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 access to water and standard pellet food.</li>
	<li>Use female C57BL/6 mice and inject them at 12 to 16 weeks of age.</li>
</ol>
2. Endotracheal injection of bleomycin
<ol>
	<li>Bleomycin preparation 
	CAUTION: Based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), bleomycin is classified as a GHS08 health hazard.
	<ol>
		<li>Prepare bleomycin under a chemical hood.</li>
		<li>To obtain the desired working concentration (0.05 U/100 &#181;L), resuspend 15 U of lyophilized bleomycin sulfate in 30 mL of sterile saline.</li>
		<li>Carefully mix the sample by inverting the tube to avoid clot formation.</li>
		<li>Properly label the tube with the date of resuspension, store it at 4 &#176;C, and use its content within 24 h.</li>
		<li>Prior to the instillation, equilibrate the bleomycin solution to room temperature. 
		NOTE: In this experiment, a single dose of 1.5 U/kg body weight of bleomycin was used to induce lung injury in C57BL/6 mice. Nevertheless, each mouse strain has a different sensitivity to bleomycin6,7. Titration of bleomycin should be performed to determine the optimal dose in the mouse strain used for the experiments.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Prepare anesthesia by dissolving 0.2 g of 2,2,2-tribromoethanol in 9 mL of sterile saline and 1 mL of absolute ethanol (at a working concentration of 20 mg/mL).</li>
		<li>Mix thoroughly by inverting the tube to avoid clot formation.</li>
		<li>Properly label the tube with the date of preparation, store it at 4 &#176;C in darkness, and use it within 3 days.</li>
		<li>Anesthetize the mice with an intraperitoneal injection of 250 &#181;L of tribromoethanol solution (at a final dose of 200 mg/kg body weight) per mouse, using a 1 mL syringe and a 26 G needle. 
		NOTE: With this dose, the mice are unconscious for at least 20 min. When necessary, adjust the dosage according to the mouse&#160;response, in consultation with the veterinarian.</li>
		<li>Monitor the mouse&#160;breathing. The respiration rate will slightly slow down. After a few minutes, pinch one of the mouse&#160;feet to check the lack of pedal reflex.</li>
	</ol>
	</li>
	<li>Endotracheal injection
	<ol>
		<li>Maintain aseptic conditions during all the procedure. Use sterile surgical instruments and materials, wear sterile gloves and avoid contact with any non-sterile surface.</li>
		<li>Lie down the anesthetized mouse on its back on a surgical platform and hold it in place by delicately fixing its legs with surgical tape strips. 
		NOTE: Gentle taping of mouse legs is recommended to avoid mouse sliding away from the surgical platform during its rotation (step 2.3.10).</li>
		<li>Place the mouse on heated mat to keep the rectal temperature stable at 37 &#176;C throughout the intervention. Measure the rectal temperature by a rectal probe.</li>
		<li>Gently hyperextend the mouse&#160;neck by placing a &#8220;pillow&#8221;, for example, a dental cotton roll, beneath its cervical region.</li>
		<li>Gently shave the throat with a razor blade.</li>
		<li>Remove hair from the surgical area with alcohol and disinfect the mouse skin several times with 1% povidone-iodine solution.</li>
		<li>Pinch the skin with a pair of anatomical forceps and make a short incision&#160;in correspondence of the mouse sternohyoid muscle, using a pair of ring-handled, curved blunt scissors. 
		NOTE: The incision of the skin is about 0.5 cm in length. The corresponding skin piece which is removed is so small that it will create no tension in the mouse neck.</li>
		<li>Stop the bleeding with cotton wool sticks.</li>
		<li>Exteriorize the trachea by blunt dissection, gently cleaning it from fat and other tissues.</li>
		<li>Rotate the surgical platform to orient the mouse with its head toward the operator. 
		NOTE: This position allows the operator, during the injection, to angle the syringe so that it follows the natural path of the trachea straight to the lungs.</li>
		<li>Place the mouse under an operating microscope to help with the visualization of the trachea. Adjust the illumination and set the magnification (between 1 and 1.2), focus, and sharpness. The trachea can be easily distinguished as a white translucent tube, and the tracheal rings are clearly visible.</li>
		<li>Mix the bleomycin solution by gently pipetting, and aspirate 100 &#181;L into a 0.5 mL syringe with a 25 G needle, avoiding bubble formation.</li>
		<li>Once the trachea in clearly visualized, carefully puncture it with the needle tip at an angle of 30&#176; (Figure 1A).</li>
		<li>Slowly instill 100 &#181;L of bleomycin or sterile saline (vehicle control) directly into the lumen of the trachea. Wait a few seconds until the entire volume travels down the needle, and then remove it from the trachea.</li>
		<li>Observe a few seconds of apnea, which occurs when the needle is correctly inserted into the trachea so that the mouse will immediately inhale the entire volume of the liquid.</li>
		<li>If the mouse is not inhaling the liquid, carefully monitor its breathing and adjust the needle position. If the mouse stops breathing, immediately remove the needle and allow the mouse to resume breathing normally before reinserting it.</li>
		<li>Safely discard the syringe and needle after the injection.</li>
		<li>Close the subcutaneous fascia and the skin wound with a 5-0 absorbable suture. 
		NOTE: When not completely reabsorbed, remove sutures 7-10 days post-surgery.</li>
	</ol>
	</li>
	<li>Animal recovery
