This article presents a unique closed-chest technique for inducing myocardial ischemia-reperfusion injury (IRI) in mice. The presented method allows mice to breathe spontaneously while remotely inducing myocardial ischemia. This provides access to the animal for studying the dynamic processes of ischemia and reperfusion in situ and in real-time via noninvasive imaging.
Acute myocardial infarction (AMI) is a prevalent and high-mortality cardiovascular condition. Despite advancements in revascularization strategies for AMI, it frequently leads to myocardial ischemia-reperfusion injury (IRI), amplifying cardiac damage. Murine models serve as vital tools for investigating both acute injury and chronic myocardial remodeling in vivo. This study presents a unique closed-chest technique for remotely inducing myocardial IRI in mice, enabling the investigation of the very early phase of occlusion and reperfusion using in-vivo imaging such as MRI or PET. The protocol utilizes a remote occlusion method, allowing precise control over ischemia initiation after chest closure. It reduces surgical trauma, enables spontaneous breathing, and enhances experimental consistency. What sets this technique apart is its potential for simultaneous noninvasive imaging, including ultrasound and magnetic resonance imaging (MRI), during occlusion and reperfusion events. It offers a unique opportunity to analyze tissue responses in almost real-time, providing critical insights into processes during ischemia and reperfusion. Extensive systematic testing of this innovative approach was conducted, measuring cardiac necrosis markers for infarction, assessing the area at risk using contrast-enhanced MRI, and staining infarcts at the scar maturation stage. Through these investigations, emphasis was placed on the value of the proposed tool in advancing research approaches to myocardial ischemia-reperfusion injury and accelerating the development of targeted interventions. Preliminary findings demonstrating the feasibility of combining the proposed innovative experimental protocol with noninvasive imaging techniques are presented herein. These initial results highlight the benefit of utilizing the purpose-built animal cradle to remotely induce myocardial ischemia while simultaneously conducting MRI scans.
Acute myocardial infarction (AMI), a prevalent global cardiovascular condition, is associated with high mortality rates and morbidity1. Despite technological advancements that enabled early and effective revascularization strategies for AMI patients, patients still experience myocardial ischemia-reperfusion injury (IRI) following these interventions2. Therefore, understanding the fundamental mechanisms and formulating approaches to mitigate IRI is crucial. IRI represents a complex pathophysiological state involving a multitude of intricate biological processes. These encompass regulated cell death, oxidative stress responses, inflammation, wound healing, fibrosis, and ventricular remodeling. Animal models, such as mice, have been of great importance for IRI research and are extensively employed due to their cost-effectiveness, rapid breeding, and the wealth of mechanistic information from transgenic models3.
This protocol presents an innovative technique to remotely induce IRI in spontaneously breathing mice with a closed chest, more closely mimicking the human pathology. Remote occlusion was achieved by utilizing a balloon catheter positioned at a distance from the myocardium to occlude the left anterior descending artery (LAD). This approach offers continued access to the beating heart at the exact moments of occlusion and reperfusion, facilitating simultaneous imaging modalities, including ultrasound or magnetic resonance imaging (MRI). These noninvasive methods could provide crucial insights into tissue responses before, during, and after occlusion and reperfusion events.
Various surgical techniques exist to induce IRI in murine models. Surgical trauma stemming from thoracotomy in open-chest models of coronary ligation triggers an immune response impacting diverse mechanisms associated with ischemia and reperfusion. Activation of the innate immune system holds the potential to influence the extent of myocardial infarction4. The proposed adapted technique provides a potential approach for exploring myocardial pre- and postconditioning and possibly reducing the impact of the innate immune response to surgical trauma in murine IRI studies by minimizing the open thorax timeframe to a maximum of 5 min. Moreover, the newly developed technique might also contribute to reducing the pro-inflammatory sequelae caused by ventilator-induced lung injury5. However, the combined effects of this new closed chest method would require further in-depth investigation.
