This study was supported by the University Synergy Innovation Program of Anhui Province (GXXT-2021-077) and the Anhui University of Science and Technology Graduate Innovation Fund (2021CX2120).

All procedures followed the guidelines of the National Institutes of Health&#39;s Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978) and were approved by the Institutional Animal Care and Use Committee at the Medical School of Anhui University of Science and Technology.
1. Managing and feeding mice
<ol>
	<li>Assign 20 healthy C57BL/6 male mice to the experimental or vehicle groups in a 1:1 ratio. Acclimatize the mice to the new environment for 1 week.</li>
	<li>Provide a constant light time of 12 h per day. Use a time control switch for precise timing.</li>
</ol>
2. Preparing the CSD suspension
<ol>
	<li>At least 1 day before nasal drips, grind the silica in an agate mortar for 0.5 h.</li>
	<li>Observe the size and shape of the crystal particles. Take representative photographs using scanning electron microscopy (SEM).
	<ol>
		<li>Use conductive tape to bind the particles to prepare the sample for SEM. Use a hair dryer to gently blow away the silicon particles that are not firmly bonded.</li>
		<li>Evacuate the sample chamber, turn on the high pressure, and capture the image. 
		NOTE: The working distance (WD) between the lens and the sample is 5.9 mm, the accelerating voltage is 2.0 kV, and the magnification (Mag) is 100,000x, using the SignalA detector. The particles are dispersed with irregular crystallization, and approximately 80% have a diameter of 1-5 &#181;m (Figure 1A).</li>
	</ol>
	</li>
	<li>Make a 20 mg/mL sterile CSD suspension. Dilute the CSD using sterile saline and mix it with an ultrasonic shaker (40 kHz, 80 W) at room temperature (RT) for 30 min.</li>
	<li>Stir and mix the CSD suspension thoroughly on a vortex mixer for 10 s before administering the nasal drips.</li>
</ol>
3. Administering nasal drips to mouse
<ol>
	<li>Rapidly anesthetize a mouse with 2% isoflurane at a dose of 3.6 mL/h in an anesthesia machine (Figure 1B, left panel). 
	NOTE: Anesthesia should be performed in a fume hood to avoid inhalation of the anesthetic by the technician. Ensure that the adequate depth of anesthesia by observing the change from rapid and irregular breathing to a slow and steady state in the mice.</li>
	<li>Drip 50 &#181;L of the CSD nasally within 4-8 s (Figure 1B, right).
	<ol>
		<li>For the nasal drip, place the head of the mouse on the researcher&#39;s metacarpophalangeal joint with the tip of the index finger.</li>
		<li>Keep the mouse in a prone position, with four fingers slightly flexed and the tip of the thumb lightly touching the lower lip of the mouse to straighten the airway. Avoid touching the pharynx to elicit the gag reflex.</li>
		<li>Aspirate 50 &#956;l of liquid using a 200 &#956;l pipette. Drop the liquid into the mouse&#39;s nasal cavity in three to four divided doses depending on the mouse&#39;s respiratory rate. Each instillation should consist of 15 to 20 &#956;l liquid. Administer the drips once every 3 days, 5x within 12 days. Treat the control mouse with an equal amount of saline.</li>
	</ol>
	</li>
	<li>Gently massage the heart area of the mouse 5x-10x for 5 s.
	<ol>
		<li>Hold the mouse&#39;s body with the palm, pinch the skin on the back of the neck with the thumb and index finger, and fix the mouse&#39;s hind limbs with the other fingers.&#160;Then, gently press the mouse&#39;s heart-beating area with the index finger of the other hand 5-10 times in a period of 5 s.</li>
	</ol>
	</li>
	<li>When the mouse&#39;s breathing has stabilized, place it in a recovery cage with a heating pad and observe until it recovers from anesthesia, then return the mouse to its home cage. Sacrifice the mouse at 31 days.</li>
</ol>
4. Collecting the lung tissues and preparing a paraffin section
<ol>
	<li>Inject 0.18 mL of 10% chloral hydrate intraperitoneally, ensuring that the mice do not respond to toe or tail stimulation (performed using the toothed forceps). Then, proceed to the next step.</li>
	<li>Fix the mouse limbs on a foam test board and spray with 75% alcohol to dampen the fur. Remove most of the thoracic ribs at the midline of the clavicle and open the thoracic cavity of the mice to expose the heart and lungs.</li>
