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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#160;</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&#160;</td>
			<td>Bruker BioSpin</td>
			<td>version 6.0.1&#160;</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&#160;</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 &#38; 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.

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 ...

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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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Research

JoVE Journal

Medicine

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.

622 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.

Closed-Chest Technique for Murine Myocardial Ischemia-Reperfusion Injury

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