This work was funded by the German Center for Lung Research (DZL). The authors would like to express a deep gratitude towards all, who contributed and supported the further development of PREFUL, in particular: Marcel Gutberlet, Till F. Kaireit, Lea Behrendt, Julian Glandorf, Sonja L&#252;diger, Tawfik Moher Alsady, Katharina B&#252;nemann, Marius Wernz, Robin M&#252;ller, Maximilian Zubke, Gesa P&#246;hler, Agilo Kern, Cristian Crisosto, Milan Speth, Arnd Obert, Julienne Scheller, Jim Wild, Edwin van Beek, Helen Marshall, Jens Gottlieb, Martha Dohna, Diane Renz, Anna-Maria Dittrich, Tobias Welte, Jens Hohlfeld, Patrick Zardo, Giles Santyr, Franz Wolfgang Hirsch, Robert Grimm, Bastian Bier, Bassem Ismail, Andr&#233; Fischer, Berthold Kiefer, Gregor Thoermer and Rebecca Ramb. Furthermore, the authors would also like to thank the radiographers and study participants. In particular, we thank Frank Schr&#246;der and Sven Thiele from the Department of Radiology (Hannover Medical School) for outstanding technical assistance in performing the MRI examinations.

Advanced Lung Imaging for Disease Detection and Monitoring

Filip Klime&#353;, Andreas Voskrebenzev, and Jens Vogel-Claussen are shareholders of BioVisioneers GmbH, a company, which has an interest in pulmonary magnetic resonance imaging methods.

