Background and purpose To test the hypothesis that systemic administration of vitamin C, through its action of stimulating collagen synthesis and crosslinking, would decrease the recurrence and improve the occlusion of experimental aneurysms treated with platinum coils.
Methods Experimental aneurysms were created in female rabbits and were embolized with platinum coils (>30% packing density). The animals were divided into three groups: group 1 (n=6) rabbits served as controls, group 2 (n=5) rabbits were fed with a vitamin C supplemented feed and group 3 (n=8) rabbits were medicated with vitamin C pills. Digital subtraction angiography was used to evaluate stability after embolization. Subjects were euthanized at 12 weeks after coil implantation, and serum vitamin C levels were then measured. Histological samples were examined with a grading system (range 0–12) based on the neck and dome features. Masson Trichrome staining was used to evaluate collagen deposition. Parametric data were analyzed with one way analysis of variance and non-parametric data were examined using a Kruskal–Wallis test.
Results There were no significant differences between groups in mean aneurysm size. Mean serum vitamin C concentration was significantly higher in group 3 and group 2 compared with group 1, while vitamin C levels between group 2 and group 3 were statistically comparable. Coil compaction was noted in one of six subjects in group 1 and in three of eight subjects in group 3. All of the remaining aneurysms in the test and control groups showed stable occlusion. There were no significant differences in histological scores or collagen deposition among groups.
Conclusions Vitamin C supplementation following platinum coil embolization does not demonstrate improvement of long term occlusion rates of aneurysms.
- Vessel Wall
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Coil embolization of saccular intracranial aneurysms has been widely accepted as a safe alternative therapy to open surgery. However, treatment of large and giant aneurysms remains problematic given ongoing low rates of initial complete aneurysm obliteration as well as high rates of late aneurysm recanalization.1 ,2 The exact mechanisms responsible for aneurysm recanalization remain unclear. Ideally, the aneurysm becomes covered by a robust endothelialized neointima and the dome is replaced by dense connective tissue. Numerous agents have been delivered or proposed for delivery in the aneurysm cavity to improve healing and some have reached clinical application, but none has shown definite efficacy to date.3–9
Collagen synthesis is a central process in wound healing, providing both increased strength for the healing wound's structural matrix as well as a surface or substrate for adhesion and attraction of cells involved in inflammation, angiogenesis and connective tissue construction to the injured area.10 ,11 Previous histologic studies of both experimental and human aneurysms treated with platinum coils show a dearth of collagen synthesis within the aneurysm cavity and along the neck/parent artery interface.12 ,13 Local delivery of collagen or collagen formation factors into aneurysm cavities has been proposed as a means for improved aneurysm healing, but this approach has not been tested clinically.14–19
We hypothesized that systemic delivery of agents that may promote collagen synthesis or crosslinking might improve healing within and around the embolized aneurysm cavity. Vitamin C, or ascorbic acid, plays an important role in collagen synthesis and crosslinking.20 ,21 Pathways affected by vitamin C include stimulation of collagen biosynthesis by promoting the activity of hydroxylases as well as increasing the steady state level of the procollagen mRNA without changing the synthesis of non-collagen proteins.22–24 If vitamin C is active in the treated aneurysm's healing response, it might improve long term occlusion rates by stimulating collagen synthesis. This collagen along the aneurysm neck may improve cell adherence with subsequent neointimal formation.
The purpose of the current study was to test the hypothesis that vitamin C supplementation following platinum coil embolization would improve histologic healing compared with subjects treated with platinum coils without subsequent vitamin C supplementation.
Materials and methods
Aneurysm creation and coil embolization
Elastase induced saccular aneurysms were created in female New Zealand white rabbits (2.5–4 kg). The Institutional Animal Care and Use Committee approved all procedures prior to study initiation. Some of the rabbits employed in this study were originally used as part of another investigation, which was entirely unrelated to the current project.25 Detailed procedures for aneurysm creation have been described in depth elsewhere.26 Aneurysms were permitted to mature for at least 21 days after creation. Animals were anesthetized and a cutdown was performed to gain access to the right common femoral artery. A microcatheter was inserted coaxially through the guiding catheter into the aortic arch. Heparin (100 U/kg) was administered intravenously. Aneurysms were embolized with platinum coils via the microcatheter.
