Non-invasive plethysmography techniques offer lung function readouts of conscious animals longitudinally. Unrestrained whole-body plethysmography (UWBP), as an extreme of non-invasiveness, provides lung function data of several awake mice/rats at the same time and at several time points. Basically, the animals are placed each into a separate closed chamber to record breathing-induced oscillations of pressure inside the chamber by employing a barometric analysis technique. This technique provides parameters such as tidal volume and respiratory frequency (Glaab et al., 2007; Vanoirbeek et al., 2010; Bates, 2017). UWBP has been employed by many researchers for longitudinal measurement of lung functions in spontaneously breathing animals, especially by reporting a parameter called enhanced pause (Penh) (Hamelmann et al., 1997; Chong et al., 1998; Finotto et al., 2001; Donaldson et al., 2002). Penh is a dimensionless parameter used for the evaluation of changes in the shape of the airflow entering and leaving a whole-body plethysmograph (Bates, 2017). In 1997, for the first time using UWBP, a study on airway hyperresponsiveness (AHR) using aerosolized methacholine challenge in conscious, spontaneously breathing mice, revealed a good correlation between Penh and readouts such as lung resistance obtained from an invasive method (Hamelmann et al., 1997). Following this pioneering study, a few more investigations confirmed good correlations between Penh and gold-standard functional parameters obtained from invasive lung function measurements (Chong et al., 1998; Berry et al., 1999; Finotto et al., 2001; Donaldson et al., 2002; Kumar et al., 2004). However, further experiments have raised serious uncertainties and questions such as: what Penh as a dimensionless index really measures and to what extent it can be related to lung mechanics (Lundblad et al., 2002; Mitzner and Tankersley, 2003; Adler et al., 2004; Bates et al., 2004; Sly et al., 2005). Using unrestrained plethysmography investigating the relationship between Penh and lung resistance, it has been shown that UWBP can determine lung resistance only when tidal volume and functional lung capacity are measured independently. Also, humidity and temperature of the gas inside the chamber should be preconditioned to the animal’s body (Lundblad et al., 2002). Since fulfilling these conditions with conscious, unrestrained animals are not possible, the authors suggested that Penh should not be used for bronchial responsiveness assessments and sent a clear warning to the community that Penh cannot represent airway or pulmonary resistance. Followed by this enlightening study, more researchers (Adler et al., 2004; Bates et al., 2004) shared their serious concerns about replacing invasive mechanical indexes such as lung resistance with Penh, since this dimensionless parameter lacks the necessary physical principles. Despite intense criticism against the application of Penh to measure airway reactivity and AHR, for further exploration of Penh, Lomask discussed the mathematics of unrestrained plethysmography for two types of whole-body plethysmographs, pressure (PWBP) and flow (FWBP) plethysmographs (Lomask, 2006). The author confirmed that PWBP that utilizes a sealed chamber poorly correlates with airway resistance at room temperature. However, the Penh values obtained by FWBP that uses a chamber with a pneumotachograph correlate with resistance. Similarly, the relationships between Penh and thoracic airflow patterns have shown that Penh of plethysmography airflow is a sensitive indicator of an increase in specific airway resistance (Frazer et al., 2011). Nevertheless, UWBP as a convenient, quick, and non-invasive method can be employed for gross screening of overall ventilatory function in rodents. However, considering the cautionary warnings toward the misusage of Penh, especially in lung studies on airway responsiveness, ventilatory function obtained from UWBP should be corroborated with independent direct measurements of pulmonary mechanics (Vanoirbeek et al., 2004; Vanoirbeek et al., 2006; Hoymann, 2007; Tarkowski et al., 2007).

