimental aneurysms, given the presence of a large variety of antibodies for immunohistochemical analysis within this species. Although the blood clot and organized tissue of the ECA sac were easily found in the bare coil group, the vascular wall was still intact with normal layers except for mild intimal hyperplasia. ECA sacs treated with HydroCoils showed significantly greater tissue reaction than ECA sacs in the bare platinum coil and sham groups. Within the HydroCoil group, normal layers of the arterial wall disappeared and were replaced by remarkable tissue proliferation, including many inflammatory cells. A few reports in the literature show that, after arterial injury, the inflammatory response is stimulated with the migration and proliferation of VSMCs, which are intermixed with infiltrating leukocytes to form an intermediate lesion. If this response continues unabated, thickening of the arterial wall may occur.22 ,23 These findings are in agreement with the histological features of the ECA sacs treated with HydroCoils in our study, which revealed dramatically increased inflammatory cells and VSMCs in the thickened arterial wall. Interestingly, our bare platinum group of animals had greater organized tissue area compared with the sham animals but this difference was not statistically significant. It is possible that the initial clot formed by placement of coils within the experimental aneurysm eventually lysed. On the other hand, it is possible that the platinum based organized tissue is mainly thrombus and thus, when the coils are removed for tissue preparation, the thrombus is disrupted leading to a falsely low value for organized tissue area.

Several reports have evaluated tissue response to hydrogel coated coils in different aneurysm animal models; however, none has described the arterial wall changes we saw after HydroCoil implantation into our rat aneurysm model.24–26 Schwartz et al27 reported that the processes of vascular remodeling after balloon injury in rats can be divided into four phases. The first phase is the burst of medial VSMC proliferation that peaks within a few days after injury. The second phase involves VSMC migration into intima, beginning at days 4–5. The third phase refers to sustained VSMC proliferation in the neointima, with cell numbers reaching a maximum at day 14. The fourth phase is characterized by extracellular matrix deposition. From their results, it seems that the processes of vascular change after hydrogel coated coil insertion may be similar to the processes of vascular remodeling after balloon injury. The significant VSMC proliferation, as well as disappearance of intima and damage of elastic lamina, was found in the ECA sacs with hydrogel coated coils on day 14 after coil insertion. Thus the fully expanded hydrogel, like the inflated balloon, can lead to vascular injury and remodeling that cannot occur with bare platinum or PGA coated coils.

Killer et al28 used a rabbit venous pouch bifurcation aneurysm created within the neck to evaluate thrombosis following bare platinum, Cerecyte, or HydroCoil insertion. They reported increased tissue organization with coated coils; however, there was no difference in the number of inflammatory cells present within the coil induced organized thrombus. In addition, Yoshino et al25 reported on the effectiveness of HydroCoils in causing an intra-aneurysmal tissue reaction in a canine venous pouch bifurcation aneurysm model. Our study differs from these findings as we used an arterial aneurysm model rather than the venous pouch used in both of these studies. In addition, we correlated the presence of greater reactive tissue with the presence of inflammatory cells, which has not been found in the previous publications.

Our study used a rat aneurysm model (an end pouch created by ligating the distal ECA) to understand the mechanisms by which HydroCoils could prevent recurrences. Although there was blood clot and organized tissue present within the ECA sac following bare coil placement, the vascular wall remained similar to that of sham animals with no evidence of cellular proliferation. ECA sacs treated with HydroCoil showed significantly greater tissue reaction than ECA sacs in the bare platinum coil and sham groups. The vascular wall remained intact and similar within the bare platinum coil and sham groups. On the contrary, ECA sacs treated with HydroCoils showed significant damage to the internal elastic lamina and associated smooth muscle cell proliferation.

Our study is limited by the fact that we chose a rat aneurysm model. Given the small size of the animal, it is difficult to create large enough aneurysms for coiling via endovascular methods that would provide in situ evaluation of the recurrence after treatment with the HydroCoils. Small aneurysm size also prevented us from placing whole coils and, instead, necessitated cutting 5 mm segments of coil. Also, tissue reaction evaluations were only carried out at one time point, day 14. Future studies are needed to examine the process at earlier time points in order to understand the VSMC proliferation process in a temporal fashion.

Conclusions

In this rat aneurysm model, HydroCoil treatment of ECA sacs led to significantly more tissue reaction as well as VSMC proliferation compared with bare platinum coils. Such a tissue response could be one plausible explanation of how aneurysm patients treated with HydroCoils have an 8.6% reduction in major recurrences, as reported in the HELPS trial.

References

Footnotes

  • Contributors Conception, design, and drafting the article: ASP, CZ, and GX. Analysis and interpretation of the data, critically revising the article, and final approval of the manuscript: ASP, CZ, NC, JG, BGT, and GX.

  • Funding This work was supported by the University of Michigan Department of Neurosurgery Research Funds. Coils were supplied by EV3 and Microvention.

  • Competing interests None.

  • Ethics approval The protocols for animal use were approved by the University of Michigan Committee on the Use and Care of Animals.

  • Provenance and peer review Not commissioned; externally peer reviewed.