Name: a mouse model of pulmonary fibrosis induced by nasal bleomycin nebulization
Uploaded: 2023-01-20T12:40:00
Description: Watch this Scientific Journal Video about A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization at JoVE.com

This work was supported by the National Natural Science Foundation of China (No. 92068108).

The Animal Studies Committee of the China-Japan Friendship Hospital (Beijing, China) approved all of the procedures involving mice that were performed as part of this study (NO.190108). Mice were kept in the sterile animal room of the China-Japan Friendship Clinical Medical Research Institute, with a room temperature of 20-25 &#176;C, relative humidity of 40%-70%, animal light intensity of 15-20 LX, and alternating light and dark for 12 h/12 h. Animals had free access to food and water.
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<ol>
	<li>Raghu, G., 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=Diagnosis+of+idiopathic+pulmonary+fibrosis.+An+official+ATS/ERS/JRS/ALAT+clinical+practice+guideline.">Diagnosis of idiopathic pulmonary fibrosis. An official ATS/ERS/JRS/ALAT clinical practice guideline.</a> American Journal of Respiratory and Critical Care Medicine. 198 (5), e44-e68 (2018).</li><li>Moore, B. B., Hogaboam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+pulmonary+fibrosis.">Murine models of pulmonary fibrosis.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 294 (2), L152-L160 (2008).</li><li>Degryse, A. 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=Repetitive+intratracheal+bleomycin+models+several+features+of+idiopathic+pulmonary+fibrosis.">Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (4), L442-L452 (2010).</li><li>Sueblinvong, V., 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=Predisposition+for+disrepair+in+the+aged+lung.">Predisposition for disrepair in the aged lung.</a> The American Journal of the Medical Sciences. 344 (1), 41-51 (2012).</li><li>Peng, 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=Bleomycin+induces+molecular+changes+directly+relevant+to+idiopathic+pulmonary+fibrosis:+a+model+for+&amp;#34;active&amp;#34;+disease.">Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for &amp;#34;active&amp;#34; disease.</a> PLoS One. 8 (4), e59348 (2013).</li><li>Izbicki, G., 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=Time+course+of+bleomycin-induced+lung+fibrosis.">Time course of bleomycin-induced lung fibrosis.</a> International Journal of Experimental Pathology. 83 (3), 111-119 (2002).</li><li>Aguilar, 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=Bone+marrow+stem+cells+expressing+keratinocyte+growth+factor+via+an+inducible+lentivirus+protects+against+bleomycin-induced+pulmonary+fibrosis.">Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis.</a> PLoS One. 4 (11), e8013 (2009).</li><li>Ortiz, L. A., 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=Mesenchymal+stem+cell+engraftment+in+lung+is+enhanced+in+response+to+bleomycin+exposure+and+ameliorates+its+fibrotic+effects.">Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.</a> Proceedings of the National Academy of Sciences. 100 (14), 8407-8411 (2003).</li><li>Rojas, 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=Bone+marrow-derived+mesenchymal+stem+cells+in+repair+of+the+injured+lung.">Bone marrow-derived mesenchymal stem cells in repair of the injured lung.</a> American Journal of Respiratory Cell and Molecular Biology. 33 (2), 145-152 (2005).</li><li>Ortiz, L. A., 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=Interleukin+1+receptor+antagonist+mediates+the+antiinflammatory+and+antifibrotic+effect+of+mesenchymal+stem+cells+during+lung+injury.">Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury.</a> Proceedings of the National Academy of Sciences. 104 (26), 11002-11007 (2007).</li><li>Foskett, A. 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=Phase-directed+therapy:+TSG-6+targeted+to+early+inflammation+improves+bleomycin-injured+lungs.">Phase-directed therapy: TSG-6 targeted to early inflammation improves bleomycin-injured lungs.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 306 (2), L120-L131 (2014).</li><li>Hecker, 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=Reversal+of+persistent+fibrosis+in+aging+by+targeting+Nox4-Nrf2+redox+imbalance.">Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance.</a> Science Translational Medicine. 6 (231), 231ra47 (2014).</li><li>Taooka, 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=Effects+of+neutrophil+elastase+inhibitor+on+bleomycin-induced+pulmonary+fibrosis+in+mice.">Effects of neutrophil elastase inhibitor on bleomycin-induced pulmonary fibrosis in mice.</a> American Journal of Respiratory &amp; Critical Care Medicine. 156 (1), 260-265 (1997).</li><li>Gharaee-Kermani, M., Ullenbruch, M., Phan, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+fibrosis.">Animal models of pulmonary fibrosis.</a> Methods in Molecular Medicine. , 251-259 (2005).</li><li>Orlando, 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=Induction+of+mouse+lung+injury+by+endotracheal+injection+of+bleomycin.">Induction of mouse lung injury by endotracheal injection of bleomycin.</a> Journal of Visualized Experiments. (146), e58902 (2019).</li><li>Moore, B. 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=Animal+models+of+fibrotic+lung+disease.">Animal models of fibrotic lung disease.</a> American Journal of Respiratory Cell and Molecular Biology. 49 (2), 167-179 (2013).</li><li>Gauldie, J., Kolb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+fibrosis:+how+far+from+effective+reality.">Animal models of pulmonary fibrosis: how far from effective reality.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 294 (2), L151 (2008).</li><li>Jenkins, R. G., 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=An+official+American+thoracic+society+workshop+report:+use+of+animal+models+for+the+preclinical+assessment+of+potential+therapies+for+pulmonary+fibrosis.">An official American thoracic society workshop report: use of animal models for the preclinical assessment of potential therapies for pulmonary fibrosis.</a> American Journal of Respiratory Cell and Molecular Biology. 56 (5), 667-679 (2017).</li><li>Moeller, A., 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+bleomycin+animal+model:+A+useful+tool+to+investigate+treatment+options+for+idiopathic+pulmonary+fibrosis.">The bleomycin animal model: A useful tool to investigate treatment options for idiopathic pulmonary fibrosis.</a> The International Journal of Biochemistry &amp; Cell Biology. 40 (3), 362-382 (2008).</li></ol>

