The authors would like to acknowledge the Cincinnati Children&#8217;s Hospital Division of Veterinary Services for their assistance. This manuscript was supported by the National Institutes of Health K12 HD028827. Anna Perl PhD taught the authors this surgical procedure.

NOTE: Animal use statement: All procedures in this study were conducted with approval and following the guidelines of the Institutional Animal Use and Care Committee (IACUC) at Cincinnati Children&#8217;s Hospital. Eight week-old C57BL/6J male mice were obtained from Jackson Laboratories (Bar Harbor, ME) and allowed to acclimate for one week prior to use. Up until surgery, animals were housed in a pathogen-free barrier facility and provided autoclaved chow and filtered water ad libdium. Each mouse cage was supplied with a dedicated air and water, and rooms were maintained on a 12 hr day-night cycle. Following recovery from surgery, mice were maintained in cages with filtered tops, provided autoclaved chow ad libidum, and provided filtered water from a water bottle.
1. Preparation of Instruments
<ol>
	<li>Make 6 skin retractors using paper clips and pins. Twist straightened paper clips on the shanks of paper pins, leave a 5 cm straight steel wire on one end and make a single 0.5 cm &#8220;U&#8221; shaped hook at the end of the wire.</li>
	<li>Make some 15 x 15 cm square surgical drapes using plastic wrap. Prepare one dressing per mouse. Put a paper tower in between each wrap.</li>
	<li>Sterilize all surgical tools along with a stack of 12 x 12 inch cork tiles, gauze, and cotton-tipped swabs.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Induce anesthesia with 2% isoflurane. Weigh animal.</li>
	<li>In a dedicated surgical preparation area shave left thorax and neck area with electrical shaver.</li>
	<li>Apply a drop of the artificial tear ointment to the mouse&#8217;s eyes.</li>
	<li>Decontaminate neck and left thorax with chlorhexidine and isopropyl alcohol. Repeat twice more.</li>
</ol>
3. Mouse Oro-tracheal Intubation and Mechanical Ventilation
<ol>
	<li>Have a non-sterile surgical technician place the mouse supine in the pre-warmed surgical area.</li>
	<li>Confirm depth of anesthesia by documenting lack of a response to paw pinch.</li>
	<li>After washing hands and donning surgical attire, mask, and hat, don sterile surgical gloves.</li>
	<li>After draping and using aseptic technique, make a 1 cm vertical incision over the anterior mid-neck to expose the larynx. Lightly retract the strap muscles with curved, serrated 10 cm forceps and expose the larynx and trachea by spreading the strap muscles with the tip of a straight scissors.</li>
	<li>Orally insert a 22 G blunt-tip angiocatheter into the mid-trachea (Figure 1A) and visually confirm placement (Figure 1B).&#160;Maintain anesthesia and ventilate using 1-3% isoflurane through rodent ventilator (225 &#181;l per stroke; 200 stokes per min). Employ a pressure limit of 15 cm H2O.</li>
</ol>
4. Mouse Pneumonectomy
<ol>
	<li>Lay the mouse in the right lateral decubitus position with the mouse&#8217;s back facing the operator (left side up). Use a self-sealing plastic wrap as a sterile drape. Cutting through the drape, use blunt tipped curved scissors to make a 2 cm long cut parallel to the ribs at the 4th and 5th intercostal space. Insert the blunt tip curved scissors and dissect the skin away from the underlying ribs and intercostal muscles.</li>
	<li>Retract the skin with four retractors to expose a square 1.5 x 1.5 cm surgical window (Figure 2A). Secure the retractors to the cork board.</li>
	<li>Dissect down to ribs using curved forceps, and use one tip of the curved forceps to enter the thoracic cavity.</li>
	<li>Using the blunt tip micro-scissors, use the lower blade to enter the thoracic cavity. Make a 0.5 cm incision between the ribs and repeat in the opposite direction.</li>
	<li>Using the two remaining retractors, open the thorax in the anterior-posterior axis and the secure the retractors to the cork board (Figure 2B).</li>
	<li>Using curved blunt-tipped forceps in the left hand, grasp the left lung and displace the upper portion of the left lung laterally and inferiorly through the thoracotomy until the left pulmonary artery and bronchus are exposed (Figure 3A,B).</li>
	<li>Holding the loaded titanium vascular microclip applicator in the right hand with the body of the applicator in the palm and curved tip pointing away from the palm (Figure 3C), slide the applicator tip into the thorax along the curvature of the posterior aspect of the left lung and clip the left bronchus and pulmonary artery (Figure 3D).</li>
	<li>Remove the applicator but keep the left lung retracted. Grasp the blunt tip micro-scissors with the right hand and cut the bronchus and pulmonary artery distal to the clip and remove left lung (Figure 3E).</li>
	<li>Remove the rib retractors.</li>
	<li>Use the curved blunt forceps to pinch up 1 cm of skin inferior to the incision but above the level of the diaphragm and insert a 24 G angiocatheter through the skin and into the left thoracic cavity (Figure&#160;4A,B).</li>
	<li>Use 5-0 prolene suture to place two interrupted sutures around the 4th and 5th ribs to close the thoracic cavity.</li>
	<li>Remove the skin retractors. Use two sets of forceps to approximate the skin along the length of the incision and glue the skin closed.</li>
	<li>Connect a 3 ml luer-lock syringe to the angiocatheter and remove residual air by applying gentle suction and withdrawing the angiocatheter.</li>
	<li>Glue the neck incision closed using two sets of forceps as before.</li>
</ol>
5. Mouse Sham Pneumonectomy
<ol>
	<li>Expose the left lung as noted in &#8220;Mouse Pneumonectomy&#8221; protocol. Lift the rib cage with curved blunt forceps to allow air into the left chest cavity (Figure 5A,B).</li>
	<li>Place a 24 G angiocatheter into the left thoracic cavity as above being careful not to injure the left lung.</li>
	<li>Using 5-0 prolene suture and being careful not to puncture the lung (Figure 5C), place two lengths of suture material into the 3rd/4th and 5th/6th rib interspaces (Figure 5D). Place both lengths of suture material before tying to lessen the risks of left lung herniation. Tie the suture material to make two interrupted stitches (Figure 5E).</li>
	<li>Glue the skin over the thoracic incision, remove residual air with the angiocatheter, and glue the neck incision as above.</li>
</ol>
6. Resuscitation, Analgesia, and Recovery
<ol>
	<li>Turn off the isoflurane, and administer 0.1 mg/kg of buprenorphine and 0.5 ml of normal saline subcutaneously.</li>
	<li>When spontaneous respirations resume, remove the endotracheal tube.</li>
	<li>Observe mouse until it is again ambulatory. Walking typically resumes several minutes after removal of the endotracheal tube.</li>
	<li>Place the mouse in a 27 &#176;C incubator (humidified, 25% oxygen) to recover O/N. 
	NOTE: We place several pellets of chow wetted with water on the cage floor for the first 24 hr after surgery.</li>
	<li>Administer 0.1 mg/kg of buprenorphine by intraperitoneal injection twice daily for three days after surgery. Take care not to open the surgical site when handling animals.</li>
</ol>
7. Mouse Monitoring
<ol>
	<li>Weigh mice at 1, 3, 5, and 7 days after surgery.</li>
</ol>