	<li>Immediately cut open the right atrium with ophthalmic surgical scissors and slowly inject 20 mL of phosphate buffer (PBS) from the heart tip at the left atrial beat with the greatest amplitude to allow the whole blood to flow. Next, remove the lower lobe of the right lung and store it at -80 &#176;C for western blotting analysis.</li>
	<li>Keep perfusing 10 mL of 4% paraformaldehyde (PFA) in the same site after the PBS injection. Collect the remaining lung and preserve the sample in 30 mL of 4% PFA for pathological analysis.</li>
	<li>After 72 h of fixation, embed specimens in paraffin.
	<ol>
		<li>Dehydrate the tissues through a graded series of ethanol (EtOH) dilutions in deionized water (60%, 70%, 80%, 90%, 100%) for 1 h each at RT. Clear the sample in two washes of xylene for 1 h each.</li>
		<li>Infiltrate the samples with the melted paraffin wax by heating to 50 &#176;C for 2 h. Repeat this process in another cylinder. Cool the wax molds with tissues for 1 h to harden.</li>
		<li>After the wax has hardened and the tissue has been embedded, use a paraffin sectioning machine to slice the tissue at 5 &#181;m. The precise sectioning and slide mounting steps were previously described11. 
		&#8203;NOTE: Sufficient tissue perfusion is indicated by developing muscle twitching and tail twisting into an &#34;S&#34; shape or flexion after receiving 10 mL of 4% PFA.</li>
	</ol>
	</li>
</ol>
5. Performing hematoxylin and eosin (HE) staining
<ol>
	<li>Heat the paraffin-embedded tissues on a hot plate (60 &#176;C) for more than 4 h to allow for adhesion to the slides and improved deparaffination.</li>
	<li>Dewax and hydrate paraffin sections. Soak the slides with samples in xylene 2x for 30 min each time. Next, dip them in anhydrous ethanol, then 95%, 85%, 75% alcohol, and deionized water for 5 min, respectively.</li>
	<li>Perform hematoxylin and eosin staining. Stain the tissues in a hematoxylin staining bucket for 10 min. Rinse them with gently running water for 5 min. Then, dip the slides in the eosin staining bucket for 10 s.</li>
	<li>Dehydrate the samples in 75%, 85%, 95%, and anhydrous ethanol for 5 min each. Clear the tissue sections by immersing them in xylene for 5 min. Seal the section with approximately 60 &#181;L of neutral resin drops. Place the cover slide over the section and carefully lower it to avoid air bubbles.</li>
</ol>
6. Performing Masson staining
<ol>
	<li>Dewax and hydrate the paraffin samples, as mentioned in step 5.2. Then, stain the cell nuclei with 50% Weigert&#39;s hematoxylin for 10 min. Soak the tissue in acidic ethanol liquefaction for 10 s and rinse the tissue slides gently with running water for nuclei bluing. 
	NOTE: Prepare the Weigert&#39;s hematoxylin staining solution immediately before use.</li>
	<li>Stain the samples with drops of Lichun red staining solution (40 &#181;L for each tissue slide) for 7 min and wash them with a weak acid working solution (30% hydrochloric acid) for 1 min to remove the unbound Lichun red dye.</li>
	<li>Dip them in 95% alcohol for 20 s and dehydrate them 2x with anhydrous ethanol for 1-3 s each. After that, clear the tissues with xylene and seal them with 60 &#181;L of neutral resin drops, as mentioned in step 5.4.</li>
</ol>
7. Performing Sirius red staining
<ol>
	<li>Dewax and hydrate paraffin sections as mentioned in step 5.2.</li>
	<li>Infiltrate the sections for 1 h with Sirius red staining solution.</li>
	<li>Stain the cell nuclei of the samples for 8-10 min with Mayer hematoxylin staining solution. Rinse them gently with running water for 10 min. Then, dehydrate and clear the tissue slides. Seal them as mentioned in step 5.4.</li>
</ol>
8. Performing immunohistochemistry
<ol>
	<li>Dewax and hydrate the paraffin samples as described in step 5.2.</li>
	<li>Infiltrate the specimens with a 3 mg/mL EDTA antigen retrieval solution of about 30 mL. Boil for 20-30 min. Wash the tissues with deionized water, and then incubate them in phosphate-buffered solution containing 0.5% Tween-20 (PBST) for 5 min.</li>
	<li>Soak the samples for 15 min with 0.3% hydrogen peroxide solution to inactivate the endogenous peroxidase in the specimens. Wash them 3x with PBST for 5 min each time. 
	NOTE: The 0.3% hydrogen peroxide solution must be made fresh in a light-proof environment.</li>
