Development of a Murine Model for Femoral Artery Anastomotic Stenosis

Summary

Here we present a murine model for femoral artery anastomosis, offering researchers a valuable animal model to study and simulate vascular anastomotic stenosis. This development is crucial for advancing our understanding of the pathophysiology underlying this condition and facilitating more accurate and effective research into vascular diseases.

Abstract

In vascular surgery, vascular anastomosis is a common reconstructive technique used to restore blood flow. However, anastomotic restenosis is a frequent postoperative complication, primarily caused by surgery-induced vascular injury, intimal hyperplasia, and inflammatory responses. The mouse femoral artery anastomosis model is widely used to investigate the mechanisms of anastomotic restenosis and vascular repair. Microscopically guided end-to-end femoral artery anastomosis allows precise simulation of vascular injury and repair processes following surgery, providing a reliable experimental tool for studying the pathological mechanisms related to restenosis. This study focuses on refining the surgical technique for femoral artery anastomosis in mice. Through refinements in surgical techniques and optimization of technical details, we have achieved a marked increase in the success rate and reproducibility of the model. Specific improvements include refined vascular handling techniques during surgery, the selection of suture materials, and the optimization of suturing methods to minimize anastomotic leakage and postoperative occlusion. The study also emphasizes the observation of intimal hyperplasia, vascular remodeling at the anastomotic site, and long-term vessel patency. Through this research, we provide a concise and efficient operational guide for performing mouse femoral artery anastomosis, offering reliable technical support for experimental studies in vascular surgery. This work lays a solid foundation for subsequent investigations into related mechanisms and evaluations of therapeutic intervention.

Introduction

Vascular anastomosis is a fundamental technique in revascularization procedures, playing a pivotal role in restoring blood flow and promoting tissue repair. However, the occurrence of intimal hyperplasia (IH) at the anastomotic site often leads to restenosis, which significantly compromises long-term vascular patency and negatively impacts clinical outcomes and patient prognosis^1,2. IH is closely associated with intraoperative vascular injury, characterized by abnormal proliferation and migration of smooth muscle cells (SMCs) and excessive deposition of extracellular matrix¹. These complex and interrelated pathological processes underline the critical need to elucidate the precise mechanisms of IH to inform preventive and interventional strategies against restenosis.

Due to their reproducibility and precise control, murine models of femoral artery anastomosis have been widely adopted in research on vascular repair and associated pathological mechanisms^3,4,5. End-to-end anastomosis in mice allows accurate simulation of post-surgical anastomotic injury, enabling dynamic observation of IH and vascular remodeling. These models provide an ideal platform to study interactions between endothelial cells and SMCs post-surgery and to evaluate the role of inflammatory responses in IH development⁶. By combining histological analysis and molecular biomarker detection, researchers can comprehensively identify key drivers of IH, offering critical insights into its underlying mechanisms and potential therapeutic targets.

The development of IH is driven by multiple factors, with hemodynamic changes being a critical contributor^1,7,8. At the anastomotic site, regions of low shear stress and abnormal oscillatory shear index (OSI) are primary stimuli for SMCs proliferation and migration^1,7. Furthermore, compliance mismatches and turbulent blood flow around the anastomosis exacerbate endothelial injury, accelerating IH progression⁸. These findings underscore the necessity of optimizing surgical techniques and selecting appropriate materials to mitigate pathological changes at the anastomotic site.

In recent years, drug-coated balloons (DCBs) have demonstrated efficacy in reducing IH. Anti-proliferative agents, such as paclitaxel, effectively inhibit SMCs proliferation and migration, significantly reducing the incidence of restenosis⁹. However, challenges persist in high-flow systems like arteriovenous grafts, where rapid fluctuations in shear stress and high blood flow rates may diminish the efficacy of DCBs¹. Future studies should focus on improving the applicability of DCBs in varied hemodynamic environments while leveraging advancements in biomaterials science to develop more personalized and effective solutions for post-surgical restenosis. In addition to localized interventions, systemic factors such as diabetes, atherosclerosis, and endothelial dysfunction significantly influence IH development¹⁰. Therefore, clinical strategies should prioritize comprehensive management of these systemic conditions to enhance overall vascular health. Concurrently, the identification and monitoring of novel biomarkers for IH progression could provide opportunities for early intervention. The integration of artificial intelligence into surgical planning offers another promising avenue, allowing for the computational design of optimized anastomotic configurations, thereby improving surgical success rates and prolonging vascular patency.

