Male Sprague-Dawley rats (180-270 g body weight) were purchased commercially (e.g., Envigo) and were housed under pathogen-free conditions at The Ohio State University Animal Facility. All procedures were humanely performed according to the NIH and the National Research Council&#39;s Guide for the Humane Care and Use of Laboratory Animals and with the approval of The Ohio State University Institutional Animal Care and Use Committee (IACUC Protocol # 2012A00000135-R2).
1. Initial setup
<ol>
	...

<ol>
	<li>Mizuta, T., Kawaguchi, A., Nakahara, K., Kawashima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+rat+lung+transplantation+using+a+cuff+technique.">Simplified rat lung transplantation using a cuff technique.</a> Journal of Thoracic and Cardiovascular Surgery. 97 (4), 578-581 (1989).</li><li>Zhai, 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=Simplified+rat+lung+transplantation+by+using+a+modified+cuff+technique.">Simplified rat lung transplantation by using a modified cuff technique.</a> Journal of Investigative Surgery. 21 (1), 33-37 (2008).</li><li>Goto, 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=Simplified+rat+lung+transplantation+using+a+new+cuff+technique.">Simplified rat lung transplantation using a new cuff technique.</a> Annals of Thoracic Surgery. 93 (6), 2078-2080 (2012).</li><li>Guo, 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=Improvements+of+surgical+techniques+in+a+rat+model+of+an+orthotopic+single+lung+transplant.">Improvements of surgical techniques in a rat model of an orthotopic single lung transplant.</a> European Journal of Medical Research. 18, 1 (2013).</li><li>Tian, D., Shiiya, H., Sato, M., Nakajima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+lung+transplantation+model:+modifications+of+the+cuff+technique.">Rat lung transplantation model: modifications of the cuff technique.</a> Annals of Translational Medicine. 8 (6), 407 (2020).</li><li>Rajab, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anastomotic+techniques+for+rat+lung+transplantation.">Anastomotic techniques for rat lung transplantation.</a> World Journal of Transplantation. 8 (2), 38-43 (2018).</li><li>Reis, A., Giaid, A., Serrick, C., Shennib, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+outcome+of+rat+lung+transplantation+with+modification+of+the+nonsuture+external+cuff+technique.">Improved outcome of rat lung transplantation with modification of the nonsuture external cuff technique.</a> Journal of Heart and Lung Transplantation. 14 (2), 274-279 (1995).</li><li>Sugimoto, 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=Experimental+orthotopic+lung+transplantation+model+in+rats+with+cold+storage.">Experimental orthotopic lung transplantation model in rats with cold storage.</a> Surgery Today. 39 (7), 641-645 (2009).</li><li>Santana Rodriguez, N., 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=Technical+modifications+of+the+orthotopic+lung+transplantation+model+in+rats+with+brain-dead+donors.">Technical modifications of the orthotopic lung transplantation model in rats with brain-dead donors.</a> Archivos de Bronconeumolog&#237;a. 47 (10), 488-494 (2011).</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> Journal of Surgical Research. 194 (2), 659-666 (2015).</li><li>Rajab, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+lung+transplantation+in+the+rat.">Techniques for lung transplantation in the rat.</a> Experimental Lung Research. 45 (9-10), 267-274 (2019).