	<li>Permeabilize the membrane of specimens for 15 min with 0.3% Triton-100 solution. Then, block with 30-40 &#181;L of 5% bovine serum albumin (BSA) for 1 h.</li>
	<li>Remove the blocking solution. Add diluted primary antibodies NF-&#954;B (dilution 1:200) and CD68 (dilution 1:1,000) and incubate the specimens overnight at 2-8 &#176;C in a microscope slide IHC wet box to prevent evaporation and light.</li>
	<li>The next day, transfer them to RT for 1 h. Then, wash them with PBST 3x for 5 min each.</li>
	<li>Incubate the samples for 1 h in rabbit anti-mouse horseradish peroxidase-labeled secondary antibodies (dilution ratio 1:500) at RT and wash them with PBST 3x for 5 min each. 
	NOTE: Both primary and secondary antibodies were diluted with 5% BSA.</li>
	<li>Incubate samples with the 3,3&#39;-Diaminobenzidine (DAB) substrate corresponding to the enzyme-labeled antibody for 5-20 min. Stop the reaction with deionized water when the optimal staining intensity is reached. 
	NOTE: The DAB solution needs to be prepared freshly and protected from light. The color development reaction should be observed in real-time under the microscope to determine when to stop staining. Positive specimens exhibit intense staining, while negative specimens do not develop color.</li>
	<li>Counterstain the samples for 30 s with Weigert hematoxylin. Next, rinse the tissues under running water for 1 min. Then, dehydrate, clear, and seal the tissue slides, as mentioned in step 5.4.</li>
</ol>
9. Performing western blotting analysis
<ol>
	<li>Lyse the lung tissues to extract proteins. Add 200 &#181;L of RIPA working solution to 20 mg of the lung tissue.</li>
	<li>Homogenize the tissue on ice for 5 min using a handheld electric grinder and incubate for 1 h on ice with gentle shaking. Afterward, centrifuge the homogenate at 4 &#176;C for 15 min at 14,800 x g.</li>
	<li>Collect the supernatant and determine the protein concentration with the BCA protein assay kit. Make the protein storage solution with RIPA at 6 &#181;g/&#181;L protein. Add 20 &#181;L of 5x loading buffer to the 80 &#181;L of the protein lysate. Disrupt the secondary protein structure by heating the protein-containing microcentrifuge tubes in a metal bath (100 &#176;C) for 20 min.</li>
	<li>After cooling, aliquot 100 &#181;L of the protein storage solution into each tube and store the samples in a -80 &#176;C refrigerator. Dilute the protein concentration to 2&#8211;3 &#956;g/&#956;L with 1x loading buffer before electrophoresis. 
	NOTE: The protein extraction process should be performed on ice. For the RIPA working solution, add 1 &#181;L of 100 mM phenylmethylsulfonyl fluoride (PMSF) to 99 &#181;L of RIPA to inhibit phosphorylated protein degradation.&#160; Prepare 1x loading buffer by diluting 5x loading buffer with RIPA at a ratio of 1:4.&#160;</li>
	<li>Add 20 &#181;g of samples to each well and run the gel. For 5% concentrated gels, use 80 V for 20 min to have the proteins electrophoresed down from the same starting point. For 10% isolated gels, run at 100 V for 1 h to allow the proteins of different molecular weights to be separated as much as possible.</li>
	<li>Pre-activate the PVDF membrane with methanol for 20 s. Transfer the proteins to the PVDF membrane using the wet transfer method with 400 mA current for 1-2 h. 
	NOTE: Ensure that the electrophoresis tank and the electrotransfer tank are horizontal. Cool the entire tank with ice, as the membrane transfer process generates much heat.</li>
	<li>Wash the membrane with TBST solution for 5 min each time, 5x. Then, block with 5% BSA or 5% skim milk for 1 h. Dilute the primary antibodies NF-&#954;B (1:1,000) and &#946;-actin (1:1,000) with 5% BSA. Submerge the strips in the antibody solution and shake the strips gently overnight at 2-8 &#176;C.</li>
	<li>Wash the strips with PBST. Next, dilute horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000) and incubate them in the diluted secondary antibody for 1 h at RT with gentle shaking.</li>
	<li>Prepare an enhanced chemiluminescence (ECL) developer, drop it on the strip, and incubate for 3 min.</li>
	<li>Expose the strip to a gel imager for 20 s. Measure the gray value of the strip to assess the protein level by system software. Use &#946;-actin as an internal control.</li>
</ol>

