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
<ol>
	<li>Software Overview (See Figure 1 for an Overview of Software Settings).
	<ol>
		<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 in drop-down box located under Exposure Time.</li>
		<li>Set the Exposure Time to 2.00 min, and No. Exposures to 1. Set the X Binning and Y Binning to 2 pixels. See Figure 1.</li>
		<li>Select Final Accumulation from the Export Options drop-down menu.</li>
		<li>In the Illumination settings, select X-Ray from the Illumination Source drop-down menu. The default KVP is set at 35.</li>
		<li>Select Autoselect in the Apply Reference File drop-down menu to apply the corresponding X-ray reference file. (See Section 2).</li>
		<li>In the Set Camera To settings, set the f-stop to 2.51.</li>
		<li>Set the FOV (Field of View) to 125.64 for PN10 mice.</li>
		<li>Set the Focal Plane to 13, and in the drop-down menu beside the Focal Plane slider, select X-Ray.</li>
		<li>For X-rays, set the Excitation Filter and the Emission Filter to 0 in the drop-down menu.</li>
		<li>Save current session by clicking New on the top next to Settings. Enter name for New Acquire Settings File, e.g., &#34;Jove Single Exposure Event for making a Protocol.&#34; See Figure 2.</li>
	</ol>
	</li>
	<li>Creating an X-ray Imaging Protocol
	<ol>
		<li>Click on the Create/Edit Protocols button on the right side of the open window.</li>
		<li>As new Protocol pop-up window appears, click the New button in the upper right-hand corner</li>
		<li>Enter the name that the protocol will save under, for example &#34;Jove Demo Protocol&#34;, and click OK. (See Figure 3).</li>
		<li>On the bottom of the Protocol pop-up window in Protocol Steps, ensure that Step 1 is highlighted in red text, Capture New Images is selected, and Do Nothing is selected in the Before Image Capture drop down menu.</li>
		<li>In Capture Setting, select the recently generated single exposure event from Step 1.1.12.</li>
		<li>Select Wait (sec) from the After Image Capture drop-down menu.</li>
		<li>Click the Edit button, type 180 into the pop-up box, and click OK to add a 3 min wait time after each 2 min acquisition.</li>
		<li>Duplicate Step 1 by right clicking the Step 1 tab and selecting Duplicate Step. Create 23 duplicates for a 2 hr observation period. (See Figure 4).</li>
		<li>On the final step (Step 24), change the After Image Capture setting to Do Nothing from Wait (sec).</li>
		<li>Click the Save button and exit the Protocol Editor.</li>
	</ol>
	</li>
</ol>
2. Illumination Reference Files
Note: Apply X-ray illumination reference files to an X-ray image in order to automatically correct for variations in detector uniformity of the X-ray images obtained throughout experiment. The procedures outlined below are specific to the Bruker In Vivo Animal Imaging Systems; other in vivo imaging systems may be used.
<ol>
	<li>Generate an illumination reference file by opening the molecular imaging software and clicking on the Capture button.</li>
	<li>Using the pop-up menu, establish the X-ray capture settings in Illumination Source (see suggested settings in section 1 above) and then select Illumination Reference in the exposure type. This can be found under the Standard Exposures drop down box. (See Figure 5).</li>
	<li>Remove all samples from the image station. Set the X and Y binning to 4 x 4 binning. Refer to Table 1: Illumination reference file exposure times to determine the accurate exposure time.</li>
	<li>Press Expose.</li>