Intratracheal injection of bleomycin results in an acute inflammatory and fibrotic response in both lungs and can be considered an effective approach to establish an experimental mouse model of human interstitial lung disease. Intratracheal administration is the most commonly used route of administration2,3,4,5,6,7,</sup...

Pulmonary fibrosis is a progressive disease process in which excessive deposition of extracellular matrix components, primarily type I collagen, in the interstitium of the lungs leads to impaired lung function1. The pathophysiology of pulmonary fibrosis is complex, and treatment options are currently quite limited. Mouse models remain an important tool to study the pathogenic mechanisms that contribute to the emergence and progression of the disease, as well as new strategies for drug development.
A variety of animal models of pulmonary fibrosis rely on intratracheal instillation of BLM2,3,4,5,6,7,8,9,10,11,12. However, the distribution of fibrotic changes that BLM causes in the lungs is not uniform, and the animals are at risk of asphyxiation during the instillation process. Although intraperitoneal injection of BLM induces relatively uniform fibrotic changes in the lung, it requires multiple doses because of insufficient drug targeting. Intratracheal aerosol administration via a laryngoscope does not require tracheotomy or puncture, and the resulting drug distribution within the lung is optimal. However, the aerosolized particles are large (5-40 &#181;m), and thus cannot reach the subpleural area of the lung tissue.
In this study, intrapulmonary administration of BLM is carried out by nasal nebulization. During nebulization, the mice breathed spontaneously and inhaled the drug particles. The aerosolized particles were 2.5-4 &#181;m in size, which enabled them not only to distribute evenly throughout the lung but also to reach the subpleural area. Under low magnification, the most significant lung histopathological features of patients with idiopathic pulmonary fibrosis (IPF) are the varying severity of lesions, inconsistent distribution, alternating distribution of different phase lesions, and the presence of interstitial inflammation, fibrotic lesions, and honeycomb lung changes, alternating with normal lung tissue. These pathological changes predominantly involve the peripheral subpleural parenchyma or lobular septum around the bronchioli. Thus, given that this approach enables BLM particles to reach the subpleural area of the lungs, this model closely simulates the clinical characteristics of the disease in humans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;bleomycin</td>
			<td>Bioway/Nippon Kayaku Co Ltd</td>
			<td>&#160;DP721</td>
			<td>Fibrosis model drugs</td>
		</tr>
		<tr>
			<td>0.9% saline for injection</td>
			<td>Baxter Healthcare(Tianjin)Co.,Ltd.</td>
			<td></td>
			<td>Bleomycin preparation</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>300481</td>
			<td>Anesthetize animals</td>
		</tr>
		<tr>
			<td>10% neutral formalin buffer</td>
			<td>TechaLab Biotech Company</td>
			<td></td>
			<td>Fix lung tissue</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>corning</td>
			<td>430790</td>
			<td>Prepare for anesthesia</td>
		</tr>
		<tr>
			<td>20 &#176;C refrigerator</td>
			<td>New-fly group</td>
			<td>BCD-213K</td>
			<td>Store drugs</td>
		</tr>
		<tr>
			<td>4 &#176;C refrigerator</td>
			<td>New-fly group</td>
			<td>BCD-213K</td>
			<td>Store drugs</td>
		</tr>
		<tr>
			<td>Adobe illustrator cc2020</td>
			<td>Adobe</td>
			<td></td>
			<td>Process images</td>
		</tr>
		<tr>
			<td>blue back liquid</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>clean bench</td>
			<td>Suzhou Sujie Purifying Equipment Co.,Ltd.</td>
			<td></td>
			<td>Bleomycin preparation</td>
		</tr>
		<tr>
			<td>differentiation fluid</td>
			<td>Beijing Chemical Works</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>METTLER TOLEDO</td>
			<td>AA-160</td>
			<td>Prepare for anesthesia</td>
		</tr>
		<tr>
			<td>eosin stain</td>
			<td>Beijing Yili Fine Chemicals Co,Ltd.</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>HIDOM</td>
			<td></td>
			<td>Mice incubation</td>
		</tr>
		<tr>
			<td>hematoxylin stain</td>
			<td>Beijing Yili Fine Chemicals Co,Ltd.</td>
			<td></td>
			<td>tissue staining</td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS) buffer</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td>Clean lung tissue</td>
		</tr>
		<tr>
			<td>Photoshop drawing software</td>
			<td>Adobe</td>
			<td></td>
			<td>Process images</td>
		</tr>
		<tr>
			<td>SCIREQ INEXPOSE</td>
			<td>EMKA Biotech Beijing Co.,Ltd.</td>
			<td></td>
			<td>Atomizing device</td>
		</tr>
		<tr>
			<td>Upright fluorescence microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td>Observe the slice</td>
		</tr>
	</tbody>
</table>

