Name: induction of mouse lung injury by endotracheal injection of bleomycin
Uploaded: 2019-04-30T12:30:00
Description: Watch this Scientific Journal Video about Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin at JoVE.com

This work was supported by a grant RF-2011-02352331 from Ministero Italiano della Salute (to Armando Gabrielli).

All animal care and experimental procedures were approved by the Italian Ministry of Health (authorization n. 456/2016-PR) and performed according to the Declaration of Helsinki conventions.
1. Mice
<ol>
	<li>After purchasing them, allow the mice to acclimate for at least 7 days before the injection. 
	NOTE: Mice were housed in the animal facility under pathogen-free conditions, were maintained under constant temperature and humidity on a 12 h light/dark cycle, and were given free acces...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+hMSCs+improve+myocardial+infarction+in+mice+because+cells+embolized+in+lung+are+activated+to+secrete+the+anti-inflammatory+protein+TSG-6.">Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6.</a> Cell Stem Cell. 5 (1), 54-63 (2009).</li><li>Scotton, C. J., Chambers, R. 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Endotracheal administration is the preferential route for delivering exogenous agents into the lungs. Since several years, the direct injection of bleomycin into the trachea has been widely used to induce pulmonary fibrosis13 and, recently, more advanced, noninvasive techniques have been developed to accomplish this14,15,16.
The method described here provides several meaningful...

Pulmonary fibrosis is a progressive pathological process characterized by the excessive deposition of extracellular matrix components, mainly type I collagen, in the lung interstitium, leading to impaired lung function. It is the hallmark of several human lung diseases with a different etiology and represents a poor clinical prognostic factor. Since current therapies are rather limited1, mouse models continue to be an essential tool both for the further investigation of the pathogenic mechanisms influencing the onset and the progression of the disease and for developing new antifibrotic strategies2,3.
To date, the administration of bleomycin has been the most commonly applied model of experimentally induced pulmonary fibrosis4. Beside multiple delivering methods (including intravenous, intraperitoneal, subcutaneous, and inhalational), intratracheal or endotracheal injections of bleomycin have emerged as the most frequently used routes4,5. The method that we describe herein has been developed to avoid the scalding effect of bleomycin on the tracheal mucosa. In fact, by exteriorizing the trachea and visualizing it through an operating microscope, it is possible to achieve the instillation of the entire volume of bleomycin solution directly into the lower airway without any spills in the upper airway. When the required surgical expertise and instrumentation are available, this method allows for the safe, robust, and reproducible induction of lung inflammation and fibrosis, as reported below.

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	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">C57BL/6 mice</td>
			<td style="width:236px;">Charles River</td>
			<td style="width:239px;">Jax Mice Stock n. 000664</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">2,2,2-Tribromoethanol (Avertin)&#160;</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:239px;">T48402</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Barraquer Micro Needle Holder</td>
			<td style="width:236px;">Lawton</td>
			<td style="width:239px;">62-3755</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Bleomycin sulfate</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>B1141000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">B&#252;rker chamber</td>
			<td style="width:236px;">Brand&#160;</td>
			<td style="width:239px;">718905</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture Flasks&#160;</td>
			<td style="width:236px;">EuroClone</td>
			<td style="width:239px;">ET7076</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Disposable razors</td>
			<td style="width:236px;">Unigloves</td>
			<td style="width:239px;">4080</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Dissecting Forceps</td>
			<td style="width:236px;">Aesculap Surgical Instruments</td>
			<td style="width:239px;">BD311R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">DPBS</td>
			<td style="width:236px;">Gibco</td>
			<td style="width:239px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Heating pad</td>
			<td style="width:236px;">2Biological Instruments</td>
			<td style="width:239px;">557023</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Isoflurane Vet</td>
			<td style="width:236px;">Merial Italia</td>
			<td>N01AB06</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Operating Microscope</td>
			<td style="width:236px;">Carl Zeiss</td>
			<td style="width:239px;">Model OPM 16</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">TrypLE Select Enzyme</td>
			<td style="width:236px;">Gibco</td>
			<td style="width:239px;">12563-029</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Vannas Micro Scissors</td>
			<td style="width:236px;">Aesculap Surgical Instruments</td>
			<td style="width:239px;">OC498R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Vicryl Plus 4/0 Absorbable Suture, FS-2 needle 19 mm</td>
			<td style="width:236px;">Ethicon</td>
			<td style="width:239px;">VCP392ZH</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of mouse lung injury by endotracheal injection of bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Lung injury was induced by a single endotracheal injection of 1.5 U/kg body weight of bleomycin sulfate in 100 &#181;L of sterile saline. Control animals received an endotracheal injection of an equal volume of saline. Two shots of hUC-MSC (2.5 x 105 in 200 &#181;L of sterile saline) were infused into the mouse tail vein, 24 h and 7 days after the bleomycin administration. Control animals received an intravenous infusion of an equal volume of sterile saline. Mice were sacrifice...

Watch this Scientific Journal Video about Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin at JoVE.com

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

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