The present study and production to this article were funded by the National Natural Science Foundation of China (Grant 81773373, 81172614 and Grant 81441089).&#160;We thank Dr. Jin Yan&#160;and Dr. Pan Yujie, of Department of Emergency, Beijing Chaoyang Hospital,&#160;and Dr. Qu Peng of Department of Ultrasound Medicine, Beijing Chaoyang Hospital&#160;for&#160;helping with the video production.&#160;

The study followed guidelines of Capital Medical University (Beijing, P.R China) for the care and use of experimental animals. All procedures were approved by the Animal Ethical Committee of Capital Medical University in China.
1. Experimental preparations
NOTE: Acclimate the female specific pathogen-free Wistar rats (weight: 200 &#177; 10 g) to the experimental environments for a week before administration (Environmental conditions: light/dark:12h/12h, temperature 22 &#177; 2 &#176;C, humidity 50 &#177; 10 %).
<ol>
	<li>Use a fresh 10 mL of PA/NPSi suspensions (nanosilica &#216;:20 &#177; 5 nm by in situ emulsion polymerization) diluted in normal saline at concentrations of 3.125, 6.25, and 12.5 mg/mL, respectively10. Before administration, sonicate the suspensions for 20-30 min and vortex for 10 min in order to prevent nanoparticles aggregation.</li>
	<li>Equally divide a total of 20 rats into four groups: one group for each concentration of PA/NPSi (0, 3.125, 6.25, and 12.5 mg/mL).</li>
	<li>To anesthetize them, place the rats in a closed container with 1.5 mL of ether (99.5%) or any other IACUC approved protocols. After 60-90 s of anesthesia, check for the lack of response to pedal reflex. Ensure that the rats are breathing.</li>
	<li>Put the anesthetized rat on the board and fix its front teeth with a sterilized line of nylon on the board too.</li>
	<li>Open its mouth and expose its fissure of glottis with the help of a surgical forceps and frontal lens.</li>
	<li>Instill the rats with 0.5 mL of PA/NPSi suspension to each rat&#39;s lung for a total of 1 mL using a&#160;fine tube with a sterilized blunt needle into the bilateral bronchus.</li>
	<li>Place the rats on a plastic board in a supine position and let the rats recover slowly in 5-10 min.</li>
</ol>
2. Ultrasound examination for pleural effusion
<ol>
	<li>Use an ultrasound system with a linear array transducer (frequency: 8 MHz) to examine rats on days 1, 3, 7 and 1410.</li>
	<li>Give anesthesia (10% chloral hydrate, 0.35 mL/100 g, i.p.) to the rats and check for the lack of pedal reflexes.</li>
	<li>Remove the hair from rats&#39; chest and upper abdomen using an electric shaver. Then place the rat on a mounting plate in a supine position.</li>
	<li>Cover the skin with the coated gel, and then place the transducer on the intercostal space and subcostal area to detect the pleural fluid. 
	NOTE: In order to detect the effusion accurately, the left and the right lateral positions were selected to perform an ultrasound examination.</li>
	<li>Put the rat on a plastic board in a supine position after ultrasound examination and let the rats recover slowly in 10 min.</li>
</ol>
3. Chest CT scanning for pleural effusion
<ol>
	<li>On days 7 and 14 post-administration, anesthetize the rats with 10% chloral hydrate (i.p). Consider it sufficient depth of anesthesia when the rat does not react to pedal reflexes. 
	NOTE: Day 7 post-administration is the most appropriate time to observe pleural effusion by CT scanning.</li>
	<li>Place the rat on a plastic sheet in a prone position and then scan its chest to investigate pleural effusion using a 64-channel CT. Use the following settings: 64 mm x 0.625 mm detector configuration, 120 kV (peak), and 350 mAs.</li>
</ol>
4. Collection of pleural effusion and isolation of nanoparticles in the pleural effusion
<ol>
	<li>After chest CT scanning of rats and under anesthesia of chloral hydrate, check the pedal reflex of the rats, shave the hair from abdomen to chest, and then disinfect the skin by iodine.</li>
	<li>Bring the rats to the surgical area.</li>
	<li>Under anesthesia, quickly cut 1-1.5 cm of the skin and abdominal muscles to the xiphoid along the midline with the intact diaphragm.</li>
	<li>Carefully open the chest and inspect bilateral pleural cavities with the help of tweezers, especially the bilateral costal phrenic angles. Collect 1-2 mL of the light-yellow pleural effusion with a 2 mL sterile syringe.</li>
	<li>Once done, sacrifice the rats with IACUC approved protocol.</li>
	<li>Centrifuge the pleural effusion in a 2 mL tube for 15 min at 300 x g in order to isolate the nanoparticles.</li>
	<li>Use a drop of the upper layer which is the bright liquid and observe under a transmission electron microscope(TEM,) at an accelerating voltage of 60-80 kV.</li>
</ol>

