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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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 importa...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#39;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)&#160;</td>
			<td>Biosharp</td>
			<td>BL601A</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological saline&#160;</td>
			<td>The First People&#39;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&#160;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-&#946;</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>Name of Material/ Equipment</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&#160;Mixers&#160;</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</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 sever...

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 ...

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Research

JoVE Journal

Medicine

650 Views.  Anhui University of Science and Technology. 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.

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

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 ...

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 ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...