Name: Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation
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This work received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

All studies were approved by the Institutional Animal Care and Use Committee at Washington University School of Medicine. Animals received humane care in compliance with the Guide for the care and use of laboratory animals, 8th edition21 prepared by the National Academy of Sciences and published by the National Institutes of Health, and the Principles of laboratory animal care formulated by the National Society for Medical Research.
1. Anesthesia and in...

Mouse Model for Studying Lung Ischemia-Reperfusion Injury

<ol>
	<li>Bharat, 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=Immunological+link+between+primary+graft+dysfunction+and+chronic+lung+allograft+rejection.">Immunological link between primary graft dysfunction and chronic lung allograft rejection.</a> Ann Thorac Surg. 86 (1), 189-195 (2008).</li><li>Daud, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+immediate+primary+lung+allograft+dysfunction+on+bronchiolitis+obliterans+syndrome.">Impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome.</a> Am J Respir Crit Care Med. 175 (5), 507-513 (2007).</li><li>Halazun, K. J., Al-Mukhtar, A., Aldouri, A., Willis, S., Ahmad, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warm+ischemia+in+transplantation:+search+for+a+consensus+definition.">Warm ischemia in transplantation: search for a consensus definition.</a> Transplant Proc. 39 (5), 1329-1331 (2007).</li><li>Warnecke, G., 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=Warm+or+cold+ischemia+in+animal+models+of+lung+ischemia-reperfusion+injury:+is+there+a+difference+.">Warm or cold ischemia in animal models of lung ischemia-reperfusion injury: is there a difference .</a> Thorac Cardiovasc Surg. 52 (3), 174-179 (2004).</li><li>Hasenauer, 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=Effects+of+cold+or+warm+ischemia+and+ex-vivo+lung+perfusion+on+the+release+of+damage+associated+molecular+patterns+and+inflammatory+cytokines+in+experimental+lung+transplantation.">Effects of cold or warm ischemia and ex-vivo lung perfusion on the release of damage associated molecular patterns and inflammatory cytokines in experimental lung transplantation.</a> J Heart Lung Transplant. 40 (9), 905-916 (2021).</li><li>Iskender, 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=Effects+of+warm+versus+cold+ischemic+donor+lung+preservation+on+the+underlying+mechanisms+of+injuries+during+ischemia+and+reperfusion.">Effects of warm versus cold ischemic donor lung preservation on the underlying mechanisms of injuries during ischemia and reperfusion.</a> Transplantation. 102 (5), 760-768 (2018).</li><li>Santos, P., Teixeira, P. J. Z., Moraes Neto, D. M., Cypel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donation+after+circulatory+death+and+lung+transplantation.">Donation after circulatory death and lung transplantation.</a> J Bras Pneumol. 48 (2), e20210369 (2022).</li><li>Moskowitzova, K., 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=Mitochondrial+transplantation+enhances+murine+lung+viability+and+recovery+after+ischemia-reperfusion+injury.">Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.</a> Am J Physiol Lung Cell Mol Physiol. 318 (1), L78-L88 (2020).</li><li>Nakata, K., 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=Functional+blockage+of+S100A8/A9+ameliorates+ischemia-reperfusion+injury+in+the+lung.">Functional blockage of S100A8/A9 ameliorates ischemia-reperfusion injury in the lung.</a> Bioengineering (Basel). 9 (11), 673 (2022).</li><li>Nakata, K., 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=Protective+effects+of+anti-HMGB1+monoclonal+antibody+on+lung+ischemia+reperfusion+injury+in+mice.">Protective effects of anti-HMGB1 monoclonal antibody on lung ischemia reperfusion injury in mice.</a> Biochem Biophys Res Commun. 573, 164-170 (2021).</li><li>Wang, Q., Li, Y., Wu, C., Wang, T., Wu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin-1+inhibition+exacerbates+ischemia-reperfusion-induced+lung+injury+in+mouse.">Aquaporin-1 inhibition exacerbates ischemia-reperfusion-induced lung injury in mouse.</a> Am J Med Sci. 365 (1), 84-92 (2023).</li><li>Gielis, J. F., 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=A+murine+model+of+lung+ischemia+and+reperfusion+injury:+tricks+of+the+trade.">A murine model of lung ischemia and reperfusion injury: tricks of the trade.</a> J Surg Res. 194 (2), 659-666 (2015).</li><li>Zhang, X., 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=Mitogen-activated+protein+kinases+regulate+HO-1+gene+transcription+after+ischemia-reperfusion+lung+injury.">Mitogen-activated protein kinases regulate HO-1 gene transcription after ischemia-reperfusion lung injury.</a> Am J Physiol Lung Cell Mol Physiol. 283 (4), L815-L829 (2002).</li><li>Sharma, A. K., 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=Adenosine+A2A+receptor+activation+on+CD4++T+lymphocytes+and+neutrophils+attenuates+lung+ischemia-reperfusion+injury.">Adenosine A2A receptor activation on CD4+ T lymphocytes and neutrophils attenuates lung ischemia-reperfusion injury.