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

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.