Development, techniques, and utility of experimental animal models of thoracic and abdominal aortic aneurysms

Desarrollo, técnicas y utilidad de modelos animales experimentales de aneurismas aórticos torácicos y abdominales

Carlos C. Bravo-Reyna1, Jacqueline Mejía-Cervantes2, Ana T. Verduzco-Vázquez2, Cristopher C. Sánchez-Rodríguez3, Leonardo Cuervo-Vargas4, Luis A. Medina-Velázquez5, Víctor Gómez-Vergara1, Carlos A. Hinojosa2, Javier E. Anaya-Ayala2,6*

1Departamento de Cirugía Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; 2Sección Angiología, Cirugía Vascular y Endovascular, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; 3Departamento de Medicina Interna, Hospital Ángeles del Pedregal; 4Departamento de Ortopedia, Hospital Regional Lic. Adolfo López Mateos, ISSSTE; 5Departamento de Física, Instituto Nacional de Cancerología; 6Dirección Médica, Hospital Ángeles Universidad. Ciudad de México, Mexico


*Correspondence: Javier E. Anaya-Ayala. E-mail: jeanayaayala@gmail.com
Date of reception: 21-08-2023
Date of acceptance: 02-02-2024
DOI: 10.24875/ACME.M24000502
Available online: 03-10-2024
Arch Cardiol Mex (Eng). 2024;94(3):360-367

Abstract

Aneurysms are clinical entities that can develop and affect human aorta; and although in most cases they have an asymptomatic course, these pathological dilatations can lead to a lethal outcome when rupture occurs, thus the establishment of predictors is crucial for death prevention. Essential events that take place in the vessel wall have been identified and described, such as inflammation, proteolysis, smooth muscle cell apoptosis, angiogenesis, and vascular remodeling. Porcine and ovine models have been useful for the development and evaluation of endovascular devices of the aorta. However, since the worldwide introduction and adoption of these minimally invasive techniques for aneurysm repair, there is lesser availability of diseased aortic tissue for molecular, cellular, and histopathological analysis, therefore over the last three decades it has been proposed various small species models that have allowed the focal induction of these lesions for the study of pathophysiological mechanisms and possible useful biomarkers as diagnostic and therapeutic targets. The present review article presents and discusses the animal models available as their applications, characteristics, advantages, and limitations for the development of preclinical studies, and their importance in the comprehension of this pathology in humans.

Keywords: Aortic aneurysms; Animals models; Extracellular matrix; Calcium chloride; Elastase; Angiotensin II

Resumen

Los aneurismas son una de las entidades clínicas que pueden desarrollarse y afectar la aorta humana. Aunque en la mayoría de los casos tienen un carácter asintomático, estas dilataciones patológicas pueden resultar letales cuando se presentan con ruptura, por lo que el reconocimiento de factores predictores de esta complicación es crucial para evitar muertes. Fisiopatológicamente se han identificado eventos esenciales que ocurren en la pared del vaso, como inflamación, proteólisis, apoptosis del músculo liso, angiogénesis y remodelación. Las grandes especies como porcinos y ovinos han sido de utilidad para el desarrollo y evaluación del desempeño de dispositivos endovasculares en la aorta, así como la remodelación; con el advenimiento y disposición de estas técnicas mínimamente invasivas para su reparación existe una menor disponibilidad de tejido aórtico para el análisis molecular, celular e histopatológico, por lo que en las últimas tres décadas se han propuesto e introducido distintos modelos que han permitido, mediante la inducción focal de estas lesiones, el estudio de los mecanismos fisiopatológicos y posibles biomarcadores de utilidad como dianas diagnósticas y terapéuticas. El presente artículo de revisión aborda tipos de modelos animales disponibles, así como sus aplicaciones, consideraciones, ventajas y limitaciones para el desarrollo de estudios preclínicos y su importancia en el entendimiento de esta patología en la especie humana.

