Name: real-time x-ray imaging of lung fluid volumes in neonatal mouse lung
Uploaded: 2016-07-18T13:00:00
Description: Watch this Scientific Journal Video about Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung at JoVE.com

This work is supported by a grant awarded to MNH by Children's Healthcare of Atlanta, the APS 2014 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) fellowship awarded to PT, and the University of Notre Dame Integrated Imaging Facility.

All experimental techniques must be conducted in accordance with Institutional Care and Use Committee guidelines.
1. X-ray Imaging Acquisition
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	<li>Software Overview (See Figure 1 for an Overview of Software Settings).
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		<li>Select the Capture button directly below the File tab.</li>
		<li>In the Settings drop-down menu, select Current Session.</li>
		<li>Select Standard Exposure ...

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	<li>Institute of Laboratory Animal Resources. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2011).</li><li>Helms, M. N., Torres-Gonzalez, E., Goodson, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tracheal+instillation+of+solutes+into+mouse+lung.">Direct tracheal instillation of solutes into mouse lung.</a> J Vis Exp. , (2010).</li><li>Hilgendorff, A., Reiss, I., Ehrhardt, H., Eickelberg, O., Alvira, 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=Chronic+lung+disease+in+the+preterm+infant.+Lessons+learned+from+animal+models.">Chronic lung disease in the preterm infant. Lessons learned from animal models.</a> Am J Respir Cell Mol Biol. 50, 233-245 (2014).</li><li>Goodson, P., 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=Nadph+oxidase+regulates+alveolar+epithelial+sodium+channel+activity+and+lung+fluid+balance+in+vivo+via+O2-+signaling.">Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O2- signaling.</a> Am J Physiol Lung Cell Mol Physiol. 302, L410-L419 (2012).</li><li>McNeilly, T. N., Tennant, P., Lujan, L., Perez, M., Harkiss, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+infection+efficiencies+of+peripheral+lung+and+tracheal+tissues+in+sheep+infected+with+Visna/maedi+virus+via+the+respiratory+tract.">Differential infection efficiencies of peripheral lung and tracheal tissues in sheep infected with Visna/maedi virus via the respiratory tract.</a> J Gen Virol. 88, 670-679 (2007).</li><li>Starcher, B., Williams, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+intratracheal+instillation+of+endotoxin+into+the+lungs+of+mice.">A method for intratracheal instillation of endotoxin into the lungs of mice.</a> Lab Anim. 23, 234-240 (1989).</li><li>Downs, C. A., Kumar, A., Kreiner, L. H., Johnson, N. M., Helms, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2O2+regulates+lung+ENaC+via+ubiquitin-like+protein+Nedd8.">H2O2 regulates lung ENaC via ubiquitin-like protein Nedd8.</a> J Biol Chem. 288, 8136-8145 (2013).</li><li>Gammon, S. 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=Preclinical+anatomical,+molecular,+and+functional+imaging+of+the+lung+with+multiple+modalities.">Preclinical anatomical, molecular, and functional imaging of the lung with multiple modalities.</a> Am J Physiol Lung Cell Mol Physiol. 306, L897-L914 (2014).</li><li>Takemura, 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=Cholinergic+regulation+of+epithelial+sodium+channels+in+rat+alveolar+type+2+epithelial+cells.">Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 304, L428-L437 (2013).</li><li>Lewis, R. 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=Dynamic+imaging+of+the+lungs+using+x-ray+phase+contrast.">Dynamic imaging of the lungs using x-ray phase contrast.</a> Phys Med Biol. 50, 5031-5040 (2005).</li><li>Nyren, S., Radell, P., Mure, M., Petersson, J., Jacobsson, H., Lindahl, S. G., Sanchez-Crespo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhalation+anesthesia+increases+V/Q+heterogeneity+during+spontaneous+breathing+in+healthy+subjects.">Inhalation anesthesia increases V/Q heterogeneity during spontaneous breathing in healthy subjects.</a> Anesthesiology. 113 (6), 1370-1375 (2010).</li></ol>

Using X-ray imaging, clear images of neonatal lungs can be analyzed for lung fluid volumes. We7,3,11, and others10, have successfully utilized X-ray imaging to determine dynamic changes in lung fluid volume in freely breathing anesthetized animal models, and this technique holds great promise to advance the study of neonatal lung injury. One major advantage in using our approach to assess lung fluid volume (as opposed to x-ray phase constrast10 for example) is that up to five PN10 mouse p...

