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 were conducted according to the guidelines issued by the National Institutes of Health&#39;s Guide for the Care and Use of Laboratory Animals (the 8th edition of the NRC) and were approved by Anhui University of Science and Technology Animal Ethics Committee.
1. Animal preparation
<ol>
	<li>House 32 male C57/BL6 mice aged 8 weeks in a laboratory with a 12 h light/dark cycle. Ensure that the mice have free access to food and water.</li>
	<li>After 2 weeks of acclimation to the environment, when the 10 week old mice weigh 23-26 g, use random numbers generated by a computer to randomly group the animals without partitioning, resulting in four groups of mice: the control group (Con), the nicotine group (Nic), the silica group (SiO2), and the nicotine combined with silica group (Nic+ SiO2).</li>
</ol>
2. Nicotine preparation
NOTE: The nicotine density is 1.01 g/mL.
<ol>
	<li>Dissolve the nicotine in anhydrous ethanol, and dilute 20x to prepare a nicotine stock solution of 50 mg/mL.</li>
	<li>Dilute the nicotine stock solution 1,000x with sterile normal saline to make a working solution at a concentration of 0.05 mg/mL. 
	&#8203;NOTE: Store nicotine away from light. The stock solution can be stored at 4 &#176;C for up to 1 week.</li>
</ol>
3. Silica preparation
<ol>
	<li>Suspend sterile crystalline silica in saline to prepare a 20 mg/mL silica suspension, and oscillate it in an ultrasonic shaking water bath for 25 min.</li>
	<li>Shake the silica suspension with a vortex oscillator for 3 min before the mice receive the nasal drip. Mix the silica suspension by pipetting up and down 2x-3x with a 200 &#181;L pipette, and then take up 50 &#181;L for the nasal drip. 
	NOTE: The silicon dioxide suspension should be mixed and administered via nasal drip as soon as possible.</li>
</ol>
4. Mouse capture and nicotine exposure
<ol>
	<li>Weigh the mice, and record the weights daily before exposure.</li>
	<li>On days 1-40, subcutaneously inject nicotine twice daily (12 h apart) into the mice in the Nic and Nic+ SiO2 groups. Use a nicotine dose of 0.25 mg/kg; for example, ensure that a mouse weighing 25 g receives 6.25 &#181;g of nicotine. For this mouse, the injection volume would be as follows: 6.25/0.05 = 125 &#181;L. Simultaneously, inject the mice in the Con group with an equal volume of saline.</li>
	<li>Prepare the required 1 mL syringes for each group, and use them to aspirate the injection volume of nicotine or saline. Before taking up the nicotine, first draw 0.1-0.2 mL of air into the syringe, and then draw up 125 &#181;L of nicotine. Carefully tap the syringe to fill the needle and the front end of the syringe with the nicotine. Place the nicotine-containing syringe in a tray protected from light. 
	NOTE: At the time of injection, 0.1-0.2 mL of air is situated behind the nicotine, guaranteeing that all the liquid is successfully administered to the mouse.</li>
	<li>Grasp the tail of the mouse with the right hand, and when the animal relaxes, use the left thumb and forefinger to press the skin on the back of the neck from the tail to the head up to the edge of the ear, applying moderate pressure. After that, release the right hand, and use the left small thumb and ring finger to pinch the tail and hind limbs, at which point the mouse will be completely immobilized by the left hand. 
	NOTE: Grasp the mouse with appropriate force. Insufficient strength makes it easy for mice to bite, whereas excessive strength may result in the asphyxiation of the mice.</li>
