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 ac...

<ol>
	<li>Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+nicotinic+ACh+receptors.">Presynaptic nicotinic ACh receptors.</a> Trends in Neurosciences. 20 (2), 92-98 (1997).</li><li>Berry, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+electronic+cigarette+use+with+subsequent+initiation+of+tobacco+cigarettes+in+US+youths.">Association of electronic cigarette use with subsequent initiation of tobacco cigarettes in US youths.</a> JAMA Network Open. 2 (2), 187794 (2019).</li><li>Masso-Silva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+e-cigarette+aerosol+inhalation+alters+the+immune+state+of+the+lungs+and+increases+ACE2+expression,+raising+concern+for+altered+response+and+susceptibility+to+SARS-CoV-2.">Chronic e-cigarette aerosol inhalation alters the immune state of the lungs and increases ACE2 expression, raising concern for altered response and susceptibility to SARS-CoV-2.</a> Frontiers in Physiology. 12, 649604 (2021).</li><li>Cisneros-Lira, J., Gaxiola, M., Ramos, C., Selman, M., Pardo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+exposure+potentiates+bleomycin-induced+lung+fibrosis+in+guinea+pigs.">Cigarette smoke exposure potentiates bleomycin-induced lung fibrosis in guinea pigs.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 285 (4), 949-956 (2003).</li><li>Sager, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+exposure+exacerbated+crystalline+silica-induced+lung+toxicity+in+rats.">Tobacco smoke exposure exacerbated crystalline silica-induced lung toxicity in rats.</a> Toxicological Sciences. 178 (2), 375-390 (2020).</li><li>Zhou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoking+aggravates+bleomycin-induced+experimental+pulmonary+fibrosis.">Cigarette smoking aggravates bleomycin-induced experimental pulmonary fibrosis.</a> Toxicology Letters. 303, 1-8 (2019).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+and+hippocampal+Epac+signaling+to+nicotine+CPP+in+mice.">Behavior and hippocampal Epac signaling to nicotine CPP in mice.</a> Translational Neuroscience. 10, 254-259 (2019).</li><li>Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+deep+sequencing+analysis+reveals+that+nicotine+restores+impaired+gene+expression+by+viral+proteins+in+the+brains+of+HIV-1+transgenic+rats.">RNA deep sequencing analysis reveals that nicotine restores impaired gene expression by viral proteins in the brains of HIV-1 transgenic rats.</a> PLoS One. 8 (7), 68517 (2013).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica+particles+disorganize+the+polarization+of+pulmonary+macrophages+in+mice.">Silica particles disorganize the polarization of pulmonary macrophages in mice.</a> Ecotoxicology and Environmental Safety. 193, 110364 (2020).</li><li>Tang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pirfenidone+ameliorates+pulmonary+inflammation+and+fibrosis+in+a+rat+silicosis+model+by+inhibiting+macrophage+polarization+and+JAK2/STAT3+signaling+pathways.">Pirfenidone ameliorates pulmonary inflammation and fibrosis in a rat silicosis model by inhibiting macrophage polarization and JAK2/STAT3 signaling pathways.</a> Ecotoxicology and Environmental Safety. 244, 114066 (2022).</li><li>Hinz, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblast:+One+function,+multiple+origins.">The myofibroblast: One function, multiple origins.</a> The American Journal of Pathology. 170 (6), 1807-1816 (2007).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA-ATB+promotes+EMT+during+silica-induced+pulmonary+fibrosis+by+competitively+binding+miR-200c.">Long non-coding RNA-ATB promotes EMT during silica-induced pulmonary fibrosis by competitively binding miR-200c.</a> Biochimica et Biophysica Acta. Molecular Basis of Disease. 1864 (2), 420-431 (2018).</li><li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suitable+silicosis+mouse+model+was+constructed+by+repeated+inhalation+of+silica+dust+via+nose.">A suitable silicosis mouse model was constructed by repeated inhalation of silica dust via nose.</a> Toxicology Letters. 353, 1-12 (2021).</li><li>Chellian, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+nicotine+withdrawal.">Rodent models for nicotine withdrawal.</a> Journal of Psychopharmacology. 35 (10), 1169-1187 (2021).</li></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 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.

