In vivo cerebral aneurysm models · Cerebral aneurysm rupture is a devastating event resulting in subarachnoid hemorrhage and is associated with signifi-cant morbidity and death

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FOCUS Neurosurg Focus 47 (1):E20, 2019

UnrUptUred cerebral aneurysms (CAs) are common in the general population, with an estimated prev-alence ranging from 2% to 6%.68 If left untreated,

aneurysms can progress and spontaneously rupture, pro-ducing a subarachnoid hemorrhage and resulting in sig-nificant morbidity and death. The pathophysiology of CA formation and rupture is not fully defined, but risk factors have been identified including increasing age, female sex, hypertension, excessive alcohol intake, and smoking.16,34,68 Studies have suggested that hemodynamic stress is a criti-cal factor in CA pathogenesis17 leading to endothelial dys-function, inflammatory cell infiltration, and arterial wall remodeling.6–7 Vascular smooth-muscle cells undergo a phenotypic switch, which exacerbates inflammation by ex-pressing inflammatory and matrix remodeling proteins,50 ultimately culminating in histological changes character-ized by disruption of the internal elastic lamina, extracel-lular matrix digestion, thinning of the media, cell loss, and aneurysm formation.

Molecular and histological analysis of human CA spec-imens has revealed significant information regarding the

pathology of CAs. Equally vital to our understanding of CA biology and treatment has been the use of CA animal models, which attempt to replicate the morphological, his-tological, and hemodynamic features observed in human CAs. These animal models provide a method for inves-tigating aneurysm formation, growth, and rupture while also providing a means of testing new treatment modali-ties. CA models have been developed in numerous species including mice, rats, rabbits, swine, sheep, canines, and primates, with each model having advantages and limita-tions such that the model selection depends on the purpose of the study. This review explores some of the more com-monly used models of CAs and compares the advantages and disadvantages of each system.

Small Animal CA ModelsThe theory behind CA formation in rats and mice is

that weakening of the cerebral blood vessels combined with hemodynamic stress will induce CA formation. Nu-merous rat and mouse models of CA formation exist and

ABBREVIATIONS BAPN = b-aminopropionitrile; CA = cerebral aneurysm; CCA = common carotid artery; DOCA = deoxycorticosterone acetate.SUBMITTED March 1, 2019. ACCEPTED April 9, 2019.INCLUDE WHEN CITING DOI: 10.3171/2019.4.FOCUS19219.

In vivo cerebral aneurysm modelsJohn W. Thompson, PhD,1,3 Omar Elwardany, MD,1,3 David J. McCarthy, MS,1,3 Dallas L. Sheinberg, BS,1,3 Carlos M. Alvarez, MD,1,3 Ahmed Nada, MD,1,3 Brian M. Snelling, MD,1,3,4 Stephanie H. Chen, MD,1,3 Samir Sur, MD,1,3 and Robert M. Starke, MD1–3

Departments of 1Neurological Surgery and 2Radiology, University of Miami; 3The University of Miami Cerebrovascular Initiative, University of Miami; and 4Marcus Neuroscience Institute, Boca Raton Regional Hospital, Boca Raton, Florida

Cerebral aneurysm rupture is a devastating event resulting in subarachnoid hemorrhage and is associated with signifi-cant morbidity and death. Up to 50% of individuals do not survive aneurysm rupture, with the majority of survivors suf-fering some degree of neurological deficit. Therefore, prior to aneurysm rupture, a large number of diagnosed patients are treated either microsurgically via clipping or endovascularly to prevent aneurysm filling. With the advancement of endovascular surgical techniques and devices, endovascular treatment of cerebral aneurysms is becoming the first-line therapy at many hospitals. Despite this fact, a large number of endovascularly treated patients will have aneurysm re-canalization and progression and will require retreatment. The lack of approved pharmacological interventions for cere-bral aneurysms and the need for retreatment have led to a growing interest in understanding the molecular, cellular, and physiological determinants of cerebral aneurysm pathogenesis, maturation, and rupture. To this end, the use of animal cerebral aneurysm models has contributed significantly to our current understanding of cerebral aneurysm biology and to the development of and training in endovascular devices. This review summarizes the small and large animal models of cerebral aneurysm that are being used to explore the pathophysiology of cerebral aneurysms, as well as the develop-ment of novel endovascular devices for aneurysm treatment.https://thejns.org/doi/abs/10.3171/2019.4.FOCUS19219KEYWORDS aneurysm; animal; model; in vivo; mice; rabbit; porcine; canine

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primarily differ in the mechanisms of vessel wall weaken-ing and hemodynamic stress induction (Fig. 1).

