# A method for MRI-guided bronchoscopy to identify obstructed airway segments

D. G. Mummy, M. J. Mcintosh, K. J. Carey, S. Kehoe, Stéphane Esnault, M. W. Johansson, M. D. Evans, R. L. Sorkness, M. Schiebler, N. N. Jarjour, et al.

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## To cite this version:

D. G. Mummy, M. J. Mcintosh, K. J. Carey, S. Kehoe, Stéphane Esnault, et al. A method for MRI-guided bronchoscopy to identify obstructed airway segments. Physiological Reports, 2025, PHYSIOL. REP., 13 (4), pp.e70119. 10.14814/phy2.70119. hal-04996463

HAL Id: hal-04996463

https://lilloa.hal.science/hal-04996463v1

Submitted on 19 Mar 2025

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## METHODS ARTICLE

# A method for MRI-guided bronchoscopy to identify obstructed airway segments

David G. Mummy<fnref n="1" />

Marrissa J. McIntosh<fnref n="2" />

Katherine J. Carey<fnref n="3" />,<fnref n="4" />

Shannon Kehoe<fnref n="3" />

Stephane Esnault<fnref n="5" />,<fnref n="6" />

Mats W. Johansson<fnref n="5" />

Michael D. Evans<fnref n="7" />

Ronald L. Sorkness<fnref n="5" />

Mark Schiebler<fnref n="4" />

Nizar N. Jarjour<fnref n="5" />

Loren C. Denlinger<fnref n="5" /> ID

Sean B. Fain<fnref n="2" />,<fnref n="8" />

<figure><img src="image_5.png" /></figure>

### Correspondence

Sean B. Fain, Roy J. and Lucille A.
Carver College of Medicine, 200
Hawkins Drive, 3966 JPP, Iowa City, IA
52242, USA.

Email: [sean-fain@uiowa.edu](mailto:sean-fain@uiowa.edu)

### Funding information

HHS | National Institutes of Health
(NIH), Grant/Award Number:
HL080412, HL115118, HL126771,
HL146689 and HL169765; National
Center for Advancing Translational
Sciences (NCATS), Grant/Award
Number: UL1RR025011; William W.
and Judith H. Busse Professorship in
Allergy and Asthma Research

### Abstract

Bronchoscopy is not conventionally guided by prior knowledge of segmental airway obstruction. Hyperpolarized gas magnetic resonance imaging (MRI) ventilation abnormalities and computed tomography (CT) air trapping are related to lung function and asthma severity but have not been used to target segmental inflammation and remodeling. We evaluate the feasibility of using bronchoscopy guided by ³He MRI and CT to reveal differences in inflammatory response, morphology, and cellular activity in poorly- (defect) versus well-ventilated (control) lung regions. Eleven participants (5 female; age, 22.8 ± 3.4 years; 9 asthma) who experienced a cold with increased lower airway symptoms underwent ³He MRI and/or CT at least 6 weeks after recovery. Differences between defect and control regions were compared. In defect as compared to control sites, bronchoalveolar lavage neutrophils (p = 0.06) and granulocytes (p = 0.08) trended towards an increase; inflammatory mediators (i.e., 15-epi-LXA4, LXA4) were also significantly different (p < 0.05) between sites. Correlations were observed between macrophages, neutrophils, and eosinophils with inflammatory mediators (i.e., 15-epi-LXA4, LXA4, LTB4). Correlations were observed for macrophages and neutrophils with 15-epi-LXA4, and eosinophils with LXA4 and leukotriene B4. Basement membrane wall

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *Physiological Reports* published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.