	<li>Using the right hand to inject, puncture the skin on the back of the neck near the ear edge of the mice in a head-to-tail direction with the syringe, and inject with nicotine at a uniform rate. Look for a semicircular skin bulge that indicates a successful injection. 
	NOTE: When the needle has punctured into the skin, there will be a feeling of resistance; the resistance disappears after the needle has been inserted. At this point, the needle needs to be slightly withdrawn to inject slowly and evenly.</li>
</ol>
5. Silica exposure in vivo
<ol>
	<li>On days 5-19, instill the silicon dioxide suspension into the nasal cavity of the mice in the SiO2 and Nic+ SiO2 groups. Rapidly anesthetize each mouse with 2% isoflurane in an anesthesia machine at a dose of 3.6 mL/h. After anesthesia, place the mouse on the palm of one hand, and expose the nasal cavity of the mouse. Instill 50 &#181;L of silica suspension into the nasal cavity within 4-8 s.</li>
	<li>To allow the silica suspension to enter the lungs as soon as possible, after instillation, press the mouse&#39;s heart gently with the index finger for 3-5 s, 3x-5x per second, to promote its respiratory rate.</li>
	<li>After the respiration of the mouse gradually becomes uniform, place the mouse in a cage to observe for 3 min.</li>
	<li>For all mice receiving the silica suspension, repeat steps 1-3. In the control group, instill 50 &#956;L of sterile saline into the nasal cavity on days 5-19.</li>
</ol>
6. Acquisition of fresh and fixed lung tissues
<ol>
	<li>On day 41, use 50 mg/kg of sodium pentobarbital to anesthetize&#160;the mice intraperitoneally at a dose of 0.1 mL/20 g according to their body weight. Fix the limbs on a foam test plate, and spray the fur with 75% alcohol.</li>
	<li>Perform&#160;cardiac perfusion to obtain fresh lung tissue, cut the midline of the abdomen to expose the chest cavity, and make a small opening at the apex of the right heart. Then, inject 20 mL of pre-cooled phosphate-buffered saline (PBS) slowly and evenly from the apex of the left heart, causing the blood to flow out from the opening created at the apex of the right heart. Take out the entire lung lobe, place it in a pre-cooled 1.5 mL centrifuge tube, and transfer to &#8722;80 &#176;C for storage pending protein extraction.</li>
	<li>Perfuse other mice with 20 mL of pre-cooled PBS followed by 10 mL of 4% paraformaldehyde at the same location to fix whole lungs; preserve each lung in 30 mL of 4% PFA.</li>
	<li>After 72 h, embed the fixed lung tissues in paraffin.
	<ol>
		<li>Immerse the lung tissue in the prepared fixative in an ultrasonic water bath at 40 &#176;C for 30 min, followed by a dehydration process in 75% ethanol, 95% ethanol, and anhydrous ethanol for 50 min each.</li>
		<li>Wash 2x with xylene for 50 min each.</li>
		<li>Place the tissue in melted paraffin wax at 55-60 &#176;C for 2-3 h so that the xylene in the lung tissue is gradually replaced by paraffin wax.</li>
		<li>Fix the lung tissue in the embedding frame with paraffin, and wait for the wax block to harden. After the wax block has hardened, use the paraffin sectioning machine to slice the lung at 4-5 &#181;m.</li>
	</ol>
	</li>
	<li>Place the lung sections in ultrapure water at 45 &#176;C. Once the paraffin has melted, load the lung section onto an adherent microscope slide.</li>
</ol>
7. Hematoxylin and eosin (HE) staining
<ol>
	<li>Bake the slides at 60 &#176;C for 2 h, and place the slides in xylene to dewax for 2 x 30 min. Subsequently, place the slides in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each. Finally, wash them for 3 x 5 min with ultrapure water.</li>
	<li>Drop 50 &#181;L of hematoxylin staining solution onto the lung tissue with a pipette gun, and stain for 5 min; then, wash the slices with running water for 5 min.</li>