Hemodynamic Stress and Vessel Wall WeakeningHemodynamic stress in the cerebral vasculature can be

increased by hypertension and/or an increase in flow rate. Using the combination of hypertension and flow rate to induce hemodynamic stress, Hashimoto et al. created the first rodent CA model in rats.31 During a series of surger-ies, hemodynamic stress was increased by ligation of the left common carotid artery (CCA) while hypertension was induced by unilateral nephrectomy, followed by subcuta-neous injections of deoxycorticosterone acetate (DOCA) and the addition of 1% sodium chloride to the drinking water. Vessel walls were weakened by feeding the rats chow containing 0.12% b-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, which prevents collagen and elas-tin cross-linking, leading to increased vessel fragility and a greater likelihood of aneurysm formation. Morimoto et al. later adapted this method for CA formation in mice and included bilateral ligation of the posterior branches of the renal arteries.51 Four months following surgery, CAs were observed at various stages of formation, located primarily

at the bifurcation of the right anterior cerebral artery and the olfactory artery. Histological analysis revealed frag-mented elastic laminin and media thinning suggestive of aneurysm formation in 78% of the treated mice. However, the CAs formed by this method are small with a few mi-croaneurysms observable by light microscopy, while other aneurysms require electron microscopy for visualization. This method of CA formation suffers from slow aneurysm formation. Other adaptations to this protocol include liga-tion of the left renal artery, unilateral nephrectomy, and bilateral ligation of the posterior branches of the renal ar-teries during the same surgery.3–6,8,10

Elastase and Angiotensin IIEarly stages of aneurysm formation are associated with

elastic lamina degeneration, which may contribute to an-eurysm progression and rupture. Given this histological finding, Nuki et al.54 stereotactically injected elastase into the cerebrospinal fluid of the right basal cistern. To induce hypertension, angiotensin II was continuously adminis-tered via a subcutaneously placed microosmotic pump. CA formation was achieved in 77% of the mice within 2 weeks of treatment. Histologically, the aneurysms demon-

FIG. 1. Cerebral aneurysm formation in rodents, hemodynamic stress, and vessel wall weakening. The procedures used for CA formations in rats and mice vary primarily in the method of inducing hypertension, increasing the flow rate, and weakening the ves-sel wall. Hypertension can be induced by a combination of a high salt diet, unilateral nephrectomy or bilateral ligation of the posterior branches of the renal arteries (not shown), and subcutaneous placement of DOCA pellets or angiotensin II–filled microosmotic pump (not shown). Increases in flow rate are accomplished by ligation of the left CCA, which causes a compensatory increase in flow rate in the contralateral internal carotid artery. Vessel wall weakening is accomplished by feeding a diet containing 0.12% BAPN, a lysyl oxidase inhibitor, or by a single stereotactic injection of elastase. Copyright Robert Starke. Published with permission.


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strated degeneration of the media layer and elastic lamina and infiltration of inflammatory cells.

