Simple low dose radiography allows precise lung volume assessment in mice

www.nature.com/scientificreports OPEN Simple low dose radiography allows precise lung volume assessment in mice Amara Khan1, Andrea Markus1, Thomas Rittmann2, Jonas Albers1,3, Frauke Alves1,3,4, Swen Hülsmann5,6 & Christian Dullin1,3,6* X-ray based lung function (XLF) as a planar method uses dramatically less X-ray dose than computed tomography (CT) but so far lacked the ability to relate its parameters to pulmonary air volume. The purpose of this study was to calibrate the functional constituents of XLF that are biomedically decipherable and directly comparable to that of micro-CT and whole-body plethysmography (WBP). Here, we developed a unique set-up for simultaneous assessment of lung function and volume using XLF, micro-CT and WBP on healthy mice. Our results reveal a strong correlation of lung volumes obtained from radiographic XLF and micro-CT and demonstrate that XLF is superior to WBP in sensitivity and precision to assess lung volumes. Importantly, XLF measurement uses only a fraction of the radiation dose and acquisition time required for CT. Therefore, the redefined XLF approach is a promising tool for preclinical longitudinal studies with a substantial potential of clinical translation. Respiratory diseases account for ~ 10% of mortality worldwide, which is set to rise due to persistent smoking, pollution and occupational irritants1. Depending on the underlying disease, the characteristics of lung dysfunction may involve inadequate oxygen exchange, breath shortening, chronic cough, chest pain and d yspnoea2,3. To improve our knowledge about the basic mechanisms regarding lung physiology, pathophysiology and treatment strategies, the use of mouse models of lung disease in translational research is inevitable. The pre-clinical assessment of pulmonary function is a valuable tool not only for investigating the pathology of respiratory and allergic diseases but also enables preclinical evaluation of the response to novel therapeutic strategies. Changes in lung function can be monitored non-invasively in restrained or unrestrained mice by double chamber plethysmography or barometric p lethysmography2,4. Other methods require invasive intubation proce5 dures some of which tend to be t erminal . Of all available techniques, unrestrained whole-body plethysmography (WBP) is extensively used in longitudinal studies due to its ease of use and high data o utput3,6–8. However, there 9–12 is also considerable concern over the validity of plethysmography techniques . The main limitation of this technique is that the functional outcomes rely on the plethysmograph pressure changes, and the relationship between chamber pressure and the lung mechanics might be non-linear. Furthermore, the accurate tidal volume estimates are significantly affected by subtle changes in temperature, pulmonary mechanism and fluctuations in barometric pressure3,10,13. Plethysmography may also be inconsistent between mouse strains requiring precise selection of the control groups11. Recently, the use of lab and synchrotron radiation based micro-computed tomography (micro-CT) has been increasingly used in preclinical studies to assess quantitative parameters including lung volumes, air spaces or lesions and mean lung d ensity14–19. These measurements have been performed in an attempt to reflect the structural and functional competence of the lung during disease progression and in response to therapy in numerous animal models of lung diseases, such as cancer, fibrosis, emphysema and t ransplantation14. Not only in clinical routine, but also in preclinical research, dose restrictions are major obstacles. Small-animal models, especially mouse models, are often utilized in preclinical research. Imaging in live mice for micro-CT based longitudinal lung studies requires a dramatically higher spatial and temporal resolution due to the small size of the mouse 1 Translational Molecular Imaging, Max-Planck-Institute for Experimental Medicine, Hermann‑Rein‑Straße 3, 37075 Göttingen, Germany. 24th Physical Institute ‑ Solids and Nanostructures, University of Göttingen, Friedrich‑Hund‑Platz 1, 37077 Göttingen, Germany. 3Institute for Diagnostic and Interventional Radiology, University Medical Center Göttingen, Robert‑Koch‑Straße 40, 37075 Göttingen, Germany. 4Clinic for Hematology and Medical Oncology, University Medical Center Göttingen, Robert‑Koch‑Str. 40, 37075 Göttingen, Germany. 5Clinic for Anesthesiology, University Medical Center Göttingen, Humboldtallee 23, 37073 Göttingen, Germany. 6These authors jointly supervised this work: Swen Hülsmann and Christian Dullin. *email: ‑goettingen.de Scientific Reports | (2021) 11:4163 | https://doi.org/10.1038/s41598-021-83319-5 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Schematic illustration of the set-up for correlative XLF, WBP and micro-CT measurements. One end of the WBP chamber has an isoflurane inlet/outlet, the other end is connected to the differential pressure sensor (DPS) which in turn is connected to a reference chamber, a Powerlab data acquisition device and a portable computer (PC). The chamber is placed inside the gantry of the micro-CT imaging system on the sample stage. The mouse is positioned inside the chamber such that the chest cavity is within the field of view (FOV). A piezoelectric (PZT) acoustic sensor that transduced the sound of the CT door interlock is used to synchronize data acquisition from WBP with XLF or micro-CT. and its fast metabolism. Consequently, this limits the use of CT based high-resolution imaging for longitudinal lung studies and also hampers the clinical application of this a pproach20–26. Therefore, the development of novel and sophisticated lung function measurement techniques with improved reliability and minimal radiation is fundamentally required. To this end, we recently established a non-invasive lung function method based on planar cinematic X-ray imaging of the chest, namely X-ray-based lung function (XLF) to measure the lung function in preclinical mouse models of allergic airway inflammation (AAI) over time27,28. Using minimal radiation dose and short exposure time, XLF showed significantly higher sensitivity than WBP for reliable assessment of lung function during AAI and response to dexamethasone treatment27. However, previously the functional parameters of XLF were not exactly relatable to commonly used lung function techniques including WBP and micro-CT and none of the XLF parameters represented the lung volume. In this study, we compared XLF determined end-inspiratory volume (EIV) with the lung volumes from micro-CT and conventional WBP to establish XLF as a biomedically relevant technique. To achieve this, we first designed a unique experimental set-up for performing correlative lung function measurements using XLF, micro-CT and WBP. Using micro-CT as a gold standard approach, XLF was found sensitive and precise to determine lung volumes, as it exhibited a higher degree (...truncated)