Name: a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation
Uploaded: 2017-07-26T15:00:00
Description: Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

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.

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			<td>15012-12</td>
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			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
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			<td height="21" style="height:21px;">Castroviejo Micro Needle Holders</td>
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			<td>Penn-Century, Inc</td>
			<td>Model MIP</td>
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			<td height="21" style="height:21px;">Hematoxylin Solution</td>
			<td>Sigma</td>
			<td>GHS132</td>
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			<td height="21" style="height:21px;">Eosin Y solution, alcoholic</td>
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			<td height="21" style="height:21px;">10 ml Pipette</td>
			<td>Thermo Scientific</td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
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			<td height="21" style="height:21px;">O.C.T Compound</td>
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			<td>Becton Dickinson</td>
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			<td height="21" style="height:21px;">Povidone iodine</td>
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			<td height="21" style="height:21px;">Phosphate buffered saline</td>
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			<td height="21" style="height:21px;">Silicone prosthesis</td>
			<td>Custom made</td>
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			<td height="21" style="height:21px;">Brightfield microscope</td>
			<td>Olympus</td>
			<td>VS120</td>
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			<td height="21" style="height:21px;">Ruler tool</td>
			<td>Adobe Photoshop</td>
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</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...

Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

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), ...

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