Thorough validation of the proposed technique was conducted by comparing it with the traditional method, which involves exposing the LAD through surgical chest dissection and ligature-induced occlusion for 30 min. Results from both techniques were compared, which included troponin measurements reflecting cardiac infarct size, assessments of the area at risk using MRI with gadolinium contrast enhancement, histological triphenyl tetrazolium chloride (TTC) staining, and the determination of final scar sizes via Sirius Red (SR) staining. The outcome demonstrates the robustness and efficacy of the proposed approach for studying ischemia-reperfusion injury in murine models.
The novel remote occlusion technique introduced in this study offers a unique platform to advance research in the field of ischemia-reperfusion injury modeling by avoiding the need for direct vessel manipulation during the initial surgery and allowing simultaneous multi-modal imaging of the early reperfused myocardium. Comprehensive characterization, including troponin-I measurements, LGE contrast MRI, TTC, and SR staining, shows that the proposed technique is equivalent to the current golden standard (i.e., ope…
The authors have nothing to disclose.
The experiments were performed at the KU Leuven core facility 'Molecular Small Animal Imaging Center' (MoSAIC). The authors would like to express their thanks to Katarzyna Błażejczyk for technical assistance. The research was supported by research grants from KU Leuven (C14/20/095) and the Research Foundation – Flanders (FWO G0A7722N). M. Algoet is supported by the Research Foundation – Flanders Fellowship Grant (FWO 11A2423N).
2-0 silk suture | Sharpoint Products | DC-2515N | |
5-0 polypropylene suture | Ethicon | 8710H | |
7-0 polypropylene suture | Ethicon | 8206H | |
ACCLARENT Balloon Inflation Device | Johnson&Johnson MedTech | BID30 | |
Acepromazine | Kela | ||
BD Vialon 18 G | BD | 381347 | |
BD Vialon 20 G | BD | 381334 | |
Betadine Solution | Purdue Pharma | 25655-41-8 | |
Buprenorphine (Buprenex Injectable) | Reckitt Benkiser Healthcare | NDC 12496-0757-1 | |
Carp Zoom Styl Knijplood 2.1 g (lead fishing weights) | Visdeal.nl | ||
Dumont #3 Forceps | Fine Science Tools | 11231-30 | |
Dumont #5 Forceps | Fine Science Tools | 11251-30 | |
Fine Scissors | Fine Science Tools | 14040-10 | |
Fucidine gel | Leo Pharma | ||
Isoflurane | Abbott | NDC 5260-04-05 | |
KD Mouse/Rat Eye Speculum | World Precision Instruments | 501897 | |
KD mouse/rat eye speculum | World Precision Instruments | 501897 | |
Ketamine | Dechra | ||
Light source | Zeiss | KL 1500 LCD | |
MRI system | Bruker BioSpin, Ettlingen, Germany | BioSpec 70/30 | |
NatriumChloride 0.9% | Baxter | ||
Nocturnal Infrared Heat Lamp | Zoo Med Laboratories, Inc. | RS-75 | |
ParaVision software | Bruker BioSpin | version 6.0.1 | |
polyethylene tubing PE-10 | SAI Infusion technologies | PE-10 | |
polyethylene tubing PE-50 | SAI Infusion technologies | PE-50 | |
remote IRI tool (PMMA) | homemade | ||
Rodent Surgical Monitor | Indus instruments | ||
Segment v4.0 | Medviso, segment.heiberg.se | R12067 | |
Self-gated gradient echo sequence | Bruker BioSpin, Ettlingen, Germany | IntraGate, ParaVision 6.0.1 | |
Slim Elongated Needle Holder | Fine Science Tools | 12005-15 | |
Sure-Seal Mouse/Rat Induction Chamber | World Precision Instruments | EZ-178 | |
Tubing Connectors, Poly, Y Shape | Westlab | 072025-0001 | |
Ultraverse 035 PTA Dilatation Catheter: 5mm x 40mm, 17 ATM RBP balloon on 130 cm long catheter | Bard Peripheral Vascular, Inc. | 00801741092671 | |
Veet (depilatory creme) | Reckitt Benkiser Healthcare | ||
Ventilator, MiniVent Model 845 | Hugo Sachs | 73-0043 | |
Vidisic | BAUSCH & LOMB PHARMA | 685313 | |
Xylazine | Bayer |
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