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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Published: September 26th, 2019

DOI:

10.3791/58197

1Department of Respiratory Medicine, Juntendo University Graduate School of Medicine, Juntendo University, 2Pharmaceutical Planning Group, Otsuka Pharmaceutical Co., Ltd.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung’s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

COPD is one of the leading worldwide causes of death1. Cigarette smoke is the most important cause of COPD, but the mechanisms of pathogenesis remain incompletely defined. COPD demonstrates two main characteristics, including progressive limitation of airflow and an abnormal inflammatory response of the lung. Emphysematous disorder frequently occurs in the lungs of COPD patients2. The pathological findings of emphysema are characterized by alveolar wall destruction3. Several animal species have been used to generate COPD models in vivo (i.e., dogs, guinea pigs, monkeys, and rodents).css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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The following methods have been approved by the Animal Care and Use Committees of Juntendo University School of Medicine. The Guidelines for Proper Conduct of Animal Experiments, Science Council of Japan, June 1, 2006 were followed. There are three main steps in this method: 1) mouse dissection, 2) lung exsanguination, and 3) fixation of lung tissues assisted by specialized equipment. Typically, lung specimens are processed to embedment after 48 h of fixation12,15

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As described previously, the specialized equipment, which generates extended constant pressure, can be divided into three parts (Figure 3A). The lower part is the point at which to insert the lung sample (Figure 4A). The lung is connected via a cannula (20 G) to the tip of formalin flow using a three-way stop cock (Figure 4B). Pressure is generated from the different surface levels .......

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The fixation procedure for rodent lungs presented here is not novel; however, this system has several advantages. Firstly, it can fix many lungs (maximum of 20) with the same condition at one time. The Society of Toxicologic Pathology states that the pressure for gravity instillation vary from 22–25 cmH2O22. Notably, several studies have performed lung fixation at a pressure of 25 cmH2O13,19,

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This work was supported in part by JSPS KAKENHI Grant Number 26461199 (T. Sato) and the Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Grant Number E2920 (T. Sato). The funder had no role in the design of the current methods and in writing the manuscript.

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Name Company Catalog Number Comments
10% formalin (formalin neutral buffer solution) Wako 060-01667
Bent forceps Hammacher HSC187-11
Cannula, size 20G Terumo SR-FS2032
Cannula, size 22G Terumo SR-OT2225C Cannula to exsanguinate lung
Forceps Hammacher HSC184-10
Kimtowel Nippon Paper Crecia (Kimberly Clark) 61000
Kimwipe Nippon Paper Crecia (Kimberly Clark) 62011
Lower container (acrylic glass material) Tokyo Science Custom-made Pressure equipment component
Roller pump Nissin Scientific Corp NRP-75 Pump machine to exsanguinate lung
Roller pump RP-2000 Eyela (Tokyo Rikakikai Co. Ltd) 160200 Pressure equipment pump
Silicone tube Ø 9 mm Sansyo 94-0479 Pressure equipment component
Somnopentyl (64.8 mg/mL) Kyoritsu Seiyaku SOM02-YA1312 Pentobarbital Sodium
Surgical scissor Hammacher HSB014-11
Suture thread, size 0 Nescosuture GA01SW
Syringe, 1 mL Terumo SS-01T
Syringe, 1 ml with needle Terumo SS-01T2613S
Syringe, 10 mL Terumo SS-10ESZ
Three-way stopcock Terumo TS-TR1K01
Upper container (acrylic glass material) Tokyo Science Custom-made Pressure equipment component

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