The authors would like to acknowledge the National Institute of Biological Sciences, Beijing for the assistance. This work was supported by Beijing Municipal Natural Science Foundation (No. Z17110200040000).

All procedures used in this protocol were carried out in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Biological Sciences, Beijing. 8 week-old CD-1 male mice were housed in a specific pathogen free (SPF) facility until the experiments were conducted. Surgeries were performed using completely anesthetized mice (i.e., without any toe pinch responses). After surgery, mice were kept in a warm, humid room with sufficient food and fresh w...

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In this protocol, we provide detailed descriptions about the measurement of pulmonary parameters after mouse left lung PNX and prosthesis implantation. ISA is now considered to be a key metric for the assessment of respiratory function in many pulmonary diseases and in injury-induced alveolar regeneration. However, although the pulmonary research community is in agreement about the utility of ISA as a useful metric, to date, there has been little consideration of the standardization of the measurement of ILV and MLI, the...

The fundamental function of the lung is the exchange of oxygen and carbon dioxide between blood vessels and the atmosphere. Lung diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and acute respiratory infections, result in decreased ISA2. Researchers studying lung disease have developed several quantitative methods to evaluate morphological changes in lungs, including MLI, ILV, number of gas exchange units, ISA, and lung tissue compliance2,3. Pioneering studies by Weibel et al.4 and Duguid et al.5 together established that ISA can be used as a direct measure of lung gas-exchange capacity in human lungs and can be used as a criterion to determine emphysema severity. A number of studies published in the last five years have used lung morphological parameters (e.g., ISA and MLI) to assess morphological and functional changes in the lungs of mice during development6 and during recovery from injury PNX1,7. ISA is calculated using Equation 18,9:
<img alt="Equation" src="/files/ftp_upload/56114/56114eq1.jpg" />
, where ILV is the internal lung volume and MLI is an intermediary parameter that represents the pulmonary peripheral airspace size10.
PNX, the surgical removal of one or more lung lobes, has been widely reported to induce alveolar regeneration in many species, including humans11, mice1, dogs12, rats13, and rabbits14,15. A study of mice lungs at fourteen days post-PNX showed that both the expansion of pre-existing alveoli and the de novo formation of alveoli contribute to the restoration of ISA, ILV, and the number of alveoli in the remaining lung tissues1. We and others have shown that the insertion of materials such as sponge, wax, or a custom-shaped prosthesis into the empty thoracic cavity following PNX (i.e., prosthesis implantation) impairs alveolar regeneration. It is now firmly established that mechanical force functions as one of the most important factors for initiating alveolar regeneration1,16,17. Such studies have highlighted the effectiveness of using ISA values from PNX-treated and Prosthesis-implanted lungs as a criterion to quantitatively evaluate alveolar regeneration.
Observer bias is known to significantly influence measured values for lung morphological parameters (e.g., MIL and ILV). Standardized protocols can be used to obviate this bias in determining both ILV and MLI, which are the two parameters used in the calculation of ISA. Here, we provide highly-detailed, standardized protocols for measuring these lung parameters. Importantly, the ability to accurately quantify ISA promises to improve the reliability and reproducibility of studies of lung function in injury-induced alveolar regeneration models and should facilitate mechanistic discoveries in multiple pulmonary diseases.

<table width="881">
	<tbody>
		<tr height="21">
			<td height="21" style="width:411px;height:21px;">Low cost cautery kit</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:136px;">18010-00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Noyes scissors</td>
			<td>Fine Science Tools</td>
			<td>15012-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel clips</td>
			<td>Fine Science Tools</td>
			<td>18374-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">I. V. Cannula-20 gauge</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>29P0601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical suture</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>F602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse intubation platform</td>
			<td>Penn-Century, Inc</td>
			<td>Model MIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td>Penn-Century, Inc</td>
			<td>Model LS-2-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO Small Animal Ventilator</td>
			<td>Kent Scientific</td>
			<td>RSP1006-05L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal pad</td>
			<td>Stuart equipment</td>
			<td>SBH130D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital sodium salt</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin Solution</td>
			<td>Sigma</td>
			<td>GHS132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin Y solution, alcoholic</td>
			<td>Sigma</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml Pipette</td>
			<td>Thermo Scientific</td>
			<td>170356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryosection machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Base Molds</td>
			<td>Fisher HealthCare</td>
			<td>22-363-553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 gauge needle</td>
			<td>Becton Dickinson</td>
			<td>305199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone iodine</td>
			<td>Fisher Scientific</td>
			<td>19-027132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP82011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infusion sets for single use</td>
			<td>Weigao</td>
			<td>SFDA 2012 3661704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Gibco</td>
			<td align="right">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tapes</td>
			<td>3M Scotch</td>
			<td style="width:136px;">8915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton pad</td>
			<td>Vinda</td>
			<td>Dr.P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone prosthesis</td>
			<td>Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brightfield microscope</td>
			<td>Olympus</td>
			<td>VS120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ruler tool</td>
			<td>Adobe Photoshop</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

We performed here an experiment with a PNX-treated group and a prosthesis implantation (Prosthesis-implanted) group. These groupings are the same as the groupings used in a previously-published study from our research group14.
The mouse PNX and prosthesis implantation procedures are shown in Figure 2. 8 week-old CD-1 male mice are used for the surgeries and for the quantifica...