	<li>Apply the reference file by selecting Auto Select from the drop down menu under Apply Reference File. (See Figure 6). The illumination reference file will now be automatically applied to all X-ray images captured with the same camera settings. Steps 2.1 - 2.4 will not need to be repeated if same camera settings are utilized in subsequent experiments. 
	Note: An illumination reference file can be applied after image acquisition, or if an error message occurs after auto-selection of reference files. Apply illumination reference files post-image capture using the following series of commands in the Navigation Panel of the molecular imaging software: Image &#62; Image Math &#62; Type: Task &#62; Calculate: Illumination correction. Select the input image (X) that you would like to apply the illumination reference file (Y) to. Rename the corrected file (Z). (See Figure 7).</li>
</ol>
3. Animal Handling
<ol>
	<li>Acquiring Animals
	<ol>
		<li>Purchase pregnant dams from commercial breeders or breed female mice in house at 12 weeks of age (or older) in accordance with institutional guidelines.</li>
		<li>House newborn mice with lactating mothers up to postnatal day (PN) 10.</li>
	</ol>
	</li>
	<li>Animal Anesthesia (PN 10)
	<ol>
		<li>Prepare a Ketamine/Xylazine cocktail to anesthetize PN 10 mice for prolonged anesthetic effects lasting up to 40 min. Add 500 &#181;l of Ketamine (100 mg/ml) to 75 &#181;l of Xylazine (100 mg/ml). Dilute 1:10 in a 0.9% saline solution to make a Ketamine (100 mg/kg)/Xylazine (10 mg/kg) anesthetic cocktail.</li>
		<li>Weigh the newborn mice.</li>
		<li>Using a 3/10 syringe with a 31 G 5/16 inch (8 mm) needle, administer 10 &#181;l/g of body weight of anesthesia with an intraperitoneal injection.</li>
		<li>Keep animals dry and insulated to prevent excessive loss of body heat.</li>
	</ol>
	</li>
</ol>
4. Tracheal Instillations
<ol>
	<li>Prepare an intratracheal saline solution comprised of 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, and 10 mM HEPES; pH = 7.4. The osmolality of this solution should be 319 mosmol/kg H2O.</li>
	<li>Mount anesthetized animals ventral side up onto a sloped surgical board using surgical tape. Ensurethat the animals&#39; heads are at the top of the incline.</li>
	<li>Perform a toe pinch to ensure that animals are anesthetized and ready for surgery. Disinfect all surgical areas, instruments, and thoracic region of the animal.</li>
	<li>Make a small (3 mm) incision in the anterior-medial ventral aspect of the neck (throat region) using a surgical scalpel, size 11. Push aside the platysma and anterior tracheal muscles using blunted forceps in order to visualize and access the trachea.</li>
	<li>Instill 3 &#181;l/g of weight (approximately 10 - 30 &#181;l final volume) of saline via the exposed trachea using a 31 G 5/16 inch (8 mm) needle. The minor incision is left open during animal imaging and usually heals itself well. Consult with local Division of Animal Resource to determine if the incision can likewise be left open. Otherwise, sutures may be required.</li>
</ol>
5. Animal Imaging
<ol>
	<li>Position the pups ventrally on a clear-bottom, removable, animal imaging tray. Center the animals so that the X-ray beam will be directly over the thoracic area.</li>
	<li>For odd numbered animal cohorts, place the first pup directly in the middle of the tray, and for even numbered cohorts place the first pup just left of center so that when the other animals are placed on the tray all of the animals are centered.</li>
	<li>Return the imaging tray to the X-ray imaging cabinet and close the cabinet door.</li>
	<li>Turn on the animal thermal control unit to maintain the anesthetized animals&#39; body temperature. Use the high setting to achieve a chamber temperature of approximately 35 - 37 &#176;C. Turn on animal anesthesia unit (vaporized isoflurane delivered via oxygen) to ensure animals are anesthetized and immobilized throughout duration of 2 hr imaging procedure.</li>
