A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Summary

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

Abstract

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

Introduction

Pulmonary fibrosis is a progressive disease process in which excessive deposition of extracellular matrix components, primarily type I collagen, in the interstitium of the lungs leads to impaired lung function¹. The pathophysiology of pulmonary fibrosis is complex, and treatment options are currently quite limited. Mouse models remain an important tool to study the pathogenic mechanisms that contribute to the emergence and progression of the disease, as well as new strategies for drug development.

A variety of animal models of pulmonary fibrosis rely on intratracheal instillation of BLM^{2,3,4,5,6,7,8,9,10,11,12}. However, the distribution of fibrotic changes that BLM causes in the lungs is not uniform, and the animals are at risk of asphyxiation during the instillation process. Although intraperitoneal injection of BLM induces relatively uniform fibrotic changes in the lung, it requires multiple doses because of insufficient drug targeting. Intratracheal aerosol administration via a laryngoscope does not require tracheotomy or puncture, and the resulting drug distribution within the lung is optimal. However, the aerosolized particles are large (5-40 µm), and thus cannot reach the subpleural area of the lung tissue.

In this study, intrapulmonary administration of BLM is carried out by nasal nebulization. During nebulization, the mice breathed spontaneously and inhaled the drug particles. The aerosolized particles were 2.5-4 µm in size, which enabled them not only to distribute evenly throughout the lung but also to reach the subpleural area. Under low magnification, the most significant lung histopathological features of patients with idiopathic pulmonary fibrosis (IPF) are the varying severity of lesions, inconsistent distribution, alternating distribution of different phase lesions, and the presence of interstitial inflammation, fibrotic lesions, and honeycomb lung changes, alternating with normal lung tissue. These pathological changes predominantly involve the peripheral subpleural parenchyma or lobular septum around the bronchioli. Thus, given that this approach enables BLM particles to reach the subpleural area of the lungs, this model closely simulates the clinical characteristics of the disease in humans.

Protocol

The Animal Studies Committee of the China-Japan Friendship Hospital (Beijing, China) approved all of the procedures involving mice that were performed as part of this study (NO.190108). Mice were kept in the sterile animal room of the China-Japan Friendship Clinical Medical Research Institute, with a room temperature of 20-25 °C, relative humidity of 40%-70%, animal light intensity of 15-20 LX, and alternating light and dark for 12 h/12 h. Animals had free access to food and water.

1. Mice

1. Ensure that all animals are acclimated to the housing facility for 7 days before nebulization.
2. Use male C57BL/6 mice, aged 8-10 weeks, for nebulization.

