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

Summary

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.

Abstract

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.

Introduction

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 compliance^2,3. Pioneering studies by Weibel et al.⁴ and Duguid et al.

Protocol

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 water. Mice were sacrificed using an overdose of anesthetic delivered by intraperitoneal injection.

1. Mouse PNX Surgery

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Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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