<footnote n="1">¹Department of Radiology, Duke University Medical Center, Durham, North Carolina, USA</footnote>

<footnote n="2">²Department of Radiology, University of Iowa, Iowa City, Iowa, USA</footnote>

<footnote n="3">³Department of Medical Physics, University of Wisconsin—Madison, Madison, Wisconsin, USA</footnote>

<footnote n="4">⁴Department of Radiology, University of Wisconsin—Madison, Madison, Wisconsin, USA</footnote>

<footnote n="5">⁵Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, University of Wisconsin—Madison, Madison, Wisconsin, USA</footnote>

<footnote n="6">⁶Institute for Translational Research in Inflammation—U1286—INFINITE, University of Lille, Lille, France</footnote>

<footnote n="7">⁷Clinical and Translational Science Institute, University of Minnesota, Minneapolis, Minnesota, USA</footnote>

<footnote n="8">⁸Roy. J Carver Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA</footnote>
thickness was similar for defect versus control sites (p = 0.9). These results support the feasibility of image-guided methods to identify airway obstruction phenotypes.

KEYWORDS

asthma, bronchial wall biopsy, bronchoalveolar lavage, CT air trapping, hyperpolarized gas MRI

# 1 | INTRODUCTION

Investigative bronchoscopic sampling of fluid and tissue has long been used for the in vivo evaluation of cellular activity and airway morphology (Godard et al., 1982; Lozewicz et al., 1990), and has been shown to be well-tolerated in asthma patients (Moore et al., 2011). Bronchoalveolar lavage (BAL), histochemical (HC) and immunohistochemical (IHC) stains and morphological measures of biopsies obtained via bronchoscopy enable a range of specific measurements which are associated with pathological response (Kelly et al., 2017). However, this technique traditionally takes place without prior knowledge of the presence or absence of airway obstruction within the region undergoing sampling. Evaluating localized cellular and morphological phenomena associated with segmental airway obstruction may provide insights into the pathophysiology of chronic obstructive lung disease and aid in the development of targeted therapies.

Hyperpolarized (HP) gas magnetic resonance imaging (MRI) provides a means of directly visualizing gas distribution in the lungs (Kauczor et al., 1997; Moller et al., 2002) without the use of ionizing radiation. Defect extent is commonly measured as the ventilation defect percentage (VDP) (1), which, in asthma, is spatially heterogeneous (Aysola et al., 2010; Zha et al., 2018), related to computed tomography (CT) structural abnormalities (i.e., air trapping, airway wall thickness) (Carey et al., 2023; Fain et al., 2008; Svenningsen et al., 2018), and associated with and predictive of exacerbations (Mummy et al., 2020; Mummy, Kruger, et al., 2018); ventilation defects have previously been shown to be spatially and quantitatively reproducible over the short (same-day, 7-day) (Mathew et al., 2008; Niles et al., 2013) and long-term (7 years) (Eddy et al., 2019). Moreover, evidence of granulocytic inflammation is highly correlated with both global (Svenningsen et al., 2018) and lobar VDP (Fain et al., 2008).

The purpose of this work was to prospectively use HP ³He MRI to obtain paired bronchoscopy samples from ventilation defect and well-ventilated lung regions within the same subject in a pilot population of patients who had experienced a recent viral-induced exacerbation. Some of

these findings have been previously presented in the form of two abstracts (Mummy et al., 2015; Mummy, Kehoe, et al., 2018).

# 2 | MATERIALS AND METHODS

## 2.1 | Participants and study design

Participants aged 16–60 years provided written informed consent to the Viral-Induced Asthma Exacerbation (VIAX) (Denlinger et al., 2011) longitudinal study. This prospective study was compliant with the Health Insurance Portability and Accountability Act (HIPAA) and was approved by the University of Wisconsin Health Sciences Institutional Review Board (IRB H-2005-0070). Image-guided bronchoscopy was performed based on the presence of a defect after recovery from a virus-induced exacerbation with the intent of sampling regions at the leading edge of airway injury. Participants underwent ¹H (standard proton) and ³He MRI, CT, and spirometry at least 6 weeks post-viral exacerbation and were excluded if they reported other respiratory complications (i.e., current smoker or significant smoking history (exceeding 5 pack years), pulmonary edema, atelectasis, upper airway obstruction). Bronchoscopy was performed to obtain BAL and bronchial wall biopsy samples within 30 days following imaging.

