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Original research
Statins are not associated with short-term improved aneurysm healing in a rabbit model of unruptured aneurysms
  1. Waleed Brinjikji,
  2. Ding Yong Hong,
  3. Daying Dai,
  4. Dana J Schroeder,
  5. David F Kallmes,
  6. Ramanathan Kadirvel
  1. Department of Radiology, Mayo Clinic, Rochester, Minnesota, USA
  1. Correspondence to Dr Waleed Brinjikji, Department of Radiology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA; brinjikji.waleed{at}mayo.edu

Abstract

Background Owing to their anti-inflammatory effects and ability to stimulate production of extracellular matrix and chemotactic migration of mesenchymal progenitor cells, statins could potentially improve aneurysm healing after endovascular treatment.

Objective To test the hypothesis that systemic administration of simvastatin would improve aneurysm healing in a rabbit model of unruptured intracranial aneurysms.

Methods Experimental aneurysms were created in female rabbits and were embolized with platinum coils. Six rabbits served as controls and six rabbits received oral administration of simvastatin. Digital subtraction angiography was used to evaluate stability after embolization. Subjects were euthanized 4 weeks after coil embolization. Histologic samples were examined with a grading system (0–12) based on neck and dome features. Aneurysm occlusion data were compared using a Student t test.

Results No significant differences in the mean aneurysm size were found between groups. No coil compaction occurred in either group. All aneurysms in both the statin and control groups showed stable occlusion. There were no significant differences in the histologic grade of occlusion in either group (statin group 2.6±0.8 vs control group 2.7±3.2, p=0.94).

Conclusions Systemic statin administration after platinum coil embolization of unruptured aneurysms in a rabbit model does not improve aneurysm occlusion rates at 4 weeks.

  • Aneurysm
  • Coil

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Introduction

Statins are commonly prescribed drugs which have been shown to have a number of beneficial health effects, including reducing the risk of cardiovascular disease and stroke. They have been found to stimulate production of extracellular matrix and stimulate chemotactic migration of mesenchymal progenitor cells and stromal osteoblasts. Some data suggest that statin therapy might improve aneurysm healing after endovascular treatment.1 A number of studies of patients receiving endovascular repair of abdominal aortic aneurysms have found that statin therapy promoted aneurysm sac regression and healing.2 ,3 One recently published study examined the effect of delivering simvastatin coated coils in a rat model of aneurysm and found that simvastatin coated coils in accelerated aneurysm healing and endothelialization over the coil.4 Furthermore, studies in untreated aneurysms have found that statins may reduce the rate of aneurysm growth.5

Because statins are so widely used and are known to be safe, it would be interesting to study the effect that statins have on healing of intracranial aneurysms after coil embolization. This study aimed to test the hypothesis that systemic statin administration after platinum coil embolization would improve histologic healing as compared with subjects treated with platinum coils without subsequent statin supplementation.

Materials and methods

Aneurysm creation and coil embolization

The Institutional Animal Care and Use Committee approved all procedures before study initiation. Some of the rabbits employed in this study were originally used as part of another investigation,6 in which we probed gene expression patterns between well-healed and poorly healed aneurysms after coil embolization. Elastase-induced saccular aneurysms were created in female, New Zealand white rabbits (2.5–4 kg). Detailed procedures for aneurysm creation have been described in depth in prior publications.7 ,8 Aneurysms were allowed to mature for a minimum of 21 days after creation. Animals were anesthetized and a cut down was performed to gain access to the right common femoral artery. A microcatheter was inserted coaxially through a guiding catheter into the aortic arch. Heparin (100 U/kg) was administered intravenously. Aneurysms were embolized with bare platinum coils via the microcatheter.

The size of the aneurysm cavity was assessed by direct comparison with radiopaque sizing devices during DSA. Appropriately sized platinum coils were placed into the aneurysm in the typical fashion. Aneurysm cavities were packed as tightly as possible in all cases. Final DSAs were performed immediately after embolization.

Statin medication

Rabbits were divided into two groups. Group 1 (n=6) rabbits comprised historic controls sacrificed at 4 weeks, who received no supplemental medication and were fed a normal diet. Group 2 (n=6) rabbits were fed a normal diet but were also given 5 mg/kg/day of oral simvastatin solution administered through oral gavage, as described previously.9

Tissue harvest and processing

At the time of sacrifice, the subjects were deeply anesthetized, and DSA was performed. Animals were euthanized with a lethal injection of pentobarbital. Harvested aneurysms were immediately fixed in 10% neutral buffered formalin. The degree of neck tissue covering was evaluated, as previously described.10 A modified histologic technique was used, and samples were embedded in paraffin and sectioned at 1000 μm intervals in coronal orientation.11 Coil fragments were removed. Sections were re-embedded in paraffin and sectioned at 4 μm intervals. Sections were stained with H&E for conventional histologic evaluation.