	<li>Add 50 &#181;L of 2% hydrochloric acid in ethanol onto the section, and rinse with distilled water for 30 s. Then, add 50 &#181;L of eosin staining solution onto the lung tissue to stain for 1 min.</li>
	<li>Dehydrate, transparentize, and seal the paraffin sections.
	<ol>
		<li>Add 100-150 mL of 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene in a combined plastic dyeing tank. Place the slides in 75%, 85%, 95%, and anhydrous ethanol for 5 min each to dehydrate.</li>
		<li>Then, place the slides in xylene for 5 min to transparentize.</li>
		<li>Finally, drop 20 &#181;L of neutral gum onto the lung section, and place a coverslip over the slide to seal it. 
		&#8203;NOTE: The above steps are carried out at room temperature.</li>
	</ol>
	</li>
</ol>
8. Masson staining
<ol>
	<li>Dewax, hydrate, and wash the paraffin sections as described in step 7.1.</li>
	<li>Stain the sections with 50 &#181;L of configured Weigert&#39;s hematoxylin staining solution for 7 min, followed by 50 &#181;L of acidic ethanol fractionation solution for 10 s, and wash with running water to turn the nuclei blue.</li>
	<li>Stain the sections with 50 &#181;L of Masson trichrome solution for 4 min, and wash with water.</li>
	<li>Rinse the sections with distilled water for 1 min, and then stain with Lichun red acid magenta for 7 min.</li>
	<li>Wash the sections with a weak acid working solution (30% hydrochloric acid) for 1-2 min, phosphomolybdic acid solution for 1-2 min, and weak acid working solution for 1-2 min.</li>
	<li>Stain the sections in aniline blue staining solution for 2 min, and then wash with a weak acid working solution for 1 min.</li>
	<li>Dehydrate the sections with 95% ethanol followed by anhydrous ethanol for 1&#160;s, transparentize with xylene for 1 s, and finally seal with neutral resin as described in step 7.4.</li>
</ol>
9. Immunohistochemistry
<ol>
	<li>Bake the slices at 60 &#176;C for 6 h. Dewax, hydrate, and wash the paraffin sections as described in step 7.1.</li>
	<li>Antigen retrieal: place the slices in a heat-resistant plastic slicing box containing enough citrate antigen retrieval solution to submerge the slices, and boil for 20-30 min.</li>
	<li>Cool to room temperature, remove the sections, and rinse with deionized water. Subsequently, soak for 2 x 5 min in PBS containing 0.5% Tween-20 (PBST).</li>
	<li>Draw a loop near the lung section on the slide with a hydrophobic pen to form a hydrophobic circle.</li>
	<li>Add 50 &#181;L of 3% hydrogen peroxide solution (3% H2O2) dropwise to the slices, and incubate for 15 min at room temperature protected from light for the inactivation of endogenous peroxidase.</li>
	<li>Soak the slices in PBST for 3 x 5 min.</li>
	<li>Add 50 &#181;L of 5% bovine serum protein (BSA)-PBST dropwise to the slices, and block for 1 h at room temperature.</li>
	<li>Discard the blocking solution, add 50 &#181;L of the diluted primary antibodies CD206 (dilution: 1:1,500), TGF-&#946;1 (dilution: 1:200), and vimentin (dilution: 1:2,000) dropwise to the slices, and incubate overnight at 4 &#176;C. 
	NOTE: In this work, all the antibodies were diluted in 5% BSA.</li>
	<li>The next day, return the slices to room temperature (40 min). Wash the slices as described in step 9.6.</li>
	<li>Dilute the secondary antibody in 5% BSA. Add 50 &#181;L of horseradish peroxidase-labeled secondary antibody (dilution: 1:500) dropwise, and incubate for 1 h at room temperature. Wash the slices as described in step 9.6.</li>