</a> J Thorac Cardiovasc Surg. 139 (2), 474-482 (2010).</li><li>Yang, Z., Sharma, A. K., Linden, J., Kron, I. L., Laubach, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+lymphocytes+mediate+acute+pulmonary+ischemia-reperfusion+injury.">CD4+ T lymphocytes mediate acute pulmonary ischemia-reperfusion injury.</a> J Thorac Cardiovasc Surg. 137 (3), 695-702 (2009).</li><li>Sayah, D. 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=Neutrophil+extracellular+traps+are+pathogenic+in+primary+graft+dysfunction+after+lung+transplantation.">Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation.</a> Am J Respir Crit Care Med. 191 (4), 455-463 (2015).</li><li>Okazaki, 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=A+mouse+model+of+orthotopic+vascularized+aerated+lung+transplantation.">A mouse model of orthotopic vascularized aerated lung transplantation.</a> Am J Transplant. 6 (7), 1672-1679 (2007).</li><li>Hsiao, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen-derived+classical+monocytes+mediate+lung+ischemia-reperfusion+injury+through+IL-1beta.">Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1beta.</a> J Clin Invest. 128 (7), 2833-2847 (2018).</li><li>Sharma, A. K., Mulloy, D. P., Le, L. T., Laubach, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+mediates+synergistic+effects+of+IL-17+and+TNF-alpha+on+CXCL1+expression+by+epithelial+cells+after+lung+ischemia-reperfusion.">NADPH oxidase mediates synergistic effects of IL-17 and TNF-alpha on CXCL1 expression by epithelial cells after lung ischemia-reperfusion.</a> Am J Physiol Lung Cell Mol Physiol. 306 (1), L69-L79 (2014).</li><li>Zanotti, G., 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=Novel+critical+role+of+Toll-like+receptor+4+in+lung+ischemia-reperfusion+injury+and+edema.">Novel critical role of Toll-like receptor 4 in lung ischemia-reperfusion injury and edema.</a> Am J Physiol Lung Cell Mol Physiol. 297 (297), L52-L63 (2009).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. , (2011).</li><li>Saito, 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=Pirfenidone+alleviates+lung+ischemia-reperfusion+injury+in+a+rat+model.">Pirfenidone alleviates lung ischemia-reperfusion injury in a rat model.</a> J Thorac Cardiovasc Surg. 158 (1), 289-296 (2019).</li><li>Tanaka, S., 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=Protective+effects+of+Imatinib+on+ischemia/reperfusion+injury+in+rat+lung.">Protective effects of Imatinib on ischemia/reperfusion injury in rat lung.</a> Ann Thorac Surg. 102 (5), 1717-1724 (2016).</li><li>Kreisel, 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=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc Natl Acad Sci U S A. 107 (42), 18073-18078 (2010).</li><li>Koletsis, E., 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=In+situ+cooling+in+a+lung+hilar+clamping+model+of+ischemia-reperfusion+injury.">In situ cooling in a lung hilar clamping model of ischemia-reperfusion injury.</a> Exp Biol Med (Maywood). 231 (8), 1410-1420 (2006).</li><li>Hermsen, 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=Genomic+landscape+of+rat+strain+and+substrain+variation.">Genomic landscape of rat strain and substrain variation.</a> BMC Genomics. 16 (1), 357 (2015).</li><li>Beck, J. 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=Genealogies+of+mouse+inbred+strains.">Genealogies of mouse inbred strains.</a> Nat Genet. 24 (1), 23-25 (2000).</li><li>Liao, W. 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=A+mouse+model+of+orotracheal+intubation+and+ventilated+lung+ischemia+reperfusion+surgery.">A mouse model of orotracheal intubation and ventilated lung ischemia reperfusion surgery.</a> J Vis Exp. (187), 64383 (2022).</li><li>Murata, T., Nakazawa, H., Mori, I., Ohta, Y., Yamabayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+after+a+two-hour+period+of+pulmonary+artery+occlusion+causes+pulmonary+necrosis.">Reperfusion after a two-hour period of pulmonary artery occlusion causes pulmonary necrosis.</a> Am Rev Respir Dis. 146 (4), 1048-1053 (1992).</li><li>Ukita, 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=A+large+animal+model+for+pulmonary+hypertension+and+right+ventricular+failure:+Left+pulmonary+artery+ligation+and+progressive+main+pulmonary+artery+banding+in+sheep.">A large animal model for pulmonary hypertension and right ventricular failure: Left pulmonary artery ligation and progressive main pulmonary artery banding in sheep.</a> J Vis Exp. 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We describe a hilar clamp technique that involves application of a slipknot on the left hilum which occludes the pulmonary artery and veins and bronchus to induce warm ischemia followed by reperfusion. After hilar clamping, the left lung can be harvested for a variety of experimental techniques such as histology, flow cytometry, bulk or single cell sequencing, and quantitative polymerase chain reaction. Additionally, the blood and spleen may be used to study systemic effects, while the non-ischemic right lung may serve a...

