Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Summary

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.

Abstract

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.

Introduction

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)⁴. However, the mouse has become the most commonly used in the construction of COPD models. This has many advantages, including its low cost, ability to be genetically modified, extensive genomic information availability, availability of antibodies, and ability to use a variety of mouse strains⁵. Presently, there is no mouse model that can mimic the full features of human COPD; thus, individual researchers must choose which model is most suitable for the specific COPD research⁶. The emphysematous mouse model is one of many COPD mouse models that are currently available. Additional models include the exacerbation mouse model, systemic co-morbidities model, and COPD susceptibility model⁷.

The emphysematous mouse model can be generated by several types of exogenous agents, including chemical agents and cigarette smoke exposure⁴. Chemical exposure (e.g., to elastase) produces a severe type of emphysema, while cigarette smoke results in mild emphysema^8,9. Cigarette smoke is believed to be the main cause for the pathogenesis of COPD; therefore, the choice of cigarette smoke as a means to create a COPD mouse model is reasonable¹⁰. Many studies have used cigarette smoke to create emphysema in the mouse. For example, Nikula et al. successfully created an emphysematous mouse model from B6C3F1 female mice by exposing them to cigarette smoke for 7 or 13 months¹¹. We have also established an emphysematous mouse model via senescence marker protein/SMP-30 KO mice¹². It is crucial to perform a lung fixation method that can properly visualize this mild emphysema model by cigarette smoke exposure.

Various methods for lung fixation have been established¹³. However, there is no gold standard method of lung tissue fixation for evaluating emphysema¹⁴. Several studies from this lab have shown that the fixation system presented here is useful by creating a stable condition for evaluating emphysema^{12,15,16,17,18}. The main advantage of the current system is that it can fix many lungs with the same condition at one time without lung collapse or deflation. The current lung fixation system uses some special equipment that allows lung specimens to be inflated at an appropriate constant pressure for a given period. This special equipment consists of three parts, including a lower container, upper container, and pump. Lung specimens are placed in the lower container that is connected to pressurized fixing agents, resulting in a 25 cmH₂O pressure difference in the level of agents between the upper and lower containers¹⁹.

Protocol

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 fixation^{12,15,16,17,18}.

1. Mouse dissection

1. Measure the body weight of the mouse, then determine the amount of pentobarbital to administer.
2. Inject pentobarbital intraperitoneally at a dosage of 70 mg/kg of body weight and confirm anesthesia by the absence of reaction to toe pinch.
3. Inject the needle at a 45° angle until it penetrates the skin and muscle. Draw the plunger and confirm an air vacuum, then inject the pentobarbital.
4. Confirm anesthesia by the absence of reflex motion.
   NOTE: Using anesthetized mouse as opposed to a euthanized mouse is recommended for fully lung exsanguination.
5. Cut the mouse skin and abdominal muscle at the medial line, aiming for the cephalic area.
6. Cut laterally to provide a wider working space.

2. Lung exsanguination

1. Expose the diaphragm layer and puncture it with forceps.
2. Open the thoracic space and cut the sternal area, allowing the lungs and heart to be seen clearly.
3. Cut the heart in left atrium and right ventricle.
4. Insert a cannula (24 G) into the right ventricle area and direct it to the cephalic area until it reaches the pulmonary artery, as shown in Figure 1.
5. Turn on the pump and allow the 1x phosphate-buffered saline (PBS) to circulate (approximately 200 mL/h) until all lung tissue changes to a white color.

3. Fixation of lung tissue

1. Remove the trachea, lungs, and heart.
2. Free all three organs by cutting the surrounding connective tissues.
3. Tie the right main bronchus with a suture thread and cut all lobes of the right lung.
4. (Optional): the lobes of the right lung consist of four parts. Cut these parts from the right main bronchus and divide the parts for processing as frozen tissue samples.
5. Insert the heart and the lobes of the left lung into fixing agents, located inside a 10 mL syringe.
   CAUTION: Fixing agents are hazardous. Wear proper protective equipment (e.g., long rubber gloves) and work in a well-ventilated room.
6. Create a vacuum condition using a 10 mL syringe to inflate the lung, as shown in Figure 2.
7. Insert a cannula (20 G) into the trachea and tie a knot.
8. Inflate the lung with fixing agents to check with for leaks, using a 1 mL syringe.
9. Transfer to lung fixation pressure equipment, as illustrated in Figure 3.
10. After fixing periods, remove the lung sample tying the trachea off with a knot.

Results

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 ...

Discussion

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 cmH₂O^13,19,

Materials

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