Since lung function data obtained from unrestrained plethysmography have no direct link to the mechanical properties of the lung, Bates et al. (2008) introduced unrestrained video-assisted plethysmography (UVAP) to non-invasively determine lung mechanical function in small animals. Reliable measurement of lung mechanical function requires a precise assessment of lung volume changes during the animal’s breathing, which is beyond the capability of UWBP (Lundblad et al., 2002; Adler et al., 2004; Bates et al., 2004). However UVAP, as an extension of UWBP, was an attempt to more precisely estimate lung volume using orthogonal video imaging (Bates et al., 2008). In UWBP, the measurements are based on the chamber pressure fluctuations due to the animal’s breathing, which results from the fact that the change in lung volume is not equal to the volume of inspired air from the chamber. Two physical processes can be introduced as the reason for this difference (Mitzner and Tankersley, 2003; Adler et al., 2004; Bates et al., 2008): (Hoymann, 2007) During inspiration, the respiratory musculature produces a necessary pressure gradient that drives the inspired air through the resistive airways, which also leads to thoracic gas compression; (Powers and Dhamoon, 2019); Due to the different temperature and humidity inside the thorax compared to the chamber, the inspired air expands inside the lungs. It has been shown that the pressure change due to the gas conditioning inside the lungs can be eliminated by heating and humidifying the air inside the plethysmography chamber to match the condition inside the lungs (Lundblad et al., 2002). Therefore, by preconditioning the air inside the chamber, the pressure fluctuations during the animal’s breathing can be related directly to the thoracic gas compression, which is also influenced by tidal volume (Bates et al., 2008). The constructed plethysmograph was a cuboidal chamber with two clear orthogonal sides for monitoring the animal, and a water jacket on the remaining sides for controlling the temperature inside the chamber. The humidity of the chamber was also controlled continuously by introducing a stream of air to the chamber after passing over a flask of hot water, except for those brief moments when lung function measurements were acquired, and the chamber was completely sealed. A pressure transducer was utilized for assessment of the pressure inside the chamber relative to the atmospheric pressure and two video cameras were fixed close to the plethysmography chamber to monitor the two orthogonal sides. Using this setup, the authors tried to simultaneously measure the pressure inside the chamber, as well as the changes in lung volume by assuming the animal’s body as an elliptical cross section in the acquired orthogonal silhouettes (Bates et al., 2008).

As the biggest advantage of this system, UVAP is able to directly and more precisely measure specific airway resistance in unrestrained and spontaneously breathing mice compared to UWBP. However, there are still downsides to this system (Reynolds and Frazer, 2011): (Hoymann, 2007) movements of the animals are problematic due to the slow sampling resolution (25 Hz, camera speed) of the cameras; (Powers and Dhamoon, 2019); controlling the conditions inside the chamber including temperature and humidity makes the system more complicated and even may induce stress to the animal. Due to the mentioned limitations, despite the solid theory related to lung mechanics behind the UVAP, this extension of unrestrained plethysmography was unable to replace the invasive methods for lung function measurements and never became a widely used method for lung mechanical function measurements in small animals.

Acoustic whole-body plethysmography (AWBP), similar to UVAP, attempts to measure tidal volume more accurately compared to UWBP. The acoustic plethysmograph proposed by Reynolds and Frazer included a main chamber, nozzle, speaker, microphone, and end stop assembly to change the volume, which represents a resonant cavity that operates at a frequency that depended on the volume of the cavity and also the dimensions of the nozzle (Reynolds and Frazer, 2006). During the breathing of the animal in the plethysmography chamber, the volume around the animal changes due to the thorax movements which influences the amplitude of the acoustic pressure inside the chamber. In this system, the acoustic pressure of the chamber is almost independent of the animal’s lung volume, due to the fact that the acoustic input impedance of the system is very large because of the large change in area from the chamber to the nasal opening (Reynolds and Frazer, 2006). Since the sound pressure level (SPL) of the plethysmograph has a direct relationship with the signal-to-noise ratio of volume measurements, the sensitivity of the AWBP can be increased with higher values of SPLs, which is tolerable for mice (Fay, 1988). The acquired acoustic pressure signal inside the chamber was related to tidal volume using a signal processing technique. Similar to UVAP, VWBP can directly measure specific airway resistance in unrestrained, spontaneously breathing animals. However, this system is susceptible to ambient noise frequencies near the excitation frequency (Reynolds and Frazer, 2011), making it impractical to use for precise assessment of lung volume changes during the animal’s breathing in a laboratory setting without acoustic insulation.

