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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
AJNR Am J Neuroradiol. Author manuscript; available in PMC 2010 August 5.
Published in final edited form as:
PMCID: PMC2917111
NIHMSID: NIHMS215147

Experience with micro-aneurysms formation at the basilar terminus in the rabbit elastase aneurysm model

Abstract

Purpose

Intracranial aneurysms have been reported to be induced in mice, rats, rabbits, and primates through carotid artery ligation. We reviewed our experience with right common carotid artery (RCCA) ligation to quantify rate of aneurysm formation in rabbits.

Materials & Methods

In 30 consecutive New Zealand White rabbits the RCCA was ligated during surgery to create elastase-induced aneurysms. The basilar artery and its bifurcation were harvested at various time points after surgery, including 8 weeks (n=5), 13 weeks (n=3), 14 weeks (n=2), 16weeks (n=4), 17 weeks (n=4), 30 weeks (n=6) and 33 weeks (n=6). All specimens were embedded in paraffin and sectioned at 5µm in a coronal orientation, to show the basilar bifurcation and its branches. All sections were stained with hematoxylin and eosin (H&E) staining. After the sections were evaluated and the photomicrographs were taken, the sections were de-stained and re-stained with Verhoeff van Gieson (VVG) staining for elastin.

Results

The internal elastic lamina (IEL) was intact and continuous at the BT in all 30 rabbits, as was the medial layer. No bulge-like, localized dilation, to suggest micro-aneurysm or nascent aneurysm formation, was observed at the BT in any subject. There were small (0.08 ± 0.02 mm in diameter) concave structures along the P1 segments in five (16.7%) of 30 rabbits. On adjacent tissue slices each of these five structures were shown to be branch vessels, with intact IELs.

Conclusion

In our rabbit model, unilateral RCCA ligation does not induce micro-aneurysm formation.

Keywords: Aneurysm, Basilar terminus, Rabbit

Introduction

Intracranial aneurysms have been reported to be induced in rats, mice and primates through carotid artery ligation15. These intracranial aneurysms form in areas of elevated blood flow, which results from the carotid artery ligation with subsequent recruitment of flow across communicating arteries. Recently, Gao et al6 demonstrated consistently elevated basilar artery flow rates following either unilateral or bilateral carotid artery ligation. In that same study the authors noted nascent aneurysm formation at the basilar terminus (BT), apparently as a result of the elevated hemodynamic stress in the basilar apex from carotid artery ligation.

We routinely ligate the right common carotid artery (RCCA) during surgery to create dilated arterial segments in the proximal RCCA. We have noted on angiography that, after RCCA ligation, the basilar artery via the right posterior communicating artery (Pcom) feeds not only the ipsilateral anterior circulation of the brain, but also supplies blood flow retrograde down the ipsilateral internal carotid artery to the external carotid artery (ECA) branches. The ECA territory in the rabbit supplies a substantial amount of soft tissue, with markedly greater demands for blood than the small, intracranial circulation. These hemodynamic factors result in markedly increased flow across the right Pcom, from the basilar to the ICA and ECA, which might predispose the experimental subjects to aneurysm formation.

Based on the recent report of micro-aneurysm formation after RCCA ligation in rabbits by Gao et al6. We prospectively performed histologic evaluation of the BT in subjects that had undergone RCCA ligation as part of elastase aneurysm surgery, to confirm the ability of RCCA ligation to induce micro-aneurysms.

Materials & Methods

Elastase-induced aneurysms were created using vessel ligation and elastase incubation in the RCCA in 30 consecutive New Zealand White rabbits. All procedures were approved by the Institutional Animal Care and Use Committee at our institution. Detailed procedures for aneurysm creation have been described elsewhere7, 8 Briefly, New Zealand White rabbits (3–4 kg) were anesthetized with an intramuscular injection of ketamine, xylazine and acepromazine (75, 5, and 1mg/kg, respectively). Using sterile technique, the right common carotid artery (RCCA) was exposed and ligated distally. A 5F sheath was advanced retrograde in the RCCA to a point approximately 3 cm cephalad to the origin of RCCA. A 3F Fogarty balloon (Baxter Healthcare Corporation, Irvine, CA) was advanced through the indwelling sheath to the origin of the RCCA at its junction with the right subclavian artery. The balloon was inflated with just enough iodinated contrast material to achieve flow arrest in the RCCA. Porcine elastase mixed with iodinated contrast material was incubated in the dead space of the RCCA, above the inflated balloon, through a microcatheter. After incubation with the elastase solution, the balloon and sheath were removed, and the RCCA was ligated below the sheath entry site.

Tissue Harvest

In selected subjects, at the time of tissue harvest, injection of the right vertebral artery was performed to assess the degree of hypertrophy of the Pcom and associated arteries (Figure 1). After deep sedation, the rabbits were euthanized. Tissues were harvested at various time points following vessel ligation, including 8 weeks (n=5), 13 weeks (n=3), 14 weeks (n=2), 16 weeks (n=4), 17 weeks (n=4), 30 weeks (n=6) and 33 weeks (n=6). The basilar artery and its branches were carefully dissected free from surrounding tissues and removed. All specimens were fixed in 10% neutral buffered formalin at least 24 hours. Gross inspection of the arterial tree was done to determine presence of posterior communicating arteries in all cases. The arteries were then embedded in paraffin for histological examination.

Figure 1Figure 1
A) Anteroposterior digital subtraction angiography (DSA) of the right vertebral atery showing the basilar artery (short arrow) and bilateral posterior communicating (Pcom) arteries (long arrows). The right Pcom artery is tortuous and dilated, nearly as ...