Critical steps 
One of the most common pitfalls during acquisition is inadequate signal scaling, which causes a loss of information during DICOM conversion by reduced precision of digital data representation. Consequently, this can lead to problems during the postprocessing stage. Another even more critical pitfall is the acquisition of multiple slices in an interleaved fashion. Thereby, the effective temporal resolution of the individual slices is critically reduced. Additionally, depending on the distance of the slices, this can have an impact on the perfusion contrast and quantification since the inflow relies on fresh spins without magnetization history. Special care is required during protocol setup, especially regarding gradient strength, asymmetric echo, bandwidth, and parallel imaging. Deviations from the suggested settings for even just one of these parameters can lead to inadequate TE and temporal resolution.
The postprocessing consists of multiple steps, which should be followed in the described order. For example, a registration after low-pass filtering is not meaningful. Consequently, failure at one step leads to a breakdown during the next steps. This makes the registration stage especially important. Since there is no single registration algorithm, depending on the respective implementation, parameters must be set empirically. Without finetuning of these parameters, a false registration will prevent the generation of any meaningful result. Another possibly time-consuming and critical step during postprocessing is segmentation. False segmentations can lead to completely wrong parameter calculations (e.g., by including non-lung regions) in the final report. Such mis-segmentations are more likely to occur with deep learning algorithms, which are accustomed to certain image appearances and are applied to images from another vendor/machine with a slightly different appearance. A visual quality check of segmentation accuracy, with potential manual correction, is therefore mandatory.
Troubleshooting 
The typical troubleshooting procedure is to follow all steps one by one and check the plausibility of the intermediate results. The procedure for the main steps is as follows: Check that the images are acquired in free breathing with the correct sequence and settings. Next, check that the dynamic range of the signals is appropriate (~50 AU in the lung parenchyma). If raw data are still available, repeat the reconstruction of the images with an appropriate scaling factor avoiding the need for a new acquisition of data. Check that the registration was performed without major artifacts and remaining motion. Next, check if small ROIs show a time series with expected ventilation- and perfusion-related modulations. Then, check if the applied filters alter the images in the expected manner (e.g., no high-frequency modulations in low-pass filtered data). Check if the synthesized respiratory and cardiac cycles are physiologic and don&#39;t show sudden jumps. Check the segmentation accuracy. Note that a search on a finer resolution level might be necessary as soon as the main step, during which the problem occurs, was identified.
Limitations 
Although the presented protocol is known to produce reproducible and sensitive results, the numbers of involved steps and parameters during acquisition and post-processing allow for nearly endless optimization and are intertwined. Therefore, a bottom-up approach should be followed by first addressing optimizations of the sequence protocol (e.g., regarding SNR and functional contrast-to-noise ratio). For the following postprocessing optimizations, a predefined ground truth in the form of a digital lung model might be useful40. As presented, this model mimics a free-breathing acquisition and includes several classes to simulate ventilation/perfusion defects. Including a known deformation due to movement, registration algorithms can also be tested directly. Despite these advantages, each model is inherently limited by the accuracy of mapping complex reality to a finite and simplified model.
The thresholds presented in this protocol were found to show reasonable results for healthy volunteers and across different patient cohorts by empirical analysis. Nevertheless, as outlined before, adjustment is likely required depending on the sequence, field strength, and cohort.
A general limitation of PREFUL is the extensive post-processing, which is not readily available as a medical product yet, although first work-in-progress versions from Siemens Healthineers and BioVisioneers are available for scientific purposes in a scientific collaboration/commercial setting. Calculations typically involve parallel processing, which poses especially high demands on CPU and RAM and might require modern workstations or server solutions to effectively process large amounts of data. Further, the time-consuming postprocessing steps currently impede an instant presentation of the results, which would be desirable for the clinical workflow.
Comparison to other methods 
There are a multitude of similar approaches like PREFUL, including the predecessor Fourier Decomposition and its other derivates such as Matrix Pencil Decomposition41 and the slightly different approach Self-gated Non-Contrast-enhanced Functional Lung MRI (SENCEFUL MRI)42. While Fourier Decomposition and similar methods operate in the frequency domain, PREFUL uses less strict Fourier-filtering and subsequent calculation of amplitudes in the time domain. Therefore, there is no requirement to select specific peaks corresponding to ventilation/perfusion. This can result in less susceptibility to respiration variability, which is known to occur in human subjects.
While PREFUL performs image sorting, SENCEFUL uses sorting of k-space lines, leading to more flexibility. Nevertheless, SENCEFUL requires sequences with self-gating capabilities, while PREFUL can be performed with a conventional spoiled gradient echo sequence. Similarly, bSSFP commonly used in Fourier Decomposition-based approaches is known for better SNR and blood flow contrast but typically requires more optimization for lung acquisition especially at 3T43. Nevertheless, other than that there is no reason to not combine PREFUL with bSSFP acquisition44.
All these signal-based approaches assume that certain unwanted signal influences, including T1, T2/T2*, diffusion, through-plane motion, and non-orthogonally perfused voxels, are negligible. While the progressed validation of PREFUL indirectly suggests that indeed such influences are not critical, Triphan et al. showed that there is a dependence on the effective T1 and TE, which is explained by the different weighting of the blood and parenchymal components depending on the TE45. In this light, the initial advantage of bSSFP to visualize blood due to T2/T1 contrast might pose an additional challenge to establish an accurate quantification in comparison to the simpler contrast mechanics of an SPGRE. Nevertheless, further studies that directly address the influence of various MR-variables, for example, as performed by Glandorf et al. for contrast media46,47, are desirable as they can directly quantify the effect on PREFUL.
Importance 
Being a free-breathing, contrast-media-free method, PREFUL shares many advantages with the previously mentioned related methods: 1) No ionizing radiation and contrast agent application, 2) No requirement for additional hardware or personnel, 3) acquisition, which depends only on minimal patient compliance. These advantages make PREFUL a convenient monitoring tool, especially for vulnerable groups such as children with chronic pulmonary disease. Although SNR is low with SPGRE sequence, the availability, and a lack of requirement for additional sequence programming/sharing further promote the dissemination of this approach.
As discussed in the introduction section, the number of studies showing good validation, reproducibility, sensitivity results, and monitoring capabilities show that the importance of this technique and corresponding dynamic parameters is on a rising trajectory and will be further supported by wide dissemination.