In head-out body plethysmography (Glaab et al., 2007), the head and body of the animal are separated by a seal in the plethysmograph, wherein the animal’s head is exposed to a continuous airflow in the head chamber, and the rest of the body is placed in the body chamber which is attached to a pressure transducer by a pneumotachograph tube (Figure 2). In the body chamber, the thoracic movements of the animal drive the flow to the pneumotachograph tube which finally reaches the differential pressure transducer, where the respiratory flow is measured and parameters such as respiratory rate and tidal volume are obtained (Hoymann, 2007). Commonly for employing this approach, the animals should be trained a few days before the lung function measurements to get used to the head-out plethysmograph (Hoymann, 2012). The introduction of head-out body plethysmography dates back to 1994, when Vijayaraghavan et al. non-invasively measured mid-expiratory flow ( E F 50 ) for the assessment of airway responsiveness in conscious mice (Vijayaraghavan et al., 1994). E F 50 is the midpoint of expiratory tidal volume, which can perfectly describe the main changes in tidal volume due to an airflow limitation caused by bronchoconstriction, edema, or accumulation of mucus (Glaab et al., 2007; Hoymann, 2012). Since then, many other research groups employed head-out plethysmography for examination of drug effects and proposed E F 50 as a meaningful, non-invasive parameter for determination of bronchoconstriction in mice and rats (Neuhaus-Steinmetz et al., 2000; Glaab et al., 2001; Glaab et al., 2002; Baelder et al., 2005; Glaab et al., 2005). In addition, validation studies by employing invasive and non-invasive PFTs showed good correlations between E F 50 and gold-standard functional parameters (Glaab et al., 2001; Glaab et al., 2002; Glaab et al., 2005). In 2005, Glaab et al. utilized head-out body plethysmography to non-invasively measure E F 50 in conscious mice which were exposed to inhalable Aspergillus fumigatus antigens, paralleled by invasive measurement of pulmonary conductance and dynamic compliance in anesthetized, orotracheally intubated mice (Glaab et al., 2005). The decrease in E F 50 and pulmonary conductance and dynamic compliance correlated well and despite the higher sensitivity of gold-standard parameters, E F 50 was sensitive enough to detect airway responsiveness in intact spontaneously breathing mice.

In conclusion, head-out body plethysmography is a non-invasive, simple, and repeatable method for lung function measurements that allows handling several conscious animals at the same time by attaching several chambers to a central system. Head-out body plethysmography offers valuable outputs such as E F 50 with physical meaning [ml/s] that directly relates to gold-standard pulmonary functional parameters such as airway resistance (Glaab et al., 2007). Despite its advantages, there is a risk of inducing the influence of stress to the results due to retainment of the animal during the measurements. However, this issue can be mitigated to some extent by training the animals beforehand and starting lung function measurements only after the animals settled down to a stable level (Glaab et al., 2007).

Double-chamber plethysmograph, as the name explains, consists of two rigid chambers that separate the animal’s body from the neck to isolate the animal’s head and nose as hermetically as possible in the front chamber, from the rest of the body in the rear chamber (Figure 2). In the front chamber, where the restrained animal is consciously and spontaneously breathing, the produced flow from the nostrils is measured, while in the rear chamber the produced airflow by volume change due to the thorax movements is measured. Either using pressure transducers or pneumotachographs, waveform signals as a function of time from each chamber are recorded, which can finally produce respiratory parameters such as tidal volume and frequency (Reynolds and Frazer, 2011; Mailhot-Larouche et al., 2018). In addition to respiratory parameters, acquiring parameters such as E F 50 and specific airway resistance (sRaw), which are sensitive to airflow obstruction, is also possible with double-chamber plethysmography (Pennock et al., 1979; Neuhaus-Steinmetz et al., 2000; Glaab et al., 2001; DeLorme and Moss, 2002; Glaab et al., 2002; Flandre et al., 2003). While measurement of airway resistance requires both waveform signals obtained from head- and body-chambers, for calculating E F 50 , just like head-out body plethysmography, only the signal from body-chamber is needed. Therefore, depending on the application, double-chamber plethysmography can be employed with or without the head-chamber (Mailhot-Larouche et al., 2018). Regardless of the advantages, double-chamber plethysmography shares the same disadvantages as head-out plethysmography, which is the requirement of restraining the animal and facing the risk of inducing the effects of stress to the obtained results. Furthermore, the reproducibility of obtained parameters such as sRaw from double-chamber plethysmography for airway responsiveness has been challenged (Duguet et al., 2000; DeLorme and Moss, 2002). However, still many researchers suggest double-chamber plethysmography as a non-invasive, easy, rapid, and reproducible technique for longitudinal assessment of respiratory function in conscious animals after challenges with aerosolized substances (Lofgren et al., 2006; Mailhot-Larouche et al., 2018).