## 2.2 | Image acquisition and analysis

Anatomical ¹H and HP ³He MRI were acquired under breath-hold conditions by using a 1.5 Tesla Signa HDx scanner (GE Healthcare, Milwaukee WI). ³He was polarized using a Polarean IGI 9600 hyperpolarizer (Polarean Inc., Durham NC) and mixed with N₂ such that the total volume of ³He and N₂ was 14% of the participant's total lung capacity. The gas mixture was prepared and delivered in a Tedlar bag (Jensen Inert Products, Coral Springs FL). Participants were positioned supine with the gas dose inhaled from functional residual capacity.

Ventilation defects were located anatomically using the corresponding slice from a ¹H MRI and registered
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<figure><img src="image_7.png" /><figcaption>FIGURE 1 (a) 3D airway tree generated from CT (VIDA Diagnostics, Coralville, IA) superposed on axial hyperpolarized (HP) gas MR. An example path through the airway tree towards an area of ventilation defect ("target") is shown. (b) MR image-guided target defect located in basal region of the right middle lobe (arrowheads) and control region in the right lower lobe (arrows) for an MRI case. The case exemplified here is illustrative of the reduced, but not fully obstructed, ventilation regions identified in many participants. (c) MR image-guided target defect located in anterior left upper lobe (arrowhead) and control region in the right lower lobe (arrows) for an MRI case. This example is illustrative of the typical fully obstructed defect case. (d) CT image-guided target air trapping defect located in the left lower lobe near the heart (arrowhead) and control region in the right middle lobe (arrow).</figcaption></figure>

CT reference (Figure 1a). Defects were visually identified as regions of no or low signal on an axial HP ³He MRI. Lower but not complete absence of gas signal was still defined as a visual defect in some cases (Figure 1b,c). In a subset of participants where MRI was not available, defects were located by proxy on CT by identifying regions of hyperlucency, indicative of air trapping (Figure 1d). MR images were segmented and quantified using custom software tools implemented in MATLAB (MathWorks, Natick MA) as described previously (Zha et al., 2018). Whole lung VDP was calculated as the ventilation defect volume normalized to the total ¹H thoracic cavity volume.

Chest CT was performed at end expiration (functional residual capacity) on a 64 slice multi-detector scanner (VCT 64, GE Healthcare). Effective CT dose was adjusted to mitigate radiation exposure and maintain image quality (Sieren et al., 2016). Air trapping was quantified from CT images as the relative area less than −850 (RA$_{-850}$) Hounsfield units (HU).

## 2.3 | Bronchoscopy and analysis

BAL and endobronchial biopsy samples of the airway wall were obtained via image-guided bronchoscopy at both defect and control sites within the same or contralateral lung of a given participant. BAL samples were analyzed for differential cell counts. Oxylipin mediators contribute to the transition between pro-inflammatory and pro-resolution states (Tavares et al., 2023; Townsend et al., 2023) thus, oxylipin levels with and without

<table><caption>TABLE 1 Oxylipin mediators obtained from bronchoalveolar lavage fluid.</caption><thead><tr><td>Oxylipin mediator</td><td>Supplier/catalogue number</td></tr></thead><tbody><tr><td>LXA4</td><td>Neogen Corporation #407010</td></tr><tr><td>LTB4</td><td>Cayman Chemical #520111</td></tr><tr><td>15-epi-LXA4</td><td>Neogen Corporation #407110</td></tr><tr><td>CysLTs</td><td>Cayman Chemical #10009291 and #500390</td></tr></tbody></table>

Abbreviations: 15-epi, 15-epimer; CysLTs, cysteinyl leukotrienes; LTB4, leukotriene B4; LXA4, eicosanoid lipoxin A4.

normalization for total protein concentration in the BAL fluid were also measured. Markers of interest are shown in Table 1 and included lipoxin A4 (LXA4) and 15-epimer (15-epi) of LXA4 (inhibit neutrophilic infiltration and production of pro-inflammatory cytokines and chemokines (Bonnans et al., 2007; Takano et al., 1997)) and leukotriene B4 (induces recruitment and activation of granulocytes and monocytes (Crooks & Stockley, 1998)). Biopsy samples were embedded in paraffin and frozen before being sectioned and mounted on slides (Kelly et al., 2017). Biopsy samples were stained with H&E and reviewed by an expert pulmonary pathologist. A subset of participants was selected for further HC and IHC staining (markers described in Table 2) based on a visual evaluation by the pathologist of the quality of tissue biopsy presentation on H&E stains. Specifically, these samples were chosen based on the quality of the sample and a preference for sections cross-sectional to the airway.