Palabras clave: Aneurismas aórticos; Modelos animales; Matriz extracelular; Cloruro de calcio; Elastasa; Angiotensina II

Introduction

An aneurysm is defined as a focal pathological dilation of more than 50% of the normal diameter of a healthy segment of a blood vessel. It most frequently affects the human aorta and is classified as thoracic aortic aneurysms (TAA) or abdominal aortic aneurysms (AAA) based on their location. These aneurysms are characterized by a progressive expansion of the vessel and are mostly asymptomatic until rupture occurs1, which to date is the most feared outcome due to its high mortality rate, reaching up to 85% to 90%. Therefore, they must be detected promptly for appropriate intervention and repair2. Some identified risk factors include advanced age, male gender (although with a higher risk of rupture in women), smoking, and systemic arterial hypertension3. The etiology of AAA is still not fully understood; it is currently known that AAA results from an inflammatory response involving proteolysis of the extracellular matrix and oxidative stress with apoptosis of vascular smooth muscle cells and angiogenesis, leading to the progressive weakening of the vessel walls due to degradation of the tunica media4. Recognized entities that lead to aneurysm formation include vasculitis, collagenopathies, infectious processes, and trauma, with degenerative etiology being the most common, accounting for up to 90% of cases, making it the most studied and described with relatively constant epidemiological patterns of the disease5-8.

Recent studies indicate a prevalence of AAA of 1% up to 2% in men aged 65 years and 0.5% in women aged 70 years9. In Mexico, in 2019, Hinojosa et al. confirmed a prevalence of 5.63% through the intentional and systematic review of computed tomography scans performed on patients at Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán10. Additionally, in 2021, a multicenter cross-sectional study conducted across 4 metropolitan areas in Mexico demonstrated a prevalence of 3.08% in imaging detection, highlighting the importance of introducing these programs into our health system11.

As endovascular treatments replace open surgical approaches as the first-line therapy12, less human tissue is available for research. Various models used for the study of AAA have enabled experiments for a better understanding of this condition. Different species have been used, offering various advantages and limitations; larger models such as pigs, sheep, and dogs have been very useful for the development of new endovascular techniques and materials, as well as the study of hemodynamic patterns and parameters associated with the presence of aneurysmal lesions13. Among smaller animals, murine species are notable for their lower cost and easy surgical and genetic manipulation, making them an ideal model for inducing various vascular lesions to understand the risk factors leading to aortic dilation14. This review article discusses available murine models, their applications, considerations, advantages, and limitations for developing preclinical studies, and their importance in understanding these conditions in human beings.

Periadventitial calcium chloride application model

It is the first method described for the induction of aortic aneurysms in murine models, in the year 200115. It is a relatively simple way to induce an aneurysm because micro-surgical manipulation of the aorta is not needed. This type of model is performed by the direct application of calcium chloride (CaCl2) to the adventitia layer16.

Tissue damage occurs because calcium ions have a high affinity for elastin in the media layer17; these ions travel inside the cells and form calcium phosphate (CaPO4) crystals via alkaline phosphatase, causing calcification and rupture of elastic fibers18. The growth of the aneurysm is time-dependent, typically occurring within the next 2-to-4 weeks; there is an increase in the presence of inflammatory cell infiltrate, where we see T lymphocytes and CD68+ macrophages localized in areas proximal to apoptotic cells, and an increase in neutrophilic infiltrate is also observed19. Damage to the elastin at extracellular matrix level results in the formation of peptides that have chemotactic qualities on monocytes and can even induce the differentiation of macrophages into M1 type, characterized by secreting pro-inflammatory cytokines. In addition to increased concentrations of metalloproteinases (MMP-2 and MMP-9), which can degrade different components of the aortic wall, observed up to 2 weeks later in all layers of the vessel20,21 (Fig. 1).

