Name: establishing a silicosis rat model via exposure of whole-body to respirable silica
Uploaded: 2022-10-28T14:30:00.000+00:00
Duration: 5 min 3 s
Description: Watch this Scientific Journal Video about Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica at JoVE.com

This work was funded by the National Natural Science Foundation of China (82003406), the Natural Science Foundation of Hebei Province (H2022209073), and the Science and Technology Project of Hebei Education Department (ZD2022127).

All animal experiments were conducted according to the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Committee on the Ethics of North China University of Science and Technology (protocol code LX2019033 and 2019-3-3 of approval). Male Wistar rats, 3 weeks of age, were used for the present study. All rats were kept in static cages with wood shavings. The animals were maintained in a 12 h/12 h light/dark cycle, and were provided with food and water ad libitum. Follow-up experiments were conducted after 1 week of adaptive feeding.
1. Animal preparation
<ol>
	<li>Upon arrival, house all the rats in a specific pathogen-free (SPF) room.</li>
	<li>Randomly divide the healthy rats into two groups (n = 10): control rats inhaling pure air and rats with silicosis inhaling silica.</li>
</ol>
2. Silica preparation
CAUTION: Silica dust inhaled by the human body can damage the lungs. Therefore, individuals must wear overalls, medical gloves, and protective masks (N95) during operations.
<ol>
	<li>Ground the silica particles (see Table of Materials) with an agate mortar for 1.5 h before each exposure. This is because freshly fractured quartz produces larger quantities of active oxygen species than aged quartz13, and silica with a diameter of 1-5 &#181;m is the most pathogenic.</li>
	<li>Weigh the silica (30 g) using an electronic balance after grinding, place it in a glass container, and bake it at 180 &#176;C for 6 h in an electric heating air-blowing drier (see Table of Materials) to eliminate pathogens from the surface of the silica particles.</li>
</ol>
3. Silica exposure to the rats
<ol>
	<li>Connect the injection and the commercially available generator systems (see Table of Materials) and place the silica (30 g) in the generator. Check whether the connection pipeline is normal, the power cord is connected, and the power supply is normal.
	<ol>
		<li>Check the water level of the spray tower and the humidifier of the waste gas treatment device (see Table of Materials) manually, and add water if it is insufficient (not up to the standard line).</li>
		<li>Add tap water to the spray tower of the waste gas treatment device and distilled water to the humidifier (Figure 1).</li>
	</ol>
	</li>
	<li>Turn on the exhaust gas discharge device (see Table of Materials) and the air source switch to confirm whether the inside of the shielding cabinet is in a negative-pressure state.
	<ol>
		<li>Confirm that the liquid mixing, powder mixing, pure gas flow control valves, and wastewater discharge valve under the inhalation chamber are closed.</li>
	</ol>
	</li>
	<li>Place a total of 10 rats in the inhalation chamber (see Table of Materials), and close the inhalation compartment and the screened cabinet doors.</li>
	<li>Set the following experimental parameters on the instrument panel or in the computer: cabinet pressure: -50 to -30 Pa; oxygen concentration: 21%; cabinet temperature: 26-30&#160;&#176;C; humidity: 30%-70%; dust entry rate: 2.0-2.5 mL/min; and cabinet dust concentration: 60 &#177; 5 mg/m3. 
	NOTE: Observe the experimental data and equipment status continuously during the experiment. The equipment failure alarm prompted timely processing.
	<ol>
		<li>Expose each animal to silica continuously for 3 h per day, 5 days per week, and allow the animals in the control group to inhale pure air.</li>
	</ol>
	</li>
	<li>On completion of the experiment, close the mixed gas flow control valve, and open the pure gas flow valve. Inject the pure gas continuously into the inhalation chamber. 