Basement membrane thickness was measured on H&E stains using ImageJ software (https://imagej.nih.gov/ij/),
<table><thead><tr><td>Stain</td><td>Primarily indicates</td><td>Supplier/catalogue number</td></tr></thead><tbody><tr><td>CD4</td><td>Helper T cells</td><td>Ventana #790-4423</td></tr><tr><td>CD8</td><td>Cytotoxic T cells</td><td>Ventana #790-4460</td></tr><tr><td>CD10</td><td>Mature granulocytes</td><td>Ventana #790-4506</td></tr><tr><td>CD20</td><td>B-cells</td><td>Ventana #760-2531</td></tr><tr><td>CD68</td><td>Monocytes and macrophages</td><td>Ventana #790-2931</td></tr><tr><td>CD117</td><td>Mast cells</td><td>Abcam #Ab15580</td></tr><tr><td>CD138</td><td>Cell proliferation</td><td>Cell-Marque #760-4248</td></tr><tr><td>Ki-67</td><td>Cell proliferation</td><td>Abcam #Ab15580</td></tr><tr><td>Synaptophysin</td><td>Neuroendocrine cells</td><td>Ventana #790-4460</td></tr><tr><td>Trichrome (Masson's)</td><td>Connective tissue, nuclei and cytoplasm</td><td>Epredia Kit #87019</td></tr><tr><td>Alcian Blue/PAS</td><td>Mucus</td><td>Newcomer Supply #1003</td></tr></tbody></table>

Note: Secondary antibody Omni-Map anti-Rabbit HRP supplied by Ventana #760-4311 was used for all analyses, except for CD20, CD68 and CD138 which used Ultra-Map anti-Mouse HRP supplied by Ventana #760-4313.

Abbreviation: PAS, Periodic Acid-Schiff.

<figure><img src="image_8.png" /><figcaption>FIGURE 2 Basement membrane analysis. Basement membrane shown on H&E samples with epithelium (left) and without epithelium (center). Approximately 20 transverse membrane measurements (yellow) were averaged for each data point (right).</figcaption></figure>

as shown in Figure 2, by two imaging scientists (KJC and SK) under pathologist guidance. In each sample, approximately 20 measurements were obtained from areas both with and without epithelium, as available. The measurements were averaged to determine the mean wall thickness for that sample. Image-based quantitative analysis of HC and IHC stained sections was performed using Aperio ImageScope software (https://www.leicabiosystems.com/digital-pathology/manage/aperio-imagescope/). Regions of interest (ROI) of a standard size were defined on H&E stains for each sample. These ROIs were transferred to equivalent areas in HC/IHC stained slides from adjacent sections by the scientist performing image-based measurements (KJC and SK). Cell counts were then obtained and normalized to total ROI area.

## 2.4 Statistical analysis

Normality was evaluated using Shapiro-Wilk tests, which showed that data was not normally distributed. Differences in BAL fluid cell counts, oxylipin levels as well as basement membrane thickness in defect versus control sites were evaluated using Wilcoxon Signed Rank tests. Univariate relationships were evaluated using Spearman correlations. Observable differences in cell counts for each participant were defined as a factor of two increase or decrease between defect and control sites. Data are presented as the mean ± standard deviation unless otherwise specified. All statistical analysis was performed using SPSS (Version 29.0, IBM). *p* values less than 0.05 were considered statistically significant.
## 3 | RESULTS