<ol>
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The authors have nothing to disclose.

We have provided the most detailed description of the mouse PNX and mouse sham PNX procedures reported to date. We have made the reader aware of several of the common pitfalls that investigators learning the procedure commonly encounter, and we have outlined several techniques developed by our laboratory to mitigate against these pitfalls. Other laboratories utilizing this model may have developed other technique modifications or use different instruments. When evaluating differences in techniques, individual investigato...

The principal function of the lung is to provide for oxygen and carbon dioxide exchange between an organism and the atmosphere. In humans, a host of congenital and acquired conditions lead to reduced lung surface area which results in impaired lung function. Although a host of therapies such as inhaled corticosteroids, bronchodilators, supplemental oxygen, and chronic mechanical ventilation are used to mitigate the consequences of impaired lung function1-3, the ideal therapy for these conditions would promote regrowth of functional lung tissue &#8211; i.e., lung regeneration.
Mammalian tissue regeneration has been well documented. The African Spiny Mouse can regenerate large areas of skin without scar formation4. The distal phalanx in humans can regenerate following injury or amputation5-7. Following pneumonectomy (PNX), compensatory lung growth occurs in mice8, rats9, dogs10, and humans11. By definition, compensatory lung growth involves not only expansion of existing airspaces, but re-septation of these enlarged airspaces with expansion of the associated microcirculation12. Gene expression analysis has demonstrated that this model recapitulates many of the signaling events of lung development13. Four weeks after mouse PNX, alveolar surface area is equivalent to that of sham operated animals14. In this manuscript, we describe the mouse PNX and sham PNX procedures.

