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 immediately sacrificed after surgery. The survival experiments represented a four-week follow-up, including troponin measurement, MRI, and SR staining.
1. Anesthesia and endotracheal intubation
<ol>
	<li>Administer pre-anesthesia medication using a mix of ketamine (7.5 mg/kg; Nimatek 100 mg/mL), xylazine (1 mg/kg; Rompun, 20 mg/mL), and acepromazine (0.1 mg/kg; Placivet, 10 mg/mL) (KXA) (see Table of Materials) with 0.9 % saline diluent intraperitoneally. This allows a smoother and less invasive intubation.</li>
	<li>Place the mouse in an induction chamber (see Table of Materials) and invoke anesthesia using 2.5% isoflurane in pure oxygen with a flow rate of 1 L/min until loss of righting and toe pinch reflex.</li>
	<li>Transfer the animal to a heated pad (&#177;38 &#176;C) and position it supine with the head towards the surgeon. Fix the tail and front paws with tape on the pad. Ensure that the front limbs are not over-stretched, as this can compromise respiration.</li>
	<li>Place a 2-0 silk suture (see Table of Materials) around the upper incisors and tape the suture on the border of the pad to pull the mouse taut. If necessary, attach a needle holder to keep tension on the suture. Maintain the animal anesthetized using 2% isoflurane in 100% oxygen with a flow of 1 L/min using a nosecone.</li>
	<li>Place a light source just rostral of the sternum, remove the nose cone, and place the isoflurane supply on the ventilator. Intubate the mouse using forceps by lifting the tongue upwards and holding a self-made laryngoscope to lift the basal part of the tongue further and visualize the vocal cords.</li>
	<li>Place a self-made tracheal tube using an 18 G catheter on a dull needle between the vocal cords. Move up the catheter, extract the dull needle, and connect the tube with the mouse ventilator (see Table of Materials).</li>
	<li>Use a modified Y-shaped connector (see Table of Materials) to connect the intubation tube to the ventilator. The correct positioning of the tracheal tube can be confirmed by judging the symmetrical chest expansion.</li>
	<li>Set the tidal volume (mL) at bodyweight (BW) (g) x 7 and ventilation rate at 53.5 x (BW (g)-0.26) strokes per min, and adjust it to the body weight of a particular mouse if necessary6 (e.g., for a 25 g mouse the tidal volume is 175 mL at 140 strokes per min).</li>
	<li>Reduce the isoflurane to 1% in 100% oxygen, as the animal will stay anesthetized due to the premedication. However, regularly assess the depth of anesthesia by performing a toe pinch. Adapt Isoflurane concentration based on anesthesia depth.</li>
	<li>Apply a drop of ophthalmic ointment to prevent dryness while under anesthesia.</li>
</ol>
2. Installation of the IRI tool
NOTE: Remove all taping and place the animal on the base plate of the purpose-built IRI tool. Details on the properties of the IRI tool are mentioned in Supplementary File 1.
<ol>
	<li>Fixate the base plate of the purpose-built remote IRI tool in a sideways position in front of the surgeon. Fix the tail and paws using tape. Fix the head using the 2-0 silk suture around the upper incisors to prevent accidental movement (Figure 1).</li>
	<li>Insert the rectal thermal probe (see Table of Materials) to monitor the body temperature and use an infrared heating lamp above the animal to maintain the temperature around 37 &#176;C. Pay attention to sufficient distance between the animal and the lamp to avoid overheating.</li>
	<li>Position the ECG needle electrodes in all four paws to observe and record heart rate and ECG waveform. Secure the rectal probe and ECG electrodes to the platform using tape.</li>
	<li>Apply hair removal cream on the left side of the thorax to remove hair and create a clear surgical field. 
	NOTE: Limit the application time and pay attention to the complete removal of the cream to prevent chemical burns.</li>
</ol>
3. Thoracotomy
<ol>
	<li>Perform aseptic preparation of the skin using Betadine Solution, afterwards place a surgical drape on the left thorax to ensure a sterile environment. Use sterilized equipment to perform the procedure. Make a vertical skin incision at the mid portion of the pectoral muscle approximately 1 cm long and 2 mm away from the left sternal border.</li>
	<li>Spit the pectoral muscle to expose the ribs underneath. Avoid accidental injury to the vessel. If bleeding occurs, use cotton applicators to stop any bleeding.</li>
	<li>Visualize the ribs and identify the intercostal spaces by observing the inflating lung through the thin, semitransparent chest wall. Open the chest cavity using surgical scissors by making a 6-8 mm incision in the third intercostal space. Ensure the incision is a minimum of 2 mm from the sternal border where the internal thoracic artery is located.</li>
	<li>Insert a small rat eye speculum (see Table of Materials) in the intercostal space. This will function as a rib retractor (Figure 1A).</li>
	<li>Gently lift the pericardium with curved forceps and pull it apart.</li>