	NOTE: In the present study, the pure gas flow (7.0-7.5 m3/h) was injected for at least 20 min until the poisonous gas in the inhalation chamber was completely replaced.
	<ol>
		<li>Close the pure air-flow valve, open the door, take the rats out, and send them back to the pathogen-free room.</li>
	</ol>
	</li>
	<li>Remove the rat rack and the branch pipe components in sequence and place them in the sink for cleaning. After rinsing, close the automatic cleaning valve and open the hatch.
	<ol>
		<li>Wipe the inner wall with a clean cloth, or turn on the pure gas to dry the tank. Finally, carry out the disinfection. After cleaning and disinfecting with 75% ethanol, close the exhaust gate and, as soon as possible, slightly open the door of the inhalation cabin to evaporate the moisture, so that the inside of the inhalation cabin remains dry.</li>
	</ol>
	</li>
	<li>Check the silica concentration in the cabinet with a comprehensive atmospheric sampler following the manufacturer&#39;s instructions (see Table of Materials) twice a week to ensure the stability of the silica concentration during the experiment. Calibrate the atmospheric sampler before sampling.
	<ol>
		<li>Use a digital single-pan analytical balance for gravimetric determination. The calculated silica concentration was 65 mg/m3 (Figure 1 and Table 1). 
		NOTE: Weigh the filter paper before and after the absorption of silica. The concentration of silica was calculated using the following formula12: 
		<img alt="Equation 1" src="/files/ftp_upload/64467/64467eq01.jpg" style="margin: auto;" /> 
		where W2 = weight of the filter paper after sampling, W1 = weight of the filter paper before sampling, and V = volume of the air.</li>
	</ol>
	</li>
</ol>
4. Acquisition and fixation of lung tissues
<ol>
	<li>Euthanize the rats by intraperitoneal injection of pentobarbital (100 mg/kg body weight) and Lidocaine (4&#160;mg/kg body weight). Assess the death by the loss of heartbeat14.</li>
	<li>At the end of the experiment, fix the right lower lung, kidney, liver, spleen, and bone with 4% paraformaldehyde for at least 24 h, embed in paraffin, and cut into 5 &#181;m sections7,15.</li>
</ol>
5. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Deparaffinize the paraffin sections in xylol (see Table of Materials) twice for 10 min each16, and rehydrate in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and distilled water for 3 min each.</li>
	<li>Stain the sections with hematoxylin (see Table of Materials) for 5 min, and then wash the sections with water10.</li>
	<li>Place the sections in 2% hydrochloric alcohol and then in distilled water until the color changes to blue.</li>
	<li>Stain the sections with eosin for 1 min, dehydrate them with 95% ethanol, make them transparent with xylene, seal them with neutral gum, and observe under a light microscope12.</li>
</ol>
6. Immunohistochemical staining
<ol>
	<li>Routinely wash the paraffin sections with water.</li>
	<li>Expose the antigens at high pressure (60 kPa) and high temperature (100 &#176;C) for 80 s and then block with an endogenous peroxidase blocker (3%) for 15 min to eliminate the endogenous peroxidases7.</li>
	<li>Incubate the samples with antibodies directed against CD68 (1:200 dilution-add 4 &#181;L of CD68 to 396 &#181;L of antibody diluent; see Table of Materials) at 4 &#176;C overnight.</li>
	<li>Incubate the samples with a secondary antibody (HRP-conjugated goat anti-mouse IgG polymer; see Table of Materials) at 37 &#176;C for 30 min, and then wash the samples with 1x PBS.</li>
	<li>Visualize the immunoreactivity with 3,3-diaminobenzidine (DAB; see Table of Materials). After applying DAB to the tissue, observe the staining of the tissue under a light microscope10. 
	NOTE: The staining time varied from a few seconds to a few minutes according to the staining time of the tissue. The staining procedure was aborted by placing the sections in water. In this study, the brown staining of the tissue represented the positive expression of CD68. All antibodies were diluted in 1x PBS.</li>
</ol>

