A Murine Model of Carotid Aneurysm Formation

Summary

The present protocol describes a standardized surgical method for the induction of carotid aneurysms in a murine model through a single surgical operation.

Abstract

Cerebral aneurysms are present in approximately 2%-5% of the population and represent a significant public health concern due to their high rates of mortality and morbidity upon rupture. Despite advancements in research, the development of effective and reproducible animal models remains a challenge. Among the available models, surgically induced aneurysms are widely utilized; however, the technical complexity of these procedures often limits their accessibility. These methods require extensive surgical expertise and are associated with higher rates of intraoperative mortality or inconsistent aneurysm formation, which can compromise the reliability of experimental outcomes. To address these limitations, this video article introduces a simplified surgical procedure for inducing carotid aneurysms in mice, performed in a single session. This streamlined approach has demonstrated an impressive 87% success rate in inducing carotid aneurysms in female mice. By reducing technical challenges and improving reproducibility, this method provides a practical and reliable alternative for cerebral aneurysm research. Its widespread adoption could facilitate standardized studies, accelerate the development of therapeutic strategies, and ultimately advance our understanding of aneurysm pathophysiology.

Introduction

Cerebral aneurysms are localized dilations in the walls of cerebral blood vessels, affecting approximately 2%-5% of the general population. Rupture of these aneurysms can result in subarachnoid hemorrhage, which carries high rates of mortality and morbidity^1,2. Understanding the pathogenesis of cerebral aneurysms and identifying potential therapeutic targets requires reliable and reproducible animal models that mimic the human disease. This article introduces a standardized surgical method for inducing carotid aneurysms in mice, offering a practical and efficient alternative to more technically demanding intracranial models³.

The development of this technique is motivated by the challenges associated with existing cerebral aneurysm models. Intracranial aneurysm models, though widely used, require complex surgical procedures and are limited by the small caliber of intracranial vessels, which hampers their utility for testing endovascular devices and other interventions^4,5. Additionally, variations in the methods used across studies -- such as differences in elastase concentrations and exposure times -- reduce reproducibility and hinder cross-study comparisons. By standardizing the procedure, this technique addresses these limitations and provides a high success rate of aneurysm induction with minimal surgical risk.

Compared to alternative methods, the carotid aneurysm model offers several distinct advantages. First, the simplified surgical procedure reduces technical barriers, making it more accessible to a broader range of laboratories. Second, the larger caliber of the carotid artery compared to intracranial vessels facilitates testing of therapeutic approaches, including endovascular devices. Previous studies have demonstrated variations of this model with promising results, but inconsistent protocols have limited reproducibility^6,7. Our approach refines these methods, achieving an 87% success rate in female mice and ensuring consistency across experiments.

In the broader context of the literature, animal models for cerebral aneurysms are critical for understanding disease mechanisms, evaluating therapeutic targets, and developing intervention strategies. Models have been developed in various species, including zebrafish, rats, rabbits, and dogs, each with unique advantages and limitations^3,8. This carotid aneurysm model complements the existing repertoire by offering a balance of technical feasibility and relevance to human disease, making it an ideal option for researchers seeking a reliable, reproducible model for preclinical studies.

This method is particularly well-suited for laboratories aiming to investigate the molecular and biomechanical underpinnings of aneurysm formation, test pharmacological agents, or evaluate endovascular devices. Researchers should consider whether the anatomical and physiological features of this model align with their specific experimental goals. By providing a standardized, reproducible protocol, this technique aims to advance cerebral aneurysm research and foster greater consistency across studies in the field.

Protocol

The animal protocol was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IS00022015). Female C57BL/6J wild-type (WT) mice, 6-8 weeks of age, were used for the experiments. The reagents and the equipment used are listed in the Table of Materials.

1. Animal preparation

1. Induce anesthesia.
   1. Place the mouse in an induction chamber and administer isoflurane gas at 3%-4% concentration for induction using an anesthesia vaporizer system (following institutionally approved protocols). Adjust the flow rate to 1.0-1.5 L/min of oxygen.
   2. Monitor the animal for signs of complete anesthesia (e.g., lack of response to external stimuli and reduced respiratory rate).
2. Transfer to surgical site.
   1. Once the mouse is fully anesthetized, transfer it to the surgical station while maintaining isoflurane anesthesia at a maintenance concentration of 1-3% and an oxygen flow rate of 0.8-1.0 L/min. Ensure the mouse is connected to the anesthesia delivery system via a nose cone.
3. Temperature maintenance.
   1. Position the mouse on a warming pad pre-set to 36 °C. Attach a temperature probe to continuously monitor body temperature throughout the procedure. Adjust the warming pad setting if necessary to maintain a stable temperature.
4. Preemptive analgesia.
   1. Administer buprenorphine extended-release injectable suspension at a dose of 3.25 mg/kg subcutaneously prior to the surgical procedure.

2. Preparation for surgery

1. Immobilize the animal.
   1. Place the mouse in the supine position. Use strips of surgical tape to gently secure each limb to the surgical platform, ensuring the body is stabilized without undue tension.
2. Shave the neck area.
   1. Using small surgical clippers, shave the fur from the neck region (approximately 2 cm x 2 cm area). Remove loose hair with sterile surgical gauze.
3. Sterilize the surgical site.
   1. Apply two rounds of povidone-iodine solution to the neck area using cotton-tipped applicators, followed by two rounds of 70% ethanol. Allow the area to air dry between applications to ensure proper sterilization.