	<li>Drop 50 &#181;L of 3,3&#39;-diaminobenzidine (DAB) solution corresponding to the enzyme-labeled secondary antibody onto the lung tissue, and incubate for 5-10 min in a black immunohistochemical wet box. Observe under the microscope until the color develops to a strong brown. Rinse the slices with distilled water to terminate the reaction. 
	NOTE: Brown staining is considered positive staining.</li>
	<li>Stain with 50 &#181;L of hematoxylin stain for 30 s, and wash with running water. Next, soak the slices in 75%, 85%, 95%, and anhydrous ethanol for 3 min each and then in xylene for 2 x 10 min. Seal the slides as described in step 7.4.</li>
</ol>
10. Western blot analysis
<ol>
	<li>Take the lung tissue out of the freezer, and weigh it. Next, add 150 &#181;L of RIPA lysis buffer containing PMSF per 20 mg of lung tissue. Lyse the mouse lungs on ice for 3-5 min using a handheld homogenizer, shake gently for 2 h at 4 &#176;C to allow adequate lysis of the lung tissue, centrifuge at 4 &#176;C and 14,800 &#215; g for 15 min, and collect the supernatant. 
	NOTE: Add 100 mM phenylmethanesulfonylfluoride (PMSF) a few minutes before using the RIPA lysis buffer. The final concentration of PMSF is 1 mM (e.g., 10 &#181;L of PMSF + 990 &#181;L of RIPA).</li>
	<li>Use a BCA protein concentration kit to determine the protein concentration of the sample by following the manufacturer&#39;s instructions. After determination, using the smallest concentration of protein lysate as a base, dilute the same batch of protein lysate with RIPA to the smallest concentration to achieve the same mass and volume. 
	NOTE: The total amount of lung tissue loading is 30 &#181;g.</li>
	<li>Add 40 &#181;L of 5x loading buffer to 160 &#181;L of protein lysate, and heat it at 100 &#176;C for 10 min.</li>
	<li>Assemble the prepared 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel in the western blotting electrophoresis system, add the SDS-PAGE electrophoresis buffer, gently pull out the electrophoresis comb, and add 20 &#181;L of protein sample per well slowly and uniformly. Switch on the power, and start the electrophoresis at 80 V for 30 min. After the proteins have reached the lower gel layer, switch to 100 V, and continue the electrophoresis for 60-70 min. 
	NOTE: Electrophoresis needs fresh electrophoresis buffer.</li>
	<li>Transfer the proteins to a PVDF membrane by wet transfer. Activate the PVDF membrane with methanol in advance (5-30 s). Take the electrophoresis gel plate out, carefully pry it open, place the separator gel into the wet transfer buffer, make the electric transfer sandwich, and assemble the wet transfer system. Fill the electrophoresis tank with transfer buffer, and transfer at 400 mA for 90 min. 
	NOTE: The transfer buffer is cooled in advance to avoid the effects of high temperatures during the wet transfer.</li>
	<li>Wash the PVDF membrane with Tris-buffered saline with 0.5% Tween-20 (TBST) for 3 x 5 min. Block the PVDF membrane with 5% skim milk diluted in TBST, and incubate on a shaker at room temperature for 60 min; then, wash with TBST for 2 min. After washing, incubate the membrane overnight at 4 &#176;C with the primary antibody vimentin (1:5,000) and GAPDH (1:10,000) diluted in 5% BSA.</li>
	<li>Place the membrane at room temperature for 30 min, and wash with TBST for 5 x 5 min. After washing, incubate the membrane with the secondary antibody (1:10,000) diluted in 5% skim milk for 60 min at room temperature.</li>
	<li>Place the membrane, protein-side up, drop the prepared ECL working solution, incubate for 1-2 min, and observe the results with a gel imager. Use the greyscale values measured by the software in the gel imager to evaluate the protein expression levels. 
	NOTE: Here, GAPDH served as an internal reference.</li>
</ol>