<ol>
	<li>Li, 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=The+burden+of+pneumoconiosis+in+China:+an+analysis+from+the+Global+Burden+of+Disease+Study.">The burden of pneumoconiosis in China: an analysis from the Global Burden of Disease Study.</a> BMC Public Health. 22 (1), 1114 (2019).</li><li>The Lancet Respiratory Medicine. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+world+is+failing+on+silicosis.">The world is failing on silicosis.</a> The Lancet. Respiratory Medicine. 7 (4), 283 (2019).</li><li>Li, T., Yang, X., Xu, H., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+identification,+accurate+diagnosis,+and+treatment+of+silicosis.">Early identification, accurate diagnosis, and treatment of silicosis.</a> Canadian Respiratory Journal. 3769134, (2022).</li><li>Adamcakova, J., Mokra, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+pathomechanisms+and+treatment+possibilities+for+lung+silicosis.">New insights into pathomechanisms and treatment possibilities for lung silicosis.</a> International Journal of Molecular Sciences. 22 (8), 4162 (2021).</li><li>Li, 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=Thalidomide+alleviates+pulmonary+fibrosis+induced+by+silica+in+mice+by+inhibiting+ER+stress+and+the+TLR4-NF-&#954;B+pathway.">Thalidomide alleviates pulmonary fibrosis induced by silica in mice by inhibiting ER stress and the TLR4-NF-&#954;B pathway.</a> International Journal of Molecular Sciences. 23 (10), 5656 (2022).</li><li>Zhang, E., 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=Exosomes+derived+from+bone+marrow+mesenchymal+stem+cells+reverse+epithelial-mesenchymal+transition+potentially+via+attenuating+Wnt/&#946;-catenin+signaling+to+alleviate+silica-induced+pulmonary+fibrosis.">Exosomes derived from bone marrow mesenchymal stem cells reverse epithelial-mesenchymal transition potentially via attenuating Wnt/&#946;-catenin signaling to alleviate silica-induced pulmonary fibrosis.</a> Toxicology Mechanisms and Methods. 31 (9), 655-666 (2021).</li><li>Li, S., 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=N-Acetyl-Seryl-Asparyl-Lysyl-Proline+regulates+lung+renin+angiotensin+system+to+inhibit+epithelial-mesenchymal+transition+in+silicotic+mice.">N-Acetyl-Seryl-Asparyl-Lysyl-Proline regulates lung renin angiotensin system to inhibit epithelial-mesenchymal transition in silicotic mice.</a> Toxicology and Applied Pharmacology. 408, 408 (2020).</li><li>Walters, E. H., Shukla, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicosis:+Pathogenesis+and+utility+of+animal+models+of+disease.">Silicosis: Pathogenesis and utility of animal models of disease.</a> Allergy. 76 (10), 3241-3242 (2021).</li><li>Langley, R. J., Mishra, N. C., Pe&#241;a-Philippides, J. C., Hutt, J. A., Sopori, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granuloma+formation+induced+by+low-dose+chronic+silica+inhalation+is+associated+with+an+anti-apoptotic+response+in+Lewis+rats.">Granuloma formation induced by low-dose chronic silica inhalation is associated with an anti-apoptotic response in Lewis rats.</a> Journal of Toxicology and Environmental Health, Part A. 73 (10), 669-683 (2010).</li><li>Jin, F., 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=Ac-SDKP+Attenuates+activation+of+lung+macrophages+and+bone+osteoclasts+in+rats+exposed+to+silica+by+inhibition+of+TLR4+and+RANKL+signaling+pathways.">Ac-SDKP Attenuates activation of lung macrophages and bone osteoclasts in rats exposed to silica by inhibition of TLR4 and RANKL signaling pathways.</a> Journal of Inflammation Research. 14, 1647-1660 (2021).</li><li>Xu, H., 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+new+anti-fibrotic+target+of+Ac-SDKP:+inhibition+of+myofibroblast+differentiation+in+rat+lung+with+silicosis.">A new anti-fibrotic target of Ac-SDKP: inhibition of myofibroblast differentiation in rat lung with silicosis.</a> PloS One. 7 (7), e40301 (2012).</li><li>Li, S., 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=Ac-SDKP+increases+&#945;-TAT+1+and+promotes+the+apoptosis+in+lung+fibroblasts+and+epithelial+cells+double-stimulated+with+TGF-&#946;1+and+silica.">Ac-SDKP increases &#945;-TAT 1 and promotes the apoptosis in lung fibroblasts and epithelial cells double-stimulated with TGF-&#946;1 and silica.</a> Toxicology and Applied Pharmacology. 369, 17-29 (2019).</li><li>Vallyathan, V., Shi, X. L., Dalal, N. S., Irr, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+free+radicals+from+freshly+fractured+silica+dust.+Potential+role+in+acute+silica-induced+lung+injury.">Generation of free radicals from freshly fractured silica dust. Potential role in acute silica-induced lung injury.</a> The American Review of Respiratory Disease. 138 (5), 1213-1219 (1988).</li><li>Khoo, S. Y., Lay, B. P. P., Joya, 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=Local+anesthetic+refinement+of+pentobarbital+euthanasia+reduces+abdominal+writhing+without+affecting+immunohistochemical+endpoints+in+rats.">Local anesthetic refinement of pentobarbital euthanasia reduces abdominal writhing without affecting immunohistochemical endpoints in rats.</a> Lab Anim. 2018 (52), 152-162 (2018).</li><li>Chooi, K. F., Rajendran, D. B. K., Phang, S. S. G., Toh, H. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dimethylnitrosamine+induced+liver+fibrosis+model+in+the+rat.">The dimethylnitrosamine induced liver fibrosis model in the rat.