	<ol>
		<li>Place the injected mouse on its side on a heating pad for recovery.</li>
		<li>Monitor the mouse&#160;breathing and observe the mouse until it starts moving and regains sternal recumbency and full consciousness.</li>
		<li>Once it is confirmed that the mouse is in good condition, return it to the original cage. Do not return it to the company of other animals until it has fully recovered.</li>
		<li>To ensure prolonged analgesia and avoid any residual post-interventional pain, administer subcutaneously buprenorphine (at a final dose of 0.05 mg/kg body weight) to mouse every 12 h&#160;post endotracheal injection.</li>
		<li>Examine the mice for 24 h after the endotracheal injection of bleomycin and do so twice a day. Monitor the mice for respiratory distress, weight loss, behavior abnormalities, and for any sign of morbidity.</li>
	</ol>
	</li>
</ol>
3. Tail vein infusion of human umbilical cord mesenchymal stromal cells
<ol>
	<li>Cell preparation 
	NOTE: The isolation, characterization, and cultivation of mesenchymal stromal cells from human umbilical cord has previously been described8,9,10. hUC-MSC should be aseptically manipulated and infused; therefore, perform all steps under a sterile hood.
	<ol>
		<li>Expand the hUC-MSC in 75 cm2 culture flasks to early passages (1&#8211;3 maximum). 
		NOTE: The hUC-MSC should be 70% confluent at the day of their infusion into mice.</li>
		<li>Wash the cells with 10 mL of sterile phosphate-buffered saline (PBS) at room temperature.</li>
		<li>Add 2 mL of trypsin and incubate the cells at 37 &#176;C for about 1 min, until they start detaching.</li>
		<li>Neutralize the trypsin by adding 8 mL of hUC-MSC complete medium containing 10% fetal bovine serum (FBS).</li>
		<li>Collect the cells by centrifugation at 350 x g for 10 min.</li>
		<li>Resuspend the pellet in sterile saline and count the cells using a B&#252;rker chamber. Prepare the cell suspension for infusion by diluting the cells to a final concentration of 2.5 x 105 in 200 &#181;L of sterile saline per mouse. Prepare an excess cell suspension to ensure there is enough volume for infusing all mice.</li>
		<li>Keep the cells on ice prior to the infusion. Infuse within a few hours, as described in section 3.3.</li>
	</ol>
	</li>
	<li>Anesthesia 
	NOTE: In order to minimize the risk of damaging the mouse tail vein during injection the mouse must not move. Therefore anesthesia has been preferred over simple mouse restraining.
	<ol>
		<li>Anesthetize the mice by 4% isoflurane inhalation in an induction chamber.</li>
		<li>Monitor the mouse&#160;breathing. The respiration rate will slightly slow down. After a few minutes, pinch one of the mouse&#160;feet to check for proper anesthetization.</li>
	</ol>
	</li>
	<li>Tail vein infusion
	<ol>
		<li>Once unconsciousness has been confirmed, place the mouse under a sterile hood for the aseptic hUC-MSC intravenous infusion.</li>
		<li>Maintain general anesthesia throughout the experiment via a facial mask with a continuous flow of 1.5% isoflurane.</li>
		<li>To promote vasodilation and allow for an easier injection, soak the mouse&#160;tail in warm water for 2 min.</li>
		<li>Mix the cell suspension by gently pipetting to make sure that the cells do not form clumps. Aspirate 200 &#181;L into a 1 mL syringe with a 26 G needle, avoiding bubble formation.</li>
		<li>Hold the mouse&#160;tail by the tip and gently straighten it.</li>
		<li>Locate the lateral vein of the mouse&#160;tail; gently scrape it with a scalpel and wipe it with 70% ethanol. 
		NOTE: Gently scraping of the tail is performed to remove hair, making the site of injection smoother and cleaner.</li>
		<li>Starting from the distal portion of the tail, insert the needle into the vein at a 15&#176; angle and slowly infuse 200 &#181;L of hUC-MSC or sterile saline (vehicle control) (Figure 1B).</li>
		<li>Monitor the successful intravenous infusion by the liquid entering the vein without resistance and by a lack of extravasation. Wait a few seconds until the entire volume travels down the needle, and then remove it from the vein.</li>
		<li>To prevent bleeding, briefly apply pressure to the entry wound with a sterile gauze.</li>
		<li>Safely discard the syringe and the needle after the infusion.</li>
	</ol>
	</li>
	<li>Animal recovery
	<ol>
		<li>Place the infused mouse on its side on a heating pad for recovery.</li>
		<li>Monitor the mouse&#160;breathing and observe the mouse until it starts moving and regains sternal recumbency and full consciousness.</li>
		<li>Once it is confirmed that the mouse is in good condition, return it to the original cage. Do not return it to the company of other animals until it has fully recovered.</li>
		<li>Examine the mice for 24 h after the tail vein infusion and every other day, to monitor their health status and detect any suffering or pathological sign early.</li>
	</ol>
	</li>
</ol>
4. Organ explant and tissue processing
<ol>
	<li>Sacrifice the mice at days 8, 14, or 21 after the bleomycin administration (Figure 1C) by overdose of injectable anesthetic.</li>
	<li>Excise the trachea and the lungs and immediately wash them in ice-cold PBS.</li>
	<li>Snap-freeze the right lungs in liquid nitrogen and store them at -80 &#176;C for a subsequent molecular analysis10.</li>
	<li>Inflate the left lungs with 4% paraformaldehyde and fix them in 10% neutral-buffered formalin solution for 24 h; then, dehydrate them in graded alcohol series, clear them in xylene, and embed them in paraffin10.</li>
</ol>