In clinics, X-ray CT as the gold-standard modality for lung medical imaging, plays an important role in the diagnostic and therapeutic workup of many lung diseases, due to its extensive availability, speed, high-resolution, and high signal-to-noise ratio for lung tissue (Simon, 2000; Tielemans et al., 2020). High-resolution computed tomography (HRCT) is the most updated optimized technique to acquire the most detailed lung images with a multidetector CT scanner (Verschakelen, 2010) (Figure 3). In addition to its traditional role in the diagnostic of diffuse parenchymal and interstitial lung diseases (DPILDs), CT techniques have been developed and utilized as non-invasive methods to measure regional ventilation in the lung for many years (Gur et al., 1979; Gur et al., 1981; Herbert et al., 1982; Snyder et al., 1984; Simon et al., 1998). Therefore, the significant potential of CT for developing new techniques has provided a magnificent opportunity to obtain not only detailed anatomical information on lung structure and pulmonary pathological patterns, but also quantitative functional characterization of the lung during the progression of pulmonary diseases (Young et al., 2019).

In preclinical research, µCT has proven to be a very powerful tool for longitudinal assessments of lung structural changes in chronic lung disease models, however, its routine implementation lags behind. Although, there are great opportunities to extract imaging-derived biomarkers not only on lung disease burdens, but also on lung function based on its ability to provide four dimensional (4D) data from different phases of the breathing cycle. Dynamic imaging of the lung using µCT to obtain 4D datasets is possible either with prospective synchronization (signal-based gating), triggering the image acquisition at certain phases of the respiratory cycle during scanning, or retrospective synchronization (image-based gating), sorting the acquired images according to their respiratory phases as a post-processing step (Liu et al., 2017). 4D-µCT of the lung using synchronization thereby not only reduces motion artifacts induced by cardiac and respiratory cycles, but also provides opportunities to extract end-inspiratory and end-expiratory images of the lungs, that can then be used to calculate (regional) tidal expansion (Ding et al., 2010; Vinogradskiy et al., 2012; Brennan et al., 2015). Using density measurements, several µCT-derived biomarkers such as mean lung density, total lung volume, areated and non-areated lung volumes have been introduced to longitudinally investigate the onset and progression of pulmonary diseases such as lung fibrosis, invasive pulmonary aspergillosis, and pulmonary cryptococcosis (De Langhe et al., 2012; Vande Velde et al., 2016; Dekoster et al., 2020). Furthermore, the distribution of air volume inside the lung can be obtained at several positive end-expiratory pressures (PEEPs) to plot a pressure-volume (P-V) curve, which can describe the static mechanical P-V relationship of the respiratory system with the assumption that the alveolar pressures are equilibrated (Marcucci et al., 2001). Calculating the distribution of regional air content and lung volume using density-based techniques has been employed by several researchers in a variety of applications to study topics such as post-pneumonectomy lung growth, special species adaptations, and respiratory distress syndrome (Hoffman and Ritman, 1985; Gattinoni et al., 1993; Olson and Hoffman, 1994; Gattinoni et al., 1995). The following sections investigate the x-ray-based techniques developed for lung function measurements in the recent years for preclinical lung research in more detail.

Application of Xenon (Xe) gas as a contrast agent for CT-based regional ventilation measurements as a non-invasive procedure for evaluation of pulmonary function dates back to 1979, when Gur et al. used ventilation rate constants to discuss the pulmonary function of normal and impaired lungs (Gur et al., 1979). Since the density of Xe is higher than air, in the presence of this gas in the airways, the density of those areas in the CT images linearly increases with the Xe concentration; therefore, by serial scanning of the same ROI in the lung during wash-in and wash-out of Xe, regional ventilation can be mapped by density measurement techniques (Simon et al., 1998; Simon, 2005). Xe-CT ventilation imaging has been employed in many lung studies to non-invasively measure regional distributions of ventilation, perfusion, and ventilation/perfusion (V/Q) ratio (Figure 4) (Hoffman et al., 1995; Simon et al., 1998; Tajik et al., 1998; Marcucci et al., 2001; Jones et al., 2003; Sauter et al., 2019). However, despite many advantages such as providing high-resolution, regional ventilation maps non-invasively, Xe-CT method includes also several limitations. As an anesthetic gas, the concentartion of Xe cannot exceed 30%–35% for ventilation measurements in humans due to the side effects, which consequently limits the maximum CT density enhancement that can be acquired (Simon, 2000). Furthermore, since Xe is soluble in blood, the maximum alveolar concentration of this gas reduces during washing; while, most of the employed models for Xe-CT ventilation measurements consider no uptake and recirculation of Xe. In addition, due to the higher density and viscosity of Xe compared to air, the regional distribution of ventilation can be different from normal respiratory gases, especially at higher inspiratory flow rates (Simon, 2005).