The Consolidated Standards of Reporting Trials diagram in Figure 3 shows that of the 66 participants enrolled in the cold virus-induced exacerbation study as detailed previously (Denlinger et al., 2011), 11 (5 females, 6 males; 9 asthma; age, 22.8 ± 3.4 years) underwent image-guided bronchoscopy. Participants in this study were recruited based on having a recent exacerbation due to a detected rhinovirus (N = 7) or respiratory syncytial virus (N = 2) in the upper airways 6–8 week prior to HP gas MRI. Table 3 provides a summary of participant demographics, pulmonary function, and imaging measurements acquired just prior to image-guided bronchoscopy. Briefly, the mean forced expiratory volume in the first second (FEV₁) and FEV₁ to forced vital capacity ratio (FEV₁/FVC) were in the normal range, while whole lung VDP (Pike et al., 2015) and RA$_{-850}$ (Mets et al., 2012) measures were typical of those seen in mild asthma or healthy normal participants. Bronchoscopy was performed 11 ± 8 days after imaging (median [min-max] = 9 [2–29] days), which was also 73 ± 21 days since the onset of cold symptoms (median [min-max] = 71 [51–129] days). All 11 participants had BAL performed in both the defect and control segments and 8 had endobronchial biopsies collected. Biopsy samples from four of these participants met quality standards and also underwent HC and IHC analysis.

<figure><img src="image_9.png" /><figcaption>FIGURE 3 CONSORT Diagram. Of the 66 participants enrolled in the Viral-Induced Asthma Exacerbation (VIAX) study, a total of 11 participants underwent image-guided bronchoscopy for BAL and tissue biopsy. Biopsy samples from four participants also underwent extensive histochemical (HC) and immunohistochemical (IHC) analysis. Participants in dashed boxes were not included in this manuscript analysis.</figcaption></figure>

<table><caption>TABLE 3 Demographics, pulmonary function, and imaging measurements.</caption><thead><tr><th>Parameter</th><th>Cohort (n = 11)</th></tr></thead><tbody><tr><td>Age (years)</td><td>22.8 ± 3.4</td></tr><tr><td>Female Sex n (%)</td><td>5 (46)</td></tr><tr><td>Asthma n (%)</td><td>9 (82)</td></tr><tr><td>FEV<sub>1</sub> (%pred)</td><td>101 ± 16</td></tr><tr><td>FEV<sub>1</sub>/FVC (%pred)</td><td>97 ± 14</td></tr><tr><td>VDP (%)</td><td>1.1 ± 1.5</td></tr><tr><td>Defect site</td><td>1.1 ± 1.9</td></tr><tr><td>Control site</td><td>0.9 ± 1.6</td></tr><tr><td>RA<sub>-850</sub> (%)</td><td>1.6 ± 1.5</td></tr><tr><td>Defect site</td><td>4.0 ± 5.8</td></tr><tr><td>Control site</td><td>1.8 ± 1.8</td></tr></tbody></table>

Note: Data is reported as mean ± standard deviation unless indicated otherwise.

Abbreviations: %pred, percent of predicted value; FEV₁, forced expiratory volume in first second; FVC, forced vital capacity; RA$_{-850}$, relative area of the lung less than -850 Hounsfield Units; VDP, ventilation defect percentage.

### 3.1 | BAL results

Figure 4 shows differences in cell counts and oxylipin mediators from BAL fluid. Neutrophil (defect: 0.27 ± 0.18 × 10⁶ cells, control: 0.17 ± 0.19 × 10⁶ cells; *p* = 0.06) and total granulocyte counts (defect: 0.32 ± 0.22 × 10⁶ cells, control: 0.19 ± 0.19 × 10⁶ cells; *p* = 0.08) had a trend for a difference in defect as compared to control sites; eosinophil cell counts were not different between sites (*p* = 0.1). Figure 4 further shows that eicosanoid lipoxin A4 (LXA4) was significantly less (defect: 69 ± 14 µg/mL, control: 76 ± 14 µg/mL; *p* = 0.05) while both 15-epimer (15-epi) of LXA4 (defect: 141 ± 52 µg/mL, control: 111 ± 61 µg/mL; *p* = 0.04) and 15-epi-LXA4 normalized to the total protein concentration (defect: 1.4 ± 0.5, control: 0.9 ± 0.3; *p* = 0.02) were significantly greater in defect compared to control sites. No differences were observed for leukotriene B4 (chemotactic for neutrophils) or cysteinyl leukotrienes (pro-inflammatory oxylipins made by eosinophils) nor were there any differences noted for several pro-inflammatory cytokines in the BAL (inclusive of Eotaxin, IFNg, IL-13, IL-5, and MCP-1; data not shown).

