An Optimized Protocol for Guidewire-Induced Aortic Valve Stenosis Model in Mice

Summary

This protocol details an optimized guidewire-induced aortic valve stenosis model in C57BL/6 mice. A custom 3D-printed vascular support platform enables precise hemorrhage control, yielding 95% 24-hour survival (19/20) and 90% overall model success at 4 weeks (18/20).

Abstract

Aortic valve stenosis (AVS) is a major cardiovascular disease with increasing prevalence in aging populations, yet the scarcity of diseased valve tissue severely limits mechanistic studies and therapeutic development. This protocol establishes and optimizes a reproducible mouse model of AVS by improving upon the Honda guidewire-induced valve injury method through controlled hemorrhage management. Male and female C57BL/6 mice undergo guidewire-induced aortic valve injury; the procedure is refined by a custom 3D-printed vascular support platform that achieves precise hemorrhage control during guidewire insertion and withdrawal. Outcome measures include 24 h survival, echocardiographic assessment at 4 weeks, and histopathological validation. The optimized protocol achieves a 95% 24 h survival rate (19 of 20 animals); among survivors, 94.7% (18 of 19) meet the predefined hemodynamic success criterion at 4 weeks, yielding an overall model success rate of 90% (18 of 20 animals), confirmed by echocardiography and histological analysis. This standardized model provides a reliable, reproducible platform for investigating mechanisms of valve degeneration and evaluating candidate therapeutic interventions.

Introduction

Valvular heart disease remains a substantial healthcare burden in both developed and developing nations, with prevalence rising in parallel with population aging¹. Although transcatheter aortic valve implantation (TAVI) has transformed the treatment of severe aortic stenosis^2,3, the cellular and molecular mechanisms that drive valve degeneration are still incompletely understood, and no approved pharmacological therapy exists to slow or reverse disease progression^4,5. A central obstacle in valve disease research is the limited a....

Protocol

NOTE: All animal procedures described in this protocol were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of West China Hospital (Protocol No. 20240311055). Male C57BL/6 mice (8–10 weeks; approximately 22–25 g) and female C57BL/6 mice (8–10 weeks; approximately 18–20 g) were used throughout. All materials, reagents, instruments, software, and custom devices used in this protocol, including the custom 3D-printed vascular support platform, are listed in the Table of Materials.

1. Pre-o....

Results

Development of a 3D-printed platform for hemorrhage control

To address perioperative hemorrhage—the principal source of morbidity and mortality in the conventional guidewire-induced AVS model—a 3D-printed vascular support platform was designed (Figure 1). The platform was fabricated by stereolithography (SLA) printing from a biocompatible (USP Class VI / ISO 10993-compliant) photopolymer resin, with a structural thickness of 1.07 mm. After printin.......

Discussion

This protocol provides a detailed description of a stable, safe animal model of aortic stenosis. A feature of this approach is an auxiliary platform that facilitates guidewire insertion into the common carotid artery. The platform is fabricated by stereolithography (SLA) printing from a biocompatible photopolymer resin (structural thickness 1.07 mm) and incorporates an inverted V-shaped apical surface with a 120° apex angle; the complete design file is freely available as a supplementary .stp file. This device ensur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D-printed vascular support platform	Custom-made	N/A	Custom 3D-printed vascular support platform; supplementary CAD file provided in .stp format.
4% Paraformaldehyde	Servicebio	G1101-3ML	Tissue fixation
5-0 nylon suture	Jinhuan Medical	F503	Skin closure by interrupted sutures
6-0 silk suture	Jinhuan Medical	RC411	Permanent ligation of distal RCCA and proximal slipknot
Acoustic coupling gel (pre-warmed)	Keppler	KL-250	Echocardiography
Biocompatible photopolymer resin	Formlabs	RS-F2-BMCL-01	SLA printing material used to fabricate the custom 3D-printed vascular support platform.
C57BL/6 mice (8–10 weeks; males 22–25 g, females 18–20 g)	GemPharmatech	C57BL/6	Both sexes used throughout; housed under standard conditions
Ethylene oxide gas	Institutional sterilization facility	N/A	Sterilization of the custom 3D-printed vascular support platform.
FreeCAD	FreeCAD Project Association	1.1.1	CAD visualization and generation of the 3D structural drawing from the .stp file.
GraphPad Prism	GraphPad Software	9.0	Statistical analysis and graphing
Guidewire, 0.014-inch diameter	Boston Scientific	H749393071900	Mechanical aortic valve injury
Hematoxylin and Eosin (H&E) staining kit	Beyotime	C0105S	Leaflet morphology and thickness
Isoflurane	RWD Life Science	R510-22-2	3% induction; 1.5–2% maintenance in 100% O2
Lidocaine	Shijiazhuang Pharmaceutical Group	H14024045	Pre-emptive local infiltration at incision site (0.5%, step 1.5)
Microsurgical scissors	RWD Life Science	F11022-11	Skin incision, arteriotomy, thoracotomy
MX550D transducer (40 MHz)	FUJIFILM VisualSonics	MX550D	Echocardiography
Nair hair removal cream	Nair	255 g	For anterior cervical and chest wall depilation
Phosphate-buffered saline (PBS)	Servicebio	G4202-100ML	Perfusion
Rotary microtome	Leica Biosystems	RM2125 RTS	Serial sectioning at 4–5 µm
Stereomicroscope (surgical)	Olympus	SZX7	Vessel isolation and microsurgery
Vevo 3100 high-resolution ultrasound system	FUJIFILM VisualSonics	Vevo 3100	Echocardiography
Von Kossa staining kit	Servicebio	G1043-20ML	Dystrophic calcification detection
Xylene	Nanjing Chemical Reagent Co., Ltd.	C0430530209	Tissue clearing during paraffin processing.