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The authors declare no conflicts of interest.

Silicosis mouse models are crucial for studying the pathogenesis and treatment of silicosis. This protocol describes a method for preparing a model of silicosis in mice through repeated nasal exposure. This method allows for the study of the pathological characteristics of silicosis induced by different exposure times. Mice were anesthetized on a ventilator, and their respiratory rate was monitored. The initial short, fast breathing rate gradually slowed and deepened over time. The anesthesia caused the mice's muscles to...

Workers are inevitably exposed to irregular crystalline silica dust (CSD), which can be inhaled and is more toxic in numerous occupational contexts, including mining, pottery, glass, quartz processing, and concrete1,2. A chronic dust inhalation condition known as silicosis causes progressive lung fibrosis3. According to epidemiological data, the incidence of silicosis has been declining globally over the past few decades, but in recent years, it has been increasing and affecting younger people4,5,6. The underlying mechanism of silicosis presents a significant challenge for scientific research due to its insidious onset and protracted incubation period. It is still unknown how silicosis develops. Furthermore, no current medications can stop the progression of silicosis and reverse pulmonary fibrosis.
The current mouse models for silicosis involve tracheal ingestion of a mixed suspension of CSD. For example, administering CSD into the lungs by adopting the cervical trachea trauma after anesthesia does not comply with repeated human exposure to dye dust7. The impact of exposure to ambient dust on individuals can be studied by exposing them to CSD in the form of aerosols, which more accurately reflects the environmental concentrations of this toxic substance8. However, environmental CSD cannot simply be inhaled directly into the lungs due to the unique physiological structure of the mouse nose9. Moreover, the equipment associated with this technology is expensive, which has caused researchers to re-evaluate the mouse silicosis model10. By inhaling CSD suspension through a nasal drip five times within 2 weeks, it was possible to build a dynamic model of silicosis. This model is consistent and safe while being easy to use. It is important to note that this study allows for repeated inhalation of CSD in mice. The mouse silicosis model created through this procedure is expected to be more beneficial for research requirements.

