Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways

Abstract

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at delivery into mouse trachea via immediate retrograde fluid motion, and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways.These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small animal models.

Variability of In Vivo Fluid Dose Distribution in Mouse Airways Is Visualized by High-Speed Synchrotron X-Ray Imaging

“…1,2,17,18 Our previous study suggested that a 20 lL dose overwhelmed the holding capacity of the mouse nose, 13 so we sought to test the effect of an intermediate dose volume. For this reason, we chose 4 lL, 10 lL, and 20 lL as the surrogate fluid volumes to examine here (n = 5 each).…”

“…Our previous fluid dosing study suggested that rapid dose deliveries better target the conducting airways and minimize dose losses via retrograde movement around the delivery and ET tubes, 13 so the maximum infusion rate of the syringe pump (*4.2 lL/ sec) was used for both volumes.…”

“…13 All images were flat-field and dark-field corrected, and image subtraction algorithms were used to locate regional For the nasal studies, each frame was subtracted from the best matched baseline image-determined by calculating the two-dimensional correlation coefficient between the current frame and each of the baseline frames-to locate regions where the image brightness had changed and so reveal fluid motion (i.e., background subtraction of the mouse anatomy). A constant threshold of 128 gray values (1/32 the dynamic range) was used to select only the large intensity changes, and a 3 · 3 median filter was applied to reduce noise.…”

“…13 In brief, 15 anesthetized female C57/Bl6 mice (*20 g) were individually secured head-high to a polyethylene imaging board 16 with their incisors hooked over a wire loop and their limbs, shoulders, and torso taped to the board to minimize body movements. The imaging area (head) was brushed with a thin layer of glycerol to eliminate air in the fur layer and minimize fur interference in the PCXI images.…”

“…6,7 We have described the advantages of PCXI for airway surface imaging in small animals, 9,10 for monitoring the post-deposition behavior of particulates on live mouse nasal and tracheal airways, 11,12 and for non-invasively tracking how a fluid bolus-typical of that used in airway gene-therapy dosing-moves through the airways of live and intact small animals. 13 Our previous fluid dosing study 13 showed the immediate fate of fluids instilled into the nose and trachea of live anesthetized mice, demonstrating that the overall distribution of the dose, the effectiveness of the delivery method, and the persistence of the delivered dose can now be visualized. The limitations of that study included a small sample size, small delivered fluid volumes, and significant motion artifacts from animal movement during the lung studies.…”