Name: Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung (Video) | JoVE Journal
Uploaded: 2016-07-18T13:00:00.000+00:00
Duration: 11 min 26 s
Description: Learn real-time X-ray imaging to assess alveolar fluid clearance in neonatal mice. This protocol enables quantitative measurement of pulmonary edema and lung fluid dynamics using image density analysis.

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5 minutes

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://scholar.google.com/scholar?hl=en&safe=off&q=%22Guide+for+the+Care+and+Use+of+Laboratory+Animals%22">Guide for the Care and Use of Laboratory Animals</a>. , 8, The National Academies Press. (2011).</li>
<li>Helms, M. N., Torres-Gonzalez, E., Goodson, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+tracheal+instillation+of+solutes+into+mouse+lung%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Chronic+lung+disease+in+the+preterm+infant.+Lessons+learned+from+animal+models%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Nadph+oxidase+regulates+alveolar+epithelial+sodium+channel+activity+and+lung+fluid+balance+in+vivo+via+O2-+signaling%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">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/pubmed?term=((Differential+infection+efficiencies+of+peripheral+lung+and+tracheal+tissues+in+sheep+infected+with+Visna%2Fmaedi+virus+via+the+respiratory+tract%5BTitle%5D)+AND+%22J+Gen+Virol%22%5BJournal%5D)">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/pubmed?term=((A+method+for+intratracheal+instillation+of+endotoxin+into+the+lungs+of+mice%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">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/pubmed?term=((H2O2+regulates+lung+ENaC+via+ubiquitin-like+protein+Nedd8%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Preclinical+anatomical%2C+molecular%2C+and+functional+imaging+of+the+lung+with+multiple+modalities%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">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/pubmed?term=((Cholinergic+regulation+of+epithelial+sodium+channels+in+rat+alveolar+type+2+epithelial+cells%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">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/pubmed?term=((Dynamic+imaging+of+the+lungs+using+x-ray+phase+contrast%5BTitle%5D)+AND+%22Phys+Med+Biol%22%5BJournal%5D)">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/pubmed?term=((Inhalation+anesthesia+increases+V%2FQ+heterogeneity+during+spontaneous+breathing+in+healthy+subjects%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">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 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.

<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

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...

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 ...