	<li>Executing X-ray imaging protocol
	<ol>
		<li>Click on Capture and select the appropriate protocol, e.g., &#34;Jove Demo Protocol,&#34; from the Protocol drop-down menu. (See Figure 8).</li>
		<li>Click the Execute Selected Protocol button on the molecular imaging software. 
		Note: A pop-up window will appear to track the status of the image acquisition, when the protocol is complete the window will disappear. The X-ray dose is low, &#60; than 0.3 mRem or about 10 times less than a dental X-ray. As with other X-Ray procedures, there is no residual radiation.</li>
		<li>When the 2 hr acquisition session is complete, remove the animals from the imaging tray and return them to their cage. Monitor animals for full recovery before returning to racks.</li>
	</ol>
	</li>
</ol>
6. Data Analysis
Note: Molecular imaging software allows for quantification and the translation of X-ray pixel intensity into rate of lung fluid clearance. The steps below outline the procedures needed to normalize X-ray images and quantify intensities in defined regions of interest (ROI).
<ol>
	<li>Design an ROI template 
	Note: A region of interest template must be created specific to the captured X-ray images during the 2 hr study, and should be used in order to compare X-ray intensities amongst experimental groups. Since small volumes of saline challenges typically accumulate in the left upper lobe of the lung4-6, the ROI(s) should focus on this portion of the lung.
	<ol>
		<li>Open the first and last x-ray image in the 2 hr set. Select the window of the first x-ray image.</li>
		<li>In the Navigation toolbar, select Manual-ROIs &#62; New ROI Set.</li>
		<li>Click ROI Ellipse and create a ROI that adequately covers the mouse&#39;s left lung.An ROI is not defined until the red, outlined ROI is dragged to a different position. A defined ROI will be outlined in blue with a number.</li>
		<li>If multiple pups are imaged, click on the red, outlined ROI and drag to the left lungs of the other mice to create more individual ROIs in the same set. Drag the red, outlined ROI to an area with a clear background to create a background ROI.
		<ol>
			<li>Select Pointer Selection to position the ROIs to lie over each mouse&#39;s left lung, directly under the second rib.</li>
		</ol>
		</li>
		<li>In the top toolbar, click Image Display.</li>
		<li>Check Overlay in the Image Display dialog to overlay the last x-ray image while maintaining the set ROI locations. If necessary, select Pointer Selection in the Navigation toolbar to adjust the positions of the ROIs and ensure adequate lung coverage in both images.</li>
		<li>In the Manual ROI dialog, select Template &#62; Save to Template. Name the template and click OK.</li>
		<li>Close both images. Select No when prompted to save changes.</li>
		<li>Apply the ROI template to each X-ray image captured to analyze fluid clearance by opening all images taken during the study. Start by selecting an open file and click Manual ROIs &#62; Template.</li>
		<li>Select the previously created ROI template from the drop down menu and click Apply to All Open Documents.</li>
	</ol>
	</li>
	<li>Export Numerical ROI Data from the Images to a Spreadsheet
	<ol>
		<li>In the upper left hand corner, click File &#62; Export Data &#62; ROI.</li>
		<li>Check As Displayed and Auto Open in Excel in the pop-up dialog.</li>
		<li>Select Export All Open Documents.</li>
		<li>Name the file and click Save.</li>
		<li>Close molecular imaging software.</li>
	</ol>
	</li>
</ol>