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Figure 1. Proposed pathophysiology of aneurysm induction with periadventitial calcium chloride application. Calcium chloride induces elastin calcification, inflammatory infiltration, and apoptosis of vascular smooth muscle cells (VSMCs). Macrophage infiltration is induced by plasminogen-mediated activation of MMP-9. Macrophages and mast cells produce TNF-α, which also contributes to VSMC apoptosis. Proinflammatory cytokines such as IL-1α and TNF-α and the consequent oxidative stress lead to the increased production of MMPs and other proteases such as elastases, which condition the rupture of the extracellular matrix18. AAA: abdominal aortic aneurysm; TAA: thoracic aortic aneurysm; IL-1α: interleukin 1 alpha; MMP: metalloproteinase; TNF-α: tumor necrosis factor alpha.

 

Although these models have the advantage of inducing localized lesions, usually performed in the infrarenal segment of the abdominal aorta, they can also be performed in the descending thoracic aorta through a lateral thoracic approach22.

Induction of descending aortic aneurysm via lateral thoracic approach, micro-surgical technique

This technique is routinely used by our research team. Wistar rats are sedated and intubated orotracheally to connect to a mechanical ventilation system23 (Fig. 2 A-D). The rat is placed in the right lateral decubitus position, and a 2 cm incision is made at the caudal edge of the scapula, followed by blunt dissection of the muscle planes until reaching the third intercostal space; an incision is performed in the intercostal space, the ribs are retracted, and the descending aorta is visualized. Two silk sutures are used (3-0) and it is gently retracted to separate it from adjacent tissues. CaCl2 (13.6 mEq of calcium/10 ml mixed with 0.5 ml of 0.9% saline solution) is locally administered with a swab to the adventitia and kept in contact for 15 to 20 minutes. The thoracic cavity is closed, the pneumothorax is reduced by negative pressure, and the lung is inflated to its full capacity (Fig. 3 A-C and Fig. 4).

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Figure 2. Cannula adapted for rat intubation (patent pending, registration No.: MX/2019/062634) (A), capnograph for verification of catheter placement (B), mechanical ventilation device (C) and catheter used to recover intrathoracic negative pressure (D).

 
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Figure 3. Left lateral thoracic approach. Thoracic aorta visualization (A), periadventitial CaCl2 application with swab under the microscope (40x) (B) and dissected aorta showing induced dilation (C) (arrow).

 
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Figure 4. Left lateral thoracic approach. Ascending aorta (A), left lung (B), anteriorly displaced thymus (C), left atrium (D), and azygos veins (E).

 

A limitation of the model is that it lacks some important pathophysiological characteristics of human aneurysms, such as intraluminal thrombus, atherosclerosis, and infrequent rupture. This type of model involves the pressurized infusion of porcine pancreatic elastase (PPE) into an aortic segment. The enzyme acts at the media layer, directly damaging the elastic lamina fibers, leading to their total loss with leukocyte invasion in an inflammatory response to the aneurysm injury. These differences are related to the intrinsic risk factors of patients with AAA16.

Induction of infrarenal aortic aneurysm via laparoscopy

Sedation and intubation are performed, and the laparoscopic machine is prepared. In the dorsal decubitus position, a 1 cm incision is performed above the umbilical scar. Blunt dissection of the abdominal layers is performed to introduce a 5 mm trocar and a 0° microscope (Storz) to visualize the abdominal cavity. Another 5 mm trocar is placed in both incisions. Intestinal loops are carefully mobilized until the pulsatile line of the abdominal aorta is observed; the retroperitoneum is dissected until the aorta is exposed, separating it from the inferior vena cava and the vagus nerve to locally administer CaCl2 with a swab (Fig. 5). It is kept in contact with the vessel’s adventitia for 20 minutes. Subsequently, trocars are removed, and the abdominal cavity is closed in layers.

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Figure 5. Laparoscopic visualization of intra-abdominal vessels Inferior hepatic border (A), inferior vena cava (B) and descending aorta (C).