The respiratory system, with its intricate mechanisms, is vulnerable to various diseases. Prominently, chronic respiratory conditions such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and chronic thromboembolic pulmonary hypertension (CTEPH) considerably reduce life expectancy1. As a result, early diagnosis, monitoring, and therapeutic response assessment have become paramount.
Pulmonary function tests (PFTs) can derive global lung function parameters like the Tiffeneau-Pinelli index, defined as the ratio of the forced expiratory volume in one second (FEV1) and forced vital capacity (FVC)2. Such parameters are well established in the clinical routine but lack regional information and require a high level of patient compliance. In this regard, imaging can offer additional insights and possibilities for more sensitive parameters. Computed tomography (CT) offers high-resolution imaging of parenchymal morphology, and recent techniques like parametric-response mapping also retrieve functional information3. Nevertheless, single photon emission computed tomography (SPECT) remains the current gold standard for depicting ventilation and perfusion (V/Q) in the lung4. Common to all, the mentioned imaging modalities require exposure to ionizing radiation, which needs special consideration in cases of monitoring and vulnerable groups. Consequently, there is an ongoing effort to promote MRI as an alternative modality.
Inherently, the lung is a challenging organ for MRI due to its low proton density and fast signal decay5. Among the multitude of approaches, the most widespread solutions include the use of hyperpolarized gas (e.g., 129Xe MRI) for ventilation6 and intravenous gadolinium-based contrast agent application for perfusion depiction7. These methods offer a high signal-to-noise (SNR) ratio and are widely considered gold standard methods in the MR community. A more recent approach avoids the application of any contrast agent and is feasible with conventional proton MR in free breathing with a total acquisition time of ~1 min/slice. Thus, potential adverse events and recently debated long-term effects of contrast agents are avoided and easier dissemination without the requirement for additional hyperpolarization and multi-nuclear hardware is enabled. Additionally, the problem of finding an adequate inflation state, which can affect the derived ventilation defect values8 is avoided by the free-breathing acquisition.
This indirect MR signal-based approach was first introduced by Zapke et al. who utilized the reciprocal relationship of proton-weighted signal S and lung volume V: S~1/V.9 It is based on the process of transforming images acquired in free-breathing to one common inflation state (typically in an intermediate position between end-expiration and end-inspiration), thereby compensating for motion and allowing to analyze the signal time series in each voxel. Thereafter, a ventilation measurement can be derived from these so-called registered images by using equation (1) by Klime&#353; et al.10:
<img alt="Equation 1" src="/files/ftp_upload/66380/66380eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1)
With the volumes/signals in inspiration (Insp), expiration (Exp), and registered state (Reg). Thereafter, the method was expanded by introducing Fourier Decomposition to differentiate between signal modulations associated with breathing frequency (ventilation) and pulse frequency (perfusion) and therefore, derive a perfectly spatially matched V/Q map from one acquisition11. This is made possible by the typical gap between breathing and heart frequencies, so that both components which are on top of each other in the time domain are effectively discriminated in the frequency domain by Fourier analysis. After the transition from low-field (0.35T) to 1.5T with an optimized balanced steady-state free precession sequence (bSSFP)12, this method started to gain more attention with several follow-up studies13,14,15.
Since breathing and pulse are subject to variability and commercially available bSSFP (gradient compensated) imaging at 1.5T can result in substantial banding artifacts (clear lines of signal void), a related method was proposed with spoiled gradient echo sequence (SPGRE) in combination with broad low-pass and high-pass filtering16,17. This captures the more complex spectrum of real breathing- and pulse-related modulations. The following calculation of the amplitude in the time domain avoids the necessity to select one specific frequency peak. Further optimization was achieved by splitting the typical one-step registration towards one reference state into two separate steps. Thereby the fact that during free-breathing a range of different respiration phases are acquired between end-inspiration and end-expiration with varying degrees of required deformation towards a fixed state is utilized. After choosing several groups and identifying the group of the individual images the following procedure is performed: 1) Registration inside the respective respiration state group, 2) Step-by-Step inter-group registration from one adjacent group to the next (e.g., 1-&#62;2, 2-&#62;3,&#8230;) to the group representing the reference group. This approach was further expanded by phase estimation for each image to establish a higher apparent temporal resolution to facilitate the analysis of ventilation and perfusion dynamics, leading to the phase-resolved functional lung (PREFUL) MR terminology to differentiate this branch from other related techniques18. Follow-up studies made use of the additional information provided by the full respiration and cardiac cycles and showed potentially increased sensitivity of such parameters19,20,21.
Validation with the gold standard SPECT revealed a dice coefficient of &#8805;67% for defect regions22, and more direct ventilation measurement with 129Xe showed ventilation defect percentages correlation &#8805;62% in a mixed COPD/CF/healthy cohort23 and 84% in a CF multi-center, multi-vendor cohort24, which also demonstrated a similar correlation to lung clearance index of PREFUL and 129Xe (r = 0.82 and r = 0.91). The perfusion analysis of the same study showed that there were no significant differences in spatial overlap with DCE among the evaluated centers25. Concordance with DCE and agreement of PREFUL results between centers was also reported for a prospective sub-study including nine centers26. A reproducibility analysis in COPD patients resulted in a coefficient of variation below 15% for all parameters27. Current studies suggest that the FVL parameter has higher predictive power and sensitivity to detect treatment changes compared to the &#34;static&#34; ventilation parameter, which only takes the end-inspiratory and end-expiratory phases into account. Responsiveness to treatment with regional flow-volume loop (FVL) measurements was demonstrated after inhaler treatment with indacaterol-glycopyrronium (IND/GLY) in COPD28. In concordance, the FVL parameter predicted graft loss in double-lung transplant patients, whereas spirometry could not (P = .02 vs. P = 0.33)29. First feasibility studies show that functional pulmonary imaging with PREFUL can be realized in free-breathing infants and neonates with standard clinical MRI hardware30,31. Glandorf et al. compared PREFUL parameters at 1.5T and 3T (SPGRE sequence) and found no significant differences for most parameters, which were highly reproducible despite the difference in field strength32. This might be an important advantage, as not every site has access to 1.5T or lower field strength scanners. Recently, the feasibility and detection of persistent symptoms after COVID-19 infection at 0.55T was demonstrated by evaluating bSSFP data with PREFUL33.
In summary, despite being a relatively novel technique, PREFUL has been extensively studied. Important criteria such as validation with more direct and established measurements, reproducibility, sensitivity for pathology, and responsiveness for treatment and progression changes were assessed. Nevertheless, still, only a few specialized centers are using this technique despite the low technological requirements. Therefore, the aim of this work is to summarize the latest methodology of PREFUL MR in written and visual form. This information can be used to establish this technique in more centers and thus, in the long-term, lead to a more mature technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced Normalization Tools (ANTs)</td>
			<td>-</td>
			<td></td>
			<td>Image registration toolbox (https://stnava.github.io/ANTs/; https://github.com/fordanic/image-registration)</td>
		</tr>
		<tr>
			<td>Forsberg</td>
			<td>-</td>
			<td></td>
			<td>Image registration toolbox</td>
		</tr>
		<tr>
			<td>MRI</td>
			<td>Siemens Healthineers AG, Munich, Germany</td>
			<td></td>
			<td>0.55T / 1.5T / 3T Scanner</td>
		</tr>
		<tr>
			<td>PREFUL App</td>
			<td>BioVisioneers GmbH, Laatzen, Germany</td>
			<td></td>
			<td>PREFUL analysis, Figures and reports</td>
		</tr>
	</tbody>
</table>