	<li>Place a small piece of cotton on top of the lung and push gently downwards.</li>
</ol>
4. LAD preparation
<ol>
	<li>The LAD appears bright red and runs from the aorta under the left auricle towards the apex. The ideal positioning for the ligature is approximately 2 mm lower than the tip of the left auricle. Use the pulmonary trunk as a marker to help identify the left auricle and optimize the LAD visualization using light influx (Figure 2).</li>
	<li>Use a tapered needle to pass a 7-0 polypropylene suture around the LAD. Do not place the needle too deep, as it can enter the left ventricle, nor too shallow, as it can damage the LAD.</li>
	<li>Remove the small piece of cotton and check if the left lung is still ventilated.</li>
	<li>Remove the rib retractor.</li>
	<li>Ensure the suture is of a minimum length of 15 cm on both ends. Cut off the needle.</li>
	<li>Place a piece of 1 mm PE-50 tubing over a piece of 15 mm PE-10 tubing (see Table of Materials). Guide both ends of the suture through the PE-10 tubing and shift the PE tubing with its thicker end against the heart.</li>
</ol>
5. Chest closure and extubating
<ol>
	<li>Apply some fusidic acid gel on the open intercostal space and gently allow the remaining intrathoracic air to escape by briefly obstructing the outflow of the ventilator. Additional fusidic acid gel can be added to the external part of the PE tube to prevent air entry into the thorax.</li>
	<li>Close the pectoral muscle with a 5-0 polypropylene X stitch above and below the level of the PE tube, and ensure the thickened part of the PE tube is under the level of the muscle.</li>
	<li>Close the skin with two 5-0 polypropylene X stitches above and two below the PE tube (Figure 1B and Figure 3).</li>
	<li>With a closed chest, the animal can breathe spontaneously again. Wean the animal from the ventilator by slowly reducing tidal volume and respiratory rate. When the animal starts breathing, spontaneously carefully disconnect the ventilator tube, but keep it in place until a stable breathing pattern is observed.</li>
	<li>Connect the isoflurane supply with a nosecone that is placed over the nose of the animal and fixed on the base plate.</li>
</ol>
6. Assembly of the remote IR tool
<ol>
	<li>Place the vertical side part in the base plate by inserting it in the slits.</li>
	<li>Use a fine hook to retrieve the two suture ends and pull them through the central hole in the side part behind the balloon (see Table of Materials).</li>
	<li>Guide one of the sutures ends above and around the balloon. Follow through with the other suture end under and around the balloon. Apply fusidic acid gel for smoother guiding of the suture.</li>
	<li>Guide the suture ends through the slits on top of the side part. On the distal part of the suture, apply a weight of 2.1 g to keep each suture end in place (Figure 1C and Figure 4). 
	NOTE: The configuration of appropriate weights is subject to change and must be tested beforehand. See Supplementary Figure 1 and Supplementary File 1 for details. Remote IRI tool design is provided in Supplementary Figure 2.</li>
</ol>
7. Induction of ischemia and reperfusion
<ol>
	<li>Induce ischemia by promptly inflating the balloon using the vascular balloon pump up to 2 bar. Lock the pump. Visually check if the balloon is inflated and the weights are lifted (Figure 4).</li>
	<li>Confirm the ischemia by observing the change in the ECG trace.</li>
	<li>Leave the balloon inflated for the set time according to the specific study protocol (30 min in the presented experiments).</li>
	<li>Stop the occlusion by simply unlocking the pump. Deflate the balloon carefully. Visually check if the balloon is deflated and weights are lowered.</li>
</ol>
8. Disassembly of the remote IR tool
<ol>
	<li>Cut the suture and tube between the animal and the side part and carefully remove the side part.</li>
	<li>Apply fusidic acid gel at the insertion site of the tubing into the thorax. Use curved forceps to gently push the skin against the tube and use another forceps to extract the tube. Cut any remaining suture close to the skin.</li>
	<li>Place an extra suture of 5-0 polypropylene to close the exit site of the tube and minimize the risk of air entry into the thorax.</li>
</ol>
9. End of narcosis and recovery
<ol>
	<li>Stop the isoflurane supply and allow the animal to wake up in a silent, warm, and oxygen-rich environment.</li>
	<li>Confirm the mouse is not in any respiratory distress by observing it until full recovery. Provide 0.3 mL of sterile saline (at body temperature) by intraperitoneal injection to prevent dehydration.</li>
	<li>For post-operative analgesia, administer an opioid analgesic (buprenorphine, 0.1 mg/kg) subcutaneously before the animal is ambulatory. Check the mice every 4-6 h for the next 24-48 h when buprenorphine is provided to ensure that the mice are responding to pain medications and to determine if additional pain medications are needed. Additional support should include a standard recommended supportive care diet gel or mash placed on the cage floor post-operatively.</li>
</ol>