<a href="https://app.jove.com/t/6603">This corrects</a> the article <a href="https://dx.doi.org/10.3791/66232">10.3791/66232</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/66232">Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury</a>. The Protocol and Representative Results&#160;sections were&#160;updated.

Erratum: Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

IRI during organ transplantation is a major risk factor for primary graft dysfunction and later episodes of graft rejection1,2. During transplantation, warm ischemia time is defined as the period of time from donor aortic cross-clamp to initiation of cold perfusion and from organ removal from ice to organ implantation. Cold storage time is defined as the period of time from the start of cold perfusion to the removal of the organ from ice3. Warm ischemia is more deleterious for later organ function than cold ischemia4,5,6, and its underlying mechanisms warrant further study in pre-clinical models. Additionally, organ transplantation from donation after cardiac death (DCD) is associated with longer warm ischemic times than traditional donation after brain death (DBD)7. While the use of DCD donors can expand the donor pool and increase lung utilization, further pre-clinical studies to evaluate the effects of warm ischemia on post-transplant lung function are needed. Below, we describe a model of warm IRI in mice via the left pulmonary hilar clamp.
Several animal models of lung hilar clamping have been developed and adapted over the last several years and may include the use of an atraumatic microvascular clamp8,9,10,11,12,13, a Rumel tourniquet14,15, or suture ligation16 as the hilar clamp. The crux of the hilar clamp is that it must be reversible and cause minimal or no damage to the hilar structures so that reperfusion can be achieved. Here, we describe our hilar clamp technique in mice that involves reversible suture ligation of the left pulmonary hilum with a slipknot. This method occludes the pulmonary arterial inflow, venous outflow, and airflow in and out of the mainstem bronchus. The main benefit of a slipknot over a vascular clamp, clip, or tourniquet is that the chest can be closed during prolonged periods of ischemia, thereby minimizing insensible fluid and heat loss in the mouse. We provide a protocol for obtaining reliable arterial blood gas (ABG) measurements and for measuring cell extravasation after hilar clamping.
This hilar clamp technique holds an important place in the wider study of lung transplantation. Compared to small animal models of orthotopic lung transplantation, the hilar clamp technique can isolate the effects of IRI without the addition of surgical anastomotic trauma or allogenicity17. Additionally, the hilar clamp technique can be more easily and quickly mastered than the mouse lung transplant. In fact, using hilar clamp techniques, several important mechanisms in the pathogenesis of IRI have been identified in the past decade, such as TLR4, NADPH oxidase, and adenosine A2A receptor14,18,19,20. In the following protocol, we present a reliable, teachable, and reproducible method of hilar clamping as a tool for studying lung IRI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medications</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% povidone-iodine solution</td>
			<td>Aplicare</td>
			<td>NDC 52380-0126-2</td>
			<td>For disinfectant</td>
		</tr>
		<tr>
			<td>Buprenorphine 1.3 mg/mL</td>
			<td>Fidelis Animal Health</td>
			<td>NDC 86084-100-30</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Cronus Pharma</td>
			<td>NDC 69043-027-18</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Heparin 1000 units/mL</td>
			<td>Sagent</td>
			<td>NDC 25021-404-01</td>
			<td>For obtaining arterial blood</td>
		</tr>
		<tr>
			<td>Isoflurane 1%-1.5%</td>
			<td>Sigma Aldrich</td>
			<td>26675-46-7</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride 100 mg/mL</td>
			<td>Vedco</td>
			<td>NDC 50989-996-06</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Puralube Vet eye ointment</td>
			<td>Medi-Vet.com</td>
			<td>11897</td>
			<td>To prevent eye dessiccation</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Akorn</td>