<table><tbody><tr><td>0.5 mL tube</td><td>Biosharp</td><td>BS-05-M</td><td></td></tr><tr><td>10% formalin neutral fixative</td><td>Nanchang Yulu Experimental Equipment Co.</td><td>NA</td><td></td></tr><tr><td>Adobe Illustrator</td><td>Adobe&amp;nbsp;</td><td>NA</td><td></td></tr><tr><td>Alcohol disinfectant</td><td>Xintai Kanyuan Disinfection Products Co.</td><td>NA</td><td></td></tr><tr><td>CD68</td><td>Abcam</td><td>ab125212</td><td></td></tr><tr><td>Citrate antigen retrieval solution</td><td>biosharp life science</td><td>BL619A</td><td></td></tr><tr><td>DAB chromogenic kit</td><td>NJJCBio</td><td>W026-1-1</td><td></td></tr><tr><td>Dimethyl benzene</td><td>West Asia Chemical Technology (Shandong) Co</td><td>NA</td><td></td></tr><tr><td>Enhanced BCA protein assay kit</td><td>Beyotime Biotechnology</td><td>P0009</td><td></td></tr><tr><td>Hematoxylin and Eosin (H&amp;amp;E)</td><td>Beyotime Biotechnology</td><td>C0105S</td><td></td></tr><tr><td>HRP substrate</td><td>Millipore Corporation</td><td>P90720</td><td></td></tr><tr><td>HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)</td><td>Proteintech</td><td>Sa00001-2</td><td></td></tr><tr><td>Iceacetic acid</td><td>West Asia Chemical Technology (Shandong) Co</td><td>NA</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td>NA</td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Science</td><td>R510-22</td><td></td></tr><tr><td>Masson&#039;s Trichrome stain kit</td><td>Solarbio</td><td>G1340</td><td></td></tr><tr><td>Methanol</td><td>Macklin</td><td>NA</td><td></td></tr><tr><td>Microtubes</td><td>Millipore</td><td>AXYMCT150CS</td><td></td></tr><tr><td>NF-&amp;kappa;B p65</td><td>Cell Signaling Technology</td><td>8242S</td><td></td></tr><tr><td>Oscillatory thermostatic metal bath</td><td>Abson</td><td>NA</td><td></td></tr><tr><td>Paraffin embedding machine</td><td>Precision (Changzhou) Medical Equipment Co.</td><td>PBM-A</td><td></td></tr><tr><td>Paraffin Slicer</td><td>Jinhua Kratai Instruments Co.</td><td>NA</td><td></td></tr><tr><td>Phosphate buffer (PBS)&amp;nbsp;</td><td>Biosharp</td><td>BL601A</td><td></td></tr><tr><td>Physiological saline&amp;nbsp;</td><td>The First People&#039;s Hospital of Huainan City</td><td>NA</td><td></td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>NA</td><td></td></tr><tr><td>PMSF</td><td>Beyotime Biotechnological</td><td>ST505</td><td></td></tr><tr><td>Polarized light microscope</td><td>Olympus</td><td>BX51</td><td></td></tr><tr><td>Precision balance</td><td>Acculab</td><td>ALC-110.4</td><td></td></tr><tr><td>Prism7.0</td><td>GraphPad&amp;nbsp;</td><td>Version 7.0</td><td></td></tr><tr><td>PVDF membranes</td><td>Millipore</td><td>3010040001</td><td></td></tr><tr><td>RIPA lysis buffer</td><td>Beyotime Biotechnology</td><td>P0013B</td><td></td></tr><tr><td>RODI IOT intelligent multifunctional water purification system</td><td>RSJ</td><td>RODI-220BN</td><td></td></tr><tr><td>Scilogex SK-D1807-E 3D Shaker</td><td>Scilogex</td><td>NA</td><td></td></tr><tr><td>SDS-PAGE gel preparation kit</td><td>Beyotime Biotechnology</td><td>P0012A</td><td></td></tr><tr><td>Silicon dioxid</td><td>Sigma</td><td>#BCBV6865</td><td></td></tr><tr><td>Sirius red staining</td><td>Nanjing SenBeiJia Biological Technology Co., Ltd.</td><td>181012</td><td></td></tr><tr><td>Small animal anesthesia machine</td><td>Anhui Yaokun Biotech Co., Ltd.</td><td>ZL-04A</td><td></td></tr><tr><td>Universal Pipette Tips (0.1&amp;ndash;10 &amp;micro;L)</td><td>KIRGEN</td><td>KG1011</td><td></td></tr><tr><td>Universal Pipette Tips (100&amp;ndash;1000 &amp;micro;L)</td><td>KIRGEN</td><td>KG1313</td><td></td></tr><tr><td>Universal Pipette Tips (1&amp;ndash;200 &amp;micro;L)</td><td>KIRGEN</td><td>KG1212</td><td></td></tr><tr><td>Vortex&amp;nbsp;mixer&amp;nbsp;</td><td>VWR</td><td>NA</td><td></td></tr><tr><td>ZEISS GeminiSEM 500</td><td>Zeiss Germany</td><td>SEM 500</td><td></td></tr><tr><td>&amp;beta;-actin</td><td>Bioss</td><td>bs-0061R</td><td></td></tr></tbody></table>