Intracranial Aneurysm Rupture ModelThe spontaneous aneurysm rupture model was in-

troduced by Makino et al.,41 who used a combination of elastase treatment to weaken cerebral blood vessels and hypertension. With this method, in a series of surger-ies, hypertension is induced by unilateral nephrectomy, implantation of a DOCA-salt pellet, and the addition of 1% NaCl to the drinking water. During the same surgery as the DOCA-salt pellet implantation, the mice receive a single injection of elastase into the right basal cistern. With this method, CA formation occurs in > 60% of the mice within 28 days of the aneurysm induction surgery. Additionally, spontaneous aneurysm rupture occurs in 50%–60% of mice within 7–11 days following surgery. Hosaka et al. later modified this model with the addition of an increased vessel flow rate and fragility induced by ligating the left CCA and the right renal artery, followed 1 week later by the injection of elastase into the right basal cistern.33 Hypertension and vessel fragility were further enhanced by angiotensin II and by chow containing 8% NaCl and 0.12% BAPN. Using this method with elastase concentrations greater than 50 mU, 100% of the mice de-velop CAs.

With elastase, CAs are made and also rupture at pre-dictable time points. With 25–30 mU of elastase, the ma-jority of mice form aneurysms at 1 week without signs of rupture.61–64 However, approximately 80% of animals will have subarachnoid hemorrhage by 4 weeks. Similar to the histological changes observed in human CAs, the aneu-rysms formed by elastase display disruption of the elastic lamina, macrophage infiltration, loss or reduction of the endothelium, and smooth-muscle cell hyperplasia. This model is utilized extensively in the literature and has been used to test pharmacological inhibitors that decrease the incidence of aneurysm progression and rupture.54,62

Surgically Created Saccular AneurysmsThe small animal size and the intracranial aneurysms

formed preclude the use of rodent CA models for endo-vascular device testing. To circumvent this issue, Frösen et al.21 and Marbacher et al.43 surgically created saccular aneurysms using a donor thoracic aorta, which was surgi-cally ligated end-to-side to the abdominal aorta in both mice and rats. These saccular aneurysms display inflam-matory cell infiltration, endothelial denudation, thrombus formation, and intimal hyperplasia. Marbacher et al.44 expanded on this model with sodium dodecyl sulfate–in-duced decellularization of the donor thoracic aorta prior to aneurysm creation. The loss of mural cells led to an un-organized luminal thrombus, increased inflammation, and wall damage resulting in aneurysm growth and rupture.44 Although mice are too small, studies have been success-fully conducted using the rat saccular aneurysm model for testing stents11,27 and coils.45

Perspectives and LimitationsRodent CA models offer a powerful tool for the inves-

tigation of aneurysm biology at a molecular, cellular, and physiological level. Excluding surgically created saccular aneurysms, rodent CA models do not require direct vessel manipulation and have an intracranial location. This leads to the question of what constitutes an aneurysm. In the early studies, aneurysm formation produced small micro-aneurysms that were rarely visible and only detectable by light or electron microscopy or histological alterations of the vessel wall. Some scientists do not believe these “mi-croaneurysms” recapitulate human CA disease. In con-trast, elastase treatment results in clear and defined out-ward bulging of the vessel walls of the circle of Willis and its major branches. Starke et al.61 defined an aneurysm as a bulge in the vessel wall whose diameter is > 150% of the diameter of the parent artery. Similarly, Nuki et al.54 de-fined an aneurysm as a bulging of the vessel wall > 150% of the diameter of the basilar artery.

Although rodent CA models replicate many of the his-tological and molecular changes found in human CAs, there are certain pathologies observed in human CAs but not in the rodent CA models. For example, in human sac-cular CAs, lipids and oxidized lipids accumulate in the aneurysm wall and are associated with cell death, vessel wall weakening, and aneurysm rupture.22,55 Similarly, the complement inflammatory system is activated in human saccular CAs and is involved in aneurysm wall degrada-tion and rupture.65

The commercial availability of genetically modified mice has made rodent CA models a vital tool in investi-gating the molecular underpinnings of CA formation, pro-gression, and rupture. Transgenic mice allow for the in-vestigation of particular proteins that are altered in human CA disease. For example, tumor necrosis factor (TNF)–a,61 monocyte chemoattractant protein (MCP)–1,4 and nuclear factor (NF)–kB p50 subunit7 knockout reduces CA formation, whereas endothelial nitric oxide synthase (eNOS)9 and SOX1740 knockout predisposes mice to CA formation. The cited studies are just a few of the many using transgenic mice to better understand CA biology in preclinical studies.