<ol>
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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=Nuclear+factor-kappaB+affects+tumor+progression+in+a+mouse+model+of+malignant+pleural+effusion.">Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion.</a> American Journal of Respiratory Cell and Molecular Biology. 34 (2), 142-150 (2006).</li><li>Shen, 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+dosage-toxicity-efficacy+relationship+of+kansui+and+licorice+in+malignant+pleural+effusion+rats+based+on+factor+analysis.">The dosage-toxicity-efficacy relationship of kansui and licorice in malignant pleural effusion rats based on factor analysis.</a> Journal of Ethnopharmacology. 186, 251-256 (2016).</li><li>Nel, A., Xia, T., M&#228;dler, L., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+potential+of+materials+at+the+nanolevel.">Toxic potential of materials at the nanolevel.</a> Science. 311 (5761), 622-627 (2006).</li><li>Maynard, A. D., 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=Safe+handling+of+nanotechnology.">Safe handling of nanotechnology.</a> Nature. 444 (7117), 267-269 (2006).</li><li>Duan, 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=Toxic+effects+of+silica+nanoparticles+on+zebrafish+embryos+and+larvae.">Toxic effects of silica nanoparticles on zebrafish embryos and larvae.</a> PLoS One. 8 (9), 74606 (2013).</li><li>Skuland, T., Ovrevik, J., L&#229;g, M., Schwarze, P., Refsnes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica+nanoparticles+induce+cytokine+responses+in+lung+epithelial+cells+through+activation+of+a+p38/TACE/TGF-&#945;/EGFR-pathway+and+NF-&#954;&#914;+signaling.">Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-&#945;/EGFR-pathway and NF-&#954;&#914; signaling.</a> Toxicology and Applied Pharmacology. 279 (1), 76-86 (2014).</li><li>Oberd&#246;rster, G., Oberd&#246;rster, E., Oberd&#246;rster, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotoxicology:+an+emerging+discipline+evolving+from+studies+of+ultrafine+particles.">Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles.</a> Environmental Health Perspectives. 113 (7), 823-839 (2005).</li><li>Song, Y., Li, X., Du, X. <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+nanoparticles+is+related+to+pleural+effusion,+pulmonary+fibrosis+and+granuloma.">Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma.</a> European Respiratory Journal. 34 (3), 559-567 (2009).</li><li>Song, 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=Nanomaterials+in+humans:+identification,+characteristics,+and+potential+damage.">Nanomaterials in humans: identification, characteristics, and potential damage.</a> Toxicologic Pathology. 39 (5), 841-849 (2011).</li><li>Zhu, 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=Polyacrylate/nanosilica+causes+pleural+and+pericardial+effusion,+and+pulmonary+fibrosis+and+granuloma+in+rats+similar+to+those+observed+in+exposed+workers.">Polyacrylate/nanosilica causes pleural and pericardial effusion, and pulmonary fibrosis and granuloma in rats similar to those observed in exposed workers.</a> International Journal of Nanomedicine. 11, 1593-1605 (2016).</li><li>Havelock, 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=Pleural+procedures+and+thoracic+ultrasound:+British+Thoracic+Society+Pleural+Disease+Guideline+2010.">Pleural procedures and thoracic ultrasound: British Thoracic Society Pleural Disease Guideline 2010.</a> Thorax. 65, 61-76 (2010).</li><li>Jha, A., Ullah, E., Gupta, P., Gupta, G., Saud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonography+of+multifocal+hydatidosis+involving+lung+and+liver+in+a+female+child.">Sonography of multifocal hydatidosis involving lung and liver in a female child.</a> Journal of Medical Ultrasound. 40 (4), 471-474 (2013).</li><li>Hikaru, 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=Histological+analysis+of+70-nm+silica+particles-induced+chronic+toxicity+in+rats.">Histological analysis of 70-nm silica particles-induced chronic toxicity in rats.</a> European Journal of Pharmaceutics and Biopharmaceutics. 72, 626-629 (2009).</li><li>Sun, 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=Cytotoxicity+and+mitochondrial+damage+caused+by+silica+nanoparticles.">Cytotoxicity and mitochondrial damage caused by silica nanoparticles.</a> Toxicology in Vitro. 25, 1619-1629 (2011).</li><li>Hikaru, 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=Silica+nanoparticles+as+hepatotoxicants.">Silica nanoparticles as hepatotoxicants.</a> European Journal of Pharmaceutics and Biopharmaceutics. 72, 496-501 (2009).</li><li>Liu, T. I., 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=Single+and+repeated+dose+toxicity+of+mesoporous+hollow+silica+nanoparticles+in+intravenously+exposed+mice.">Single and repeated dose toxicity of mesoporous hollow silica nanoparticles in intravenously exposed mice.</a> Biomaterials. 32, 1657-1668 (2011).</li><li>Ding, M., 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=Diseases+caused+by+silica:+Mechanisms+of+injury+and+disease+development.">Diseases caused by silica: Mechanisms of injury and disease development.</a> International Immunopharmacology. 2, 173-182 (2002).</li><li>Shen, 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+dosage-toxicity-efficacy+relationship+of+kansui+and+licorice+in+malignant+pleural+effusion+rats+based+on+factor+analysis.">The dosage-toxicity-efficacy relationship of kansui and licorice in malignant pleural effusion rats based on factor analysis.</a> Journal of Ethnopharmacology. 186, 251-256 (2016).</li><li>Ji, J. 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=Twenty-eight-day+inhalation+toxicity+study+of+silver+nanoparticles+in+Sprague-Dawley+rats.">Twenty-eight-day inhalation toxicity study of silver nanoparticles in Sprague-Dawley rats.</a> Inhalation Toxicology. 19 (10), 857-871 (2007).</li></ol>