X-ray body plethysmography was another attempt to cover the limitations of UWBP for accurate measurements of tidal and end-expiratory volumes using single projection x-ray imaging, with the purpose of assessing airway resistance in conscious, spontaneously breathing mice (Lai-Fook et al., 2008). The proposed plethysmograph by Lai-Fook et al. included a transparent plastic tube with a cone-shaped end which was connected to a thin-walled copper cylinder. The x-ray source and sensor were located in the plastic tube to acquire single projection images of the animal’s thorax using a single x-ray pulse of 10 ms exposure time to minimize image blur due to respiratory and cardiac cycles. The plethysmograph also included a heat lamp in the transparent plastic tube and a pressure transducer, thermistor, and humidity gauge in the thin-wall copper cylinder for controlling the air condition inside the chamber and measuring the pressure. Since evaluation airway resistance requires both tidal and end-expiratory volumes, single projection x-ray images and pressure oscillations inside the chamber were used to estimate these lung volumes in spontaneously breathing mice. Pressure oscillations inside the plethysmography chamber were assumed as sinusoidal variations. The biggest advantage of x-ray body plethysmography was the capability of measuring lung volumes in a way that allows separate estimations of airway resistance and compliance; however, the high cost of the x-ray system, slow collection of x-ray images throughout a breathing cycle, and the manual segmentation of the images, halted the way of seeing this system as an efficient screening tool (Bates et al., 2008; Reynolds and Frazer, 2011).

In further exploration for finding a non-invasive method for lung function measurements and address drawbacks of plethysmography techniques, Dullin et al. (2016) introduced an imaging-based technique called x-ray lung function (XLF) method. XLF is a non-invasive technique for lung function measurements that employs low-dose planar cinematic x-ray imaging to monitor the animal’s breathing during the measurements. In this in vivo approach for lung function measurements, a video of 2D radiographs of the chest movements during spontaneously breathing of the unrestrained, anesthetized animal was captured to measure the average x-ray transmission of the lungs in each frame of the recorded video. The intensity fluctuations of the animal’s chest movements normalized by the average background signal in the acquired 2D movie were described using an x-ray transmission function (XTF) over time. After filtering, the breathing cycles of the XTF for each animal were parameterized using a third-order polynomial and the average of each parameter ( b 1 - b 5 ) over all of the breathing cycles were used to characterize the lung function by the ratio between inhalation and exhalation times as well as the maximum air content in the lung. Applying the XLF technique to an ovalbumin-induced experimental allergic airway disease mouse model mimicking severe acute asthma (SAA), Dullin et al. showed a significantly higher sensitivity for XLF in detecting the elasticity reduction of the lungs in comparison to UWBP (Dullin et al., 2016). XLF using the XTF parameters ( b 1 - b 5 ), showed shorter relative inspiration periods and reduced air flows in the lungs, which both pointed to a reduction in elasticity related to the inflammation in SAA. UWBP using parameters such as MaxSlope and MinSlope showed the same trend in air flow reduction for SAA mice compared to the controls. Furthermore, the authors also assessed the efficacy of dexamethasone as the common treatment of SAA (Figure 5), and correlated the results with UWBP, and further with post-mortem histology, broncho-alveolar lavage (BAL), and synchrotron phase contrast CT. Similarly, in comparison to UWBP, XLF with higher accuracy showed the improvement of lung function parameters in the treated mice with dexamethasone.

For further evaluation on reliability and sensitivity of XLF technique, the same research group used XLF and propagation synchrotron phase-contrast computed tomography (pSRμCT) for quantification of lung remodeling in an allergic airway inflammation (AAI) mouse model (Markus et al., 2017). In the lung function measurements using XLF, the breathing frequency during acquisition was adjusted to one breathing cycle in 1,400 msec using the level of anesthesia and overall for each mouse 21 breathing cycles were recorded. Using the same parameterization technique explained above, XTF parameters ( b 1 - b 5 ) were used to characterize the lung function of each animal. After each lung function measurement, the mice were euthanized for in situ lung imaging by synchrotron pSRμCT, to assess whether the XLF findings in the recovered mice are associated with subtle structural changes. Based on the pSRμCT results, the authors showed the persistence of airway remodeling after the resolution of the inflammatory response. In addition, they found a high degree of correlation between the pSRμCT volume ratio and XLF results, which showed a significant air trapping in AAI mice in comparison to controls, most probably due to the reduction of elasticity in the lungs induced by allergic exposure. The authors concluded that the persistent loss of lung elasticity in AAI mice even after a few months’ recovery can be related to the loss of elastic fibers. The results of this study once again confirmed the reliability and sensitivity of XLF as a non-invasive, in vivo technique for longitudinal lung function measurements in AAI mouse models.