Relationships between pro-resolving oxylipins and granulocyte counts are presented in Figure 5. We observed inverse correlations between average BAL eosinophil concentration with normalized LXA4 (*ρ* = -0.70, *p* = 0.04), and between the average normalized 15-epi-LXA4 with neutrophils (*ρ* = -0.80, *p* = 0.01) and macrophages (*ρ* = -0.77, *p* = 0.02); average eosinophils and normalized leukotriene B4 trended towards an association (*ρ* = -0.59, *p* = 0.09).
<figure><img src="image_10.png" /><figcaption>FIG URE 4 Bronchoalveolar Lavage (BAL) Cells and Lipid Proteins. Differential cell and lipid and protein levels from BAL samples in paired control versus defect sites. There were trends to increases in neutrophil (p = 0.06) and total granulocyte counts (p = 0.08) in regions of defect versus controls. Immunomodulatory lipid proteins, including LXA4 was reduced in defect versus control sites (p = 0.05), while both 15-epi-LXA4 (p = 0.01) and 15-epi-LXA4/totprot (p = 0.02) were significantly increased. 15-epi, 15-epimer; CysLTs, cysteinyl leukotrienes; Eos, eosinophils; Gran, total granulocytes; LTB4, leukotriene B4; LXA4, eicosanoid lipoxin A4; Mac, macrophages; Neut, neutrophils; totprot, normalized to total protein concentration.</figcaption></figure>

**3.2 | Biopsy findings**

Figure 6 shows CD4, Periodic Acid-Schiff (PAS), and Masson's Trichrome stains of paired defect-control biopsy samples in two participants. The CD4 stain shows a substantial increase in CD4 cells (stained brown) in defect versus control site. PAS shows increased mucin and goblet cell hyperplasia (blue and purple) in the defect site. Finally, Trichrome stain shows qualitative evidence of squamous metaplasia, loss of epithelial cilia, and collagen deposition (blue) characteristic of chronic airway injury and fibrosis. Basement membrane wall thickness was similar for tissue biopsies from defect versus control sites (6.97 ± 1.71 microns vs. 6.43 ± 2.54 microns, respectively; *p* = 0.9). Both participant 42 and 126 had evidence of elevated CD8, with participant 126 also demonstrating elevated CD4, CD10, and CD68, in defect as compared to control sites. There were no observable differences for participant 96 and 110 between defect and control sites for the cell marker stains evaluated.

**4 | DISCUSSION**

We performed a novel image-guided bronchoscopy approach using HP ³He MRI to sample sites of ventilation defect paired with well-ventilated control sites within the lungs of the same patient in a pilot cohort of patients who had recently experienced a viral-induced exacerbation. Prospectively identified areas of ventilation defects which are associated with airway inflammation and obstruction exhibited qualitative evidence of goblet cell hyperplasia and squamous metaplasia; trends suggesting greater numbers of neutrophils and granulocytes in defect regions; and significant differences in immune cell modifying proteins in defect regions compared to well-ventilated control regions. In some instances, *p*-values suggest mean differences that do not meet the traditional *p* < 0.05 threshold. These trends are maintained and reported to guide future work as our study is almost certainly underpowered due to the challenges of recruiting patients to imaging and bronchoscopy within
<figure><img src="image_11.png" /></figure>

**FIGURE 5** Relationships for average cell and lipid concentrations. Top left: BAL macrophage concentration inversely correlated with 15-epi-LXA4/totprot ($\rho$ = -0.77, *p* = 0.03). Top right: Neutrophils inversely correlated with 15-epi-LXA4/totprot ($\rho$ = -0.80, *p* = 0.01). Bottom left: Eosinophils inversely correlated with LXA4/totprot ($\rho$ = -0.70, *p* = 0.04). Bottom right: There was a trend to an inverse correlation between eosinophils and LTB4/totprot ($\rho$ = -0.59, *p* = 0.09). 15-epi, 15-epimer; Eos, eosinophils; Gran, total granulocytes; LTB4, leukotriene B4; LXA4, eicosanoid lipoxin A4; Mac, macrophages; Neut, neutrophils; totprot, normalized to total protein concentration.