In the study of post-surgical IH and associated pathological mechanisms, the femoral artery anastomosis model stands out for its precision and reproducibility¹¹. This model, employing microsurgical techniques to create end-to-end anastomosis of the femoral artery in mice, accurately mimics localized surgical trauma at the anastomotic site. The advantages of this model become particularly evident when compared to models such as wire-induced injury or other alternatives. A major technical advantage of the femoral artery anastomosis model is its ability to induce highly localized and controlled vascular injury¹². The surgical trauma allows for a focused impact on the anastomotic region, closely mimicking the injury patterns encountered in clinical vascular surgery. In contrast, wire-induced injury models, while simpler in technique, often result in extensive endothelial denudation, making it difficult to replicate localized trauma observed in real-life anastomotic surgeries¹³. Furthermore, the variability in the depth and extent of wire-induced damage across different trials potentially diminishes the reproducibility of results. The extensive and diffuse nature of the damage in wire injury models makes it less relevant for investigating the localized IH that is specifically associated with anastomotic regions.

In this study, utilizing a murine model of femoral artery anastomosis, we systematically refined surgical techniques to enhance model success rates and ensure long-term patency of the anastomotic site. Leveraging this established foundation, our study delved into the molecular and cellular mechanisms underlying IH, including regulatory pathways that govern SMCs migration and proliferation, as well as the role of inflammatory mediators in the progression of IH. Through this research, we aim to contribute novel theoretical insights into the mechanisms of post-anastomotic restenosis and establish an experimental foundation for the development of therapeutic strategies specifically targeting IH.

Protocol

This study was approved, and the animals were handled in accordance with the Guidelines for the Management and Use of Laboratory Animals in China. The research strictly adhered to the ethical requirements of animal experiments, with approval from the Animal Ethics Committee (Approval Number: SWMU20221109-019). Here, 8-week-old healthy C57BL/6 mice of either gender, weighing between 20-22 g, were utilized for the present study. The animals were housed at the Laboratory Animal Center of Southwest Medical University (SWMU).

1. Preoperative procedures

1. Anesthetize mice with 3% isoflurane inhalation in accordance with institutionally approved protocols. After the onset of anesthesia, verify the complete loss of reflexes, such as the lack of response to toe-pinch, to ensure deep anesthesia before proceeding with the surgery. After inducing anesthesia, reduce the concentration of isoflurane to 1%-1.5% to maintain the anesthetic state at O₂ volume 300 mL/min, ensuring that the animals remain in deep anesthesia throughout the surgical procedure.
2. Position the mouse supine on a surgical platform, extending the hind limbs without overstretching. Apply ophthalmic ointment to the eyes of the animals to prevent corneal drying and protect ocular surfaces. Additionally, provide thermal support throughout the procedure using a heating pad to maintain the animals' body temperature and ensure their comfort and well-being.
3. Apply depilatory cream to the thigh area for approximately one minute to remove hair, then thoroughly clean the area with warm sterile water (~37 °C) to remove any residual cream and stray hairs.
4. Prep the surgical site with an iodine-based scrub to remove surface contaminants, followed by 70% alcohol to eliminate residual iodine and further disinfect the area.

2. Vascular anastomosis of the femoral artery

NOTE: After the initial preparation of the animal, strictly maintain sterile conditions. Specifically, sterilize all surgical instruments thoroughly using an autoclave prior to use. Additionally, don new sterile gloves before handling the mouse and placing it on the surgical surface.