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The authors have nothing to disclose.

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.

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

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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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Immunology and Infection

17.2K Views.  Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)-Istituto Nazionale Ricovero e Cura Anziani (INRCA). Dit protocol kan met succes worden toegepast om belangrijke ontstekings- en fibrotische respons in beide longen te induceren door endotracheale injectie van Bleomycine als om nieuwe ontstekingsremmende en antifolbrotische systemische therapieën door staartaderinfusie te valideren. Het belangrijkste voordeel van deze techniek is dat directe injectie van Bleomycine in de muispijplucht zorgt voor een snelle targeting van de longen zonder morsen in de bovenste luchtwegen, waardoor een veilige, r...

Video: Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Medicine

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is accompanied by the activation of inflammatory cells such as macrophages and neutrophils, as well as the release of inflammatory factors including TNF-&#945;, IL-1&#946;, and IL-6. Early inflammation caused by activated pulmonary interstitial macrophages (IMs) in response to IL-33 stimulation is known to play a vital role in the pathological process of IPF. This protocol describes the adoptive transfer of IMs stimulated by IL-33 into the lungs of mice to study IPF development. It involves the isolation and culture of primary IMs from host mouse lungs, followed by the adoptive transfer of stimulated IMs into the alveoli of bleomycin (BLM)-induced IPF recipient mice (which have been previously depleted of alveolar macrophages by treatment with clodronate liposomes), and the pathological evaluation of those mice. The representative results show that the adoptive transfer of IL-33-stimulated macrophages aggravates pulmonary fibrosis in mice, suggesting that the establishment of the macrophage adoptive transfer experiment is a good technical means to study IPF pathology.

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

This protocol describes the isolation of pulmonary interstitial macrophages (IMs) and their adoptive transfer after IL-33 stimulation of the lung alveoli in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis (IPF).

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is ...

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.

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 ...

Biology

Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis

Interstitial lung disease (ILD) represents a broad spectrum of disorders characterized by the progressive and often irreversible scarring of the lung parenchyma, the most common being idiopathic pulmonary fibrosis (IPF). Several animal models of IPF have been developed, with the bleomycin murine model being the most widely used. Bleomycin is a chemotherapeutic known to induce DNA damage in the alveolar epithelium, resulting in acute lung injury and pulmonary fibrosis in humans. Rodent models of IPF use bleomycin administration via various methods, the most common being intratracheal (IT). Recently, the oropharyngeal aspiration (OA) technique has been shown to be equally efficacious as IT for multiple fibrosing agents, with considerably fewer side effects and an easier route of delivery. This protocol details the OA method of bleomycin delivery into the murine lung and highlights examples of potential downstream applications for data quantification. This methodology offers a simple, quick, and safe way to utilize this widely used animal model for studying the molecular mechanisms underlying IPF.

This protocol demonstrates the oropharyngeal aspiration technique for use in the bleomycin murine model of pulmonary fibrosis.

Interstitial lung disease (ILD) represents a broad spectrum of disorders characterized by the progressive and often irreversible scarring of the lung ...

Adenocarcinoma Engraftment: A Method to Implant Cancer Cells into the Murine Lungs

Source: Stevens, L.E. et al, <a href="https://www.jove.com/v/56650/pre-conditioning-airways-mice-with-bleomycin-increases-efficiency">Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment.</a> J. Vis. Exp. (2018).
This video describes a method to engraft lung adenocarcinoma cells in murine lungs by preconditioning the airways with fibrosis-inducing agent&#8211;bleomycin. This method significantly enhances orthotopic engraftment of lung cancer cells into murine lungs

Source: Stevens, L.E. et al, Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment. J. ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

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