</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>Lin, 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=Five-year+update+on+the+mouse+model+of+orthotopic+lung+transplantation:+Scientific+uses,+tricks+of+the+trade,+and+tips+for+success.">Five-year update on the mouse model of orthotopic lung transplantation: Scientific uses, tricks of the trade, and tips for success.</a> Journal of Thoracic Disease. 4 (3), 247-258 (2012).</li><li>Song, 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=Standardization+of+bronchoalveolar+lavage+method+based+on+suction+frequency+number+and+lavage+fraction+number+using+rats.">Standardization of bronchoalveolar lavage method based on suction frequency number and lavage fraction number using rats.</a> Toxicological Research. 26 (3), 203-208 (2010).</li><li>Chang, J. E., Kim, H. J., Yi, E., Jheon, S., Kim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+ischemia-reperfusion+injury+in+a+rat+lung+transplantation+model+by+low-concentration+GV1001.">Reduction of ischemia-reperfusion injury in a rat lung transplantation model by low-concentration GV1001.</a> European Journal of Cardio-Thoracic Surgery. 50 (5), 972-979 (2016).</li><li>De Backer, J. 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=Study+of+the+variability+in+upper+and+lower+airway+morphology+in+Sprague-Dawley+rats+using+modern+micro-CT+scan-based+segmentation+techniques.">Study of the variability in upper and lower airway morphology in Sprague-Dawley rats using modern micro-CT scan-based segmentation techniques.</a> Anatomical Record. 292 (5), 720-727 (2009).</li><li>Nelson, 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=Method+of+isolated+ex+vivo+lung+perfusion+in+a+rat+model:+lessons+learned+from+developing+a+rat+EVLP+program.">Method of isolated ex vivo lung perfusion in a rat model: lessons learned from developing a rat EVLP program.</a> Journal of Visualized Experiments. (96), e52309 (2015).</li><li>Suzuki, H., Fan, L., Wilkes, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+obliterative+bronchiolitis+in+a+murine+model+of+orthotopic+lung+transplantation.">Development of obliterative bronchiolitis in a murine model of orthotopic lung transplantation.</a> Journal of Visualized Experiments. (65), e3947 (2012).</li><li>Jia, Y., 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=Treatment+of+acute+lung+injury+by+targeting+MG53-mediated+cell+membrane+repair.">Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.</a> Nature Communications. 5, 4387 (2014).</li><li>Kim, J. L., 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=Pegylated-Catalase+Is+Protective+in+Lung+Ischemic+Injury+and+Oxidative+Stress.">Pegylated-Catalase Is Protective in Lung Ischemic Injury and Oxidative Stress.</a> Annals of Thoracic Surgery. , (2020).</li><li>Beal, E. 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=D-Ala(2),+D-Leu(5)]+Enkephalin+Improves+Liver+Preservation+During+Normothermic+Ex+Vivo+Perfusion.">D-Ala(2), D-Leu(5)] Enkephalin Improves Liver Preservation During Normothermic Ex Vivo Perfusion.</a> Journal of Surgical Research. 241 (2), 323-335 (2019).</li><li>Akateh, 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=Intrahepatic+Delivery+of+Pegylated+Catalase+Is+Protective+in+a+Rat+Ischemia/Reperfusion+Injury+Model.">Intrahepatic Delivery of Pegylated Catalase Is Protective in a Rat Ischemia/Reperfusion Injury Model.</a> Journal of Surgical Research. 238, 152-163 (2019).</li></ol>