			<td>NDC 59399-110-20</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Tools and Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Argent High Temp Fine Tip Cautery Pen</td>
			<td>McKesson</td>
			<td>231</td>
			<td>To coagulate blood vessels</td>
		</tr>
		<tr>
			<td>Curved mosquito clamp</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Fine curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>15040-11</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Intubation clamp set-up</td>
			<td>Fine Science Tools</td>
			<td>18374-44, 18144-30</td>
			<td>For holding mouse vertically by the tongue during intubation. See Supplementary Figure 1A.&#160;</td>
		</tr>
		<tr>
			<td>Magnetic rib retractors</td>
			<td>Fine Science Tools</td>
			<td>18200-01, 18200-10</td>
			<td>For retraction of thoracotomy. Magnetic fixator and retractor should be connected by micro latex tubing below.</td>
		</tr>
		<tr>
			<td>Optical Grade Plastic Optical Fiber Unjacketed, 500&#956;m</td>
			<td>Edmund Optics</td>
			<td>02-532</td>
			<td>To make the introducer for the endotracheal tube. See Supplemental Figure 1B. A 1.5-inch length of this optical fiber should have a piece of silk tape secured to one end. It can then be used as an introducer for the endotracheal tube. The end of the introducer should be curves slightly.</td>
		</tr>
		<tr>
			<td>Power Pro Ultra clipper</td>
			<td>Oster</td>
			<td>078400-020-001</td>
			<td>To clip hair</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14370-22</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Small animal heating pad</td>
			<td>K&#38;H Pet Products</td>
			<td>Thermo-Peep Heated Pad</td>
			<td>To maintain normothermia</td>
		</tr>
		<tr>
			<td>Small animal ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0000</td>
			<td>For ventilation (TV 0.35 cc, PEEP 1 cm H2O, RR 100-105/min, FiO2 100%)</td>
		</tr>
		<tr>
			<td>Spearit Micro Latex Rubber Tubing (1/8 in outside diameter, 1/16 in inside diameter)</td>
			<td>Amazon.com</td>
			<td>https://www.amazon.com/Rubber-Tubing-CONTINUOUS-Select-Length/dp/B00H4MT7V0?th=1</td>
			<td>For retraction of thoracotomy</td>
		</tr>
		<tr>
			<td>Stat Profile Prime Critical Care Blood Gas Analyzer</td>
			<td>Nova Biomedical</td>
			<td>https://novabiomedical.com/prime-plus-critical-care-blood-gas-analyzer/index.php?gad=1&#38;gclid=Cj0KCQjwmICoBhDx 
			ARIsABXkXlInZX--R3ezBkc304nS_GVGI9Z2T3Esr33 
			2aM8WGPiUVhicPQZ 
			Wj2AaAqhDEALw_wcB&#160;&#160;</td>
			<td>For retraction of thoracotomy</td>
		</tr>
		<tr>
			<td>Straight clamp</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Straight forceps</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Wild Heerbrugg</td>
			<td>no longer produced</td>
			<td>For intubation and surgical procedure; recommend replacement with Leica surgical microscopes</td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#189; cc syringe with &#189; inch 29G needle</td>
			<td>McKesson</td>
			<td>942665</td>
			<td>For injecting ketamine/xylazine intraperitoneally</td>
		</tr>
		<tr>
			<td>&#189; inch 31G needle on a 1 cc tuberculin syringe</td>
			<td>McKesson</td>
			<td>16-SNT1C2705</td>
			<td>For aspiration of arterial blood from left ventricle</td>
		</tr>
		<tr>
			<td>1-inch 20G IV catheter</td>
			<td>Terumo</td>
			<td>SROX2025CA</td>
			<td>For endotracheal tube (ETT)</td>
		</tr>
		<tr>
			<td>1-inch silk tape</td>
			<td>Durapore</td>
			<td>3M ID 7100057168</td>
			<td>To tape ETT to nose and to secure limbs</td>
		</tr>
		<tr>
			<td>3/10 cc syringe with 5/16 inch 31G needle</td>
			<td>McKesson</td>
			<td>102-SN310C31516P</td>
			<td>For antibody injection into the inferior vena cava</td>
		</tr>
		<tr>
			<td>6-0 monofilament suture on a P-10 needle</td>
			<td>McKesson</td>
			<td>S697GX</td>
			<td>For closure of thoracotomy, muscle layer, and skin</td>
		</tr>
		<tr>
			<td>6-0 silk tie</td>
			<td>Surgical Specialties Look</td>
			<td>SP102</td>
			<td>To make slipknot for hilar clamp</td>
		</tr>
		<tr>
			<td>Pointed cotton-tipped applicators</td>
			<td>Solon</td>
			<td>56225</td>
			<td>To manipulate lung and for blunt dissection</td>
		</tr>
	</tbody>
</table>

