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.

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 p...

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,

Materials

List of materials used in this article

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