1. Under a stereomicroscope, make an incision approximately 1.5 cm long along the axis of the femur in the mid-thigh region using a microscalpel. After incising the skin, perform blunt dissection to separate the subcutaneous tissue until the femoral artery and femoral vein are exposed. The femoral artery is located laterally to the femoral vein, and the nerve is positioned superiorly and laterally to the artery.
2. Carefully dissect the tissue to fully expose a 1 cm section of the femoral artery. Using hemostatic forceps, gently separate the muscles and deep fascia to expose the nerves, artery, and vein. During this process, the nerve is located on the outermost layer, the artery is in the middle, and the vein is on the inside.
3. Gently separate the femoral artery and femoral vein using blunt dissection. Be cautious to use fine forceps to separate the connective tissue to fully expose the femoral artery. Place a sterile pad beneath the femoral artery to prevent accidental needle injury during the procedure. Subsequently, gently clamp the femoral artery with forceps to induce a hematoma.
4. Using small hemostatic forceps, clamp the proximal and distal ends of the femoral artery. Use fine scissors to neatly and symmetrically transect the femoral artery.
5. Draw up 400 μL of 1% heparinized saline (1% heparin in normal saline) in a 1 mL syringe for injection at the femoral artery anastomosis. Add the solution as 3-4 drops at a time, with repeated rinses to remove blood clots from the artery.
6. To provide support for the vessel during subsequent suturing and simulate the potential damage caused by a guidewire intervention, insert a 1 cm long 6-0 nylon surgical suture into the femoral artery. Ensure proper alignment and smoothness of the femoral artery to facilitate the suturing process.
7. Use a 12-0 nylon surgical suture for anastomosis and adjust the needle angle under the microscope to ensure that the needle exits from the inner side to the outer side of the vessel.
8. Create eight puncture sites, four on the proximal and four on the distal ends of the femoral artery.
   1. Ensure that the puncture sites are located at the anterior, posterior, left, and right points of the vessel, with corresponding sites at the distal and proximal ends. When selecting these points, ensure the vessel is accessible, avoid critical anatomical structures, and maintain good blood flow during anastomosis.
   2. Make the puncture diameter small enough to minimize damage to the vessel but large enough for necessary procedures. A 12-0 suture needle is typically used for puncture.
9. Cut four lengths of 12-0 nylon surgical suture, each 3-4 cm long, and thread each through the corresponding puncture hole. Begin with tying a loose knot to avoid tangling the sutures.
10. Remove the 6-0 nylon surgical suture used for vessel support and securely tie the knots. Release the hemostatic forceps after the anastomosis is completed.
11. Gently scrape the femoral artery with curved forceps from the proximal to the distal end to check for patency, ensuring that blood flows freely through the vessel. Additionally, carefully inspect the anastomosis site for any signs of obvious leakage.

3. Postoperative suture

1. Suture the skin of the lower limb by using a 6-0 nylon surgical suture, employing an interrupted suture to ensure precise alignment and secure approximation of the tissue.
2. Maintain sterile conditions post-surgery. After surgery, administer 0.125 mL of sterile saline solution via subcutaneous injection to maintain hydration. Monitor the animals in a warm, quiet environment for at least one hour, checking their vital signs and behavior regularly to ensure proper recovery. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return the animals that have undergone surgery to the company of other animals until they are fully recovered.

4. Postoperative observation and sampling

1. Regularly inspect the surgical site for any signs of inflammation, excessive swelling, or discharge. Document the animal's condition and provide appropriate care as needed.
2. At 4 weeks post-surgery, humanely euthanize the mice in accordance with approved ethical guidelines. For euthanasia, administer an overdose of pentobarbital sodium (150 mg/kg, intraperitoneal injection), ensuring a painless and humane endpoint. Once unconsciousness and cessation of reflexes are confirmed, initiate tissue collection.
   NOTE: In the preliminary studies, we found that harvesting the samples during the 3^rd week or earlier was not conducive to the formation of intimal hyperplasia. Conversely, collecting samples at the 5^th week led to excessive intimal hyperplasia. This excessive growth not only impeded the observation of subsequent experiments but also posed potential health risks to the mice. So the 4^th week was selected here.
3. Collect femoral artery samples, centering on the anastomosis site, extending approximately 1 cm in both directions from the anastomosis. Using fine scissors, excise the femoral artery along with all surrounding muscle tissue.
4. After excision, rinse the sample in PBS to remove residual blood, then fix it in 10% neutral formalin for 1-2 days for further preservation and analysis.
   NOTE: A 10% neutral formalin is a classic choice for tissue fixation, effectively cross-linking proteins and preserving the integrity of tissue structure, making it particularly suitable for the long-term storage of tissues. In contrast, 4% paraformaldehyde (PFA) is a milder fixative that is better suited for the finer preservation of intracellular structures (such as nucleic acids and proteins) and is commonly used for immunohistochemistry or immunofluorescence analysis. The primary aim of this study is to observe the histological changes in blood vessels (such as intimal hyperplasia and vascular remodeling) rather than the precise localization of intracellular molecules or proteins. Therefore, the fixation effect of formalin is sufficient to meet the experimental needs. If the research requires higher molecular fidelity (such as the preservation of RNA or proteins), PFA may be a better choice.

