An optimized Protocol for the Assessment of Cystic Fibrosis-Like Lung Disease by Correlative µCT Imaging and Scanning Electron Microscopy

W. Wagner1, M. Finke1, H. Horstmann2, H.-U. Kauczor1, M. Wielpütz1 and W. Stiller1

1Department of Diagnostic and Interventional Radiology, Translational Lung Research Center Heidelberg (DZL), University of Heidelberg, Germany

2Department Medical Cell Biology, Institute for Anatomy and Cell Biology, University of Heidelberg, Germany

Introduction In previous studies, we monitored CF-like lung disease progression longitudinally in mice over-

- -Tg). An intermodal approach was now used to characterize the development and progression of morphologic abnormalities of main conducting

ENaC-Tg mice in greater detail with higher spatial resolution. Methods

-Tg and wild-type littermate control lungs were perfusion fixed in situ to preserve airway lumen, luminal mucus, and airway mucus plaques in their orthotopic region (Fig. 1 and supplemental data). Global ex vivo of interest were subjected to ultra-high-resolution synchrotron radiation-based tomography (~325 nm voxel size -srCT-). Results were cross-validated by histology and the same specimens examined by µCT were subjected to scanning electron microscopy (SEM) for direct cross validation. See supplemental data for a detailed depiction of the optimized vascular perfusion fixation protocol. Results

obstruction and atelectasis, which were noted in previous in vivo studies (Fig. 2&3). Airway wall thickening was observed in the proximal bronchial tree and particularly in the bronchoalveolar duct junctions. Mucus plaques were localized in main conducting airways, distal airways and terminal bronchioles, as distal as the bronchoalveolar duct junction in the -Tg group (Fig. 3). Partially versus fully obstructed distal airways were defined (Fig. 2). Atelectatic areas distal

-Tg groups patchy areas of hyperinflated parenchyma was revealed, as a possible microscopic correlate to air trapping as reported previously (Fig. 3). On a sub-micron level srCT showed perturbed distal lung parenchyma architecture with hyperdialated alveolar ducts and overinflated sacculi distal to mucus obstructed air ways (Fig. 3). Cross-validation between data obtained from µCT, srCT and SEM was performed. A distinction between air way epithelium and overlaying adherent mucus plaque was replicable (Fig. 4). Conclusion We present a novel optimized fixation protocol for the assessment of cystic fibrosis like lung disease using correlative µCT and scanning electron microscopy. Our study demonstrates the spatial arrangement of luminal mucus plaques throughout the bronchial tree, including terminal bronchioles and bronchoalveolar duct junctions in a mouse model of CF-like lung disease. Characteristic CF abnormalities of conducting airways and distal lung parenchyma were confirmed on a sub-micron level. Perturbed acinar architecture as a hallmark of emphysematous

lung destruction was identified distal to obstructed airways. The results suggest high and ultra-high-resolution micro-tomography to be a suitable tool to study small airways disease in mouse models. Figures:

Figure 1: Vascular perfusion fixation. To preserve airway mucus in orthotopic location the trachea was tied at RLC and lungs were fixed via the pulmonary artery at a constant physiologic pressure. No positive pressure was applied to the airway tree.

Figure 2: Global ex vivo measurements were p (A-C) -Tg (D-E) mice at ages 0.5 (post natal), 14 days and

~ 40 days (considered adult). Scale bar tic distances = 1000 m. n = 6-8 mice per group. No air way mucus was observed at d0.5, 14 day old Tg mice showed airway mucus obstruction mostly in distal airways. Adult mice showed strong mucus plugging in central airways.

Figure 3: Characteristic CF lung disease abnormalities. All images from 14 day old mice. -Tg mouse lung showing multiple hallmarks of CF lung disease. Arrowheads indicate

an atelectatic area of the middle lobe. White arrow shows a focal subpleural infiltrate. Red arrows point to air way wall adherent mucus plaques in all images. (B) Red asterisks mark distended areas with perturbed distal lung architecture as a possible correlate to air trapping and an early sign of emphysematous lung destruction. These areas were predominantly found distal to mucus obstructed airways (C). Wall adherent mucus plaques were identified as distal

-Tg , E=WT, F=TG SEM).

Figure 4: Cross- -Tg by µCT, srCT and SEM. -Tg mouse. Images A-E were obtained from the same specimen. µCT analysis of critical point dried whole lung samples were used to identify regions of interest. Cross-validation was between data obtained from µCT, srCT and SEM. A distinction between air way epithelium and overlaying adherent mucus plaque was replicable.

Supplemental Material: Fixation to preserve mucus - RLCvasc (Residual Lung Capacitiy, vasculature) Materials:

3 forceps bent, serrated 1 forceps, super fine, sharp/pointed 2 Micro scissors, sharp! Silk suture Venous catheters (Abbocath, BISI: 26G (smallest, white), 24G (blue) 22G (yellow)) Swabs Cork boards Trays Pins, sharp Set-up for Inflation fixation (stand + tubed syringe) LED light source (Schott) Stereo microscope

Pre-flush solution:

Ri Dextran 70 (Carl Roth, part number: 9228.2) Heparin 25000 IU Lidocaine 2% Funnel and Folded filter or sterile filters

Fixative solution:

Polyethylene glycol (PEG) 400 (Carl Roth) Ethanol, 99% Formaldehyde, 37% ddH2O

1. buffer preparation need to be freshly prepared, especially the pre-flush, even if stored at 4°C, it is growing fungi! Use it up within a few days! 1L of Pre-flush:

final concentration 1L

Dextran 70 50 g ~ 5 % It will take up to 2h for the Dextran to dissolve!

Filter the dextran- Heparin 1 ml ~ 5 IU/ml Lidocaine 10 ml 0,02 %

1L of Fixative:

final concentration ddH2O 500 ml PEG 250 ml 25 % ethanol 100 ml 10 % formaldehyde 100 ml 3,7%

Can be stored at 4°C for a few days.

2. bench preparation1. collect all the material that is needed 2. rinse the tubed syringe of the inflation fixation stand with ddH2O. It needs to be free of any

dust/precipitate/particles 3. plug the appropriate venous catheter to the tube 4. add ca. 40 ml of pre-flush to the syringe 5. flush the tube and catheter with pre-flush, make sure there are no air bubbles in the system6. mark the 20 cm H2O column on the syringe 3. Mouse prep. 1. inject an overdose of Ket/Xyl 2. as soon as it is deeply anesthetized, start the preparation of the trachea 3. tie the trachea to preserve a residual volume of air in the lungs 4. open the chest, as you would do to e.g. lavage the lungs, but do not exsanguinate the mouse5. cut off the rib cage (gives you better access to the heart for the perfusion and avoids

distortions) 6. remove the thymus 7. move to the perfusion stand 8. cannulate right ventricle and cut left atrium (see below) 9. perfuse with pre-flush 10. after ~ 15 min switch from pre-flush to fixative. Perfusion with fixative should run ~ 30 min11. to avoid drying of the tissue add some drops of pre-flush and later fixative on top of the lung Cannulation of right ventricle and cutting of left atrium

Reference Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography. Vasilescu DM, Knudsen L, Ochs M, Weibel ER, Hoffman EA. J Appl Physiol (1985). 2012 Jan;112(1):159-66. doi: 10.1152/japplphysiol.00550.2011. PMID: 21817110 http://jap.physiology.org/content/112/1/159.long