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			<td style="width:263px;">Company</td>
			<td style="width:263px;">Catalogue number</td>
			<td style="width:263px;">Comments (optional)</td>
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			<td height="101" style="height:101px;width:263px;">6-inch Vascular Clip Applicator</td>
			<td style="width:263px;">Teleflex Medical (WECK)</td>
			<td style="width:263px;">137062</td>
			<td style="width:263px;"></td>
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		<tr height="101">
			<td height="101" style="height:101px;width:263px;">Horizon Small Titanium Red Clip</td>
			<td style="width:263px;">Teleflex Medical (WECK)</td>
			<td style="width:263px;">1201</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:263px;">Narrow Pattern 12cm Curved Forceps</td>
			<td style="width:263px;">Fine Science Tools</td>
			<td style="width:263px;">11003-12</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:263px;">Curved Serated 10 cm Graefe Forceps&#160;</td>
			<td style="width:263px;">Fine Science Tools</td>
			<td style="width:263px;">11052-10</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Castroviejo Needle Holder</td>
			<td style="width:263px;">Fine Science Tools</td>
			<td style="width:263px;">12565-14</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:263px;">Straight 9 cm Strabismus Scissors (Blunt Tip)</td>
			<td style="width:263px;">Fine Science Tools</td>
			<td style="width:263px;">14075-09</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:263px;">Straight 8.5 cm Hardened Fine Scissors</td>
			<td style="width:263px;">Fine Science Tools</td>
			<td style="width:263px;">14090-09</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:263px;">Straight, Blunt Tip Cohan-Vannas Spring Scissors</td>
			<td style="width:263px;">Fine Science Tools</td>
			<td style="width:263px;">15000-12</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Skin Glue</td>
			<td style="width:263px;">Gluture</td>
			<td style="width:263px;">32046</td>
			<td style="width:263px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">22 ga angiocatheter</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">24 ga angiocatheter</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:263px;">3 mL luer lock syringe</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">4 short retractors</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">2 long retractors</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:263px;">5-0 Prolene on curved cutting needle</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:263px;">8698G</td>
			<td style="width:263px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">0.5 mL syringe on 27 ga needle</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:263px;">Normal Saline</td>
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			<td height="21" style="height:21px;width:263px;">Buprenorphine</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:263px;">Press-n-Seal Wrap</td>
			<td style="width:263px;">Glad Products Company</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">12 inch X 12 inch cork board stack</td>
			<td style="width:263px;">Office Depot</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:263px;">70% Ethanol</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:263px;">Betadine</td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
			<td style="width:263px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse Ventilator</td>
			<td style="width:263px;">Hugo Sachs Elektronnik&#160;</td>
			<td style="width:263px;">Minivent Type 845</td>
			<td style="width:263px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Isoflurane Vaporizer</td>
			<td style="width:263px;">OHMEDA</td>
			<td style="width:263px;">Excel 210 SE</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">artificial tear ointment</td>
			<td style="width:263px;">puralube</td>
			<td style="width:263px;">NDC: 17033-211-38</td>
			<td style="width:263px;"></td>
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mouse pneumonectomy model of compensatory lung growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

A plot of PNX and sham operated mouse weights is provided in Figure 6. In our hands, survival is consistently 95 - 100% for both PNX and sham pneumonectomy. For descriptions of how the right lung re-grows in this model and the expected time course, we refer the reader to manuscripts of Gibney et al.15 and Wang et al.14
Several common pitfalls must be avoided to successfully perform the mouse PNX and mouse sham pneumonectomy procedures.</...

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Mouse Pneumonectomy Model of Compensatory Lung Growth

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

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Research

JoVE Journal

Medicine

12.6K Views.  Cincinnati Children's Hospital Medical Center. Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.