Angiographic evaluation

All angiograms obtained at sacrifice were compared with post-embolization angiograms and assessed for changes in coil configuration or aneurysm filling. Sacrifice angiograms were then categorized as stable, progressive occlusion, or coil compaction/recanalization as compared with the post-embolization angiograms. Stable and progressive occlusions were categorized as positive outcomes, whereas compaction was deemed a negative result.

Histologic evaluation

Sections were viewed by a single experienced reviewer, paying particular attention to the thickness of the cellular layer across the neck of aneurysms and the collagen matrix deposition within the aneurysm dome. An ordinal grading system was used to evaluate histologic healing.10 This scale was devised based on findings at the neck and in the dome. Neck coverage was included on both gross and microscopic inspection. Tissue coverage across the neck was graded, on gross and microscopic inspection, according to tissue thickness and tissue type. The scores from the gross and microscopic neck inspections were averaged to yield a single neck score. Micro-compaction assessment was based on the shape of the coil mass across the neck, from convex to concave. Histologic characteristics in the dome were categorized according to the density of cellular infiltration and presence of organized tissue. These scores (the neck, microcompaction, and dome) were summed to obtain a total score representative of the aneurysm's pathology.

Statistical analysis

All statistical analyses were performed using the SAS-based statistical software package JMP V.10.0 (http://www.jmp.com, Cary, North Carolina, USA). Continuous variables were compared using a Student t test and categorical variables were compared using Fisher's exact test.

Results

Angiographic findings

Aneurysm sizes and coil packing densities are summarized in table 1. There were no significant differences in aneurysm neck size (p=0.72), aneurysm width (p=0.78), and aneurysm height (p=0.29) between groups. The packing density was similar between groups as well (p=0.90). There were no instances of coil compaction or recanalization in either group. All aneurysms in both the test and control groups showed stable occlusion. There were no differences in coil compaction on angiography between groups (p=1.00).

Table 1

Angiographic results

Quantitative histology

The histological scoring data for control and statin administered groups are shown in table 2. There were no significant differences in the mean gross neck score, micro-neck score, or dome score between groups. The control group did have a higher neck compaction score than the statin group (1.0±1.1 vs 0.0±0.0, p=0.06) although this difference was not statistically significant. There was no difference in the total histologic healing score between the statin (2.6±0.8) and control (2.7±3.2) groups (p=0.94).

Table 2

Histologic results

Qualitative histology

Microscopic evaluation showed that all six statin-group aneurysms had poorly organized thrombus filling the majority of the aneurysm dome. A small amount of organized loose connective tissue was primarily located at the periphery of the aneurysm dome. A neck remnant was present in each aneurysm. Unorganized thrombus and fibrin were present at the neck–vessel interface in all cases and was concave in relation to the aneurysm dome. Dense, diffuse mixed inflammatory cell infiltration was noticed surrounding coil loops within the dome tissue in one of six aneurysms; inflammatory reaction was minimal in the remaining five aneurysms. The histologic features mentioned above were similar to those in the control group. A comparison of the statin and non-statin groups at 4 weeks showed no qualitatively improved histologic healing. An example case from the statin group is shown in figure 1.

Figure 1

Aneurysm healing in a rabbit receiving simvastatin therapy. Pretreatment (A), immediate post-treatment (B), and 4-week follow-up (C) angiograms show almost complete obliteration of the aneurysm on the immediate post-treatment angiogram with a tiny amount of contrast filling the aneurysm sac (B). At 4 weeks after treatment there is no residual filling of the aneurysm sac and the aneurysm is completely occluded (C). (D) Gross specimen of the aneurysm taken from the perspective of the vessel lumen into the aneurysm sac demonstrates some endothelialization of the coils at the parent vessel–neck interface (red circle). (E) H&E specimen of the aneurysm shows loose connective tissue and thrombus in the aneurysm sac. There is a small amount of endothelial tissue across the aneurysm neck.

Discussion

Our study demonstrated that daily simvastatin administration after bare platinum coil embolization did not improve the healing of experimental aneurysms in a rabbit model of unruptured aneurysm. In general, there was no difference in quantitative histologic measures of healing between the two groups. There was a lack of cellularity and fibrosis in the aneurysm cavity, as well as minimal neointimal coverage at the neck in statin-treated rabbits, similar to that seen among our historical control group. These negative results suggest that clinical trials of systemic statin administration to improve aneurysm healing in coiled unruptured aneurysms are probably not warranted.