murine left pulmonary hilar clamp model of lung ischemia reperfusion injury

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows for&#160;detailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

We are interested in studying the effects of ischemia-reperfusion injury on outcomes following lung transplantation with the goal of understanding underlying cellular and molecular mechanisms. Neutrophils that enter the lung contribute to ischemia-reperfusion injury, or IRI. We've recently discovered that B cells proceed neutrophils in entering the lung, and they play a role in the recruitment of neutrophils following IRI.Left lung transplantation in the mouse is technically challenging, and requires full-time, dedicated training over several months to achieve consistent results. Additionally, it introduces allergenicity and surgical anastomotic trauma. Thus, we need a more accessible technique that can isolate the study of ischemia-reperfusion injury.Using our hilar clamp model, we have found that B-cells enter the lung early after ischemia-reperfusion injury, and they recruit classical monocytes in a B-cell receptor into TLR4 dependent fashion. This ultimately results in increased neutrophil extravasation into the lung. Our protocol involves reversible suture ligation of the left pulmonary hilum with a slip knot.The pliable and unobtrusive nature of the suture allows for chest closure, which is useful for studying longer periods of warm ischemia, as chest closure minimizes heat and insensible fluid losses in the mouse.

After left hilar clamping, partial pressure of oxygenation in the arterial blood (PaO2) attributed to the left lung is ~100 mmHg, significantly lower compared to the ~500 mmHg following sham thoracotomy (Figure 7A, n=6-7). Of note, sham thoracotomies were performed in B6 mice with ABG measurement taken after 4 min of right hilar clamping, representing values attributed to the left lung alone. H&#38;E staining of the hilar clamped left lung demonstrated infiltrating inflammatory ce...

Watch this Scientific Journal Video about Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation at JoVE.com

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse models.

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft ...

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