<ol>
	<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>

This article is part of a special issue on Multimodal Pre-Clinical Imaging, sponsored by Bruker Biospin.

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 pups can be simultaneously studied using an imaging system that is common place in research facilities and cores.
Instilling an appropriate lung fluid volume, as to not drown or collapse the lung, is critical to the successful implementation of this protocol and may need to be experimentally explored before X-ray imaging protocols can be applied. The sensitivity of this assay allows for the detection of very small volumes of instilled saline via X-ray detection. We have been able to discern differences in the X-ray opacity of neonatal lungs instilled with 10 &#956;l volumes of saline solution. The difference in X-ray opacities are even more pronounced when sodium channel inhibitors are introduced into the alveolar airspace because the saline challenges cannot be absorbed and the lungs continue to secrete fluid into the airspace. In the event that an inappropriately high volume of saline is introduced, placing animals into the imaging chamber with oxygen flowing into the chamber via the anesthesia ports can facilitate respiration in flooded lungs.
Our results using X-ray imaging are comparable to alveolar fluid clearances measured using more conventional approaches, such as lung wet-to-dry weight ratios and Evan&#39;s Blue for the determination of protein concentration4. We now demonstrate that this approach can be applied to the neonatal mouse pup. This X-ray imaging technique for determining lung fluid volumes can easily be combined with additional imaging modalities. For instance, fluorescent markers or bioluminescent probes can be simultaneously instilled into the alveoli and assessed. (Detection of fluorescent and luminescent probes has been described8, and is beyond the scope of this report). The ability to co-register the volume of lung fluid (using X-ray imaging) alongside the ability to detect fluorescent biomarkers is one of several advantages of using this dynamic assay and the commercial system for measuring lung fluid clearance. Other benefits of utilizing this approach for determining clearance and relative lung fluid volume includes the ability to conduct longitudinal studies (thus decreasing the number of animals needed to achieve statistically significant observations), and the ability to detect small changes in lung fluid volume in freely breathing, anesthetized, neonatal mouse pups. One limitation of using an in vivo imaging approach, however, is that the anesthesia can alter the distribution of gas and blood flow within the lungs. Mismatches in ventilation and perfusion (V/Q) and shunting have been shown to increase under anesthesia in healthy adult volunteers12, thus reducing oxygenation of the body. This adverse effect, however, can be compensated for by increasing the inspired oxygen concentration. From a technical viewpoint, variability between imaging systems in X-ray flux energy may require optimization of each system prior to performing imaging studies. For example, on a system with an X-ray source with more flux and/or a detector with superior quantum efficiency, a higher f/stop and lower binning state might provide better image quality when assessing small change in X-ray impedance.

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.

<table><tbody><tr><td>Preclinical Imaging System (In- Vivo MS FX PRO)</td><td>Bruker; Billerica, MA</td><td></td><td></td></tr><tr><td>Ketamine</td><td>Ketaset; Fort Dodge Animal Health, IA</td><td>26637-411-01</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories; Shenandoah, IA</td><td>4821</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (with Calcium and Magnesium)&amp;nbsp;</td><td>Lonza; Walkersville, MD</td><td>17-513F</td><td></td></tr><tr><td>Sodium chloride</td><td>Amresco; Solon, OH</td><td>241</td><td></td></tr><tr><td>Potassuim chloride</td><td>Fisher Scientific; Fair Lawn, NJ</td><td>P217-3</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich; St. Loius, MO</td><td>C5080</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich; St. Loius, MO</td><td>H3375</td><td></td></tr><tr><td>0.3 ml insulin syringe with 31 G x 5/16&quot; (8 mm) needle</td><td>BD Insulin Syringe; Franklin Lakes, NJ</td><td>328438</td><td></td></tr></tbody></table>