phase-resolved functional lung mri for pulmonary ventilation and perfusion (v/q) assessment

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time series of each voxel is analyzed with regard to the cardiac and breathing frequency components.
Using a standard 2D spoiled gradient-echo sequence with a temporal resolution of ~300 ms, an image-sorting algorithm was developed to produce phase-resolved functional lung imaging (PREFUL) with an increased temporal resolution. Thus, it is feasible to evaluate regional flow volume loops (FVL) during tidal volume breathing and depict the propagation of the pulse wave during the cardiac cycle. This method can be applied at 1.5T or 3T with standard MR hardware without the necessity for sequence programming, as the described protocol can be implemented with the default SPGRE sequence on most systems.
PREFUL ventilation MRI has been validated using 129Xe and 19F gas imaging with good regional agreement. Perfusion-weighted PREFUL MRI has been validated using SPECT as well as dynamic contrast enhanced (DCE) MRI. PREFUL has been tested in a dual center dual vendor setting and is currently applied in several ongoing multicenter trials. Furthermore, it is feasible across a range of field strengths (0.55T-3T) and different age groups, including newborns.
Quantitative V/Q PREFUL MRI has been used in patients with cystic fibrosis, chronic obstructive pulmonary disease, chronic thromboembolic pulmonary hypertension, and corona virus disease-2019 to quantify disease and monitor treatment change after therapy. Furthermore, PREFUL V/Q imaging has been shown to predict transplant loss due to chronic lung allograft dysfunction in patients after lung transplantation. In summary, PREFUL MRI is a validated technique for quantitative ventilation and pulmonary pulse wave/perfusion imaging for regional pulmonary disease detection, quantification, and treatment monitoring with potential added value to the current clinical routine.

