Name: establishing a silicosis rat model via exposure of whole-body to respirable silica
Uploaded: 2022-10-28T14:30:00
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 li...

<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. 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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. 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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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 &#160;</td>
			<td>HOPE Industry and Trade Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Generator systems&#160;</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&#160;</td>
			<td>HOPE Industry and Trade Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe</td>
			<td></td>
			<td></td>
			<td>&#160;No specific brand is recommended.</td>
		</tr>
		<tr>
			<td>Light microscope&#160;</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 ...

Using this protocol of silicosis rat model, it is possible to closely mimic the pathological process of human silicosis. This technique is easy to perform cost effectively with good repeatability, and there is no trauma to the animal. The established rat model in this study contributes to early identification, accurate diagnosis, and treatment of silicosis.To begin, place the silica particles in an agate mortar and grind for 1.5 hours before each animal exposure. Then weigh 30 grams of the ground silica in an electronic balance and place it in a glass container. Bake at 180 degrees Celsius for six hours in an electric-heating, air-blowing dryer to eliminate pathogens from the surface of the silica particles.Set up the silica exposure controller by connecting the injection and the generator systems. Then place the weighed silica in the generator and confirm that the pipeline connection is proper. The power cord is connected, and the power supply is normal.After ensuring the valves of the inhalation chamber are closed, turn on the exhaust gas discharge device and the air source to confirm that the inside of the shielding cabinet is in a negative pressure state. Check the silica concentration in the cabinet with a calibrated atmospheric sampler following the manufacturer's instructions. For gravimetric determination, use a digital, single-pan, analytical balance and weigh down the silica.Place the rat in the cage, then place 10 rats in the inhalation chamber and close the inhalation compartment and the screened cabinet doors. Then proceed with the silica inhalation experiment as planned. On completion of the experiment, close the mixed gas flow control valve and open the pure gas flow valve to inject the gas into the inhalation chamber.After 20 minutes, close the pure air flow valve. Open the door and take the cage out of the inhalation chamber. Then take the rats out of the cage and take them back to the pathogen-free room.Next, 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. Then wipe the inner wall with a clean cloth to dry the tank.Finally, disinfect the tank with 75%ethanol and close the exhaust gate. Next, quickly open the door of the inhalation cabin to evaporate the moisture so that the inside of the inhalation cabin remains dry. The rats exposed to silica for two and 24 weeks revealed alveolar wall thickening after two weeks of silica inhalation, whereas the alveolar structure disappeared, and large areas of fibrosis formed after 24 weeks.Further, the silica particles were seen trapped in the macrophages of the lung lobes observed through a polarized light microscope. Immunohistochemical staining of CD 68 in the lung revealed the dynamic evolution of silicotic nodules from two to 24 weeks. As the exposure time increased, the area of the nodules also gradually increased, and the adjacent silicotic nodules fused into larger nodules.Multiple fibrotic lesions of varying sizes were formed in the lungs of rats exposed to silica compared with the control rats. However, no significant differences in the kidney, liver, and spleen were found between the rats with silicosis and the control. But interestingly, bone loss was observed in rats with silicosis.The most important thing in this protocol is ensuring that the silica is well-ground, and the concentration is constant before starting the procedure.

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Research

JoVE Journal

Medicine

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 ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

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 ...

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.