The size of the aneurysm cavity was assessed by direct comparison with radiopaque sizing devices during digital subtraction angiography (DSA). The volumetric occlusion rate was calculated as follows: Volume=π (dome)2 (length)/6. In this study, we assumed the value of the aneurysm dome in this formula was equal to that of aneurysm width. Volumetric occlusion was calculated in real time, during aneurysm embolization, using the AngioCalc tool (http://www.angiocalc.com/index.aspx). Appropriate sized platinum coils (Heraeus Precious Metals GmbH and Co, Germany; diameter 0.0008 inches) were placed into the aneurysm, as in typical practice. Before and after each successive coil was placed, volumetric occlusion was calculated. Aneurysm cavities were packed as tightly as possible (>30% packing density) in all cases. Final DSAs were performed immediately following embolization.
Vitamin C medication
Rabbits were divided into three groups. Group 1 (n=6) rabbits served as controls, while group 2 (n=5) and group 3 (n=8) animals were fed a diet containing vitamin C (1732 ppm) or were medicated with a vitamin C pill (80 mg/kg, once/day), respectively, for 12 weeks following embolization. Vitamin C content (1732 ppm) in the diet is equal to 80 mg vitamin C per kg body weight per day in rabbits, based on food intake.
Vitamin C measurement
Arterial blood was collected from the rabbits just prior to aneurysm embolization and at the time of sacrifice. Vitamin C level in blood was measured calorimetrically using a commercial ascorbic acid assay kit (AbCam, Cambridge, Massachusetts, USA).
Tissue harvest and processing
At the time of sacrifice, subjects were deeply anesthetized, blood was obtained and DSA was performed. Animals were then euthanized with a lethal injection of pentobarbital. Harvested aneurysms were immediately fixed in 10% neutral buffered formalin. The degree of aneurysm neck tissue coverage was evaluated, as previously described.27 Using a modified histology technique,28 samples were embedded in paraffin and sectioned at 1000 μm intervals in a coronal orientation. Coil fragments were then carefully removed. Sections were re-embedded in paraffin and sectioned at 5 μm intervals. Sections were stained with H&E for conventional histologic evaluation and Masson Trichrome for collagen deposition.
All sacrifice angiograms 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 compared with the post-embolization angiograms. Stable and progressive occlusions were categorized as positive outcomes while compaction was deemed a negative result.
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.27 This scale was devised based on findings at the neck and in the dome. Neck coverage was included both on gross and microscopic inspection. Tissue coverage across the neck was graded, on gross inspection as well as microscopic findings, which included 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 on 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.
Collagen deposition analysis
Collagen deposition or fibrosis was quantitatively analyzed using Masson Trichrome staining using a recently described and experimentally verified image analysis technique based on Photoshop software (Adobe Systems, San Jose, California, USA).14 A fibrosis ratio, the total area of fibrosis within the aneurysmal cavity and neck divided by the total area of the aneurysmal cavity and neck, was calculated for each aneurysm.
Collagen gene expression analysis
Collagen-I gene expression level was measured by real time reverse transcription PCR (RT-PCR). Briefly, total RNA was isolated from the formalin fixed paraffin embedded tissue sections using RNeasy FFPE kit (Qiagen, California, USA). First strand cDNAs were synthesized from 500 ng of total RNA using Superscript III First Strand Synthesis (Invitrogen, California, USA). Quantitative real time RT-PCR was performed for collagen-I and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using IQ SYBR Green Supermix in an iCycler (Bio-Rad, Hercules, California, USA). The primer sequences for collagen-1 and GAPDH were constructed as described previously.29 Thermal cycling parameters were 90°C for 8 min and 30 s (DNA polymerase activation), followed by 40 cycles of amplifications at 95°C for 30 s and 60°C for 1 min. Relative levels of mRNA expression were normalized in all of the samples analyzed, with respect to the levels of GAPDH amplification.