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 had successfully cleared the saline challenge. Specifically, the X-ray intensity of this animals ROI increased from 187.67 to 515. Thus, there is a reverse correlation between pixel density and lung fluid volume; that is to say, the larger the relative value, the less fluid there is in the lungs. It may be helpful to understand that more X-ray energy is absorbed (hence a larger reported value) when there is less fluid attenuating the X-ray. In Figure 10, the PN 10 mouse lung was tracheally instilled with a compound containing oxidized glutathione (reconstituted in saline described in 2.1) that inhibited alveolar fluid clearance of the saline challenge by blocking epithelial sodium channel activity; the numerical value of this animal&#39;s ROI will decrease from the pre-instill and post-instilled X-ray imaged files, indicative of increasing X-ray opacity. Specifically, the net intensity of the animal approximately 5 min post-instillation was - 64, and decreased to - 182. Again, note the inverse relationship between the ROI pixel intensity and amount of fluid in the lungs; increased fluid in the upper left lobe of the lung attenuates X-ray absorbtion.
Evaluating net intensity of the ROI enables quantitative evaluation of changes in the rate of lung fluid clearance, albeit acquisition software also allows the investigators to express data in terms of g/cm3 if desired. Moreover, the investigators can use each animal as its own control and normalize all X-ray intensities to an initial time point (Io), such as t = 5 min and report net changes in X-ray opacity (i.e., a measure of change in lung fluid volumes).
<img alt="Figure 1" src="/files/ftp_upload/52751/52751fig1.jpg" /> 
Figure 1. Exposure Settings. This screen shot illustrates the appropriate exposure settings utilized in this protocol. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52751/52751fig2.jpg" /> 
Figure 2. Settings File. This screen shot illustrates a key step in generating a file setting that will be used in a protocol. A pop up window (as shown) will request a new name for the acquisition settings file. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52751/52751fig3.jpg" /> 
Figure 3. Imaging Protocol. This screen shot illustrates a key step in determining whether a new imaging protocol has been successfully created. A pop up window (as shown) will appear and a new protocol name will be requested for the generated protocol. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52751/52751fig4.jpg" /> 
Figure 4. Protocol Steps. This screen shot illustrates a shortcut to duplicate an acquisition settings file, insert a new step, or to delete a step within an imaging protocol. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52751/52751fig5.jpg" /> 
Figure 5. Illumination Reference. This screen shot exhibits the illumination reference command and appropriate settings in the animal imaging software appropriate for creating an illumination reference file. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52751/52751fig6.jpg" /> 
Figure 6. Auto Select. This screen shot exhibits the Auto Select function and appropriate settings in the animal imaging software appropriate for applying an illumination reference file. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52751/52751fig7.jpg" /> 
Figure 7. Illumination Correction. This screen shot illustrates the appropriate application of an illumination reference file generated after animal imaging. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/52751/52751fig8.jpg" /> 
Figure 8. Execute Protocol. This screen shot illustrates how to execute a selected protocol. <a href="https://www.jove.com/files/ftp_upload/52751/52751fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/52751/52751fig9.jpg" /> 
Figure 9. X-ray images of cleared lungs. Representative image of PN 10 lungs prior to receiving a saline challenge (pre-instill; left panel); 5 min post-instillation (middle panel), and 2 hr after the saline challenge had cleared from the otherwise healthy lung (right panel). <a href="https://www.jove.com/files/ftp_upload/52751/52751fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/52751/52751fig10.jpg" /> 
Figure 10. X-ray Images of Flooded Lungs. Representative image of PN 10 lungs prior to receiving a saline challenge (pre-instill; left panel) containing glutathione disulfide , which inhibits paracellular solute transport; 5 min post-instillation of glutathione disulfide (middle panel), and 2 hr after inhibiting paracellular transport which leads to alveolar flooding (right panel). <a href="https://www.jove.com/files/ftp_upload/52751/52751fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>No filter=</td>
			<td>10 sec exposure</td>
		</tr>
		<tr>
			<td>0.1mm =</td>
			<td>15 sec exposure</td>
		</tr>
		<tr>
			<td>0.2mm =</td>
			<td>20 sec exposure</td>
		</tr>
		<tr>
			<td>0.4mm =</td>
			<td>30 sec exposure</td>
		</tr>
		<tr>
			<td>0.8mm=</td>
			<td>30 sec exposure</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">The size of the X-ray filter correlates to a specific exposure time for creating an illumination reference file.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1. Illumination Reference File. This file reports the appropriate exposure times for generating illumination reference files based on X-ray filters used in imaging studies.

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 ...

real-time-x-ray-imaging-of-lung-fluid-volumes-in-neonatal-mouse-lung

Nanyang Technological University

Research

JoVE Journal

Medicine

8.6K Views.  Emory University. We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology. 

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

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Cancer Research

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Immunology and Infection

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Developmental Biology

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

Bioengineering

Preserving Fungal Spore Localization in Lungs for Fluorescent Microscopy Analysis

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...