Since none of the functional parameters ( b 1 - b 5 ) of XLF described pulmonary air volume, Dullin’s research group developed a unique experimental setup to simultaneously perform either XLF or µCT with WBP (Khan et al., 2021). They replaced the animal’s bed inside the gantry of the µCT scanner with a custom-made plethysmography chamber, which included a differential pressure sensor and isoflurane inlet-outlet for anesthesia (Figure 6). The original approach in XLF for calibration of x-ray transmission over time included a background selection for normalization, which required a large field of view (FOV) that led to low image resolution (Dullin et al., 2016). In this study, the XTF function was modified by applying a new approach for background correction, an adaptive moving filter, which provided the possibility of using a smaller FOV during the XLF measurements and consequently a better image resolution at the lung region (Khan et al., 2021). The relative x-ray transmission function (rXTF) was used for analysis of the breathing cycles and quantitative functional parameters such as end-inspiration lung volume (EIV), the relative x-ray transmission at end-expiration, as well as the decay rate of the expiration phase. The results showed a strong correlation between the acquired lung volume by rXTF function in XLF technique and those extracted from the µCT data; however, the XLF data were obtained by only 7% of the x-ray dose and 13% of the acquisition time used in µCT, which shows the capability of XLF as a reliable technique for in vivo, non-invasive lung volume measurements in longitudinal studies on lung diseases.

Since 2D radiographs of the chest movements are used by XLF protocol, the acquired imaging data could only be used for monitoring the animal’s breathing during the measurements and no anatomical information could be obtained. To address this issue, Dullin et al. proposed a new technique based on XLF to quantify lung function in the raw data of retrospectively gated lung µCT scans, which is called retrospective gating-based x-ray lung function measurement (rgXLF) (Dullin et al., 2022). For assessment of the newly developed technique, they applied the rgXLF protocol on mdx mice, the most commonly used mouse model for studying Duchenne muscular dystrophy (DMD). The authors employed the same strategy for parameterization of the breathing pattern that Khan et al. (Khan et al., 2021) used in their study. The comparison of functional parameters between XLF and rgXLF revealed a strong correlation, with almost the same k-values of the expiration phase and similar heart rates. In a comparison between control and mdx mice, both cross sections and 3D lung reconstruction showed the differences in the shape diaphragm between the mice (Figure 7). Therefore, rgXLF with a low x-ray dose, short acquisition time, and minimum voxel size of 40 µm showed the ability for longitudinal lung function measurements and also providing anatomical information without the requirement for additional scanning.

The continuous search for finding new imaging-based techniques to obtain regional lung function and biomechanics data led to employing synchrotron radiation sources to produce highly coherent, high flux x-rays that are required for phase contrast x-ray imaging (PCXI) [see (Bayat et al., 2001), (Bayat et al., 2009), (Porra et al., 2004), (Monfraix et al., 2004), (Bayat et al., 2022a), (Cercos-Pita et al., 2022), (Bayat et al., 2022b)]. PCXI is a high-resolution imaging technique, capable of differentiating between soft tissues by enhancing the contrast of biological interfaces and also providing dynamic motions of lung tissue. Recently, Bayat et al. throughly reviewed the present methods for synchrotron radiation-based imaging that have been used for regional lung function measurements [see (Bayat et al., 2020)]. The synchrotron radiation-based imaging methods included free propagation-based phase-contrast lung imaging (PBI), speckle-based lung imaging, 4D lung imaging, and K-edge subtraction (KES) imaging as well as their applications in preclinical animal models.