Parametric data were analyzed with one way analysis of variance (ANOVA). If there was a main effect, then post hoc analysis was conducted with a Tukey post hoc test. Non-parametric data were examined using a Kruskal–Wallis test, a non-parametric test of independent samples, similar to ANOVA.
Aneurysm sizes and coil packing densities are summarized in table 1. There were no significant differences in aneurysm size or packing density among groups (ANOVA, p>0.05). Coil compaction was noted in one of six subjects in group 1 and in three of eight subjects in group 3. All of the remaining aneurysms in the test and control groups showed stable occlusion. There were no differences in coil compaction between groups (Kruskal–Wallis test, p>0.05).
Vitamin C levels
Serum vitamin C levels were significantly higher in group 3 (45.4±9.0 mM, p<0.001) and group 2 (38.6±8.9 mM, p=0.005) compared with group 1 (21.1±3.6 mM). There was an increase in the vitamin C level in group 3 compared with group 2, but this difference did not reach statistical significance (ANOVA, p>0.05).
Group 1 (control)
There was a thin membranous tissue covering the neck orifice in the aneurysms (four of six subjects). Microscopy demonstrated that poorly organized thrombus filled the majority of the aneurysm cavity in four of six aneurysms. Collagen deposition was minimal and scant in these subjects. Loose connective tissue, associated with a thin collagen matrix, filled the aneurysmal cavity in three of six subjects (figure 1). An endothelial lined thin neointimal layer that bridged the aneurysm neck was noted in three of six aneurysms. Unorganized fibrin was noticed across the neck in the remaining three subjects. There was mild scattered chronic inflammation infiltrating the organized loose connective in all of the aneurysm samples.
Group 2 (vitamin C received via feed)
Three of five aneurysms had organized loose connective tissue, which completely filled the aneurysm cavity. These same three subjects also had endothelial lined thin layer of neointima which completely crossed the neck/parent artery interface. In the other two subjects, loose connective tissue completely filled the aneurysm cavity; thin layers of both neointima and unorganized fibrin were present at the neck interface in these latter two subjects (figure 2). Collagen deposition was minimal in all subjects. Microscopic coil compaction at the neck interface was present in all five subjects.
Group 3 (vitamin C received via pill)
Gross examination showed that three of eight subjects had a thin membranous tissue partially covering the neck orifice. These three subjects had organized loose connective tissue which filled the aneurysm cavity completely, and had an endothelial lined thin layer of neointima and unorganized fibrin at the parent artery/neck interface. A separate three subjects had an endothelial lined thin layer of neointima completely crossing the neck interface. One of these latter three subjects had organized, loose, connective tissue filling the cavity while the other two had large areas of unorganized thrombus within the cavity. One of eight subjects had unorganized fibrin crossing the neck; loose connective tissue and unorganized thrombus were noted within the aneurysmal cavity in this subject (figure 3). The remaining subject had a poorly organized thrombus filling respective dome and covering the parent artery/neck interface. Microcompaction of coils and minimal collagen deposition was noted in all subjects.
The histological scoring data for the control and vitamin C administered groups are shown in table 2. There were no significant differences in the mean gross neck score, micro-compaction, dome score or total score among the three groups. There was no difference in the fibrosis ratio between the test and control groups.
Collagen-I gene expression
Real time RT-PCR experiments were performed to determine the effect of vitamin C on collagen-I gene expression level. There was no significant difference in collagen-I gene expression in vitamin C medicated rabbits and control rabbits (fold change 1.08 ± 0.96, n=5 for both control and vitamin C pilled groups).