</a> Journal of Visualized Experiments. 112 (112), (2016).</li><li>Valentin, J., Frobert, A., Ajalbert, G., Cook, S., Giraud, M. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+quantification+of+chronic+myocardial+infarct+in+rats.">Histological quantification of chronic myocardial infarct in rats.</a> Journal of Visualized Experiments. 118 (118), (2016).</li><li>Zhang, H., 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=silicosis+decreases+bone+mineral+density+in+rats.">silicosis decreases bone mineral density in rats.</a> Toxicology and Applied Pharmacology. 348, 117-122 (2018).</li><li>Zhang, 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=Targeting+the+RAS+axis+alleviates+silicotic+fibrosis+and+Ang+II-induced+myofibroblast+differentiation+via+inhibition+of+the+hedgehog+signaling+pathway.">Targeting the RAS axis alleviates silicotic fibrosis and Ang II-induced myofibroblast differentiation via inhibition of the hedgehog signaling pathway.</a> Toxicology Letters. 313, 30-41 (2019).</li><li>Li, S., 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+perturbs+primary+cilia+and+causes+myofibroblast+differentiation+during+silicosis+by+reduction+of+the+KIF3A-repressor+GLI3+complex.">Silica perturbs primary cilia and causes myofibroblast differentiation during silicosis by reduction of the KIF3A-repressor GLI3 complex.</a> Theranostics. 10 (4), 1719-1732 (2020).</li><li>Gao, X., 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=Pulmonary+silicosis+alters+microRNA+expression+in+rat+lung+and+miR-411-3p+exerts+anti-fibrotic+effects+by+inhibiting+MRTF-A/SRF+signaling.+Molecular+therapy.">Pulmonary silicosis alters microRNA expression in rat lung and miR-411-3p exerts anti-fibrotic effects by inhibiting MRTF-A/SRF signaling. Molecular therapy.</a> Nucleic Acids. 20, 851-865 (2020).</li><li>Cai, W., 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=Differential+expression+of+lncRNAs+during+silicosis+and+the+role+of+LOC103691771+in+myofibroblast+differentiation+induced+by+TGF-&#946;1.">Differential expression of lncRNAs during silicosis and the role of LOC103691771 in myofibroblast differentiation induced by TGF-&#946;1.</a> Biomedicine &amp; Pharmacotherapy. 125, (2020).</li><li>Cai, W., 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=Transcriptomic+analysis+identifies+upregulation+of+secreted+phosphoprotein+1+in+silicotic+rats.">Transcriptomic analysis identifies upregulation of secreted phosphoprotein 1 in silicotic rats.</a> Experimental and Therapeutic. 21 (6), (2021).</li><li>Li, 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=Minute+cellular+nodules+as+early+lesions+in+rats+with+silica+exposure+via+inhalation.">Minute cellular nodules as early lesions in rats with silica exposure via inhalation.</a> Veterinary Sciences. 9 (6), 251 (2022).</li><li>Mao, N., 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=Glycolytic+reprogramming+in+silica-induced+lung+macrophages+and+silicosis+reversed+by+Ac-SDKP+treatment.">Glycolytic reprogramming in silica-induced lung macrophages and silicosis reversed by Ac-SDKP treatment.</a> International Journal of Molecular Sciences. 22 (18), 10063 (2021).</li><li>Hamilton, R. F., Thakur, S. A., Holian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica+binding+and+toxicity+in+alveolar+macrophages.">Silica binding and toxicity in alveolar macrophages.</a> Free Radical Biology and Medicine. 44 (7), 1246-1258 (2008).</li><li>Park, 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=Exposure+to+crystalline+silica,+silicosis,+and+lung+disease+other+than+cancer+in+diatomaceous+earth+industry+workers:+a+quantitative+risk+assessment.">Exposure to crystalline silica, silicosis, and lung disease other than cancer in diatomaceous earth industry workers: a quantitative risk assessment.</a> Occupational and Environmental. 59 (1), 36-43 (2002).</li><li>Honnons, S., Porcher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+experimental+model+for+silicosis.">In vivo experimental model for silicosis.</a> Journal of Environmental Pathology, Toxicology and. 19 (4), 391-400 (2000).</li><li>Lakatos, H. F., 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=Oropharyngeal+aspiration+of+a+silica+suspension+produces+a+superior+model+of+silicosis+in+the+mouse+when+compared+to+intratracheal+instillation.">Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation.</a> Experimental Lung Research. 32 (5), 181-199 (2006).</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>Hoy, R. F., Chambers, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica-related+diseases+in+the+modern+world.">Silica-related diseases in the modern world.</a> Allergy. 75 (11), 2805-2817 (2020).</li><li>Davis, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+silicosis:+current+concepts+and+hypotheses.">Pathogenesis of silicosis: current concepts and hypotheses.</a> Lung. 164 (3), 139-154 (1986).</li><li>Moss, O. R., James, R. A., Asgharian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+exhaled+air+on+inhalation+exposure+delivered+through+a+directed-flow+nose-only+exposure+system.">Influence of exhaled air on inhalation exposure delivered through a directed-flow nose-only exposure system.</a> Inhalation Toxicology. 18 (1), 45-51 (2006).</li></ol>