In this report, we have intervened at several critical steps in a rat lung transplantation protocol to optimize the procedure.&#160;While various cuffing techniques for rat lung transplantation has been reported1,2,3,4,5,6,7,8,9...

For over three decades, researchers have been modifying and improving rat lung transplantation models so that the data generated are more consistent and more reflective of the actual clinical condition. In our laboratory&#39;s time performing this model, we have determined four areas of improvement: cuffing techniques for anastomoses, identification of the recipient&#39;s 4th intercostal space, lung inflation and wound closure during the recipient&#39;s procedure, and the harvesting of samples for analysis.
Cuffing technique modifications for anastomoses can improve the entire transplantation procedure by shortening handling time of the donor lung1,2,3,4,5,6 and making the anastomosis procedure faster and technically easier for the microsurgeon. While it is critical to use the proper sized cuffs to supply the necessary blood and airflow to the transplanted lung, there is limited guidance regarding how one should choose the size of cuffs for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br)5,7,8,9. Since the diameters of the PV, PA, and Br are related to the body weight of the donor and recipient rats, we propose that the cuff size be based on body weight. This report provides a size guide for cuffs based on a rat&#39;s body weight (180 g to over 270 g) that serves to optimize blood and air supply to the transplanted lung (Table 1).
While a newer microsurgeon can successfully and easily procure a donor lung during the donor procedure, transplanting the lung during the recipient&#39;s procedure is more complicated and is dependent on the microsurgeon&#39;s experience. Attempts to find the 4th intercostal space to access the recipient&#39;s left lung is one of the more difficult steps that holds some subjectivity and can increase the procedure time. Therefore, we introduce a simple and objective method to assist in the identification of the 4th intercostal space location by using chest measurements and the palpitations of the heart to find the correct area chest wall to dissect4,5,6,10,11,12.
We also propose an improvement to donor lung inflation, which is a potential source of injury to the organ. The donor lung is deflated until reperfusion starts. While suturing the 4th intercostal space, the donor lung is commonly inflated by increasing PEEP from 2 cmH2O to 6 cmH2O. In order to minimize lung injury from overinflation, we propose a technique where three 6-0 nylon sutures are placed around the 4th rib inferior to the 5th rib with simple double knots. When it is time for wound closure, the ends of the three sutures are held with hemostats in both hands, the wound is closed all at once by pulling up on each side, and PEEP is immediately reduced to 2 cmH2O. In this way, the lung is allowed to expand for the shortest time possible10.
At the conclusion of an experiment, the researcher often wants to collect many types of samples for many types of analysis from each transplant. For example, snap frozen tissue, formalin fixed tissue, tissue for wet-to-dry weight ratio to determine pulmonary edema, and bronchoalvelolar lavage (BAL) fluid can all be used to assess how well the transplant went. The traditional method of collecting BAL fluid allows for a mixed pooled sample from both the recipient&#39;s native lobes and the donor&#39;s transplanted lobe13,14,15. To overcome this, we present a method of clamping the hilar areas that can yield more precise insight into the transplanted and native lungs&#39; condition. Additionally, the volume of PBS used to collect BAL fluid from each side of the lungs is important to consider because BAL fluid contains numerous soluble factors such as cytokines and chemokines that are measured by concentration. Normalizing the volume of the fluid instilled to the estimated volume of lung capacity can help with comparison. With four lobes on the right side and one lobe on the left side, each of the rat&#39;s five lobes has a different volume and surface area16. According to a previous study on volume measurement of lung lobes by Backer et al., of the total volume of the whole lung the volume of the right lobes is 63% (4400 mm3) and the left lobe is 37% (2500 mm3). Therefore, we recommend that the volume of PBS used to collect BAL fluid should be calculated as twice the tidal volume (7.2 mL/kg) multiplied by 63% for the right lung and 37% for the left lung. By using this approach, one can better control for variables like body weight and timing10,16.
