Name: Author Spotlight: A Novel Standardized Technique for Real-Time Biomedical Imaging of Acute Myocardial Injury
Uploaded: 2024-03-22T13:40:00
Description: Read this Scientific Article Protocol about Author Spotlight: A Novel Standardized Technique for Real-Time Biomedical Imaging of Acute Myocardial Injury at JoVE.com

The experiments were performed at the KU Leuven core facility &#39;Molecular Small Animal Imaging Center&#39; (MoSAIC). The authors would like to express their thanks to Katarzyna B&#322;a&#380;ejczyk 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&#160;- Flanders Fellowship Grant (FWO 11A2423N).

This animal protocol was approved by and is in accordance with the guidelines and regulations set forth by the Ethical Committee for Animal Experimentation (ECD) at The Catholic University of Leuven. All policies developed by the local ECD follow the regulations of the European Union concerning the welfare of laboratory animals as declared in Directive 2010/63/EU. All tests described below have been performed on 8-12 week-old (body weight = 20-23 g) male C57BL/6 mice. The acute experiments represent animals that were imm...

Closed-Chest Technique for Murine Myocardial Ischemia-Reperfusion Injury

<ol>
	<li>Bryda, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+The+impact+of+rodents+on+advances+in+biomedical+research.">The mighty mouse: The impact of rodents on advances in biomedical research.</a> Mo Med. 110 (3), 207-211 (2013).</li><li>Fishbein, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phase+acute+myocardial+infarct+size+quantification:+Validation+of+the+triphenyl+tetrazolium+chloride+tissue+enzyme+staining+technique.">Early phase acute myocardial infarct size quantification: Validation of the triphenyl tetrazolium chloride tissue enzyme staining technique.</a> Am Heart J. 101 (5), 593-600 (1981).</li><li>Ib&#225;&#241;ez, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+therapies+for+myocardial+ischemia/reperfusion+injury.">Evolving therapies for myocardial ischemia/reperfusion injury.</a> J Am Coll Cardiol. 65 (14), 1454-1471 (2015).</li><li>D&#261;browska, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+to+surgery+and+infection.">The immune response to surgery and infection.</a> Cent Eur J Immunol. 39 (4), 532-537 (2014).</li><li>Muthuramu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+ligation+of+the+left+anterior+descending+coronary+artery+in+mice:+a+model+of+post-myocardial+infarction+remodelling+and+heart+failure.">Permanent ligation of the left anterior descending coronary artery in mice: a model of post-myocardial infarction remodelling and heart failure.</a> J Vis Exp. (94), e52206 (2014).</li><li>Nickles, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+ventilation+causes+airway+distension+with+proinflammatory+sequelae+in+mice.">Mechanical ventilation causes airway distension with proinflammatory sequelae in mice.</a> Am J Physiol-Lung C. 307 (1), L27-L37 (2014).</li><li>Reed, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction.">Acute myocardial infarction.</a> Lancet. 389 (10065), 197-210 (2017).</li><li>Ritti&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+picrosirius+red-polarization+detection+of+collagen+fibers+in+tissue+sections.">Method for picrosirius red-polarization detection of collagen fibers in tissue sections.</a> Methods Mol Biol. 1627, 395-407 (2017).</li><li>Schwarte, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+ventilation+of+mice.">Mechanical ventilation of mice.</a> Basic Res Cardiol. 95 (6), 510-520 (2000).</li><li>Vaneker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+ventilation+induces+a+Toll/Interleukin-1+receptor+domain-containing+adapter-inducing+interferon+&#946;-dependent+inflammatory+response+in+healthy+mice.">Mechanical ventilation induces a Toll/Interleukin-1 receptor domain-containing adapter-inducing interferon &#946;-dependent inflammatory response in healthy mice.</a> Anesthesiology. 111, 836-843 (2009).</li><li>Van Allen, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+volatile+anesthetics+in+cardioprotection:+A+systematic+review.">The role of volatile anesthetics in cardioprotection: A systematic review.</a> Med Gas Res. 2, 22 (2012).</li><li>Kumar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mouse+coronary+anatomy+and+myocardial+infarction+consequent+to+ligation.">Distinct mouse coronary anatomy and myocardial infarction consequent to ligation.</a> Coron Artery Dis. 16 (1), 41-44 (2005).</li><li>Bayat, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+heart+failure+after+myocardial+infarction+in+mice.">Progressive heart failure after myocardial infarction in mice.</a> Basic Res Cardiol. 97 (3), 206-213 (2002).</li></ol>

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...

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&#160;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&#160;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.

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			<td>Zeiss</td>
			<td>KL 1500 LCD</td>
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			<td>MRI system&#160;</td>
			<td>Bruker BioSpin, Ettlingen, Germany</td>
			<td>BioSpec 70/30</td>
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			<td>NatriumChloride 0.9%</td>
			<td>Baxter</td>
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			<td>Nocturnal Infrared Heat Lamp</td>
			<td>Zoo Med Laboratories, Inc.</td>
			<td>RS-75</td>
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			<td>ParaVision software&#160;</td>
			<td>Bruker BioSpin</td>
			<td>version 6.0.1&#160;</td>
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			<td>polyethylene tubing PE-10</td>
			<td>SAI Infusion technologies</td>
			<td>PE-10</td>
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			<td>polyethylene tubing PE-50</td>
			<td>SAI Infusion technologies</td>
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			<td>remote IRI tool (PMMA)</td>
			<td>homemade</td>
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			<td>Rodent Surgical Monitor</td>
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			<td>Segment v4.0</td>
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			<td>Self-gated gradient echo sequence&#160;</td>
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			<td>Ultraverse 035 PTA Dilatation Catheter: 5mm x 40mm, 17 ATM RBP balloon on 130 cm long catheter</td>
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			<td>Veet (depilatory creme)</td>
			<td>Reckitt Benkiser Healthcare</td>
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remotely triggered lad occlusion using a balloon catheter in spontaneously breathing mice

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.