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</ol>

All authors declare they have no conflicts of interest.

A dual-exposure animal model is necessary to investigate the role and the potential mechanisms of concurrent exposure to nicotine and crystalline silicon dioxide. This model was achieved in this work through the subcutaneous injection of nicotine and the nasal drip of silica. To ensure a successful nicotine injection, the operator has to become familiar with grasping the mice, as grasping the skin at the back of the neck could be painful for them. Therefore, allowing the mice to adapt gradually to the grasping is important. In addition, a critical step in silica exposure in mice is the nasal drip. To increase the success of the procedure and the survival rate of the mice, it is essential to practice the nasal drip beforehand.
When performing the injection, the syringe should be skimmed over the mouse's head to avoid a strong struggle when the mouse sees the syringe. The mouse's tail should be firmly grasped with the right hand, while the left hand should be used to push the skin up to the ear margin at the back of the neck carefully and gently and to pinch the skin there. After releasing the mouse's tail with the right hand, the tail and hind limbs should be stabilized using the left thumb and ring finger to prevent biting. Using a 1 mL syringe in the right hand, the needle should be inserted into the loose skin above the neck at a 30&#176; angle in a head-to-tail direction. The needle should be withdrawn slightly after the resistance is lost, and the injection should be administered slowly and evenly. On days 5-19, mice in the dual-exposed group were exposed to nicotine and silica. It is recommended that the nicotine be injected at 08:00 a.m., followed by the nasal drip of silicon dioxide at 14:00 p.m., and another injection of nicotine at 20:00 p.m. The two nicotine injections must be given 12 h apart to avoid the effects of repeated grasping on the mice.
Performing subcutaneous injections and nasal drip presents several technical challenges. For subcutaneous injections, the mice should be grasped with appropriate force. If the skin at the back of the neck is pinched too tightly, the mouse's airway will be blocked, quickly leading to suffocation. For optimal strength, the skin at the back of the neck should be pinched until the eyeballs protrude slightly. At this time, the mouse feels the lowest pain and breathes smoothly. In addition, a 1 mL syringe that is emptied of air should be used to draw the nicotine injection from the tube, followed by a further 0.1-0.2 mL of air. Before injecting, the bubbles should be gently flicked out. Due to the small volume of the injection, the nicotine left at the tip of the needle end may lead to an insufficient dose. The key parts of the injection are inserting the needle quickly, injecting the nicotine slowly, and removing the needle gently. For nasal drip, in this study, the nasal cavity of the mice was fully exposed under deep anesthesia, followed by the slow and uniform instillation of a silicon dioxide suspension. It is also important to gently press the thoracic cavity after silica exposure to avoid coughing or suffocation.
This model has certain limitations. The 1-5 &#181;m silica particles used in the experiments were not collected from the environment and were pure silica particles. In contrast, in actual occupational settings, workers may be exposed to a variety of hazardous materials, not just silica particles, such as a mixture of coal and silica dust in a ceramic factory or a coal mine. We used nasal instillation silica to achieve low doses and repeated multiple exposures to simulate the chronic exposure of workers. In the dual-exposure mouse model, although the nicotine does not come from cigarette smoke exposure, the injected nicotine directly enters the bloodstream, allowing for a more focused exploration of the role of nicotine in the silicosis process and the avoidance of the effects of other components of cigarette smoke.
Scientific research on silicosis has typically utilized single-factor mouse models, either through the inhalation or bronchial perfusion of silica, to assess the role of silica in the development of disease. An alternative approach is the nasal instillation of silicon dioxide, which is simple and easy and allows for the establishment of repeated and chronic silica exposure models13. The well-established silicon dioxide nasal drip model causes minimal damage to the mice and could be combined with other factors to create a multi-factorial-exposed animal model. Additionally, the main methods of nicotine exposure in mice include nicotine in drinking water, subcutaneous injection of nicotine, nicotine infusion via osmotic minipumps, and tobacco smoke exposure14. The subcutaneous injection used in the dual-exposure model allows the dosage and timing of nicotine to be precisely set, ensuring that each mouse is administered the same dose.
In short, the animal model of nicotine in combination with silica exposure replicates a realistic chronic exposure pattern, and this model can be utilized for further investigations on the impacts of nicotine on inflammation and fibrosis in the development of silicosis. Additionally, this model serves as a foundation for forming a dual-exposure animal model with multiple doses and time frames.

