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.

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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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 ...

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854 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

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

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. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...