In the present study, we evaluated the effect of systemic administration of vitamin C on the healing of aneurysms embolized with platinum coils. Our results suggest that systemic vitamin C supplementation does not improve the long term histological healing of rabbit aneurysms following platinum coil embolization, as evidenced by lack of cellularity and fibrosis in the aneurysm cavity as well as dearth of neointimal coverage at the neck of the aneurysm. These negative results were found even though adequate levels of vitamin C were achieved in both test groups. Thus clinical trials of vitamin C to improve coil stability likely are not warranted.
The dose of vitamin C was selected based on our preliminary dose escalation study where four groups of aneurysms were purposely loosely packed with coils (packing density <25%) and animals in each group were fed a diet containing 0, 866, 1732 and 4330 ppm vitamin C, respectively, for 12 weeks. These parts per million correspond to 0, 40, 80 and 200 mg of vitamin C per kg body weight based on the daily food intake of rabbits. At the end of 12 weeks, serum vitamin C levels were measured at sacrifice. Our analysis showed that there were no significant differences in histological scores among control and vitamin C fed groups, but mean total histologic score and serum vitamin C levels were higher in group 3 compared with the other groups. Since we did not observe significant changes in histological healing after aneurysms loosely embolized with coils in the dose study, we embolized aneurysms with coils packed as tightly as possible; the goal was to achieve 30% packing density by volume, in the present study. The selected vitamin C dose in the present study (1732 ppm; equivalent to 80 mg/kg body weight) was the dose that produced the highest serum vitamin C concentrations and histological healing among the groups in the dose study.
Local delivery of collagen fibers into the aneurysm cavity has been shown to improve the healing of aneurysms.17 Instead of employing local delivery to the aneurysm cavity, we hypothesized that systemic delivery of vitamin C might improve collagen deposition in the coil embolized aneurysms. We have previously reported a relatively small study in rabbit aneurysms suggesting the efficacy of vitamin C supplementation.30 In the current study, we substantially enhanced the methodology compared with the prior study. First, we controlled packing density across groups, since previous reports have shown improved healing with higher packing density and poorer healing in the aneurysms with reduced packing densities.31 Second, we compared two methods of vitamin C medication delivery and confirmed that adequate levels were achieved. In this large and well controlled study, we failed to demonstrate improved aneurysmal healing, as evidenced by lack of positive influence of vitamin C on gene expression and deposition of collagen. Even though vitamin C has been shown to increase collagen synthesis and to decrease inflammatory responses in the wound healing process, vitamin C supplementation has not been demonstrated to increase wound closure rate in otherwise healthy individuals.32–34 These findings suggest that vitamin C may only affect specific facets of the wound healing response.
The present study had several limitations. We used an elastase induced rabbit aneurysm model. This rabbit aneurysm is similar to human aneurysm histologically, morphologically and hemodynamically12 ,35; aneurysms created using elastase share some molecular features with human intracranial aneurysms15 ,36 and they mimic the healing seen in human aneurysms after embolization with platinum coils.12 However, rabbits can synthesize their own vitamin C while humans cannot. Employing animals that cannot synthesize their own vitamin C would be a more controlled model to address the effects of vitamin C supplementation. We acknowledge that the ordinal scale for grading the histological findings has not been validated. Nevertheless, this method has been widely used by several investigators.37–39
Vitamin C supplementation following platinum coil embolization may not improve long term occlusion of aneurysms.
We express our gratitude to Dr Arash Ehteshami Rad and Dr Soon Chan Kwon for their generous help with the study.
Contributors DFK and RK designed the study; DD performed the histological analysis; DY-H and IR performed the animal experiments; IR and RK performed the molecular biological experiments; DD, D-YH, IR, DAL and RK collected and analyzed the data; DD, DAL, DFK and RK wrote the manuscript.
Funding This work was supported in part by the American Heart Association Scientist Development grant (AHA 09SDG2160146–01) and the National Institutes of Health grant (R01NS076491).
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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