In all, in this report we will demonstrate a few modifications to the standard experimental model of rat lung transplantation that can make the procedure more efficient and increase the capability of generating more accurate and plentiful data from each experiment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 Gauge angio-catheter</td>
			<td>BD</td>
			<td>382277</td>
			<td></td>
		</tr>
		<tr>
			<td>14 Gauge angio-catheter</td>
			<td>B. Braun</td>
			<td>4251717-02</td>
			<td></td>
		</tr>
		<tr>
			<td>16 Gauge angio-catheter</td>
			<td>B. Braun</td>
			<td>4252586-02</td>
			<td></td>
		</tr>
		<tr>
			<td>18 Gauge angio-catheter</td>
			<td>B. Braun</td>
			<td>4251679-02</td>
			<td></td>
		</tr>
		<tr>
			<td>20 Gauge angio-catheter</td>
			<td>B. Braun</td>
			<td>4252527-02</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 silk suture</td>
			<td>Surgical Specialties Corp.</td>
			<td>SP116</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 nylon suture</td>
			<td>AD Surgical</td>
			<td>S-N618R13</td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 nylon suture</td>
			<td>AD Surgical</td>
			<td>S-N718SP13</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 nylon suture</td>
			<td>AD Surgical</td>
			<td>XXS-N807T6</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine Spray</td>
			<td>Avrio Health L.P</td>
			<td>UPC 367618160039</td>
			<td></td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>VWR</td>
			<td>MSPP-023326</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo micro dissecting spring scissors</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-5668</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery</td>
			<td>Macan</td>
			<td>MV-7A</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal intubation kit</td>
			<td>Kent Scientific</td>
			<td>ETI-MSE</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsted-mosquito hemostat</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-7112</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresnius Medical Care</td>
			<td>C504701</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Life Technologies</td>
			<td>B328446</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol Swabs</td>
			<td>BD</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hikma Pharmaceuticals PLC</td>
			<td>NDC 0413-9505-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dieffenbach Bulldog Clamp</td>
			<td>World Precision Instruments</td>
			<td>WPI14117</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder/Forceps, Curved</td>
			<td>Micrins</td>
			<td>MI1542</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder/Forceps, Straight</td>
			<td>Micrins</td>
			<td>MI1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfadex Plus (Organ Preservation Solution)</td>
			<td>XVIVO Perfusion AB</td>
			<td>REF# 19950</td>
			<td></td>
		</tr>
		<tr>
			<td>PhysioSuite</td>
			<td>Kent Scientific</td>
			<td>PS-MSTAT-RT</td>
			<td>Used to check SpO2 and heartbeat</td>
		</tr>
		<tr>
			<td>Retractor</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-6560</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>PP Pharmaceuticals LLC</td>
			<td>NDC 63323-186-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Small Animal Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-MVG-Module</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile&#160; Cotton Gauze Pad</td>
			<td>Fisherbrand</td>
			<td>22-415-469</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>Leica</td>
			<td>M500-N w/ OHS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 5mL</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas-Tubingen Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Korn Pharmaceuticals Corp</td>
			<td>NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Yasargil Clamp</td>
			<td>Aesculap, Inc</td>
			<td>FT351T</td>
			<td>Used to clamp bronchus</td>
		</tr>
		<tr>
			<td>Yasargil Clamp</td>
			<td>Aesculap, Inc</td>
			<td>FT261T</td>
			<td>Used to clamp hilum</td>
		</tr>
		<tr>
			<td>Yasargil Clamp Applicator</td>
			<td>Aesculap, Inc</td>
			<td>FT484T</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat lung transplantation model of warm ischemia/reperfusion injury: optimizations to improve outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