 

Elastase-induced model

This model involves the pressurized infusion of porcine pancreatic elastase (PPE) into an aortic segment. The enzyme acts at the media layer, directly damaging the elastic lamina fibers, leading to their total loss with leukocyte invasion in an inflammatory response to the aneurysm injury. Subsequently, there is usually the development of an intraluminal thrombus, which promotes aneurysm growth due to the endogenous secretion of proteases that activate plasmin, MMPs, and the recruitment of neutrophils24-26.

For this procedure, the anesthetized rat, prepared in the inguinal region, is placed in the dorsal decubitus position. Under a microscope, an oblique incision is performed 1 mm away from the inguinal fold; blunt dissection separates the adipose and muscle tissue from the neurovascular bundle, dissecting the vessel’s adventitia and separating the femoral artery origin the vein. A catheter is, then, inserted into the femoral artery, reaching the aorta below the birth of the left renal branch. Then, through laparotomy, the inferior vena cava and abdominal aorta are dissected, the catheter location is verified, and the abdominal aorta is clamped below the origin of the left renal branches and 1 cm below, leaving a 1 cm distance, to proceed with the intraluminal infusion of PPE27. Other proposed techniques include the intra-adventitial28 and periaortic application of elastase29.

The aneurysm growth can go from 300% up to 421% of the initial diameter within the first week, which is a limitation due to the higher risk of rupture in a short time. However, after 2-to-3 weeks of elastase induction, the aneurysm stabilizes through fibrosis and cellular turnover30.

Angiotensin II model

The murine model involves the infusion of angiotensin II (Ang II) at a dose of 500 or 1000 ng/min/kg for 28 days using an infusion minipump implanted in the neck of genetically modified mice with apolipoprotein E deficiency (Apo E -/-), which led to the development of aneurysms in the thoracic portion in 20% and abdominal portion in 33% of the cases. Immunohistochemistry has demonstrated the presence of macrophages in the adventitia as well as damage to smooth muscle cells, which showed new lesions characterized by cells with lipid inclusions31. Another advantage of this model is the formation of an intraluminal thrombus and vascular remodeling, specific characteristics observed in human AAA32.

In 2020, Yue et al. proposed a model combining intraluminal infusion of PPE into the infrarenal aorta via open surgical approach and subcutaneous Ang II, resulting in a higher rate of growth and rupture with the possibility of extending into the iliac arteries. This technique was used to study the expression of pro-inflammatory cytokines, finding elevated mRNA levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), chemokine ligand 5 (CCL5), and interferon-γ33.

Recent variants of chemical induction models

  • – Transforming growth factor-β (TGF-β) neutralization: Other models have used TGF-β neutralization through the injection of blocking antibodies, promoting the development of more severe AA in the Ang II model34 and the elastase model35.

  • – β-aminopropionitrile (BAPN): Lysyl oxidase (LOX) is an enzyme that causes chemical bonding between collagen and elastin chains. In 2019, it was described that BAPN fumarate salt can be used to inhibit LOX. This model was used in combination with the previously described Ang II and PPE models. A current technique combines periadventitial application of elastase with oral BAPN36. The results demonstrate the formation of a focal fusiform aneurysm with intramural thrombus and extracellular matrix remodeling. When monitored for 14 weeks, a progressive expansion to 8 times its original size was observed, with rupture occurring in half of the cases37. In 2022, Pellenc et al. described a model with oral BAPN along with isosorbide dinitrate, where subcutaneous infusion of Ang II could histologically reproduce human AAA, leading to a less favorable prognosis and survival in the animals38.