Histologic Processing

The fixed vessels were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. Vessels were then sectioned at 5µm in a coronal orientation, to show the basilar bifurcation and its branches. The first section was collected once the investigator observed the bifurcation of basilar artery, and then all of the serial sections were collected until the bifurcation disappeared.

Staining

H&E

All the sections of each sample were stained with H&E for conventional Histopathologic evaluation. After the H&E staining sections were evaluated and the photomicrographs were taken, the slides were placed in 70% alcohol with 1%HCL to remove the H&E stain. The slides were then washed in tap water for 10 minutes and then stained with Verhoeff Elastic Van Gieson (VVG).

Verhoeff Elastic Van Gieson staining

The destained H&E sections were placed in Verhoeff’s staining solutions for 15 minutes. They were rinsed in running distilled water for 10 minutes, after which they were differentiated in 2% ferric chloride using the aid of a microscope. After being rinsed in running distilled water, the slides were rinsed in 95% alcohol for 30 seconds, counterstained in filtered Van Gieson’s solution for 1 minute 30 seconds. Finally, the slides were dehydrated in methanol and acetone, cleared in xylene, and mounted with Shandon EZ mount medium.

Results

Typical angiographic findings after RCCA ligation are shown in Figure 1. When injecting the vertebral artery, brisk flow is seen across the tortuous and dilated right posterior communicating artery to fill the right cerebral hemisphere, which represents only a small amount of flow compared to the large volume that travels retrograde in the ICA to fill the ECA branches (Figure 1). Gross examination showed posterior communicating arteries to be present and asymmetrically enlarged on the right side in all cases (Figure 2).

Figure 2
A photo of basilar artery and its branches, showing both posterior communicating arteries are present (arrow) and that the right side Pcom (long arrow) is larger than the left Pcom (short arrow).

Histologic evaluation of the basilar terminus.

The internal elastic lamina (IEL) was intact and continuous at the BT in all (30/30) rabbits, as was the medial layer in all cases (Figures 3A–D). No bulge-like, localized dilation associated with missing IEL, to suggest micro-aneurysm or nascent aneurysm formation, was observed at the BT in any subject. There were small (0.08 ± 0.02 mm in diameter) concave structures along the P1 segments or at the BT area in five (16.7%) of 30 rabbits (Figure 4A). These concave structures had intact IELs that were contiguous with the adjacent vessel wall as well as intact, but thinned, media (Figure 4B). Each of these five concave structures was shown to be a branch vessel (Figures 4C and 4D) on adjacent tissue slices.

Figure 3Figure 3Figure 3
Typical photomicrographs of the basilar terminus (BT) showing the internal elastic lamina (IEL) is intact and continuous at the BT. Panel A (H&E; original magnification 40X) showing the basilar artery and its bifurcation. Panel B (VVG, original ...
Figure 4Figure 4Figure 4Figure 4Figure 4
Photomicrographs of the basilar tip (BT) from a 30-week subject. Panel A (Verhoeff Van Gieson (VVG) stain, original magnification 40X) showing the basilar artery, BT and two concave structures along the P1 segments, labeled as 1 and 2(arrows) for correlation ...

Discussion

Increased flow and hemodynamic stress have been shown to be important factors in the formation of aneurysms in various animal models2, 5, 6, 9, 10. Previous reports indicate that increased hemodynamic stress reliably resulted in micro-aneurysm formation in small animals2, 11 Recently, Gao et al6 reported that uni- or bilateral CCA ligation induced micro-aneurysm formation at the BT in rabbits. A relationship between aneurysm formation and elevated basilar artery flow was noted in that study, with higher indices of aneurysm formation seen with bilateral as compared to unilateral CCA occlusion.

A review of consecutive animals subjected to chronic, unilateral CCA occlusion in our own laboratory failed to uncover any evidence of micro-aneurysm formation along the BT. We did detect small, concave structures along the P1 segments or at the BT histologically in a minority of cases, but each of these were shown to be branch vessels rather than nascent aneurysms. Evidence that these structures represented branch vessels included intact IELs and direct contiguity to branches identified on adjacent histologic sections. Thus, at least in the New Zealand White rabbits used in our laboratory, elevated flow does not appear to induce nascent aneurysm formation.

Multiple prior reports are available regarding hemodynamic alterations leading to aneurysm formation in animal models. Hashimoto et al1 reported that the occurrence of aneurysms in the circle of Willis induced by unilateral CCA ligation and hypertension was low, although hypertension could increase the incidence of aneurysm formation. Gao et al6, however, demonstrated that the occurrence of intracranial aneurysm was 100% using unilateral or bilateral ligation of CCA even without hypertension induction. Although hemodynamics plays a big role in aneurysm formation, the precise mechanism still remains to be determined.

There are several potential reasons to explain the disparate results from our series and that of Gao et al6. Different lineages of the New Zealand White rabbit may have differential susceptibility to aneurysm formation. Further, in some cases Gao et al6 performed bilateral CCA ligation, where as we performed only unilateral ligation. Gao group6, however, did report micro-aneurysm formation even with unilateral ligation. We did not perform a “nascent aneurysm index,” as reported by Gao6, since we saw no evidence of IEL loss in any of our subjects. Finally, it remains possible that, at least in some of their cases, branch arteries artifactually simulated micro-aneurysms.

There are several limitations in current study: First, the comparison of aneurysm formation induced through bilateral ligation of CCA and/or hypertension were not performed. Second, we did not measure the hemodynamic change at the BT after unilateral RCCA ligation.

Conclusion

Unilateral RCCA ligation does not induce micro-aneurysm formation at the BT in rabbits.

Supplementary Material

Title page

Acknowledgement

This project was partially supported by NIH grant NS42646

Footnotes

These data were presented at the ASNR 47th Annual Meeting & NER Foundation Symposium, Vancouver, Canada, May 16 - May 23, 2009

References

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