Large Animal CA ModelsLarge animal CA models have been made in numer-

ous species but are primarily formed in rabbits, dogs, and swine.15 Aneurysm formation in large animals requires di-rect vessel manipulation through either microsurgical or endovascular intervention, and these aneurysms are typi-cally formed using the CCA. Therefore, these models are extracranial in location and suffer from the effects of sur-gical creation at the aneurysm neck and dome.15 Despite these weaknesses, each model has particular characteris-tics that are either advantageous for or detrimental to the purposes of a particular study.

Rabbit Aneurysm ModelsVenous Graft Aneurysm

To simulate arterial bifurcation aneurysms, a technique was developed to create venous pouch aneurysms using a jugular venous graft at a surgically induced bifurca-tion at the end-to-side anastomosis of the left CCA to the


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right CCA.20 Marbacher et al.42 modified this technique to create a more complicated aneurysm by grafting large, wide-necked, bilobar, and bisaccular venous pouches to this surgically created bifurcation. Most recently, a very broad-necked aneurysm model was developed by longitu-dinally opening a segment of the jugular vein and graft-ing this patch to the CCA.59 A limitation of these venous pouch models is their inability to replicate the histological changes observed in human aneurysms. A beneficial qual-ity of this model is the ability to tailor aneurysms in terms of both dome and neck size.

Arterial AneurysmsThe most common model of aneurysm creation in rab-

bits is the saccular aneurysm created by enzymatic weak-ening of the arterial wall. A reliable method was devel-oped whereby aneurysms were produced by occluding the CCA with an endovascular balloon and incubating the aortic arch–brachiocephalic trunk bifurcation with elas-tase.18 Subsequently, the same group refined their tech-nique whereby the CCA was surgically exposed, cannulat-ed, and occluded with an endovascular balloon, and then the CCA was incubated with elastase above the balloon prior to its distal ligation (Fig. 2).1 While early techniques for endovascular incubation with elastase were effective at producing aneurysms with adequate patency duration, the retrograde flow of elastase would often cause damage to the trachea and other organs. To curtail this complica-tion, the technique was modified further so that a micro-catheter was introduced distal to the balloon and proximal

to the CCA ligation to prevent retrograde elastase flow.38 Finally, to dissolve the collagen in the tunica media, colla-genase was added to the elastase in endovascular models, which resulted in aneurysms that are nearly histologically identical to human CAs, showing fragmentation and dimi-nution of the internal elastic lamina and increased levels of smooth-muscle cells.36,37,44,45 Although aneurysms cre-ated with this method show long-term patency more than 24 months, these aneurysm fail to grow and rupture and demonstrate a homogeneity in vessel wall makeup and thickness. Therefore, it fails to replicate the more com-plex, heterogenic aneurysm vessel environment of athero-sclerosis and wall thinning, as well as the inflammatory cell infiltration and de-endothelialization associated with aneurysm rupture.

Hemodynamic Stress–Induced Aneurysms of the Posterior Circulation

Studies by Hassler in 196332 and later by Gao et al.23 demonstrated that aneurysms can be formed in the pos-terior circulation of rabbits using hemodynamic stress alone without hypertension or vessel wall weakening. In this model, hemodynamic stress is increased in the basilar artery by unilateral or bilateral ligation of the carotid ar-teries. Using this technique, Hassler and Gao found histo-logical changes in the arterial wall of the basilar terminus resembling nascent aneurysm formation characterized by a loss of internal elastic lamina, media thinning, and an outward bulge of the vessel lumen. This model has been expanded with the addition of the aneurysm risk factors of hypertension and estrogen deficiency.66 Hypertension is induced by unilateral nephrectomy combined with a high salt diet, and estrogen deficiency is induced by bi-lateral oophorectomy. The combining of hemodynamic stress with hypertension and estrogen deficiency induced changes in the circle of Willis, such as vessel length and tortuosity, as well as aneurysm lesion formation and vas-cular damage.