The authors have nothing to disclose.

Sonography is the most convenient tool for determining pulmonary diseases, due to its excellent sensitivity to the free fluid in the pleural cavity11. That is because sonography can immediately detect the contrast in acoustic impedance of air and fluids in the lung12. Besides, sonography is more flexible in a small animal&#39;s model than CT. Nevertheless, the air in the lung reflected the sound wave and impeded from observing the intrapulmonary changes after nanoparticles ...

Pleural effusion is a very common clinical manifestation of pulmonary diseases with a variety of causes. Having a useful animal pleural effusion model is very important to study these pulmonary diseases, the roles of the two pleural membrane layers, the mechanisms of pleural effusion, and its treatment. However, some reported pleural effusion models mainly focus on the malignant pleural effusion or biological factors rather than the nanoparticles in the environment1,2. Here, we introduce a new model of pleural effusion that is simple, safe and effective.
With the development of nanotechnology and the extensive use of nanoproducts, there is a concern about the potential hazards of nanomaterials to the environment and the human health3,4. Nanomaterials introduce risk factors and potentially lead to novel hazards within the workplace or through environmental contamination. In vitro and in vivo studies show that nanomaterials can result in multi-organ damage to the lungs, the heart, the liver, the kidney, and the nervous system, as well as the reproductive and immune systems5,6. Additionally, some studies reported that the specific toxicity of nanomaterials was due to their unique physicochemical properties3,4,7.
We have reported that a group of workers with occupational exposure to nanomaterials clinically presented with pleural and pericardial effusion, pulmonary fibrosis and granuloma8,9. Silica nanoparticles were isolated in these patients&#39; pleural effusion9. In order to reproduce and verify the pleural effusion induced by the inhaled nanoparticles in human, we conducted the experiment by instilling the polyacrylate/nanosilica (PA/NPSi) via the respiratory tract in rats, which mimicked human respiration in a real environment, and found that intratracheal instillation of PA/NPSi could result in pleural effusion in rats. Here, we introduce how to make pleural effusion in rats by intratracheal instillation of PA/NPSi, and how to isolate nanoparticles in the pleural effusion. This model may be useful for the further study of nanotoxicology and pleural effusion diseases.