3. Stage I: Common carotid artery ligation

1. Confirm anesthesia.
   1. Perform a toe-pinch test using blunt forceps. Lack of withdrawal or reflex movement confirms adequate anesthesia.
2. Perform midline incision.
   1. Using a scalpel blade, make a 1 cm vertical incision along the midline of the neck, starting from just below the jawline. Cut through the dermis and underlying fascia.
3. Expose the submandibular gland.
   1. Use blunt forceps to mobilize the submandibular gland laterally, taking care not to damage surrounding tissues. Gently lift the gland and separate it from the sternum by blunt dissection.
   2. Secure the gland with a small sterile retractor or clip to maintain a clear surgical field.
4. Expose the sternocleidomastoid (SCM) muscle.
   1. Visually identify the ipsilateral sternocleidomastoid (SCM) muscle. Use blunt forceps to lift the muscle gently and thread a 5-0 braided silk suture underneath. Reflect the muscle laterally by gently pulling the suture to expose the carotid sheath.
5. Isolate the carotid artery.
   1. Open the carotid sheath using sharp forceps. Carefully separate the carotid artery from the adjacent jugular vein and vagus nerve.
   2. To improve visibility, insert a sterile silicone cuff or retractor beneath the carotid artery. Avoid causing trauma to the artery or surrounding structures.
6. Ligate the carotid artery.
   1. Using sharp forceps, scoop the carotid artery and thread a 9-0 suture underneath. Tie a secure double knot to ligate the artery, ensuring proper tension to occlude blood flow without damaging the vessel. Trim the suture ends to minimize tissue irritation.
7. Elastase treatment.
   1. Bathe the carotid artery in 10 U/mL porcine pancreatic elastase solution diluted in 1× PBS, maintained for 10- 20 min.
   2. Keep the tissue moist throughout the incubation period to prevent desiccation. Immediately after application, gently dab away any excess elastase solution to minimize damage to surrounding tissues.
8. Close the incision.
   1. Approximate the skin edges using sterile nylon suture (5-0) or surgical staples. Apply gentle pressure with sterile gauze to ensure hemostasis.

4. Post-operative care

1. Monitor the animal.
   1. Observe the animal daily for clinical signs of complications such as premature aneurysmal rupture (evidenced by sudden lethargy or hematoma), stroke, seizures, or significant weight loss (>10% of baseline weight).
   2. Promptly euthanize animals (following institutionally approved protocols) exhibiting severe symptoms to minimize pain and distress.
      NOTE: Buprenorphine extended-release provides up to 72 h of analgesia; additional post-operative analgesics are administered only if needed.
2. Wound care
   1. Immediately after the surgery, apply 4% lidocaine gel to the incision site for local pain relief. Apply a thin layer of antibiotic ointment (e.g., bacitracin) to prevent infection. Repeat wound care daily for at least 3 days or until the wound shows signs of healing.
3. Recovery environment
   1. Place the animal in a clean recovery cage with soft bedding. Ensure access to warmed fluids or a hydration gel to aid recovery. Monitor until the animal is fully awake and mobile, then return it to its standard housing.

Results

   A total of 15 female wild-type (WT) mice underwent the aneurysm induction procedure following the standardized protocol described (Figure 1). Two weeks post-surgery, aneurysm formation was successfully achieved in approximately 87% of the mice, as confirmed by gross morphological examination (Figure 2 and Figure 3). These results highlight the robustness and reproducibility of the technique, demon...

Discussion

The carotid aneurysm model induced by ligation of the carotid artery was initially described in the 1970s by Yasargil and Matsuda, who utilized vein grafts to induce aneurysm development^9,10. Subsequent modifications to this technique, including the addition of elastase treatment and carotid ligation, have enabled the generation of larger carotid aneurysms in a more efficient and expedited manner^6,11. Des...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-0 absorbable braided suture	Ethicon / Ethilon	J463G
5-0 non-absorbable monofilament suture	Ethicon / Ethilon	8698G
9-0 non-absorbably monofilament suture	Ethicon / Ethilon	2829G
alcohol prep pads	Medline	CUR45585RB
animal pocket pro shaver	Wahl	9861-900
Dumont #5 Forceps	Roboz	RS-5058
Dumont Titanium Tweezers; Pattern #5	Roboz	RS-5050
Graefe Forceps	Roboz	RS-5138
Graefe Tissue Forceps	Roboz	RS-5155
lidocaine, 4%, topical	Medline	52565-009-50
micro-clip straight, 5 mm	Roboz	RS-5420
micro-dissecting scissors	Roboz	RS-5610
micro-sclip straight, 8 mm	Roboz	RS-5424
mosquito forceps, 4"	Roboz	RS-7100
Nitrile gloves	Fisherbrand	19-130-1597
Penicillin-Streptomycin	Thermo Fisher	15140122
Porcine pancreatic elastase	Worthington	LS002274
povidone-iodine	Medline	MDS093943
scissors, extra-fine	Roboz	RS-5880
scissors, fine	Roboz	RS-5840
Sterile 0.9% saline solution	Baxter	2B1324X
Sterile Cotton Applicator	Dynarex	4303
Sterile Disposable Surgical Towels, Blue	Medline	MDT2168202H
Tegaderm Transparent Film Dressing	3M	1624W