Name: a mouse model of pulmonary fibrosis induced by nasal bleomycin nebulization
Uploaded: 2023-01-20T12:40:00
Description: Watch this Scientific Journal Video about A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization at JoVE.com

This work was supported by the National Natural Science Foundation of China (No. 92068108).

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 &#176;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.
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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 administration2,3,4,5,6,7,</sup...

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 BLM2,3,4,5,6,7,8,9,10,11,12. 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 &#181;m), 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 &#181;m 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;bleomycin</td>
			<td>Bioway/Nippon Kayaku Co Ltd</td>
			<td>&#160;DP721</td>
			<td>Fibrosis model drugs</td>
		</tr>
		<tr>
			<td>0.9% saline for injection</td>
			<td>Baxter Healthcare(Tianjin)Co.,Ltd.</td>
			<td></td>
			<td>Bleomycin preparation</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>300481</td>
			<td>Anesthetize animals</td>
		</tr>
		<tr>
			<td>10% neutral formalin buffer</td>
			<td>TechaLab Biotech Company</td>
			<td></td>
			<td>Fix lung tissue</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>corning</td>
			<td>430790</td>
			<td>Prepare for anesthesia</td>
		</tr>
		<tr>
			<td>20 &#176;C refrigerator</td>
			<td>New-fly group</td>
			<td>BCD-213K</td>
			<td>Store drugs</td>
		</tr>
		<tr>
			<td>4 &#176;C refrigerator</td>
			<td>New-fly group</td>
			<td>BCD-213K</td>
			<td>Store drugs</td>
		</tr>
		<tr>
			<td>Adobe illustrator cc2020</td>
			<td>Adobe</td>
			<td></td>
			<td>Process images</td>
		</tr>
		<tr>
			<td>blue back liquid</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>clean bench</td>
			<td>Suzhou Sujie Purifying Equipment Co.,Ltd.</td>
			<td></td>
			<td>Bleomycin preparation</td>
		</tr>
		<tr>
			<td>differentiation fluid</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>METTLER TOLEDO</td>
			<td>AA-160</td>
			<td>Prepare for anesthesia</td>
		</tr>
		<tr>
			<td>eosin stain</td>
			<td>Beijing Yili Fine Chemicals Co,Ltd.</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>HIDOM</td>
			<td></td>
			<td>Mice incubation</td>
		</tr>
		<tr>
			<td>hematoxylin stain</td>
			<td>Beijing Yili Fine Chemicals Co,Ltd.</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS) buffer</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td>Clean lung tissue</td>
		</tr>
		<tr>
			<td>Photoshop drawing software</td>
			<td>Adobe</td>
			<td></td>
			<td>Process images</td>
		</tr>
		<tr>
			<td>SCIREQ INEXPOSE</td>
			<td>EMKA Biotech Beijing Co.,Ltd.</td>
			<td></td>
			<td>Atomizing device</td>
		</tr>
		<tr>
			<td>Upright fluorescence microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td>Observe the slice</td>
		</tr>
	</tbody>
</table>

a mouse model of pulmonary fibrosis induced by nasal bleomycin nebulization

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.

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&#38;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...

Watch this Scientific Journal Video about A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization at JoVE.com

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

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.

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...

In this study, inter-pulmonary administration of Bleomycin is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimic clinical disease characteristics. After anesthetizing the mouse press its toes with the index finger and thumb to ensure the limb withdrawal reflects has disappeared. Once the instrument is calibrated, secure the anesthetized mouse with a soft mesh cover.Using a pipette, add the working BLM solution to the top atomizing head of the exposure tower, and run the nebulizer for 30 minutes to achieve stable atomization. Now double click the FlexiWare 8 icon. Select the Experimentation Session and click the New Study button.Edit the Experiment Name, Title, and Owner. Then choose the IX-4DIO template. Input the operator and click the Confirm button.Click Next In the Welcome window. In the Pump Selection window, choose the Pump tab and click Next. It leads to the window named, Test set-up-Pump 4.Click the Next button and the Pump Output Adjustment window will pop up, and click Next again. Then click the Finish button in the Results-Pump 4 window. Click on the settings for Continuous 1L/min.Select DIO1 and set the Duty Cycle to 25%Then click OK and set the Duty Cycle percent for DIO2, DIO3, and DIO4. Now start the procedure by clicking the top green button and stop by clicking the red button. Click X at the top right corner to quit.The BLM administered mice lungs displayed 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, compared to the control group. The Ashcroft scores for the BLM mice on the 9th, 16th and 23rd days, were higher than those in the control group. The aerosolized the particle with 2.5 to four micrometer in size, which enable Bleomycin to distribute evenly throughout the lung and that reach the subpleural area.

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