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.

Lung injury was induced by nebulized BLM, and the control animals were nebulized with the same volume of normal saline. The mice were nebulized once a day for 3 days, 30 min per day, using a BLM concentration of 7 U/mL. Mice were sacrificed on days 9, 16, and 23 after BLM administration for H&#38;E staining (Figure 2B). Diffused pneumonic lesions with loss of the normal alveolar architecture, septal thickening, enlarged alveoli, and increased infiltration of inflammatory cells into the interstitial and p...

Watch this Scientific Journal Video about A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization at JoVE.com

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

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 ...

In this study, inter-pulmonary administration of Bleomycin is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimic clinical disease characteristics. After anesthetizing the mouse press its toes with the index finger and thumb to ensure the limb withdrawal reflects has disappeared. Once the instrument is calibrated, secure the anesthetized mouse with a soft mesh cover.Using a pipette, add the working BLM solution to the top atomizing head of the exposure tower, and run the nebulizer for 30 minutes to achieve stable atomization. Now double click the FlexiWare 8 icon. Select the Experimentation Session and click the New Study button.Edit the Experiment Name, Title, and Owner. Then choose the IX-4DIO template. Input the operator and click the Confirm button.Click Next In the Welcome window. In the Pump Selection window, choose the Pump tab and click Next. It leads to the window named, Test set-up-Pump 4.Click the Next button and the Pump Output Adjustment window will pop up, and click Next again. Then click the Finish button in the Results-Pump 4 window. Click on the settings for Continuous 1L/min.Select DIO1 and set the Duty Cycle to 25%Then click OK and set the Duty Cycle percent for DIO2, DIO3, and DIO4. Now start the procedure by clicking the top green button and stop by clicking the red button. Click X at the top right corner to quit.The BLM administered mice lungs displayed diffused pneumonic lesions with loss of the normal alveolar architecture, septal thickening, enlarged alveoli, and increased infiltration of inflammatory cells into the Interstitial and Peribronchiolar areas, compared to the control group. The Ashcroft scores for the BLM mice on the 9th, 16th and 23rd days, were higher than those in the control group. The aerosolized the particle with 2.5 to four micrometer in size, which enable Bleomycin to distribute evenly throughout the lung and that reach the subpleural area.

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Biology

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...