2. Nasal bleomycin nebulization

1. Bleomycin preparation
   CAUTION: BLM is a chemical poison, which kills tumors but may also damage normal cells and normal tissues.
   1. Prepare BLM solution at a clean bench.
      1. To obtain the working concentration (7 U/mL), resuspend 15 U of BLM hydrochloride in 2.14 mL of normal saline. Carefully mix the resuspended BLM until it is completely dissolved.
   2. Store the working solution at 4 °C or on ice and use within 1 day. Before nebulization, bring the BLM solution to room temperature.
2. Anesthesia
   1. Prepare for anesthesia by dissolving 0.1 g of pentobarbital sodium in 10 mL of normal saline. Store the working anesthesia solution in a dark place at 4 °C and use within 3 days.
   2. Anesthetize the mice by injecting the pentobarbital sodium into the abdomen, using a 1 mL syringe with a 26 G needle, at a final dose of 75 mg/kg body weight.
      NOTE: In this study, the mice did not respond to this dose for at least 30 min. If necessary, adjust the dose in consultation with the vet according to the mouse's response.
   3. After a few minutes, press the anesthetized mouse's toes with the index finger and thumb to ensure that the limb withdrawal reflex has disappeared.
3. Preparation of the nebulization system
   1. After calibrating the instrument, secure the anesthetized mouse with a soft mesh cover (Figure 1A), add the working BLM solution to the top atomizing head of the exposure tower with a pipette (Figure 1B), and run the nebulizer for 30 min to achieve stable atomization.
      NOTE: The workflow is shown in Figure 1C,D. For detailed operation steps of the software, refer to Supplemental Figure S1 and Supplemental Figure S2, which illustrate steps 2.3.1.1-2.3.1.9.
      1. Double-click on the flexiWare 8 icon (Supplemental Figure S1A), click on experimentation session (Supplemental Figure S1B), and click on the New Study button (Supplemental Figure S1C).
      2. Edit the experiment name (Supplemental Figure S1D) and edit the Title and Owner (Supplemental Figure S1E).
      3. Choose the IX-4DIO template (Supplemental Figure S1F), input the operator (Supplemental Figure S1G), click on the Confirm button (Supplemental Figure S1H) | Next button (Supplemental Figure S1I).
      4. Choose the Pump and click on next (Supplemental Figure S1J) | Next button (Supplemental Figure S2A) | Next button (Supplemental Figure S2B) | Finish button (Supplemental Figure S2C).
      5. Click on the Settings for Continuous-1Lmin (three dots, Supplemental Figure S2D) | DIO1, set the Duty Cycle (%) to 25%, and then click OK (Supplemental Figure S2E).
      6. Click DIO2, set the Duty Cycle (%) to 25%, and then click OK (Supplemental Figure S2F).
      7. Click DIO3, set the Duty Cycle (%) to 25%, and then click OK (Supplemental Figure S2G).
      8. Click DIO4, set the Duty Cycle (%) to 25%, and then click OK (Supplemental Figure S2H).
      9. Click the top green button (left) to start operating, and click the red button (right) to stop work. Click the x at the top-right corner to quit (Supplemental Figure S2I).
   2. In the BLM group, atomize 12 mice at a time, for four times in total, up to a total of 48 mice. Atomize six mice at a time in the normal control group with normal saline.
4. Animal recovery
   1. After treatment in the nebulizer for 30 min, move the mouse to a recovery cage equipped with a heating pad.
   2. Observe the mice until they are fully conscious.
   3. Once the mouse is confirmed to be in good condition, which means that it can move freely, return it to its original cage. Do not place it with other animals until it has completely recovered.
   4. Check physiological indexes, including breathing rate, survival, and any symptoms, twice during the 24 h following treatment with BLM.