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A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

The overall goal of this procedure is to accurately measure the internal surface area of a mouse lung after pneumonectomy and prosthesis implantation. This method helps to analyze the morphological and the physiological functions of the lung in mechanical force-induced adult lung regeneration. The main advantage of this technique is to accurately quantify internal lung surface area, which promise to increase the reliability and the reproducibility of lung function status.Prepare the mouse for surgery on an intubation platform with its ventral side accessible. Then pull out the tongue and inspect the vocal chords with a small animal laryngoscope that is compatible with guiding catheters. Then gently insert a 20 gauge intravenous intubation canula into the trachea at an interior angle.Next, put the mouse in a right lateral recumbent position and connect the canula to a mechanical ventilator. Observe the chest to judge the insertion of the canula into the trachea. Set the inspiratory pressure to 12 centimeters of water and set the respiratory rate to 120 breaths per minute.Now, clean the exposed skin with at least three alternating scrubs of Betadine and 70%ethanol. Then begin the surgery with a two to three centimeter posterolateral thoracotomy incision into the fifth intercostal space. Penetrate the musculature using Noyes spring scissors.Next, make a 1.5 centimeter incision to expose the left lung. As needed throughout the surgery, use a high temperature cauterizer to stop bleeding. Then, lift 1/3 of the left lung lobe from the chest with blunt tip forceps, and use a cotton swab to pull out the entire left lung.Now, identify the pulmonary artery and bronchi of the left lung lobe, and tightly ligate the bronchi and vessels at the hilum with a soaked surgical suture. Next, cut out the left lung lobe three to four millimeters from the ligation, being careful not to cut off the suture, which could result in pneumothorax. Next, close the chest wall with one suture.Then, stitch the muscle layer and the skin layer sequentially, using five to six interrupted sutures with three to four millimeter gaps. Be sure to keep the surgical suture needle away from the heart. Cardiac puncture is lethal.Lastly, disinfect the surgical area with povidone-iodine. Then, place the mouse on a 38 degrees Celsius thermal pad. Keep using the ventilator until spontaneous breathing returns.Perform the previously described operation, but just after removing the lung, prepare to insert the prosthesis. Using blunted forceps, grip the center of the ellipsoid silicone prothesis. Next, while holding the rib with forceps to expose the thoracic cavity, very gently insert the prothesis.Enter at a 45 degree angle between the frontal plane of the prosthesis and the thoracic surface. Using blunt forceps, ensure that the prosthesis is fully within the left empty thoracic cavity. Now, close the chest and monitor the mouse as previously described.For this procedure, assemble a custom inflation tube that consists of a plunger removed from a 10 milliliter serological pipette, a 40 centimeter long flexible tube with a needle adapter, a flow rate control valve, and an 18 gauge needle. Once assembled, secure the pipette to a sideboard with tape. The distance between the top of the pipette and the bench must be at least 30 centimeters.After euthanizing the mouse by anesthetic injection, secure it to a sterilized board and carefully open the chest to cut out the sternum and expose the lung lobes. Then, expose the trachea. Next, fill the inflation tube with freshly made 4%PFA and remove all the bubbles from the tube.Then, insert the needle into the trachea. To avoid leakage, clip the trachea with vessel clips. Now, inflate the lung with fixative at a constant transpulmonary pressure of 25 centimeters of water.Incubate the lungs at room temperature for two hours for full expansion. After the inflation, take note of the volume displacement of fixative. Now, ligate the trachea and gently dissect out the lungs intact from the surrounding connective tissues.Use scissors and be very gentle. Next, incubate the lungs in a 50 milliliter conical tube filled with 4%PFA for 12 hours at four degrees Celsius with gentle shaking on a shaker. Then proceed with processing and staining as described in the text protocol.To quantify the MLI, use images of the agent used in sections collected under bright-field at 20x magnification. From three different lung sections, randomly select a total of 15 non-overlapping views, each about one square millimeter, and then vent them. Only use areas without arteries and veins, major airways, or alveolar ducts.For the analysis, draw a 10 by 10 grid over each selected area using a ruler tool. The grid lines are thus about 100 microns apart. Then, to find an intercept along the line as the length between two adjacent alveolar epithelia.For example, the double-headed arrows illustrate the lengths. Count all these lengths along each grid line, and the MLI is the average value of the intercept lengths. Using the described protocols, PNX-treated mice were compared with prosthetic implanted mice.The mice used were eight-week-old CD-1 males, and their left lung lobes were resected. The prosthesis mimics the size and shape of the left lung lobe and was inserted into the chest after the left lung lobe was removed. 14 days after the surgery, a custom made inflation tube was used to determine the internal volume of the remaining right lung.The average volume of the right lung in the five PNX-treated mice was approximately 1.4 milliliters, which was significantly greater, 1.05 milliliters for the five prosthesis-implanted mice. The MLI values in the remaining right lungs were significantly greater as well. Using these parameters, the internal lung surface area was calculated and was found to be significantly smaller in the prosthesis-implanted mice.After watching this video, you should have a good understanding of how to calculate the internal lung surface area for the mechanical force-induced alveolar regeneration mouse model.

a-standardized-method-for-measuring-internal-lung-surface-area-via

Research

JoVE Journal

Developmental Biology

12.9K Views.  Peking University. The overall goal of this procedure is to accurately measure the internal surface area of a mouse lung after pneumonectomy and prosthesis implantation. This method helps to analyze the morphological and the physiological functions of the lung in mechanical force-induced adult lung regeneration. The main advantage of this technique is to accurately quantify internal lung surface area, which promise to increase the reliability and the reproducibility of lung function status.Prepare the mo...