<figure><img src="image_12.png" /></figure>

a window that is shortly after viral-induced asthma exacerbation. Future studies may benefit from further study of these trends.

Lipoxin protein levels including LXA4 and 15-epi-LXA4 were significantly different in defect versus control sites. Oxylipin mediators are derived from plasma cell membranes and contribute to the transition between pro-inflammatory and pro-resolution states (Tavares et al., 2023; Townsend et al., 2023). Lipoxins play an important role in the downregulation of inflammation, with 15-epi-LXA4 having been previously shown to facilitate the resolution of acute lung inflammation (Sekheri et al., 2020). In addition, the number of neutrophils and granulocytes trended towards an increase in defect regions. The presence of granulocytes in asthmatic airways is linked to increased disease severity and airway inflammation, and exacerbations (Fahy, 2009). Together, these findings are consistent with the immunomodulatory role of these proteins and strengthen earlier retrospective results linking ventilation defects with local inflammatory response (Fain et al., 2008).

In a subset of 4 participants, we conducted extensive analysis of a suite of HC and IHC markers of inflammation, injury, and cellular proliferation. In one such participant, PAS staining exhibited a visually striking increase in mucin and goblet cell hyperplasia in the defect region that

included quantitative increases in CD4, CD8, CD10, and CD68 staining in the same biopsy sample. The presence of CD4 or CD8 cells in bronchial wall biopsies of asthma patients has been linked to a longitudinal loss of lung function measured via spirometry (den Otter et al., 2016). Another participant demonstrated an obviously stratified epithelial layer with squamous metaplasia and a marked increase in collagen in the tissue deep to the basement membrane in the tissue biopsy from the defect site, alterations which are characteristic of chronic airway injury and repair. Interestingly, this participant showed no observable differences in quantitative stains for cellular activity from the same site, which suggests that the observed pathophysiology in this participant may be the result of past chronic airway injury and inflammation.

The difference in inflammatory cell counts and mediators between defect and control sites as well as evidence of abnormal pathology within defect regions demonstrate the utility of functional image-guided bronchoscopy, suggesting the potential for image-guided treatment interventions. In fact, asthma patients treated using image-guided bronchial thermoplasty using HP gas MRI (Hall et al., 2020; Svenningsen et al., 2021) have similar improvements in quality of life as compared to patients treated using standard bronchial thermoplasty protocols, but significantly reduced number of radiofrequency
<figure><img src="image_13.png" /><figcaption>**FIGURE 6** Paired Biopsy Samples from ventilation defect site (left) and control site (right) in the same participant. Top row: Samples from participant 126 (mild asthma) stained for CD4, which is preponderant in defect versus control samples. Middle row: Samples from participant 126 stained with Alcian Blue—Periodic Acid Schiff (PAS). Mucin appears pink and purple. Note increase in mucin and goblet cell hyperplasia in defect versus controls samples. Bottom row: Samples from participant 96 (no prior lung disease) stained with trichrome. Note stratified epithelium with squamous metaplasia and highlighting of collagen (blue), in defect versus control samples. Original magnification ×20 on all images.</figcaption></figure>

activations. Future clinical practice which combines the ability to identify and localize abnormally functioning airway segments and evaluate the underlying pathology to treat with phenotype-specific regimens and image-guided interventions could result in improved outcomes in asthma patients.

Important limitations to this work include the small sample size, especially in the N=4 subset of participants in which HC/IHC staining was performed. Histology sections were further limited in sample size in this study given the airway epithelium integrity was not intact in most of the biopsy samples acquired. Moreover, such qualitative differences are also prone to sampling bias within a given tissue section. Although the generalizability of these findings is limited, there is still value in comparing paired defect and control sites for sections where control and defect epithelium are both intact as shown in Figure 6.