Silica exposure in workers is inevitable in some occupational settings, and once exposed to silica, the deterioration progresses even after removal from the environment. In addition, most of these workers smoke, and traditional cigarettes contain thousands of chemicals, with the key addictive component being nicotine1. E-cigarettes are becoming increasingly popular in younger age groups2; these e-cigarettes act as a nicotine delivery system and increase nicotine access, thus increasing lung susceptibility and pneumonia3. Cigarette smoke also accelerates pulmonary fibrosis in bleomycin-exposed mice4 and increases pulmonary toxicity and fibrosis in silica-exposed mice5,6. However, whether nicotine can affect the inflammatory and pulmonary fibrosis process caused by silica remains to be investigated.
The silicosis mouse model established by the one-time inhalation of a high dose of silica into the trachea is traumatic to mice. Although this method quickly provides a silicosis model, it does not match the reality of an environment where workers are repeatedly exposed to silica. Therefore, we established a silica-exposed mouse model by repeatedly giving a low dose of silica suspensions via a nasal drip; this dose can cause inflammation and fibrosis in mice.
To circumvent the effects of other cigarette components, this mouse model was subcutaneously injected with nicotine into the loose skin of the neck for determining the effect of the addictive component, nicotine, on silicosis. By administering subcutaneous injections, accurate dosing can be achieved, thus making it possible to create nicotine exposure models and observe dose-toxicity responses, as well as addiction. A nicotine addiction model has been developed in male mice, with a nicotine injection dose of 0.2-0.4 mg/kg7,8. In that model, to meet the drug-seeking needs of the addicted mice, two subcutaneous injections were administered at intervals of 12 h. This mouse nicotine addiction model is useful for simulating human smoking habits and exposure to silica.
Single-factor animal models have limitations in disease studies, whereas the method described here involves a two-factor mouse model of nicotine and silica co-exposure. Prior to the silica exposure, the mice were pre-exposed to nicotine to replicate nicotine exposure in people who smoke. Subsequently, silica exposure took place from day 5 to day 19 to imitate silica exposure in a working environment for individuals with a history of smoking.
Alveolar macrophages are known to play a significant role in the regulation of lung inflammation and fibrosis. Macrophages cannot break silica down upon its inhalation of silica, leading to macrophage polarization or apoptosis9 and the release of cytokines such as tumor necrosis factor-alpha (TNF-&#945;) and transforming growth factor beta (TGF-&#946;). M1 macrophages, which are identified by the presence of the surface marker CD86, are the primary instigators of the inflammatory response in silicosis, while M2 macrophages, which are marked by CD206, are responsible for the fibrotic phase of the condition10. In dual-exposed mice, nicotine induced the polarization of macrophages toward the M2 phenotype in silica-injured lungs, thus promoting pulmonary fibrosis. Furthermore, TGF-&#946;1 is key to the induction of fibrosis and EMT11; the increased expression of TGF-&#946;1 accelerated the progression of lung fibrosis through EMT. This model successfully analyzed the effects of nicotine on silicosis and further highlighted the importance of nicotine cessation.