Author Spotlight: A Novel Standardized Technique for Real-Time Biomedical Imaging of Acute Myocardial Injury

Remotely Triggered LAD Occlusion Using a Balloon Catheter in Spontaneously Breathing Mice

Scope of our research is to develop a new standardized technique for marine ischaemia-reperfusion injury. This allows us to obtain real-time biomedical imaging, creating new insights into the mechanistic effects of acute myocardial injury. The most challenging parts in the development of this model was to create a well-controlled direct access to the coronary vessels.This needs to happen in a living, spontaneously breeding animal one meter further away. Its capacity to reduce surgical trauma, to enhance control over ischemia time and to improve experimental consistency underscores its significance. Additionally, our technique allows us to obtain real-time imaging during acute myocardial infarction or myocardial ischemia-reperfusion injury.

Validation of the ability to induce ischemia has been performed by four tests: Triphenyl tetrazolium chloride (TTC) and Sirius Red (SR) staining, cardiac troponin I measurement, and Late gadolinium enhancement (LGE) MR imaging. Statistical significance was evaluated using the Mann-Whitney non-parametric test, considering the limited sample sizes. Statistical significance was attributed to p &#60; 0.05.
Acute experiments (TTC staining, n = 15) had no technical failure, and all animals ...

Read this Scientific Article Protocol about Author Spotlight: A Novel Standardized Technique for Real-Time Biomedical Imaging of Acute Myocardial Injury at JoVE.com

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 ...

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Left Anterior Descending Coronary Artery Ligation for Cardiac Monitoring in Mice

To begin, secure the base plate in a sideways position in front of the surgeon. After endotracheal intubation, place the anesthetized mouse on the base plate of the purpose-built ischemia reperfusion injury tool. Use tape to fix the mouse's tail and paws.Then fix the head using the 2-0 silk suture around the upper incisors, to prevent accidental movement. Insert the rectal thermal probe to monitor the body temperature. Use an infrared heating lamp above the mouse to maintain the temperature around 37 degrees Celsius.Position the ECG needle electrodes in all four paws to record heart rate and ECG wave form. After thoracotomy, place a rib retractor in the intercostal space, followed by a small piece of cotton on top of the lung to protect it. Next, use a tapered needle to pass a 7-0 polypropylene suture around the left anterior descending coronary artery.Remove the small piece of cotton and verify the continued ventilation of the left lung. Remove the rib retractor and cut off the needle. Position a segment of one millimeter PE-50 tubing over a 15 millimeter segment of PE-10 tubing.Direct both ends of the suture through the PE-10 tubing and shift the tubing with its thicker end against the heart. Apply fucidic acid gel to the exposed intercostal space and briefly obstruct the outflow of the ventilator to allow the remaining intrathoracic air to escape. Suture the pectoral muscle using a 5-0 polypropylene X stitch above and below the level of the PE tube.Close the skin with two 5-0 polypropylene X stitches above and two below the PE tube. Next, gradually reduce the tidal volume and respiratory rate to wean the mouse off mechanical ventilation. When the mouse starts breathing carefully disconnect the ventilator tube.

Development and Utilization of a Remote Ischemia-Reperfusion Injury Tool in Mice

After left anterior descending coronary artery preparation and extubating the anesthetized mouse, insert the vertical side part and the slits on the base plate of the purpose-built ischemia-reperfusion injury tool. Using a fine hook, retrieve the two suture ends and pull them through the central hole on the side part behind the balloon. Guide one end of the suture above and around the balloon, and subsequently navigate the opposite end under and around the balloon.Direct the ends of the suture through the slits on top of the side part. On the distal part of the suture, attach a 2.1 gram weight to keep each suture end in place. To induce ischemia, quickly inflate the balloon using the vascular balloon pump up to two bars.After locking the pump, confirm the balloon inflation and check that the weights are lifted. Confirm the ischemia by observing the change in the ECG trace. After 30 minutes, unlock the pump to stop the occlusion.Confirm that the balloon is deflated and weights are lowered. Cut the suture and tubing between the mouse and the side part, then carefully remove the side part. Apply gel at the insertion side of the tubing into the thorax.Using curved forceps, gently push the skin against the tube, and with another forceps, extract the tube. Cut any remaining suture close to the skin. Place an additional 5-0 polypropylene suture to seal the exit side of the tube and minimize the risk of air entry into the thorax.In this study, myocardial viability after ischemia-reperfusion injury was investigated. Triphenyl tetrazolium chloride staining differentiated viable from non-viable myocardial regions in mice. No significant differences were observed between the standard and remote ischemia-reperfusion injury techniques.Late gadolinium enhancement magnetic resonance imaging revealed no notable differences in the area at risk between standard and remote ischemia-reperfusion injury techniques. The sirius red staining differentiated the necrotic core from viable tissue. The calculated scar sizes revealed no notable differences in the area at risk between the two ischemia-reperfusion injury methods.Using a custom-built remote occlusion tool within the MRI scanner, contrast differences between non-infarcted and infarcted regions were observed. ECG traces before and after occlusion confirmed successful occlusion of the left anterior descending coronary artery.