3. Lung tissue processing

1. At 9, 16, and 23 days after BLM administration, sacrifice six mice by injecting them in the abdomen with pentobarbital sodium (final dose 100 mg/kg body weight).
2. Remove the trachea and lungs and immediately rinse with cold phosphate-buffered saline.
3. Fix the left lung in 10% neutral formalin buffer for 24 h before paraffin embedding.
4. Hematoxylin-eosin staining (H&E staining)
   1. Dehydrate the fixed lung tissue in a dehydrator.
   2. Place the dehydrated lung tissue block in a wax-dissolving box at 56 °C for 1 h.
   3. For embedding, pour the melted paraffin into the mold box, and then quickly place the paraffin-impregnated tissue block at the bottom of the mold box, cool, and solidify.
   4. Fix the embedded wax block on the microtome, adjust the slicing table up and down to an appropriate position, push the blade, adjust the scale, and cut a slice with a thickness of 4 µm. Use tweezers to place the slice in a display box filled with water to uncurl (front side up).
   5. Patch and bake the slices. After the wax slice has completely uncurled, place the glass slide against the wax slice at a 135° angle, use tweezers to move the wax slice onto the glass slide, lift the glass slide, and adjust the position of the wax slice as needed. Place the slides with mounted wax slices on a constant-temperature table at 47 °C for 1 min to again allow the slices to uncurl; once the slices are completely flat, place the slides in a 62 °C incubator for 1 h.
   6. For dewaxing, place the baked slides in xylene for 20 min twice.
   7. For rehydration, place the slides in a graded alcohol series to rehydrate the lung tissue slices: 100% alcohol for 2 min, 95% alcohol for 1 min, 90% alcohol for 1 min, 85% alcohol for 1 min, 75% alcohol for 1 min, and distilled water for 3 min.
   8. Stain the sections in hematoxylin for 5 min. Wash for 1 min twice, then place in a 0.5% hydrochloric acid alcohol differentiation solution for 5 s, wash with water for 1 min twice, place in Blue staining solution for 8-10 s, rinse twice with tap water, and stain with eosin for 30 s.
   9. For dehydration and destaining, place the stained sections in 75% alcohol for 1 min, 85% alcohol for 1 min, 90% alcohol for 1 min, 95% alcohol for 1 min, 100% alcohol for 1 min, and then xylene for 1 min twice, and remove all remaining liquid with absorbent paper.
   10. For mounting, pace a drop of neutral gum sealing solution in the middle of the slice and mount a coverslip.
5. Morphological grading standard of mouse lung tissue
   1. Using the method described by Ashcroft et al., have an investigator blinded to the experimental group randomly select three microscopic fields from each sample, and observe the degree of pulmonary fibrosis at 100x magnification: grade 0, normal lung; grade 1, slightly swollen alveoli, localized mild fibrosis; grade 2, marked fibrosis (thickness of alveolar wall greater than three times normal), with fibrous foci; grade 3, continuous areas of fibrosis (thickness of alveolar wall three times greater than normal); grade 4, fusion of fibrous foci, with the fibrotic area accounting for less than 10% of the lung tissue; grade 5, the fibrous foci are fused, the fibrosis area is 10% to 50%, and the alveolar structure is significantly damaged; grade 6, large continuous fibrous foci (greater than 50% of the lung tissue); grade 7, the alveolar space is filled with fibrous tissue, pulmonary bullae are present; grade 8, complete fibrosis.
   2. Assign a score corresponding to the grade to each field of view (e.g., grade 4 equals four points), and score each group of six samples separately.

Representative Results

Lung injury was induced by nebulized BLM, and the control animals were nebulized with the same volume of normal saline. The mice were nebulized once a day for 3 days, 30 min per day, using a BLM concentration of 7 U/mL. Mice were sacrificed on days 9, 16, and 23 after BLM administration for H&E staining (Figure 2B). Diffused pneumonic lesions with loss of the normal alveolar architecture, septal thickening, enlarged alveoli, and increased infiltration of inflammatory cells into the interstitial and peribronchiolar areas were observed in the lungs of mice that received BLM compared with the mice in the control group. The severity of fibrotic changes in each lung tissue section was assessed as the mean severity score (Ashcroft score) of the observed microscopic fields. The Ashcroft scores for the BLM-D9, BLM-D16, and BLM-D23 mice were higher than those of the mice in the control group (Figure 2C).

Figure 1: Nebulization system. (A1,A2) Soft restraints: restraining devices available for use with mice. Compress the mesh toward the plastic part of the restraint and hold it with one hand, present the opening created to the mouse, and gently push its nose forward into the restraint. Once the animal is placed in the restraint so that its nose slightly protrudes from the plastic portion, release the mesh. Place a clamp behind the animal. Allow a few minutes for the mouse to adjust to the restraint. (B) Exposure system: the main body is mounted on top of the base unit-modular, nose-only exposure towers, including mouse restraints. (C) Illustration of the complete hardware and tubing layout. For a detailed description of the software operation steps, refer to the supplementary materials. (D) Working diagram of the nebulization experiment. Please click here to view a larger version of this figure.