a silicosis mouse model established by repeated inhalation of crystalline silica dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

The potential pathogenesis of silicosis in mice was investigated using the proposed method. We found that the body weight of the mice in the experimental group decreased significantly relative to the control group and that the body weight recovered slowly after cessation of exposure. Due to the optimized dose used here, no mortality was observed in silica-exposed mice in this experiment. The technical roadmap of repeated nasal drip to CSD is shown in (Figure 1). The previously described proc...

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A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

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Research

JoVE Journal

Medicine

2.1K Views.  Anhui University of Science and Technology. In het beschreven silicosemodel van muizen dat wordt gegenereerd door intralaserinfusie van kristalsilicastof, simuleerden alle CSD-suspensie meerdere keren het optreden en de ontwikkeling van menselijke silicose in grote mate. Deze methode is tijdbesparend en niveaubesparend, maar ook praktisch en effectief. Veroorzaakt verder geen mechanisch letsel aan de bovenste luchtwegen als gevolg van de operatie.Deze methode zou ook kunnen worden gebruikt om ziektemodellen van virussen, bacteri...

Video: A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

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 ...

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.

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 ...

Immunology and Infection

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent models continue to be an essential tool for studying human silicosis. For silicosis, the classic pathological process needs to be inducible via the inhalation of freshly generated quartz particles, which means specifically inducing human occupational disease. This study described a technique to establish and effectively mimic the pathological dynamic evolution process of silicosis. Further, the technique had good repeatability with no surgery involved. The inhalation exposure system was fabricated, validated, and used for toxicology studies on respirable particle inhalation. The critical components were as follows: (1) bulk dry SiO2 powder generator adjusted with an air-flow controller; (2) 0.3 m3 whole-body inhalation exposure chamber accommodating up to 3&#160;adult rats; (3) a monitoring and control system for regulating oxygen concentration, temperature, humidity, and pressure in real-time; and (4) a barrier and waste disposal system for protecting laboratory technicians and the environment. In summary, the present protocol reports the inhalation via the whole body, and the inhalation chamber created a reliable, reasonable, and repeatable rat silicotic model with low mortality, less injury, and more protection.

This study describes a technique to establish a silicosis rat model with the inhalation of silica through the whole body in an inhalation chamber. The rats with silicosis could closely mimic the pathological process of human silicosis in an easy, cost-effective manner with good repeatability.

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent ...

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Biology

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 ...

Citric Acid-Induced Mouse Model for Cough Hypersensitivity

Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity&#160;via&#160;continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

This protocol describes the development of a mouse model with cough hypersensitivity, which can serve as an ideal model for studying the mechanisms of chronic cough.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable ...

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 ...

Establishing a Mouse Model of Pleural Disseminated Lung Cancer

Source: Yasui, H., et al. <a href="https://app.jove.com/v/61593">Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination.</a> J. Vis. Exp. (2021)
This video demonstrates a method to generate a mouse model of pleural dissemination by injecting lung cancer cell suspension into the thoracic cavity of an immunodeficient mouse.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

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