5. Dehydration and embedding of the femoral artery

1. After fixation, place the samples in embedding boxes. Rinse the femoral artery samples with running water for 6-8 h before being processed for tissue dehydration using an automated tissue processor.
2. Pour melted paraffin into embedding molds, carefully pick the femoral artery up using heated forceps, and vertically embed it into the mold containing the molten paraffin. Separate the lid and bottom of the embedding box, with the bottom placed on top of the embedding mold. Add a small amount of melted paraffin to fix it in place, serving as the base for the paraffin block. Air bubbles should be meticulously avoided.
3. When the wax block has cooled to the point where a transparent wax film forms on the surface, place it on a freezing table to rapidly cool.
4. Remove the embedded wax block from the embedding mold and trim the excess paraffin encircling the tissue block carefully using a sharp blade.

6. Preparation of paraffin sections of the femoral artery

1. Mount the blade on the microtome, ensuring the knife is sharp for sectioning.
2. Fix the paraffin block onto the holder and adjust the block relative to the blade to the appropriate position for sectioning.
3. Trim the block to ensure the embedded tissue can be completely sectioned. Set the thickness of the initial sections to 15-20 µm.
4. Adjust the section thickness to approximately 4 µm and proceed with sectioning.
5. Lift the sections gently using a brush and transfer them with specialized fine tweezers to a slide box filled with warm water (approximately 45 °C) to facilitate floating.
6. Transfer the spread sections onto microscope slides. Position the slides at a 45° angle to drain excess water. Afterward, place the slides in an oven to dry, typically at 37 °C for 2 h, followed by baking at 60 °C for 1 h.

7. Hematoxylin-Eosin staining

1. Hydrate the sections using a series of graded ethanol concentrations, including absolute ethanol, 95%, 80%, and 70% ethanol, with each step taking approximately 5 min. Subsequently, rinse the sections with distilled water to remove any residual ethanol.
2. Stain the sections with hematoxylin for approximately 8-10 min and thoroughly wash 3x with running water to remove excess stain.
3. To differentiate the stained sections, apply 1% hydrochloric acid alcohol solution for a brief period of 5-7 s.
4. To intensify the blue color, treat the sections with a 1:400 ammonia solution for 1 min.
5. After soaking in 75% ethanol, stain the sections with eosin for 36 s. Following staining, sequentially dehydrate the sections with an ascending series of ethanol concentrations (80%, 90%, 95%, and 100%), with each step lasting for 3-5 s.
6. Finally, bake the sections at 37 °C in an oven for 10 min, apply a couple of drops of sealing agent on a glass slide, cover the specimen with a coverslip to secure them in place, and allow it to air dry naturally.

Results

In vascular anastomosis surgery, mechanical injury to the vessel wall can activate intimal cells and trigger proliferation. Changes in blood flow velocity and direction after the anastomosis can also stimulate the proliferation of intimal cells. The vascular remodeling process and the long-term instability of blood flow can also persistently stimulate intimal cells, ultimately leading to thickening.

To confirm the success of the femoral artery anastomosis model, hematoxylin and eosin staining ...

Discussion

Vascular anastomosis is a crucial technique in vascular reconstruction surgery, with its animal model playing a key role in studying the mechanisms of postoperative restenosis. This model offers a controlled approach to investigating vascular pathological changes, particularly in understanding the origin of over-proliferating cells in the neointima during restenosis. The source of proliferating smooth muscle cells (SMCs) becomes a crucial issue when severe arterial injury occurs after vascular anastomoses, resulting in t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-0 Nylon Suture with Needle	Ningbo Chenghe	240102
12-0 Nylon Suture with Needle	Ningbo Lingqiao	22064
Electro-heating standing-temperature incubator	Shanghai Boxun	HPX-9272MBE
Eosin Staining Solution	Servicebio	G1005-2
Formaldehyde Solution	KESHI	50-00-0
Hematoxylin Staining Solution	Servicebio	G1005-1
Heparin Sodium	Solarbio	H8060
MAGSCANNER KF-PRO-002	KFBIO	KFPBL00200107003
Mounting medium	Wuxi Jiangyuan	220810
OLYMPUS SZ2-ILST	OLYMPUS CORPORATION	SN 9B40828
Paraffin embedding machine	YAGUANG	YB-7LF
Phosphate-Buffered Saline	Solarbio	P1010