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>

<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 border="0" cellpadding="0" cellspacing="0" style="width:1024px;" width="1023">
	<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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Immunology and Infection

16.9K Views.  Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)-Istituto Nazionale Ricovero e Cura Anziani (INRCA). 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.

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

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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 ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...