Fusiform AneurysmRecently, Avery et al.12 developed a carotid artery fu-

siform aneurysm in rabbits. In this model, the right CCA is exposed and wrapped in gauze and isolated from sur-rounding tissue by placing the CCA-wrapped section into a cradle. The gauze is then soaked in elastase and CaCl2 for 20 minutes. With this method, fusiform aneurysms, which were defined as vessel dilations greater than 50% of the proximal artery diameter, were formed in 100% of the animals at 6 weeks after aneurysm creation surgery. Histologically, these aneurysms demonstrate an almost complete loss of the internal elastic lamina, a reduction in the tunica media, and a thickening of the tunica intima. The long-term patency of this model was not investigated past 6 weeks.

Canine Aneurysm ModelsVenous Pouch Model

The first reliable aneurysm model was developed in 1954 by German and Black, who used a venous pouch graft to create saccular aneurysms in dogs.24 This tech-

FIG. 2. Rabbit elastase aneurysm model. Aneurysm formation in rabbits consists of exposing the right CCA. After gaining arterial access, a bal-loon is advanced to the origin of the CCA (A). As the balloon is inflated, elastase is simultaneously injected, filling the artery (B). The artery is incubated with elastase for 20 minutes. The elastase and balloon are then removed, and the distal portion of the CCA is ligated, forming the aneurysm. Residual elastase and hemodynamic forces will cause the aneurysm to maturate over a period of several weeks following surgery. A digital subtraction angiography study shows a newly formed aneurysm (C). Copyright Robert Starke. Published with permission.


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nique has remained in use to date.62,70 The technique involves exposing the external jugular vein and section-ing a suitable length; one side of the venous segment is closed with a suture to create a venous pouch, which is then sutured to an arteriotomy created at any location of the investigator’s choice. Several modifications of this technique have been described including those for gi-ant, wide-necked, and fusiform aneurysm creation (Fig. 3).29,35, 57, 71,72

Generally, the CCA or cervical internal carotid artery is selected for the creation of aneurysms because of their similarity in caliber and blood flow to human cerebral vessels and the ability of the animal to tolerate the surgi-cal procedure.26,47,60,69 The canine’s CCA is approximately 4 mm in diameter, similar to the human internal carotid artery, and the relatively long CCA in dogs (10–12 cm) affords easy surgical access.

Hemodynamic Stress and Arterial Wall InjuryMore recently, Wang et al. described a novel method of

CA formation by inducing hemodynamic stress in com-bination with arterial wall weakening.67 In this model, a new branch in the CCA is surgically constructed by at-taching the proximal segment of one CCA to the proximal sidewall of the contralateral CCA.49,61,67 Hypertension is induced, and elastase is delivered externally to the apex of the newly created bifurcation.

Swine Aneurysm ModelsThe procedure for aneurysm production in swine is

similar to that described by German and Black for an-eurysm formation in canines.24 This method was slightly

modified by using a longer venous segment and a side-to-side anastomosis to construct giant aneurysms, which were more prone to rupture if left untreated than smaller sized aneurysms.56

Elastase was introduced by Goericke et al.25 to create saccular aneurysms. As in rabbit models, the CCA is ex-posed and occluded at the origin, and elastase is injected into the CCA and incubated for a period of time. As in both the rodent and rabbit CA models, elastase weakens the arterial wall, triggering an initial inflammatory re-sponse as well as activation of endogenous proteinases to break down elastin and collagen, resulting in vascular dilation.30

Perspectives and LimitationsLarge animal CA models offer broad utility for inves-

tigating endovascular therapeutic interventions, healing, and endovascular training. Among the large animal mod-els, the venous pouch aneurysm model allows for the se-lection of aneurysm size, morphology, and location, and the aneurysm can be created in vessels with a caliber and blood flow similar to those of human cerebral vessels. However, the venous pouch model suffers in terms of the surgical trauma and suture material involved in aneurysm formation, nonarterial aneurysm composition, and an arti-ficial neck. Despite these drawbacks, large animal models allow for testing of endovascular devices as well as endo-vascular training. However, the preferred CA model for endovascular training is still up for debate.