At birth, the newborn lung must transition from a fluid secreting to a fluid reabsorbing organ to establish adequate ventilation and oxygenation of the body. The mechanisms that facilitate (or hinders) effective clearance of lung fluid at the time of birth remain unclear. Modeling the rate of alveolar fluid clearance in C57BL/6 newborn mouse pups will lead to a better understanding of regulatory factors that can enhance or attenuate the rate of fluid absorption. It could also be applied to other neonatal models of acute lung injury or infection, and could lead to novel therapeutic strategies for newborn infants with respiratory distress.
Since newborn lungs are minuscule compared to adult lungs, conventional measures of alveolar fluid clearance that rely on lavage or gravimetrical measurements may not be suitable to accurately study lung fluid clearance in neonatal lung models. In this protocol, we demonstrate an assay that allows for the accurate determination of alveolar fluid clearance rates in postnatal day 10 C57BL/6 mouse pups using a small animal imager. One major benefit of using a fluoroscopic approach is that the animals are imaged in vivo. They are freely breathing and may recover from this minimally invasive assay for future observation and study. The overall goal of this method is to model pulmonary edema in the newborn lung, and evaluate the rate of alveolar fluid clearance in neonatal lung. This technique was developed, in part, as a reduction strategy to decrease the number of animals needed, yet maximize experimental output. This technique also allows for superior detection of lung fluid volumes using X-ray imaging and requires proficiency in basic animal restraint and handling1; small animal surgeries and tracheal instillation2, a small animal imager, and basic image analysis software. Investigators who wish to evaluate lung fluid volumes in vivo (freely breathing anesthetized animal models) may find this procedure suitable for their application. Lastly, this protocol could augment other existing models of neonatal lung injury used in the mechanistic study of bronchopulmonary dysplasia, including hyperoxia-induced lung injury, mechanical ventilation, and models of lung inflammation3.

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			<td style="width:241px;">Bruker; Billerica, MA</td>
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			<td height="20" style="height:20px;">Ketamine</td>
			<td>Ketaset; Fort Dodge Animal Health, IA</td>
			<td>26637-411-01</td>
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			<td height="20" style="height:20px;">Xylazine</td>
			<td>Lloyd Laboratories; Shenandoah, IA</td>
			<td>4821</td>
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			<td height="20" style="height:20px;">Dulbecco&#39;s Phosphate Buffered Saline (with Calcium and Magnesium)&#160;</td>
			<td>Lonza; Walkersville, MD</td>
			<td>17-513F</td>
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			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>Amresco; Solon, OH</td>
			<td>241</td>
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			<td height="20" style="height:20px;">Potassuim chloride</td>
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			<td>Sigma-Aldrich; St. Loius, MO</td>
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			<td height="20" style="height:20px;">0.3 mL insulin syringe with 31Gx5/16&#34; (8mm) needle</td>
			<td>BD Insulin Syringe; Franklin Lakes, NJ</td>
			<td>328438</td>
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real-time x-ray imaging of lung fluid volumes in neonatal mouse lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

The left panels in Figures 9 - 10 are of PN 10 mouse lungs imaged at baseline (pre-instilled). These images show successful instillation of saline challenges into the left lobe of the neonatal lungs. In Figure 9, the mouse lung was tracheally instilled with the saline solution defined above (see section 2.1). The middle and right panels of Figure 9 are X-ray images from the same mouse obtained 5 min and 2 hr post instillation; this animal...

Watch this Scientific Journal Video about Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung at JoVE.com

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

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