<table><tbody><tr><td>Acuson S2000 Color Doppler ultrasound system</td><td>Siemens Medical Solutions, Mountain View ,CA</td><td></td><td></td></tr><tr><td>&amp;nbsp;Polyacrylate/nanosilica</td><td>Fudan University,Shanghai, China</td><td></td><td>made by order with nanosilica(20&amp;plusmn;5)nm</td></tr><tr><td>10% chloral hydrate</td><td>Beijing Chemical Works</td><td>302-17-0</td><td></td></tr><tr><td>Light speed 16 spiral computed tomography</td><td>GE Healthcare, US</td><td></td><td></td></tr><tr><td>Specific pathogen-free Wistar</td><td>Animal Center of Lianhelihua (Beijing, China)</td><td></td><td>Wistar rats</td></tr></tbody></table>

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.

Using a thoracic ultrasound, we found no pleural effusions on day 1 in all groups. However, on day 3, the pleural effusion appeared in the 6.25 and 12.5 mg/kg&#8729;mL groups. The effusion was mainly in the right costal phrenic angle, while the pericardial effusion only presented in 12.5 mg/kg&#8729;mL group. Furthermore, on day 7, both pleural effusion (Video 1) and pericardial effusion (Video 2) were detected in 6.25 mg/kg&#8729;mL group (<strong class=...

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

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 ...

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Medicine

7.8K Views.  Capital Medical University. Deze methode kan inzicht geven in de toxiciteit van nanomaterialen en kan worden toegepast voor de studie van longtoxiciteit. Deze techniek werd afgeleid om de effecten van polyacrylaat/nanosilica-geïnduceerde pleurale effusie te bestuderen. Voordat u begint met de instillatie, sonicate een verse, 10-milliliter, polyacrylaat / nanosilica suspensie in normale zoutoplossing gedurende 20 tot 30 minuten, gevolgd door 10 minuten vortexen, alvorens de suspensie te verdunnen tot 3.125, 6,25 en 12,5...

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

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

Biology

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Immunology and Infection

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

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 ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

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

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent models continue to be an essential tool for studying human silicosis. For silicosis, the classic pathological process needs to be inducible via the inhalation of freshly generated quartz particles, which means specifically inducing human occupational disease. This study described a technique to establish and effectively mimic the pathological dynamic evolution process of silicosis. Further, the technique had good repeatability with no surgery involved. The inhalation exposure system was fabricated, validated, and used for toxicology studies on respirable particle inhalation. The critical components were as follows: (1) bulk dry SiO2 powder generator adjusted with an air-flow controller; (2) 0.3 m3 whole-body inhalation exposure chamber accommodating up to 3&#160;adult rats; (3) a monitoring and control system for regulating oxygen concentration, temperature, humidity, and pressure in real-time; and (4) a barrier and waste disposal system for protecting laboratory technicians and the environment. In summary, the present protocol reports the inhalation via the whole body, and the inhalation chamber created a reliable, reasonable, and repeatable rat silicotic model with low mortality, less injury, and more protection.

This study describes a technique to establish a silicosis rat model with the inhalation of silica through the whole body in an inhalation chamber. The rats with silicosis could closely mimic the pathological process of human silicosis in an easy, cost-effective manner with good repeatability.

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent ...

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

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 ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...

Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis

Interstitial lung disease (ILD) represents a broad spectrum of disorders characterized by the progressive and often irreversible scarring of the lung parenchyma, the most common being idiopathic pulmonary fibrosis (IPF). Several animal models of IPF have been developed, with the bleomycin murine model being the most widely used. Bleomycin is a chemotherapeutic known to induce DNA damage in the alveolar epithelium, resulting in acute lung injury and pulmonary fibrosis in humans. Rodent models of IPF use bleomycin administration via various methods, the most common being intratracheal (IT). Recently, the oropharyngeal aspiration (OA) technique has been shown to be equally efficacious as IT for multiple fibrosing agents, with considerably fewer side effects and an easier route of delivery. This protocol details the OA method of bleomycin delivery into the murine lung and highlights examples of potential downstream applications for data quantification. This methodology offers a simple, quick, and safe way to utilize this widely used animal model for studying the molecular mechanisms underlying IPF.

This protocol demonstrates the oropharyngeal aspiration technique for use in the bleomycin murine model of pulmonary fibrosis.

Interstitial lung disease (ILD) represents a broad spectrum of disorders characterized by the progressive and often irreversible scarring of the lung ...

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

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