<table><tbody><tr><td>10% formalin neutral fixative</td><td>Nanchang Yulu Experimental Equipment Co.</td><td></td><td></td></tr><tr><td>alcohol disinfectant</td><td>Xintai Kanyuan Disinfection Products Co.</td><td></td><td></td></tr><tr><td>BSA, Fraction V</td><td>Beyotime Biotechnology</td><td>ST023-200g</td><td></td></tr><tr><td>CD206 Monoclonal antibody</td><td>Proteintech</td><td>60143-1-IG</td><td></td></tr><tr><td>Citrate Antigen Retrieval Solution</td><td>biosharp life science</td><td>BL619A</td><td></td></tr><tr><td>dimethyl benzene</td><td>West Asia Chemical Technology (Shandong) Co</td><td></td><td></td></tr><tr><td>Enhanced BCA Protein Assay Kit</td><td>Beyotime Biotechnology</td><td>P0009</td><td></td></tr><tr><td>GAPDH Polyclonal antibody</td><td>Proteintech</td><td>10494-1-AP</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-Mouse IgG(H+L)</td><td>Proteintech</td><td>SA00001-1</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>ImmPACT[R] DAB EqV Peroxidase (HRP) Substrate</td><td>Vector Laboratories</td><td>SK-4103-100</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></td><td></td></tr><tr><td>Nicotine</td><td>Sigma</td><td>N-3876</td><td></td></tr><tr><td>phosphate buffered saline (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></td><td></td></tr><tr><td>PMSF</td><td>Beyotime Biotechnological</td><td>ST505</td><td></td></tr><tr><td>Positive fluorescence microscope</td><td>OlympusCorporation</td><td>BX53+DP74</td><td></td></tr><tr><td>Prestained Color Protein Molecular Weight&amp;nbsp;Marker, or Prestained Color Protein Ladder</td><td>Beyotime Biotechnology</td><td>P0071</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>SDS-PAGE gel preparation kit</td><td>Beyotime Biotechnology</td><td>P0012A</td><td></td></tr><tr><td>Silicon dioxide</td><td>Sigma</td><td>#BCBV6865</td><td></td></tr><tr><td>TGF-&amp;beta;</td><td>Bioss</td><td>bs-0086R</td><td></td></tr><tr><td>Vimentin Polyclonal antibody</td><td>Proteintech</td><td>10366-1-AP</td><td></td></tr><tr><td>&lt;strong&gt;Name of Material/ Equipment&lt;/strong&gt;</td><td>Company</td><td>Catalog Number</td><td></td></tr><tr><td>0.5 mL Tube</td><td>Biosharp</td><td>BS-05-M</td><td></td></tr><tr><td>Oscillatory thermostatic metal bath</td><td>Abson</td><td></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></td><td></td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td></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>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></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</td><td>KIRGEN</td><td>KG1011</td><td></td></tr><tr><td>Universal Pipette Tips</td><td>KIRGEN</td><td>KG1212</td><td></td></tr><tr><td>Universal Pipette Tips</td><td>KIRGEN</td><td>KG1313</td><td></td></tr><tr><td>Vortex&amp;nbsp;Mixers&amp;nbsp;</td><td>VWR</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name of Material/ Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe Illustrator</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td></td></tr><tr><td>Photoshop</td><td></td><td></td><td></td></tr><tr><td>Prism7.0</td><td></td><td></td><td></td></tr></tbody></table>

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.