The lower part of Figure 2 illustrates the consequence of proper and inadequate scaling with a corresponding effect on the dynamic range. Figure 6 shows the inhomogeneous signal distribution, which is representative of scans without and with coil normalization. Avoiding a low dynamic range and images without coil normalization is recommended.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66380/66380fig06.jpg" /> 
Figure 6: Exemplary images after acquisition without coil correction (incorrect) and with coil correction (correct). Note the artificial signal enhancement at the body boundaries near the coil elements. <a href="https://www.jove.com/files/ftp_upload/66380/66380fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 7 illustrates successful and failed automatic segmentation. Note that the failed segmentation does not include all lung voxels, which will falsify further analysis and statistics. Special care is required for cases with infiltrates as such voxels can be misclassified as vessels due to their high signal by AI models or not segmented at all.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66380/66380fig07.jpg" /> 
Figure 7: An example of automated segmentation resulting in a failed (Subject A: 83-year-old male with COPD) and a successful result (Subject B: 30-year-old female healthy control). The first row shows the images, which were used as input for the AI models. The second row shows the results of the first segmentation stage consisting of finding the lung boundary. The third row shows the final result after the exclusion of vessels. As shown by the blue arrows, the algorithm was challenged by the high-signal lung variances causing wrong lung boundary detection. Note that the images were normalized by maximal signal, which led to different results due to missing coil normalization of the scan performed on subject A. Red regions show the ROIs, which were automatically detected for perfusion-phase sorting. Abbreviations: COPD = chronic obstructive pulmonary disease; ROIs = regions of interest. <a href="https://www.jove.com/files/ftp_upload/66380/66380fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 8 and Figure 9 show representative parameter maps for a healthy control (age 30, female) and a COPD patient (age 60, male). Note that the healthy control shows a more homogeneous ventilation and perfusion and thus, fewer defect voxels. The corresponding reports of the ROI statistics can be found in Table 4 and Table 5.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66380/66380fig08.jpg" /> 
Figure 8: PREFUL parameter maps of a 30-year-old female healthy control. The perfusion (1st row), regional ventilation (2nd row), flow-volume loop correlation metric (3rd row), and thresholded V/Q maps (4th row). Note the homogeneous distribution of the parenchymal values and low defect percentages. Abbreviations: PREFUL = phase-resolved functional lung; V = ventilation; Q = perfusion. <a href="https://www.jove.com/files/ftp_upload/66380/66380fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/66380/66380fig09.jpg" /> 
Figure 9: PREFUL parameter maps of a 60-year-old male COPD patient. The perfusion (1st row), regional ventilation (2nd row), flow-volume loop correlation metric (3rd row), and thresholded V/Q maps (4th row). Note the heterogeneous distribution of the parenchymal values and high defect percentages. Abbreviations: COPD = chronic obstructive pulmonary disease; PREFUL = phase-resolved functional lung; V = ventilation; Q = perfusion. <a href="https://www.jove.com/files/ftp_upload/66380/66380fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 4: Exemplary report of PREFUL parameters obtained for a healthy control (30-year-old female). Note the low coefficient-of-variation and defect values, which are in line with the parameter maps presented in Figure 8 for the same subject.&#160;See also Table 5 and Figure 9. Abbreviation: PREFUL = phase-resolved functional lung. <a href="https://www.jove.com/files/ftp_upload/66380/66380_Table_4_rev.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Exemplary report of PREFUL parameters obtained for a COPD patient (60-year-old male). Note the high coefficient-of-variation and defect values, which are in line with the parameter maps presented in Figure 9 for the same subject.&#160;See also Table 4 and Figure 8. <a href="https://www.jove.com/files/ftp_upload/66380/66380_Table_5_rev.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Material:&#160;Animated Explanation of the PREFUL Algorithm.&#160;<a href="https://www.jove.com/files/ftp_upload/66380/JOVE_All_Screen_Recordings.mp4" target="_blank">Please click here to download this file.</a>

Author Spotlight: Enhancing Diagnostic Strategies and Biomarker Development for Comprehensive Lung Function Analysis

Here, we describe the implementation of phase-resolved functional lung MRI as a contrast-agent-free proton MR technique for the assessment of pulmonary ventilation and perfusion dynamics. Validated and applicable across different field strengths and age groups, it could enhance clinical decision-making in the future by aiding in disease quantification and therapy monitoring.