Four-dimensional x-ray velocimetry (4DxV) is a PCXI-based technique that can capture the expansion/contraction of lung tissue throughout a breathing process and also measure the airflow inside the airways. Therefore, any regional structural change of lung parenchyma and alteration of airflow inside the airways due to obstructive/restrictive lung diseases can be detected using this technique (Dubsky et al., 2010; Dubsky et al., 2012; Fouras et al., 2012). Since most of the 4DxV techniques were developed and validated in synchrotron radiation facilities, a more compact and accessible experimental setup was required to make 4DxV mapping more commonly-used in lung research laboratories (Tuohimaa et al., 2007; Bravin et al., 2012; Krenkel et al., 2016). In 2020, Murrie et al. introduced a dynamic in vivo 4DxV imaging system using a liquid-metal-jet microfocus X-ray source for regional lung function measurements in β-ENaC mice, a mouse model of cystic fibrosis (CF) (Figure 8) (Murrie et al., 2020). Mice were anesthetized, intubated, and ventilated during scanning. The results of 4DxV analysis, the expiratory time constant, showed a dramatic decrease in regional lung expansion of the left lung for β-ENaC mice in comparison to healthy controls, which correlated directly to the reduction of aeration due to the patchy CF-like airway obstructions in this region. This reduction of aeration directly indicates the regional reduction of lung function for the β-ENaC mouse, which can be considered as a biomarker for early detection of an obstructive airway disease (Figure 9). The proposed 4DxV imaging system is capable of regional imaging of lungs and airways with 60 µm resolution d 30 frames per second for pulmonary functional imaging and obtaining 3D ventilation maps. However, ionizing radiation measurements showed 1.47–1.74 Gy radiation dose delivered to each animal during image acquisition, which was below the lethal radiation dose to damage lung tissue (7.5 Gy for BALB/C mice and 8.3 Gy for C57BL/6 mice (Okunieff et al., 1996)), but still high enough to be considered as the endpoint for the animal.

Search for finding a simpler way than WBP and XLF to monitor respiratory dynamics, resulted in introduction of an optical technique by Svetlove et al. called optical respiratory dynamics tracking (ORDT) (Svetlove et al., 2022). This technique is especially useful for tracking diaphragm function of mice with neuromuscular diseases such as Duchenne muscular dystrophy (DMD). ORDT is a simple method for monitoring respiratory dynamics of anesthetized mice using camera tracing of chest surface markers. For developing this optical technique, the authors utilized a camera with the ability to produce images with 600 × 400 pixels resolution for 10 s at 100 frames/sec. They placed four paper markers with black cross-hair pattern on the thoracic-abdominal region of the mice with double-sided tape and tracked the movements of these markers in the acquired video with Linear Assignment Problem algorithms. The expiration constant was computed in XLF software. To assess the performance of ORDT technique, the authors used mdx mouse model to investigate the irregularities in the breathing pattern of the mice due to respiratory muscle weakness, which is one of the common characteristics of DMD. In comparison with the data acquired by XLF, the results obtained by ORDT showed significantly steeper expiration for mdx mice compared to the controls by calculating the expiration constant (k), which most probably shows the higher sensitivity of ORDT compared to XLF in capturing the change of respiratory dynamics in dmx mice. Furthermore, unlike XLF technique, ORDT was also able to show the differences between fast and slow expiratory phases in mdx mice, while healthy controls had almost the same fast and slow phases. Compared to the alternative methods for longitudinal assessment of diaphragm function in mice, e. i. WBP and XLF, ORTD is easier to perform, completely non-invasive (no ionizing radiation), cheaper, and can be performed by commonly available tools and equipment (Svetlove et al., 2022). Furthermore, since this optical technique directly assess the dynamics of the body surface, it has a greater potential in detecting abnormal breathing patterns.

In the past decades, alongside CT and nuclear medicine, magnetic resonance imaging (MRI) has been employed to evaluate chronic lung diseases in terms of gas exchange (Ohno et al., 2022). Pulmonary functional imaging with MRI includes measurements of ventilation, perfusion, as well as respiratory motion and mechanics (Wielpütz and Kauczor, 2012), which dates back to 1990s when hyperpolarized (HP) noble gas MR imaging and oxygen-enhanced MRI were introduced for the first time (Albert et al., 1994; Edelman et al., 1996; Kauczor et al., 1998).

Due to the abundance of hydrogen atoms in soft tissues (water and fat protons H 1 ) which contain polar nuclei, MRI of lung tissues results in high-quality images. However, in an inflated lung that approximately 80% filled with air, MRI is challenging due to low proton density and abundant air-tissue interfaces, which reduce signal-to-noise ratio and increase magnetic susceptibility effects, resulting in very low inherent signal that is available for lung imaging (Velde et al., 2014; Dubsky and Fouras, 2015; Kumar et al., 2016). Inhalation of HP noble gases by improving the MRI signal overcomes this issue (Dubsky and Fouras, 2015). Contrast agents such as helium-3 ( H e 3 ) and xenon-129 ( X e 129 ) are the most commonly-used non-radioactive noble gases for HP MRI (Ohno et al., 2022), which have been studied extensively for evaluation of diseases burden and efficacy assessment of therapeutics (Ruppert et al., 2000; Altes et al., 2001; Fain et al., 2006; Fain et al., 2007; Kirby et al., 2012; Chang, 2013). Physical methods of polarization such as spin-exchange optical pumping (SEOP) and metastability exchange (ME) can increase the polarization up to 4-5 order of magnitude above the thermal equilibrium which compensates for the low density of the inhaled noble gas inside the lung (Fain et al., 2010; Adamson et al., 2017). SEOP can be used for polarization of both H e 3 and X e 129 ; however, ME can be employed only for H e 3 applications (Adamson et al., 2017).