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			<td>10% formalin neutral fixative</td>
			<td>Nanchang Yulu Experimental Equipment Co.</td>
			<td></td>
			<td></td>
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			<td>alcohol disinfectant</td>
			<td>Xintai Kanyuan Disinfection Products Co.</td>
			<td></td>
			<td></td>
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			<td>BSA, Fraction V</td>
			<td>Beyotime Biotechnology</td>
			<td>ST023-200g</td>
			<td></td>
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			<td>CD206 Monoclonal antibody</td>
			<td>Proteintech</td>
			<td>60143-1-IG</td>
			<td></td>
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			<td>Citrate Antigen Retrieval Solution</td>
			<td>biosharp life science</td>
			<td>BL619A</td>
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			<td>dimethyl benzene</td>
			<td>West Asia Chemical Technology (Shandong) Co</td>
			<td></td>
			<td></td>
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			<td>Enhanced BCA Protein Assay Kit</td>
			<td>Beyotime Biotechnology</td>
			<td>P0009</td>
			<td></td>
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			<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>
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		<tr>
			<td>HRP substrate</td>
			<td>Millipore Corporation</td>
			<td>P90720</td>
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			<td>HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)</td>
			<td>Proteintech</td>
			<td>SA00001-1</td>
			<td></td>
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		<tr>
			<td>HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)</td>
			<td>Proteintech</td>
			<td>SA00001-2</td>
			<td></td>
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			<td>ImmPACT[R] DAB EqV Peroxidase (HRP) Substrate</td>
			<td>Vector Laboratories</td>
			<td>SK-4103-100</td>
			<td></td>
		</tr>
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			<td>Masson&#39;s Trichrome Stain Kit</td>
			<td>Solarbio</td>
			<td>G1340</td>
			<td></td>
		</tr>
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			<td>Methanol</td>
			<td>Macklin</td>
			<td></td>
			<td></td>
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		<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>
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		<tr>
			<td>Prestained Color Protein Molecular Weight&#160;Marker, or Prestained Color Protein Ladder</td>
			<td>Beyotime Biotechnology</td>
			<td>P0071</td>
			<td></td>
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			<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>
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			<td>Oscillatory thermostatic metal bath</td>
			<td>Abson</td>
			<td></td>
			<td></td>
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			<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>
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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 ...

The mouse model employed in this study effectively simulates the effect of chronic nicotine ingestion and repeated exposure to silica on lung fibroses through epithelial mesenchymal transition in human beings. The jury portion model was achieved by subcutaneous injection of nicotine and the nasal drip of silica in a simple and a reliable way. Suspend sterile crystalline silica in saline to prepare a 20 milligrams per liter suspension.Oscillate it in an ultrasonic shaking water bath for 25 minutes. Place the silica suspension with a vortex oscillator for three minutes. Aspirate the suspension to mix it, and then use 50 microliters for the nasal drip.To begin silica exposure, first, place the anesthetized mouse on the palm of one hand and expose the nasal cavity of the mouse. Inject 50 microliters of the silica suspension from one nostril within four to eight seconds. To allow for quick lung penetration of the suspension, gently press the mouse's chest with the index finger for three to five seconds, repeating the press three to five times each second to maintain its respiratory rate.Once the mouse displays uniform respiration, place it under observation in a cage for three minutes. Prepare nicotine syringes by drawing air until the mark of 0.2 milliliter into a one milliliter syringe. Then, draw up 125 microliters of nicotine.Tap the syringe to fill the needle and the front end with nicotine, and then place it in a tray protected from light. With the right hand, grasp the mouse's tail. When the mouse has relaxed, Use the left thumb and forefinger to apply moderate pressure on the skin from the tail to the edge of the ear.Now, release the right hand and use the left, small, and ring fingers to pinch the tail and hind limbs to immobilize the mouse. Hold the injection syringe in the right hand and puncture the skin on the back of the neck in a head to tail direction. Inject the nicotine at a uniform rate.Fix the anesthetized mouse on a foam test plate and spray the fur with 75%alcohol. Make an incision along the midline of the abdomen exposing the chest cavity. Make a small cut at the apex of the right side of the heart.Inject 20 milliliters of pre-cooled PBS slowly and uniformly from the apex of the left heart, causing blood to flow out from the opening created at the apex of the right heart. Remove the entire lung lobe and refrigerate it at minus 80 degrees Celsius. Perfuse the remaining mice with 10 milliliters of 4%para-formaldehyde.After the PBS perfusion, preserve the lungs in 30 milliliters of the para-formaldehyde solution. After 72 hours, immerse the lungs in the prepared fixative and place them in an ultrasonic water bath. Tempered at 40 degrees Celsius for 30 minutes.Next, perform dehydration by placing the tissue in 75%ethanol, then in 95%ethanol, and then in anhydrous ethanol for 50 minutes each. Immerse the lung tissue in xylene for 50 minutes twice to wash the tissue. Place the tissue in melted paraffin wax for two to three hours at 55 to 60 degrees Celsius.Once the wax has hardened, use a sectioning machine to obtain the lung slices of four to five micrometer thickness. Heat the sections at 45 degrees Celsius in ultrapure water. Place the lung sections onto an adherent microscope slide once the wax melts.Hematoxylin and eosin staining studies showed that mice exposed to nicotine combined with silica had significantly more severe lung damage than those exposed to either nicotine or silica alone. Masson's staining revealed increased collagen fiber deposition in the lungs exposed to nicotine silica combination relative to the other groups. Alveolar structures were destroyed in the silica-exposed mice.Immunohistochemical studies revealed increased CD two zero six positive macrophages and the pro-fibrotic factor, TGF beta one positive cells in the mice exposed to the combination of nicotine and silica. Vimentin expression was higher in mice subjected to the dual expression relative to the other groups. Immunohistochemical studies and protein quantification suggest severe epithelial mesenchymal transformation.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.

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604 visualizações.  Anhui University of Science and Technology. O modelo de camundongo empregado neste estudo simula efetivamente o efeito da ingestão crônica de nicotina e da exposição repetida à sílica sobre as fibroses pulmonares através da transição mesenquimal epitelial em seres humanos. O modelo de porção do júri foi obtido pela injeção subcutânea de nicotina e gotejamento nasal de sílica de forma simples e confiável. Suspender a sílica cristalina estéril em soro fisiológico para preparar uma suspensão de 20 miligramas por litro...