Phase-Resolved Functional Lung MRI for Pulmonary Ventilation and Perfusion (V/Q) Assessment

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time ...

phase-resolved-functional-lung-mri-for-pulmonary-ventilation

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866 Views.  Hannover Medical School. Here, we describe the implementation of phase-resolved functional lung MRI as a contrast-agent-free proton MR technique for the assessment of pulmonary ventilation and perfusion dynamics. Validated and applicable across different field strengths and age groups, it could enhance clinical decision-making in the future by aiding in disease quantification and therapy monitoring.

Video: Author Spotlight: Enhancing Diagnostic Strategies and Biomarker Development for Comprehensive Lung Function Analysis

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased oxygen demand; otherwise, a diffusion limitation may occur. The increase in DLCO with exercise is due to increased capillary blood volume (Vc) and membrane diffusing capacity (Dm). Vc and Dm increase secondary to the recruitment and distension of pulmonary capillaries, increasing the surface area for gas exchange and decreasing pulmonary vascular resistance, thereby attenuating the increase in pulmonary arterial pressure. At the same time, the recruitment of intrapulmonary arteriovenous anastomoses (IPAVA) during exercise may contribute to gas exchange impairment and/or prevent large increases in pulmonary artery pressure.
We describe two techniques to evaluate pulmonary diffusion and circulation at rest and during exercise. The first technique uses multiple-fraction of inspired oxygen (FIO2) DLCO breath holds to determine Vc and Dm at rest and during exercise. Additionally, echocardiography with intravenous agitated saline contrast is used to assess IPAVAs recruitment.
Representative data showed that the DLCO, Vc, and Dm increased with exercise intensity. Echocardiographic data showed no IPAVA recruitment at rest, while contrast bubbles were seen in the left ventricle with exercise, suggesting exercise-induced IPAVA recruitment.
The evaluation of pulmonary capillary blood volume, membrane diffusing capacity, and IPAVA recruitment using echocardiographic methods is useful to characterize the ability of the lung vasculature to adapt to the stress of exercise in health as well as in diseased groups, such as those with pulmonary arterial hypertension and chronic obstructive pulmonary disease.

To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...

A Standardized Workflow for Hyperpolarized 129Xe MRI to Study Pulmonary Function

Acquiring Hyperpolarized 129Xe Magnetic Resonance Images of Lung Ventilation

Hyperpolarized 129Xe MRI comprises a unique array of structural and functional lung imaging techniques. Technique standardization across sites is increasingly important given the recent FDA approval of 129Xe as an MR contrast agent and as interest in 129Xe MRI increases among research and clinical institutions. Members of the 129Xe MRI Clinical Trials Consortium (Xe MRI CTC) have agreed upon best practices for each of the key aspects of the 129Xe MRI workflow, and these recommendations are summarized in a recent publication. This work provides practical information to develop an end-to-end workflow for collecting 129Xe MR images of lung ventilation according to the Xe MRI CTC recommendations. Preparation and administration of 129Xe for MR studies will be discussed and demonstrated, with specific topics including choice of appropriate gas volumes for entire studies and for individual MR scans, preparation&#160;and delivery of individual 129Xe doses, and best practices for monitoring subject safety and 129Xe tolerability during studies. Key MR technical considerations will also be covered, including pulse sequence types and optimized parameters, calibration of 129Xe flip angle and center frequency, and 129Xe MRI ventilation image analysis.

Author Spotlight: Standardization and Best Practices for Advancing Lung Imaging Using 129Xe MRI

Hyperpolarized 129Xe magnetic resonance imaging (MRI) is a method for studying regionally resolved aspects of pulmonary function. This work presents an end-to-end standardized workflow for hyperpolarized 129Xe MRI of lung ventilation, with specific attention to pulse sequence design, 129Xe dose preparation, scan workflow, and best practices for subject safety monitoring.