The authors declare no conflict of interest.

As the leading cause of silicosis, silica plays a decisive role in molding. The silica particles inhaled by patients with pneumoconiosis are fresh, free silica particles produced by mechanical cutting. Silica can generate reactive oxygen species either directly on freshly cleaved particle surfaces or indirectly through its effect on the macrophages25. Therefore, the grinding of silica particles is of high importance. In the proposed protocol, silica was ground with agate mortar for more than 90 mi...

Silicosis, which is caused by the inhalation of silica, is the most serious occupational disease in China, accounting for more than 80% of the total number of occupational disease reports every year1. The etiology of silicosis is clear, and it can be prevented and controlled, but no effective treatment method is available2. Many drugs have been proven to be effective in basic studies, but they have imprecise clinical effects3,4. Therefore, the pathological and physiological mechanisms of silicosis still need to be explored.
Many studies have used a one-time infusion of silica into the trachea of rats or mice to investigate the pathogenesis of silicosis5,6. Although these rodent silicotic models could be obtained in a short time7, these methods still had challenges, such as animal trauma and high mortality. Some studies have involved instilling stored silica into the lungs to induce a nonspecific lung reaction, but did not mention silicotic nodules in mice8. Furthermore, away from acute silicosis, chronic exposure to silica in occupational environments induced significantly lower pulmonary inflammation and elevated the levels of anti-apoptotic markers, rather than pro-apoptotic markers, in the lungs9. Therefore, a reliable, reasonable, and repeatable animal model is needed to explore the pathogenesis of silicosis further.
The present study describes a method to mimic the disease process of patients with silicosis through silica inhalation via the whole body, air-delivered particles in an inhalation chamber, which comprised an air-delivered silica generator, a whole-body chamber, and a waste disposal system. This method is simple, easy to operate, and effectively mimics the pathological dynamic evolution process of silicosis. Also, many possible mechanisms and the pathogenesis of silicosis are identified using this method10,11,12. The proposed protocol is anticipated to help further investigations in the related field of silicosis research.