A major disadvantage of the rabbit elastase aneurysm model is that it lacks an inflammatory response and does not spontaneously rupture, but it does have coagulation and

FIG. 3. Venous pouch aneurysms. Illustration depicting the surgical creation of sidewall, bifurcation, and terminal aneurysms using the left and right CCA and a segment of the external jugular vein (EJV). Copyright Robert Starke. Published with permission.


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thrombolysis profiles similar to those of humans, which is critical for testing new materials for use in endovascular devices for aneurysm occlusion.13 A major disadvantage of the swine CA venous graft model is a tendency for spon-taneous thrombosis and healing with or without emboli-zation.28,39, 52,58 Another disadvantage of the large animal models is the presence of viable mural vascular smooth-muscle cells, which are significantly reduced or absent in human CA tissue. Marbacher et al.44 demonstrated that decellularized aneurysm grafts formed an unorganized luminal thrombus and had increased inflammation and wall damage resulting in aneurysm growth and rupture. Therefore, the healing response in CA models with normal cellularization of the aneurysm wall would be enhanced and thereby potentially enhance the healing response in device studies.

Silicone Aneurysm ModelsThe recent advancement in and accessibility to 3D

printed technologies has allowed for the fabrication of pa-tient-specific true-to-scale arterial replicas.19,37,46 These 3D printed models serve as education tools for presurgical as-sessment or can be further processed using silicone-cast-ing technology to form a hollow, silicone-walled artificial vasculature. There is an increasing volume of literature in which artificial CA models have been used for endovascu-lar device testing and training, surgical clip ligation train-ing, presurgical assessment, and fluid dynamics studies,2 and they have also been surgically implanted into swine and cadaveric human heads for neurosurgical training.14,53 Although this model offers an excellent alternative to ani-mals in endovascular training, it does not fully replicate the natural arterial biology, which may greatly affect test-ing results.

Clinical Translation and ConclusionsAnimal models of CA have been and continue to be an

invaluable tool for investigating the molecular, cellular, and physiological aspects of CA pathophysiology as well as for testing novel endovascular devices. Ideally, the CA model will replicate the hemodynamic forces, wall sheer stresses, and cellular and tissue responses observed in human CAs. However, no animal model perfectly replicates the human disease being investigated. Therefore, each investigator must consider the strengths and weaknesses of each model in order to best replicate the aspect of CAs that is being investigated. In general, rodent CA models are useful for investigating the molecular and cellular mechanisms of aneurysm formation, growth, and rupture with the goal of finding druggable targets for therapeutic intervention and translational potential. In contrast, large CA animal models are primarily used in the development and refine-ment of new endovascular therapies and in the assess-ment of novel therapeutic interventions, as was done with Gamma Knife radiosurgery.48 Large animal models also allow for the investigation of aneurysm healing following therapy. No current CA model perfectly replicates human CA disease. Therefore, further work is needed to create a CA model that more closely replicates the histological and pathophysiological features of human CA disease.

AcknowledgmentsThis work was supported by a National Research and Educa-

tion Foundation (NREF) Young Clinician Investigator Award, Joe Niekro Research Grant, Bee Foundation Award, Brain Aneurysm Foundation Award, and Miami Clinical and Translational Science Institute Award (R.M.S.). The project described was supported by grant no. UL1TR002736, Miami Clinical and Translational Science Institute, from the National Center for Advancing Trans-lational Sciences and the National Institute on Minority Health and Health Disparities. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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DisclosuresDr. Snelling has direct stock ownership in RIST Neurovascular.

Author ContributionsConception and design: Starke, Thompson. Drafting the article: Thompson, Elwardany, McCarthy, Sheinberg, Alvarez, Nada, Snelling, Chen, Sur.

CorrespondenceRobert M. Starke: Miami University, Lois Pope LIFE Center, Miami, FL. [email protected].


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In vivo cerebral aneurysm models · Cerebral aneurysm rupture is a devastating event resulting in subarachnoid hemorrhage and is associated with signifi-cant morbidity and death