Figure 2: Nasal bleomycin nebulization induces pulmonary fibrosis in mice. (A) The timeline for nebulization and sacrifice. The control (CTL) group included six animals, and the six animals in the model group were sacrificed on the 9^th day, 16^th day, and 23^rd day after nebulization; these mice were designated as BLM-D9, BLM-D16, and BLM-D23, respectively. (B) Representative microscopic images (1x and 10x magnification) of H&E staining of lung sections obtained from C57BL/6 mice. At 9, 16 and 23 days after nasal nebulization with sterile saline (saline) or BLM, the lung tissue of the mice in the CTL group was observed under a light microscope. The lung tissue structure was clear without swelling. The structure was intact and not damaged, and the alveolar cavity was clean without fibrous tissue filling. However, 9, 16, and 23 days after nasal BLM nebulization, the lung tissue showed uniform and extensive inflammatory infiltration, and the thicknesses of the alveolar wall and alveolar septum were significantly increased. The width was greater than three times the normal width, the alveolar structure was clearly destroyed, pulmonary bullae were present, the alveolar cavity was filled with fibrous tissue, and a large amount of extracellular matrix and fused fibrous foci could be seen. The normal ordered alveolar structure gradually deformed and distorted, eventually forming uniform and extensive fibrotic foci, and the nebulized BLM particles reached the subpleural area of the lung tissue, resulting in the formation of fibrous foci. Arrowheads: interstitial inflammation, fibrotic lesions, and honeycomb lung changes involve the subpleural area of the lung tissue. Scale bars = 0.5 mm and 50 µm. (C) Evaluation of fibrotic changes by the Ashcroft scale in mouse lungs. The lung sections stained with H&E were assigned Ashcroft scores. The severity of fibrotic changes in each section was assessed as the mean severity score for the observed microscopic fields. Three fields per section were analyzed. Abbreviations: CTL = control; BLM = bleomycin; H&E = hematoxylin and eosin. Please click here to view a larger version of this figure.

Supplemental Figure S1: Setting up the nebulizer system software. See protocol steps 2.3.1.1-2.3.1.4 for the description of the screenshots. Please click here to download this File.

Supplemental Figure S2: Setting up the nebulizer system pumps and starting operation. See protocol steps 2.3.1.1-2.3.1.9. Please click here to download this File.

Discussion

Intratracheal injection of bleomycin results in an acute inflammatory and fibrotic response in both lungs and can be considered an effective approach to establish an experimental mouse model of human interstitial lung disease. Intratracheal administration is the most commonly used route of administration^{2,3,4,5,6,7,8,9,10,11,12}. After BLM enters the trachea, the drug is evenly distributed in the lung tissue by means of upright rotation, causing consistent fibrosis throughout the lung. BLM can be administered via the trachea once or multiple times. There are three ways to deliver the drug to the animal's trachea: 1) direct bronchial intubation followed by instillation of BLM¹³; 2) anesthetization, surgical incision of the neck skin, blunt separation of the muscle tissue to expose the bronchi, and then injection of BLM^14,15; 3) atomization of an aqueous BLM solution that is then inhaled by the animals¹³. The first method requires a high level of intubation skill and runs the risk of the animal dying if the intubation fails repeatedly. In the second method, exposure of the muscle tissue to air can lead to infection; hence, it is essential to maintain a clean environment during the experiment and minimize the amount of time that the tissue is exposed to air. Although both methods are simple to execute, the extent of the lesions that they generate is relatively limited compared with human lesions. Thus, these methods do not fully reflect the physical findings of IPF^16,17,18 or the usual histopathological patterns of interstitial pneumonia, meaning that the clinical significance of such models is questionable. The model established by intratracheal BLM administration has provided excellent information regarding important molecules and cells involved in the fibrogenic process, but does not respond appropriately to any interventions that are known to be effective treatments for human disease^18,19. The lack of an effective correlation between treatment responses in the model and in humans may be due to a variety of reasons.

The use of nebulization in our model resulted in uniform distribution of lesions to all areas of the lung, including the subpleural area of the lung tissue, as shown by the arrowheads in Figure 2B. Inhalation of atomized drugs using a nebulizer box can promote uniform distribution of drugs in the lungs, but this method requires a long inhalation time, the drug dose is difficult to control, and the device is difficult to operate; thus, the success rate is low. Compared with the nebulization method used in our study, the former consumes more BLM and results in systemic exposure, while this method involves exclusively oral and nasal exposure. In this study, appropriate nebulization measurements and times were explored, and a fibrosis model was successfully created. The dose can be accurately controlled. The working concentration of bleomycin used was 7 U/mL. Bleomycin was atomized at a fixed proportion in 1 L of air/min, and the concentration of the drug in the atomized gas was known. The atomized drug was evenly distributed in the atomization tower, and 12 mice inhaled the same concentration of aerosolized bleomycin. Furthermore, the mouse pulmonary fibrosis model generated via nasal BLM nebulization resulted in more uniform and extensive fibrosis lesions, and the nebulized particles reached the subpleural area of the lung tissue, resulting in the formation of subpleural fibrous foci, which is more in line with the clinical characteristics of pulmonary fibrosis.