<table><tbody><tr><td>Air detector (compressive atmospheric sampler)</td><td>Qingdao Xuyu Environmental Protection Technology Co. LTD</td><td></td><td></td></tr><tr><td>Anatomical table</td><td></td><td></td><td>&amp;nbsp;No specific brand is recommended.</td></tr><tr><td>Antibody of CD68</td><td>Abcam</td><td>ab201340</td><td></td></tr><tr><td>DAB</td><td>ZSGB-BIO</td><td>ZLI-9018</td><td></td></tr><tr><td>Electric heating air-blowing drier</td><td>Shanghai Yiheng Scientific Instrument Co., LTD</td><td></td><td></td></tr><tr><td>Electronic balance</td><td>OHRUS</td><td></td><td></td></tr><tr><td>Embedding machine</td><td>leica</td><td></td><td></td></tr><tr><td>Exhaust gas discharge device &amp;nbsp;</td><td>HOPE Industry and Trade Co. Ltd.</td><td></td><td></td></tr><tr><td>Generator systems&amp;nbsp;</td><td>HOPE Industry and Trade Co. Ltd.</td><td></td><td></td></tr><tr><td>Gloves (thin laboratory gloves)</td><td>The secco medical</td><td></td><td></td></tr><tr><td>Hematoxylin and eosin</td><td>BaSO Diagnostics Inc.</td><td>BA4025</td><td></td></tr><tr><td>HOPE MED 8050 exposure control apparatus</td><td>HOPE Industry and Trade Co. Ltd.</td><td></td><td></td></tr><tr><td>Inhalation chamber&amp;nbsp;</td><td>HOPE Industry and Trade Co. Ltd.</td><td></td><td></td></tr><tr><td>Injection syringe</td><td></td><td></td><td>&amp;nbsp;No specific brand is recommended.</td></tr><tr><td>Light microscope&amp;nbsp;</td><td>olympus</td><td></td><td></td></tr><tr><td>Object slide</td><td>shitai</td><td></td><td></td></tr><tr><td>PV-6000 (HRP-conjugated goat anti-mouse IgG polymer)</td><td>Beijing Zhongshan Jinqiao Biotechnology Co. Ltd</td><td>s5631</td><td></td></tr><tr><td>Silicon dioxide</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Slicing machine</td><td>leica</td><td>RM2255</td><td></td></tr><tr><td>Waste gas treatment device</td><td>HOPE Industry and Trade Co. Ltd.</td><td></td><td></td></tr><tr><td>Wet box</td><td>Cooperative plastic Products Factory</td><td></td><td></td></tr><tr><td>Xylol</td><td>Tianjin Yongda Chemical Reagent Co., LTD</td><td></td><td></td></tr></tbody></table>

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.

Using the proposed method, some potential mechanisms and the pathogenesis of silicosis were explored in rats. The schematic of the inhalation chamber is shown in Figure 1. The chamber consisted of an air-delivered silica generator, a whole-body chamber, and a waste disposal system, as previously described17. The pulmonary functions, levels of inflammatory factors in the serum and lung, collagen deposition, and myofibroblast differentiation were reported in the previou...

Watch this Scientific Journal Video about Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica at JoVE.com

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

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 ...

establishing-silicosis-rat-model-via-exposure-whole-body-to

Research

JoVE Journal

Medicine

1.2K Views.  North China University of Science and Technology. 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.

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

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

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 ...

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

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

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

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

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

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

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

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

Biology

Establishing a Mouse Model of Pleural Disseminated Lung Cancer

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

Opracowanie mysiego modelu rozsianego raka płuc w opłucnej

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...