A mouse model to study nicotine combined with silica exposure was established to investigate the potential role of nicotine in the progression of silicosis in mice. Figure 1 depicts the experimental procedure for using a dual-exposure mouse model, which paired a nicotine injection with the nasal instillation of a silica suspension. The pathological changes of the mice in each group were observed using HE staining. The mice exposed to nicotine combined with silica had significantly more severe lung damage than those exposed to nicotine or silica alone. Lymphocytes increased near the lymphatic vessels in the lungs of the nicotine-exposed mice, forming inflammatory cell clusters. Masson staining revealed a significant increase in collagen fiber deposition in the lungs exposed to nicotine combined with silica compared to the lungs in the other groups, and this was supported by the Masson staining quantification (Figure 2). The alveolar structure was destroyed in the silica-exposed mice, and the number of macrophages increased. However, after exposure to nicotine combined with silica, there was significant inflammatory cell infiltration, and fibroblast nodules appeared. In addition, accumulated&#160;macrophages, especially M2 macrophages, present&#160;in the double-exposed group. M2 macrophages are essential for the advanced fibrotic stage of silicosis.
Additionally, a significant increase in the pro-fibrotic factor TGF-&#946;1 was observed by immunohistochemical (IHC) staining in the lungs of dual-exposed mice, especially in inflammatory cells near lymphatic vessels (Figure 3). TGF-&#946;1 secreted by macrophages promotes the EMT of epithelial cells and pulmonary fibrosis12. Compared with mice exposed to silica alone, vimentin levels were significantly elevated in the lungs of dual-exposure mice. Both IHC staining and protein quantification indicated severe EMT in dual-exposed mice (Figure 4). The combined evidence suggests that chronic silica exposure promotes EMT after the upregulation of TGF-&#946;1, leading to an increase in fibroblasts and progressive fibrosis. At the same time, the addition of nicotine accelerates the process of pulmonary fibrosis by aggravating EMT, allowing lung fibrosis to appear earlier. This model is designed to explore the impact of nicotine on pulmonary fibrosis, which is a consequence of&#160; chronic silica exposure in humans.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65127/65127fig01v2.jpg" /> 
Figure 1: Design of an experimental model of combined exposure to nicotine and silica in mice. Continuous injection of nicotine for 1-40 days and continuous nasal instillation of silica for 5-19 days. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65127/65127fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65127/65127fig02.jpg" /> 
Figure 2:&#160;Nicotine promotes the formation of fibroblastic masses in the lungs of silica-exposed mice. (A) HE staining was used to visualize pathological changes in the lungs. The dual-exposed mice had severe inflammatory cell infiltration and fibrosis. The green arrows indicate inflammatory cell masses. Scale bar = 50 &#181;m.&#160;(B) Masson staining was used to show collagen fibers in the lungs. Scale bar = 50 &#181;m. (C) Quantification of collagen fibers in the lungs. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65127/65127fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65127/65127fig03v2.jpg" /> 
Figure 3:&#160;Increased CD206-positive cells and TGF-&#946;1 in the lungs of nicotine and silica dual-exposed mice. (A) IHC staining of CD206 was used to observe the distribution and expression of macrophages in the dual-exposed mice. CD206-positive macrophages increased in the lungs of mice after combined nicotine and silica exposure. The short arrows represent CD206-positive cells. Scale bar = 50 &#181;m.&#160;(B) TGF-&#946;1, a promoter of fibrosis, was elevated in dual-exposed mice. The long arrows represent TGF-&#946;1-positive cells. Scale bar = 50 &#181;m. (C,D) AOD&#160;of CD206 and TGF-&#946;1. AOD = IOD (integrated optical density)/area. The AOD reflects the concentration of CD206 and TGF-&#946;1 per unit area. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. Abbreviations: TGF-&#946;1 = transforming growth factor-beta; IHC = immunohistochemistry; AOD = average optical density. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65127/65127fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65127/65127fig04.jpg" /> 
Figure 4: Promotion of lung fibrosis by nicotine in silica-exposed mice through the aggravation of EMT. (A) The expression of vimentin in each group was observed by IHC staining. Vimentin was strongly expressed in the lungs of the mice exposed to nicotine and silica. Scale bar = 50 &#181;m.(B) Western blot of vimentin expression in each exposure group, with an increase in vimentin in the lung tissues of double-exposed mice. (C) The AOD value of vimentin was significantly more elevated in the double-exposed group compared to the other groups. (D) The relative protein expression level of vimentin compared to GAPDH. The vimentin expression in the double-exposure group was significantly higher than in the nicotine- or silica-exposure groups. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. Abbreviations: EMT = epithelial-mesenchymal transition; AOD = average optical density. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65127/65127fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition at JoVE.com

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

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 ...

using-nicotine-silica-exposed-mouse-model-to-promote-lung-epithelial

Nanyang Technological University

Research

JoVE Journal

Medicine

702 Views.  Anhui University of Science and Technology. Het muismodel dat in deze studie wordt gebruikt, simuleert effectief het effect van chronische nicotine-inname en herhaalde blootstelling aan silica op longfibroses door epitheliale mesenchymale overgang bij mensen. Het juryportiemodel werd bereikt door subcutane injectie van nicotine en het neusinfuus van silica op een eenvoudige en betrouwbare manier. Suspendeer steriel kristallijn silica in zoutoplossing om een suspensie van 20 milligram per liter te bereiden.Oscilleer het gedurende...

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

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Developmental Biology

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Immunology and Infection

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 ...

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.

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 ...

In Vitro Model of Murine Airway Epithelial Cells and Chronic Smoke Exposure

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Author Spotlight: Development and Characterization of an In Vitro Model to Study Chronic Cigarette Smoke Exposure and Its Impact on Airway Epithelial Cells in COPD Research

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

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

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