Endovascular reconstruction models in larger species

Endovascular repair of both thoracic and abdominal aorta is a widely used procedure in humans, which is still being studied due to the development of increasingly novel, complex, and adaptable devices, determining long-term outcomes, and their advantages and disadvantages vs open surgical management. These include various types of AAA, such as dissection, saccular, fusiform, and even rupture39. For this purpose, larger species models are used vs those previously described, such as sheep, pigs, and dogs. Since the 1990s, the latter have been recommended for testing arterial valves40. Breeds like the German Shepherd have been used for introducing branched devices used in complex aortic arch approaches where a hybrid technique, both endovascular and open, is performed41. However, despite the canine model’s similarity to humans in graft endothelialization processes, it has the main disadvantage of tending towards hypercoagulability, making it difficult to evaluate this characteristic during recovery39. The porcine model has been proposed for thoracic aorta repair; meeting specifications such as weight can achieve an internal aortic diameter of 2 cm42. Additionally, the REBOA (Resuscitative endovascular balloon occlusion of the aorta) has been tested in traumatic injuries, finding differences between total and partial occlusion, with the latter being superior by allowing some distal flow43. However, due to a longer torso, the device used in humans may be insufficient to cover the injury area and less tolerant to anesthesia39.

Regarding sheep, this model offers the advantage of being anatomically similar to humans regarding sizes, along with a coagulation system and cascade physiologically corresponding to our species39. In their study, Doorschodt et al. describe the CAR (customized aortic repair) method for AAA repair, which involves using a bone-shaped balloon while the rest of the sac is filled with polymer, demonstrating its potential for AAA treatment. However, the authors mention the need to validate this technique in humans44, and current literature mentions a high mortality rate when using this technique45.

The use of these models is for academic purposes, collecting information about the technique and materials, as well as to train vascular surgeons to avoid complications such as endoleaks, stent migration, endotension, or device failure42,45.

Discussion

The preclinical phase of the study of AAA has gained importance and is a fundamental part via the use of animal models as the basis for research into the pathophysiological mechanisms related to the development of this entity. Over the past 3 decades, it has gained more relevance due to the decreased availability of human aortic tissue owing to less invasive management since the implementation of endovascular techniques12.

Each model offers advantages in terms of histological similarity to human aneurysms, especially the more recent ones that combine chemical induction techniques. These allow for the follow-up of animals to evaluate not only the growth but also the outcome of the AAA. Similarly, some pathophysiological limitations have been found that, although deviating from the pathophysiological and molecular study purpose, have been used for testing endovascular techniques, such as the decellularized xenograft model39,46 and the anterior aortic patch model, where a saccular aneurysm is created using autologous or artificial tissue. These techniques use larger species compared to murines, as previously mentioned4.

A crucial aspect is the translation of research to its application in contemporary clinical practice. Its relevance is primarily based on the timely detection of aneurysms using both structural and molecular imaging methods aimed at proteins and cells related to inflammation, vascular remodeling, and angiogenesis47-49, which can currently improve the relatively uncertain prognosis of these patients. Likewise, understanding the involved pathophysiological mechanisms allows for the description of potential therapeutic targets.

Multiple studies have focused on analyzing LDL and Ang II type 1 receptors implicated in the pathogenesis of atherosclerosis50,51, being therapeutic targets used today through statins, beta-blockers, and calcium antagonists, although these drugs have had limited effects on controlling AAA progression. Recently, type 2 sodium-glucose cotransporter inhibitors have been used in animals, showing benefits in cardiovascular disorders and proving to be highly expressed in the aorta51.

Conclusions

Aneurysms constitute one of the most frequent aortic diseases, with a significant knowledge gap existing from their pathophysiological bases to predictors of success and failure of contemporary treatments. The development of animal models with both smaller and larger species has been useful in understanding this entity and has guided studies in recent years toward more precise detection programs for the proper management and follow-up of this disease, both conservatively and invasively.

Funding

None declared.

Conflicts of interest

None declared.

Ethical disclosures

Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

Confidentiality of data. The authors declare that no patient data appear in this article.

Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Use of artificial intelligence for generating text. The authors declare that they have not used any type of generative artificial intelligence for the writing of this manuscript, nor for the creation of images, graphics, tables, or their corresponding captions.

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