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 authors have no conflict of interest to disclose.

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.

<table><tbody><tr><td>2-0 silk suture</td><td>Sharpoint Products</td><td>DC-2515N</td><td></td></tr><tr><td>5-0 polypropylene suture</td><td>Ethicon</td><td>8710H</td><td></td></tr><tr><td>7-0 polypropylene suture</td><td>Ethicon</td><td>8206H</td><td></td></tr><tr><td>ACCLARENT Balloon Inflation Device</td><td>Johnson&amp;amp;Johnson MedTech</td><td>BID30</td><td></td></tr><tr><td>Acepromazine</td><td>Kela</td><td></td><td></td></tr><tr><td>BD Vialon 18 G</td><td>BD</td><td>381347</td><td></td></tr><tr><td>BD Vialon 20 G</td><td>BD</td><td>381334</td><td></td></tr><tr><td>Betadine Solution</td><td>Purdue Pharma</td><td>25655-41-8</td><td></td></tr><tr><td>Buprenorphine (Buprenex Injectable)</td><td>Reckitt Benkiser Healthcare</td><td>NDC 12496-0757-1</td><td></td></tr><tr><td>Carp Zoom Styl Knijplood 2.1 g (lead fishing weights)</td><td>Visdeal.nl</td><td></td><td></td></tr><tr><td>Dumont #3 Forceps</td><td>Fine Science Tools</td><td>11231-30</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14040-10</td><td></td></tr><tr><td>Fucidine gel</td><td>Leo Pharma</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>NDC 5260-04-05</td><td></td></tr><tr><td>KD Mouse/Rat Eye Speculum</td><td>World Precision Instruments</td><td>501897</td><td></td></tr><tr><td>KD mouse/rat eye speculum</td><td>World Precision Instruments</td><td>501897</td><td></td></tr><tr><td>Ketamine</td><td>Dechra</td><td></td><td></td></tr><tr><td>Light source</td><td>Zeiss</td><td>KL 1500 LCD</td><td></td></tr><tr><td>MRI system&amp;nbsp;</td><td>Bruker BioSpin, Ettlingen, Germany</td><td>BioSpec 70/30</td><td></td></tr><tr><td>NatriumChloride 0.9%</td><td>Baxter</td><td></td><td></td></tr><tr><td>Nocturnal Infrared Heat Lamp</td><td>Zoo Med Laboratories, Inc.</td><td>RS-75</td><td></td></tr><tr><td>ParaVision software&amp;nbsp;</td><td>Bruker BioSpin</td><td>version 6.0.1&amp;nbsp;</td><td></td></tr><tr><td>polyethylene tubing PE-10</td><td>SAI Infusion technologies</td><td>PE-10</td><td></td></tr><tr><td>polyethylene tubing PE-50</td><td>SAI Infusion technologies</td><td>PE-50</td><td></td></tr><tr><td>remote IRI tool (PMMA)</td><td>homemade</td><td></td><td></td></tr><tr><td>Rodent Surgical Monitor</td><td>Indus instruments</td><td></td><td></td></tr><tr><td>Segment v4.0</td><td>Medviso, segment.heiberg.se</td><td>R12067</td><td></td></tr><tr><td>Self-gated gradient echo sequence&amp;nbsp;</td><td>Bruker BioSpin, Ettlingen, Germany</td><td>IntraGate, ParaVision 6.0.1</td><td></td></tr><tr><td>Slim Elongated Needle Holder</td><td>Fine Science Tools</td><td>12005-15</td><td></td></tr><tr><td>Sure-Seal Mouse/Rat Induction Chamber</td><td>World Precision Instruments</td><td>EZ-178</td><td></td></tr><tr><td>Tubing Connectors, Poly, Y Shape</td><td>Westlab</td><td>072025-0001</td><td></td></tr><tr><td>Ultraverse 035 PTA Dilatation Catheter: 5mm x 40mm, 17 ATM RBP balloon on 130 cm long catheter</td><td>Bard Peripheral Vascular, Inc.</td><td>00801741092671</td><td></td></tr><tr><td>Veet (depilatory creme)</td><td>Reckitt Benkiser Healthcare</td><td></td><td></td></tr><tr><td>Ventilator, MiniVent Model 845</td><td>Hugo Sachs</td><td>73-0043</td><td></td></tr><tr><td>Vidisic</td><td>BAUSCH &amp;amp; LOMB PHARMA</td><td>685313</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td></td><td></td></tr></tbody></table>

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.

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

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

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.

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 ...

remotely-triggered-lad-occlusion-using-balloon-catheter-spontaneously

Research

JoVE Journal

Medicine

870 Views.  KU Leuven. 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.

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

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...