Prior experimental studies have examined the effect of statin administration on the healing of intracranial aneurysms. In a study of a coiled aneurysm model of rats, Liu et al12 found that systemic administrations of rosuvastatin promoted endothelialization of the aneurysm neck by inducing endothelial progenitor cell) proliferation and migration. One recently published study examined the effect of delivering simvastatin-coated coils in a rat model of aneurysm and found that this resulted in accelerated aneurysm healing and endothelialization over the coil in addition to increased accumulation of smooth muscle cells and collagen connective matrix at the aneurysm dome.4 A number of mechanisms have been proposed to explain why statins might improve aneurysm healing in these rat models, including (1) increased proliferation, migration and adherence of endothelial progenitor cells to the aneurysm neck,13 (2) positive effects on cell survival by reducing apoptosis,14 and (3) statin-induced release of endothelium-related cytokines at the aneurysm neck.15

A few published clinical studies have examined the effect of statins on aneurysm healing. In a retrospective study examining the effect of statins on aneurysm healing in patients with ruptured aneurysms receiving endovascular coiling, one group found that statins were associated with a lower rate of aneurysm recurrence after endovascular coiling.7 A number of studies of patients receiving endovascular repair of abdominal aortic aneurysms have found that statin therapy promoted aneurysm sac regression and healing.2 ,3 Proposed mechanisms for improved healing in both intracranial and abdominal aortic aneurysms include attenuation of matrix metalloproteinase (MMP)-6 and MMP-9 activity in the aneurysm sac,16 decreased inflammation in the aneurysm wall due to the anti-inflammatory effect of statins, and reduced activity of proteases.17

A number of factors may explain why our study did not yield similar results to these prior experimental and clinical studies. First and foremost are the intrinsic differences in the rabbit and rat model of intracranial aneurysms. We chose to use the rabbit model as it closely mimics the aneurysm healing process seen in humans.7 ,18 It is for this reason that the elastase rabbit model has been extensively used in the premarket approval of many aneurysm treatment devices.7 ,18 Second, we chose to use simvastatin as it has been extensively tested in rabbit models of atherosclerosis.19–21 However, rosuvastatin is more potent than simvastatin.22 Changing the statin agent administered to a more potent agent might have produced different results. Lastly, while prior clinical studies have shown a positive effect of statin therapy in ruptured intracranial aneurysms and abdominal aortic aneurysms treated with endovascular methods, the biology of these aneurysms differs substantially from that of unruptured intracranial aneurysms. For example, prior studies have shown that stable unruptured aneurysms do not exhibit increased expression of MMPs, while ruptured intracranial aneurysms and abdominal aortic aneurysms do.23 ,24 In addition, unruptured aneurysms have significantly lower expression of a number of markers of inflammation than ruptured aneurysms.25 Such differences might attenuate the potential beneficial effects of statins in healing of unruptured aneurysms.

Limitations

Our study has limitations. First, we did not evaluate the concentration of statin drugs and byproducts in rabbits at the time of sacrifice. Thus, we are unable to determine if the treated rabbits had inadequate or subtherapeutic levels of simvastatin administered. No dose escalation study was performed. It is possible that a higher dose of systemic statin administration or local administration of statin medication to the aneurysm dome might have yielded a therapeutic effect. In addition, we used a group of historical controls rather than a standard control group, which could potentially have biased the results of our study. However, the person who graded the histologic healing of the treated aneurysms was unaware that the animals in this study had received statin therapy. Another limitation of this study is that while the rabbit model simulates the biology of unruptured aneurysms, it does not simulate the biology of ruptured aneurysms. It is possible that the anti-inflammatory effect of statins is beneficial only for ruptured aneurysms. We had only one follow-up time-point in this study. We chose to sacrifice the rabbits at 4 weeks as a number of prior studies have shown that the most biologically active time-period of the aneurysm healing process is within the first 4 weeks.18 Lastly, the histologic scale used in this study has not been validated. However, it has been used in prior studies.26 ,27

Conclusion

In conclusion, our study comparing the effects of statins on aneurysm healing in an experimental model of unruptured aneurysms found no effect at 4 weeks after treatment. These negative results suggest that clinical trials of systemic statin administration to improve aneurysm healing in coiled unruptured aneurysms may not be warranted. Further studies examining the role of statins in the healing of unstable and ruptured aneurysms should still be considered.

References

Footnotes

  • Contributors All authors participated in drafting the article and revising it critically for important intellectual content, made substantial contributions to conception and design, acquisition of data, and analysis and interpretation of data. All authors provided final approval of the version to be published.

  • Funding NIH grant NS076491, Brain Aneurysm Foundation Grant.

  • Competing interests None declared.

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

  • Data sharing statement The authors are happy to share any data upon request. Contact the corresponding authors for any requests.