The overall goal of this procedure is to determine there rate of alveolar fluid clearance in neonatal mice using real time x-ray imaging technology. This is accomplished by first setting up an x-ray imaging acquisition protocol with appropriate illumination reference files. The second step is to model pulmonary adema by tracheally instilling saline in anesthetized pups.Next the pups are imaged using the acquisition protocol. The final step is to quantify and compare x-ray image densities as a measure of alveolar fluid volume. Using molecular image software to study treatments and other conditions.Modeling alveolar fluid clearance using this protocol can increase our understanding to ultimately develop therapies for pre-term and newborn lung injury, frequently seen in the Neonatal Intensive Care Unit. Visual demonstration of this method is critical, since newborn lungs are very small and tracheal installation of neonatal mice can be very difficult to master. Demonstrating the procedure will be Phi Trac, a student from my laboratory.To operate the x-ray imaging software, first, establish the settings for a single exposure event. Select the Capture button directly below the File tab. Then in the Settings drop down menu, select Current Session.Then, under Exposure Time, select Standard Exposure from the drop down box. Set the Exposure Time to two minutes and Number of Exposures to one. Then set the X Binning and Y Binning to two pixels.Under the Illumination settings, select X-ray in the Illumination Source drop down menu. The default KVP is set at 35. Now toggle Auto Select in the Apply Reference File drop down menu to apply the corresponding x-ray reference file.In the Set Camera To settings, make the F Stop 2.51. Set the Field of View to 125.64 for PN10 mice or as needed. Set the Focal Plane to 13.Next, from the drop down menu beside the Focal Plane slider, select X-ray. For x-rays, set the Excitation Filter and the Emission Filter to zero in the drop down menu. Save the current session by clicking New on the top next to Settings.Enter a name for New Acquired Settings File, eg, Jove Single Exposure Event for Making a Protocol. To establish the imaging protocol, click on the Create Edit Protocols button on the right side of the open window. As the new protocol pop up window appears, click the New button in the upper right hand corner.Enter the name that the protocol will be saved under, for example, Jove Demo Protocol, and click okay. On the bottom of the Protocol pop-up window in Protocol Settings, check for the following. Step One should be highlighted in red text.Capture New Images should be selected. And in the Before Image Capture drop down menu, Do Nothing should be selected. Now, under the Capture Setting, select the recently generated single exposure event.This is step one. Then, to add a three minute wait time after each two minute acquisition, select Wait from the After Image Capture drop down menu. Then, click the Edit button and change the value to 180 seconds and click OK.To duplicate step one, right click the Step One tab, and select Duplicate Step.Create 23 duplicates for a two hour observation period. For the final step, being step 24, change the After Image Capture setting from Wait to Do Nothing. Then, click the Save button and exit the Protocol Editor.To anesthetize new post-natal day 10 mice, deliver the anesthetic via a 310 syringe with a 31 gauge, eight millimeter needle with an introperiteneal injection. Position the mice ventral side up onto a sloped surgical board. Secure them with surgical tape so their heads are at the top of the incline.Now, move the pups to a clear bottom removable animal imaging tray, being sure to center the animals so the x-ray will view the thoracic area. Then, return the tray to the x-ray imagining cabinet. Be sure that the thermal control unit of the chamber is set to physiological levels.Set the anesthesia vaporizer dial setting to 2 percent. To image the animals in the software, from the Protocol drop down menu, click on Capture and select the appropriate protocol, e. g, Jove Demo Protocol.Next, click the Execute Selected Protocol button on the molecular imaging software. A pop-up window will display the program's status. After the two hour imaging session, remove the animals from the imaging tray and return them to their cage.To normalize x-ray images and quantify intensities and to find regions of interest, first design a region of interest or ROI template specific to the study to use on all the tested groups. Begin by opening the first and last x-ray image in the two hour set. Then select the window of the first x-ray image.On the navigation toolbar, select Manual ROIs and then New ROI Set. Choose to make an ROI elipse and create an ROI that adequately covers the mouse's left lung. The left upper lobe makes an ideal ROI for saline challenges.When the ROI is dragged to a different position, it becomes defined by the software. This is indicated by the region's border turning blue and gaining an identification number. Additional ROIs can be made by dragging the blue ROI.Now select pointer selection to position the ROIs over each mouse's left lung, directly under the second rib. Then, in the top toolbar, click Image Display. Check Overlay in the Image Display dialogue to overlay the last x-ray image while maintaining the set ROI locations.If necessary, choose pointer selection in the navigation toolbar to adjust the positions of the ROIs to improve their coverage. In the Manual ROI dialogue select Template and Save To Template. Then name the template and click OK.Now apply the ROI template to each captured x-ray image.First open all the files and then select any open file and click Manual ROIs and Template. Select the saved ROI template and then from the drop down menu click Apply to All Open Documents. Now export the numerical ROI data from the images to a spread sheet.In the upper left hand corner click File, Export Data and ROI. Check the As Displayed and Auto Open In Exel toggles in the pop-up dialogue. Then select Export All Open Documents, name the file and click Save.PN10 mouse lungs were imaged after an installation of saline challenges into the left lobe of the neonatal lungs. Shown here is the baseline appearance of a lung. The same lung was imaged five minutes after the lung was tracheally instilled with the saline solution.After two hours the animal had successfully cleared the saline challenge as indicated by a decrease in x-ray opacity in the upper lobe of the left lung. As such, the numerical ROI increased in value. A different PN10 mouse lung was tracheally instilled with a compound containing oxidized glutathione that inhibited alveolar fluid clearance of the saline challenge.So the numerical value of this animal's ROI decreased from the pre-instilled and post-instilled x-ray imaged files, indicative of increasing x-ray opacity. The lungs were flooded and stayed flooded. Once mastered, this protocol can be completed in multiple pups for quick and reliable measurements of lung fluid clearance.The pups typically make a full recovery from this minimally invasive procedure, allowing longitudinal studies of the lung. Therefore, application of this protocol to animal models of pediatric disease is translationally significant.

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

Research

JoVE Journal

Medicine

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A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

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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.

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

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

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.

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

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.

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

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

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

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

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

We are developing a dynamic adaptive exposure technique using our scanning beam digital X-ray system. Rather than exposing an object uniformly, the exposure is adapted depending on the opacity of the object. Here we show an experiment on an anthropomorphic phantom that resulted in a dose saving of 30%.

X-ray Dose Reduction through Adaptive Exposure in Fluoroscopic Imaging

Learn real-time X-ray imaging to assess alveolar fluid clearance in neonatal mice. This protocol enables quantitative measurement of pulmonary edema and lung fluid dynamics using image density analysis.

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

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