In order to measure pulmonary edema, the wet-to-dry weight ratio was calculated. The donor&#39;s native lobe, the transplanted lobe, and the recipient&#39;s native lobe were collected as described in the protocol and weighed immediately for wet weight, dried at 60 &#176;C for 48 h, and then weighed again for the dry weight. An increased wet-to-dry weight ratio would be indicative of pulmonary edema. Our results indicate that the transplanted lobe did have a significant increase in wet-to-dry ...

Watch this Scientific Journal Video about A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes at JoVE.com

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

This protocol identifies four key areas to help improve the methodology and have reliable lung transplantation. First is a reproducible cuffing technique. Second is accurately and reproducibly identifying the fourth intercostal space, a thorough closure of the wound, and then how to recover and obtain samples for analysis.These techniques help develop a reliable method of lung transplantation to work on molecular mechanisms and develop therapeutic interventions. Begin with the surgical device setup by turning on the heart rate/oxygen saturation monitoring equipment and warming up the board to 42 degrees Celsius. Also, turn on the ventilation and anesthesia machine to prewarm the isoflurane evaporator.Fill an anesthesia syringe with 10 milliliters of liquid isoflurane and mount the syringe onto the ventilation and anesthesia machine. Next, turn on the surgical microscope with the height and focus adjusted to the preferences and start the electrocautery device. Likewise, prepare and lay out the surgical tools.Determine the correct gauge size of the angio-catheter to be used to make the cuffs based on the body weight of the rat by referring to table one. After preparing the cuffs for the pulmonary artery, bronchus, and pulmonary vein, place the angio-catheter of the appropriate size on a sterile surface under the surgical microscope. Then, use a rib-back surgical blade number 11 to cut the angio-catheter to a 90 degree angle to form a two millimeter long cuff body with a one millimeter width and height at the top of the cuff body.After preparing the wrap for the surgery, make a three to four centimeter-long midline skin incision, mid neck, with the scissors, and carefully dissect out the subcutaneous tissues and muscles using forceps. For the endotracheal intubation, thread a 4-0 silk suture around the trachea. Then, insert 16 gauge angio-catheter into the trachea and tie the suture around the trachea tightly with a double knot, followed with a single knot to hold the angio-catheter in place.Connect the angio-catheter to the ventilator to maintain a surgical plane of anesthesia in the rat with 1 to 2%isoflurane. Use the scissors to perform a laparo-sternotomy, as a combined midline and transverse incision. Then, inject 1, 000 units per kilogram of heparin with an insulin syringe via the inferior vena cava, or IVC, and allow 10 minutes for systemic circulation.While the lungs are are ventilating, cut the right and left auricles with micro dissecting spring scissors and then introduce an 18 gauge angio-catheter through the pulmonary artery to gravity flush the lungs with 20 milliliters of preservation solution in a syringe hanging at 28 centimeters of water, connected to the tubing and angio-catheter. Disconnect the ventilator from the endotracheal tube to connect to a five milliliter syringe filled with a proper volume of air based on body weight. After one hour of the warm ischemia, excise the heart-lung block with the micro dissecting spring scissors and forceps and place on a sterile gauze dampened with ice cold PBS on a sterile Petri dish on ice.Carefully trim the left lung hilar area with Vannas-Tubingen spring scissor and procure the left pulmonary vein, pulmonary artery, and bronchus. Use a mosquito hemostat to grab the cuff tab. Then, take the distal end of the pulmonary vein, pulmonary artery, or bronchus through the appropriate cuff body and evert the extra tissue around the cuff before securing with 8-0 nylon suture.Once done, use Vannas-Tubingen spring scissors to trim the extra tissue and the cuff around the cuff body. To prepare the recipient rat, draw a line on the chest of the rat from the suprasternal notch to the xiphoid process. At the middle of this line, draw a line along the left side of the chest that measures half of the measurement from the suprasternal notch to the xiphoid process.Using the fiber optic cable connected to an LED light from the endotracheal intubation kit, visualize and intubate the recipient with a 16 gauge angio-catheter. Once the lungs are pulled out, place a sterile gauze around the left lung while holding it with Dieffenbach bulldog clamp and apply a Yasargil clamp on the left lung hilar area as proximally as possible. When the rat is prepared, proceed for the pulmonary vein anastomosis by placing 7-0 nylon suture around the recipient's pulmonary vein.Incise the recipient's pulmonary vein using the Vannas-Tubingen spring scissors by transversely cutting the upper and lower segmental veins as distally as possible, and flush out blood with 0.2 milliliters of one international unit per milliliter heparinized saline using an insulin syringe. Place the donor lung wrapped with an ice-cold, wet, sterile gauze into the thoracic cavity, and insert the donor's cuffed pulmonary vein into the recipient's pulmonary vein before securing the anastomosis with the prepositioned 7-0 nylon suture. For the bronchial or bronchus anastomosis, place a 7-0 nylon suture around the recipient's bronchus before incising the recipient's bronchus by transversely cutting the upper and lower segmental airways as distally as possible.After inserting the donor's cuffed bronchus into the recipient's bronchus, secure the anastomosis with the prepositioned 7-0 nylon suture. Next, perform the pulmonary artery anastomosis as described earlier. To allow the reprofusion and ventilation of the transplanted donor lung, remove the Yasargil clamp on the hilum, followed by closing the thoracotomy incision with a 6-0 nylon suture.Collect the BAL fluid from the native or transplanted lungs as desired. The wet to dry weight ratio of the lobes was calculated to measure the pulmonary edema. The transplanted lobe showed a significant increase in the wet to dry weight ratio compared to the donor's or recipient's native lobe, indicating no pulmonary edema.In our opinion, the two critical steps of this procedure are to one, accurately use anatomic landmarks to identify the fourth intercostal space, and then two, select and appropriately use the cuffs to have a reproducible cuffing technique. A rat lung transplantation model that is both reproducible and consistent is a basis for molecular investigations into ischemia reprofusion and transplantation in general.

a-rat-lung-transplantation-model-warm-ischemiareperfusion-injury

Research

JoVE Journal

Medicine

2.8K visualizações.  The Ohio State University Wexner Medical Center. Este protocolo identifica quatro áreas-chave para ajudar a melhorar a metodologia e ter transplante pulmonar confiável. A primeira é uma técnica de cuffing reprodutível. A segunda é identificar com precisão e reprodutibilidade o quarto espaço intercostal, um fechamento completo da ferida e, em seguida, como recuperar e obter amostras para análise.Essas técnicas ajudam a desenvolver um método confiável de transplante pulmonar para trabalhar mecanismos moleculares e desenvolv...