The biggest advantage of HP H e 3 MRI is the visualization of areas in the lung which are actively involved in ventilation such as terminal respiratory bronchioles and adjacent alveoli (Santyr et al., 2009; Fain et al., 2010). H e 3 , with low solubility and a high diffusion coefficient, is the most commonly-used HP noble gas for pulmonary functional imaging due to its large gyromagnetic ratio which offers the strongest signal (Santyr et al., 2009). Due to this unique ability, HP H e 3 MRI has been used to study many animal models of lung diseases such as asthma, pulmonary fibrosis and emphysema (Holmes et al., 2005; Mata et al., 2007; Thomas et al., 2009; Stephen et al., 2010). For early detection of bleomycin-induced pulmonary fibrosis progression in Sprague-Dawley rats, Stephen et al. employed HP H e 3 MR imaging and pulmonary function testing by a plethysmography chamber for validation purposes (Stephen et al., 2010). To evaluate lung function and structure of the animals, they used apparent diffusion coefficient (ADC) and fractional ventilation as the most commonly-used metrics. While PFTs showed no significant differences between the treatment groups, fractional ventilation and the ADC value in small airways and alveoli declined for the fibrotic rats, suggesting that the metrics of H e 3 MRI are more sensitive measures to monitor the progression of pulmonary fibrosis in animal models compared to the parameters of lung function tests. Despite showing the feasibility of measuring the ventilation changes in fibrotic rats, this method had limited success most probably due to the fact that ventilation is an indirect measure of the severity of pulmonary fibrosis (Adamson et al., 2017). In the case of emphysema, it has been shown that H e 3 ADC is sensitive to alveolar damage due to emphysema in both humans (Salerno et al., 2002; Swift et al., 2005) and animal models (Woods et al., 2004; Jacob et al., 2008; Rodríguez et al., 2009). Also, Xu et al. hypothesized and proved that the transverse diffusion coefficient ( D T ) of H e 3 apparent diffusion, measured at sub-milli-second diffusion times, is a more sensitive metric for detecting the alveolar damage in the elastase-instillation model of emphysema in rats compared to the longitudinal diffusion coefficient ( D L ) (Xu et al., 2012). Nevertheless, despite many advantages, due to the extremely limited quantity of H e 3 available worldwide, the cost of using this contrast agent for pulmonary functional imaging is very high which currently limited its application (Fain et al., 2010).

On the other hand, X e 129 as a by-product of the liquid air industry can be found in abundance which consequently leads to a relatively cheaper price compared to H e 3 (Santyr et al., 2009; Fain et al., 2010). In addition, compared to H e 3 , X e 129 has higher solubility in tissues and blood which results in a higher ability for quantitative modeling of gas exchange (Kaushik et al., 2013; Qing et al., 2014a; Qing et al., 2014b). To address this issue, due to a lower gyromagnetic ratio and more challenging polarization protocols compared to H e 3 , the application of HP X e 129 MRI lagged behind (Fain et al., 2010). However, recent protocols for HP X e 129 MRI employs a mixture of X e with X e 129 isotope enriched up to 85%, which improves the MR signal and solves the problem of low gyromagnetic ratio (Kirby and Parraga, 2013; Mugler and Altes, 2013). In recent years, HP X e 129 MR imaging has been employed in many lung disease studies with different animal models such as radiation-induced lung injury (RILI), allergic inflammation models of asthma, bleomycin models of pulmonary fibrosis, etc. (Cleveland et al., 2014; Doganay et al., 2016; Li et al., 2016; Lilburn et al., 2016; Sharma et al., 2017).

Despite the potential of pulmonary functional MRI (PfMRI) using inhaled hyperpolarized gases ( H e 3 ; X e 129 ), the development and application of PfMRI have been hampered due to technological challenges such as limited access to MRI and the requirement for multinuclear capabilities which consist of a dedicated X e 129 coil and the polarizer (Kooner et al., 2022). By addressing these technological challenges, further validation, and standardization of imaging protocols and analysis, PfMRI can play an important role in clinical care of many chronic lung diseases such as asthma and interstitial lung diseases to detect symptoms, guide therapy interventions and investigate treatment response (Kooner et al., 2022).