Repeated nebulization once a day for 3 days, 30 min per day, at a concentration of 7 U/mL of BLM, was performed for the following reasons. First, when the mice underwent nebulization once a day for 2 days, their lungs recovered rapidly; when a third round of nebulization was performed on the 9^th day, the mice lungs recovered by day 23. Thus, administering BLM by nebulization only once a week, even for 7 weeks, did not result in a robust model of pulmonary fibrosis. Furthermore, mice that underwent nebulization on 3 consecutive days and those that received a dose of 1.5 U/kg by laryngoscope exhibited a similar weight loss rate and magnitude. Therefore, 3 days of nebulization was selected as the optimal administration timeline. Second, nebulization was performed for 30 min per day because the mice were deeply anesthetized for only 1 h. It takes approximately 30 min to place the mice in the atomization device and wait for each mouse to enter a state of deep anesthesia, leaving 30 min for nebulization. In addition, when the mice were nebulized for 1 h the first day and 30 min the next day, their lungs had recovered by day 21. Therefore, nebulization for 30 min each day was considered to be the optimal timing. Third, the dose of 7 U/mL was calculated based on the dose of BLM that is typically instilled through a laryngoscope and the parameters of the nebulization device. However, a disadvantage of this study is that BLM is expensive, and a large amount is required for each nebulization session. In addition, the equipment needs to be operated in a fume hood to reduce environmental pollution.

In conclusion, nasal bleomycin nebulization can cause significant lung tissue damage and changes that reflect pulmonary fibrosis, intratracheal nebulization can ensure a more even distribution of BLM within the lungs, resulting in consistent fibrosis. The changes observed in this model were more diffused and similar to the changes seen in human pulmonary fibrosis than those observed in other mouse models. Nasal nebulization with a BLM solution does not require tracheal puncture, which reduces animal pain and trauma, and thus may be a preferable method for replicating pulmonary fibrosis in a mouse model. This method can also be used to deliver drugs other than bleomycin to create various experimental models of lung disease.

Materials

Name	Company	Catalog Number	Comments
bleomycin	Bioway/Nippon Kayaku Co Ltd	DP721	Fibrosis model drugs
0.9% saline for injection	Baxter Healthcare(Tianjin)Co.,Ltd.		Bleomycin preparation
1 mL syringe	BD	300481	Anesthetize animals
10% neutral formalin buffer	TechaLab Biotech Company		Fix lung tissue
15 mL centrifuge tube	corning	430790	Prepare for anesthesia
20 °C refrigerator	New-fly group	BCD-213K	Store drugs
4 °C refrigerator	New-fly group	BCD-213K	Store drugs
Adobe illustrator cc2020	Adobe		Process images
blue back liquid	Beijing Chemical Works		tissue staining
clean bench	Suzhou Sujie Purifying Equipment Co.,Ltd.		Bleomycin preparation
differentiation fluid	Beijing Chemical Works		tissue staining
Electronic balance	METTLER TOLEDO	AA-160	Prepare for anesthesia
eosin stain	Beijing Yili Fine Chemicals Co,Ltd.		tissue staining
Heating pad	HIDOM		Mice incubation
hematoxylin stain	Beijing Yili Fine Chemicals Co,Ltd.		tissue staining
phosphate buffered saline (PBS) buffer	Hyclone	SH30256.01	Clean lung tissue
Photoshop drawing software	Adobe		Process images
SCIREQ INEXPOSE	EMKA Biotech Beijing Co.,Ltd.		Atomizing device
Upright fluorescence microscope	Olympus	BX53	Observe the slice