More robust methods of biopsy tissue sampling and handling are currently being used for ongoing studies in severe asthma. In addition, HP ³He MRI was not acquired in three participants and thus, regions of CT air trapping were chosen as defect sites. Previous work, however, has demonstrated a spatial relationship between MRI ventilation defects and CT air trapping (Carey et al., 2023; Fain et al., 2008), hence, regions of air trapping identified using CT are likely a good estimate and surrogate for ³He MRI ventilation defect regions. Hyperpolarized MRI studies in the lungs have also largely replaced ³He gas with ¹²⁹Xe gas, which has slightly different volumes of distribution that could have small but measurable effects on airway defect size (Stewart et al., 2018). Finally, as MRI and CT air trapping were acquired only following an exacerbation, it is difficult to discern whether the ventilation defects selected for image-guided bronchoscopy in these patients
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

were related to active airway inflammation in response to the viral-induced exacerbation or whether they were the consequence of prior airway injury. We would also like to note that this study was performed before CT-image guided bronchoscopy was implemented into routine clinical practice.

## 4.1 | Conclusion

In this study, we have demonstrated the feasibility and utility of bronchoscopy guided by functional imaging. In 11 participants who had recently experienced a virus-induced exacerbation, lung regions with abnormal airway function identified via HP gas MRI exhibited an increased presence of inflammatory cells and mediators as compared to well-functioning lung regions. Results in this pilot population suggest that ventilation patterns observed via HP gas MRI provide a valuable tool for the identification and evaluation of localized airway phenomena associated with regional reductions in lung function. Advancing the understanding of airway activity specifically associated with localized regions of airway obstruction in future investigations may help refine the definition of asthma phenotypes and guide the development and evaluation of personalized, targeted therapies.

### AUTHOR CONTRIBUTIONS

DGM was responsible for developing the image guidance workflow, image processing, analysis, and interpretation and for preparing the first draft of the manuscript. KJC was responsible for data collection, image analysis and pathology interpretation. MJM was responsible for statistical analysis, interpretation of the results and writing and editing the manuscript. SK was responsible for data collection and biopsy sample analysis. SE was responsible for developing methodology and recommended cell-marker stains for tissue pathology, and for processing and analysis of BAL lipid protein analysis. MWJ was responsible for developing methodology and recommended cell-marker stains for tissue pathology, and interpretation of the results. MDE was responsible for statistical analysis and design. RLS was responsible for developing the study design and interpretation of the results. MS was responsible for interpretation of the results. NNJ was responsible for developing the study design, supervising the bronchoscopy protocol and interpretation of the results. LCD was responsible for developing the study design, performing bronchoscopy procedures and interpretation of the results. SBF was responsible for developing the study design, interpretation of the results, and direction of all aspects of the study,

and writing and editing the manuscript. All authors had an opportunity to review and revise the manuscript and approved its final submitted version.

### ACKNOWLEDGMENTS

The authors thank Dr. Scott Aesif (Cleveland Clinic, OH) for pathology collection, processing and interpretation.

### FUNDING INFORMATION

This study was funded by National Heart, Lung, and Blood Institute (NHLBI) grant R01 HL080412 (Jarjour) with institutional resources funded by National Center for Advancing Translational Sciences (NCATS) grant 1 UL1RR025011. Secondary analyses were supported by R01 HL115118 (Denlinger) with effort contributed by The William W. and Judith H. Busse Professorship in Allergy and Asthma Research. SBF is supported by the National Institutes of Health (NIH) R01 HL126771, R01 HL146689, and R01 HL169765.

### CONFLICT OF INTEREST STATEMENT

DGM and SBF serve as scientific advisors for Polarean LLC. SBF also receives research funding from Polarean LLC, Siemens Healthineers INC, and GE Healthcare INC for development of pulmonary CT and MRI methods. MWJ has received research funding from Hoffmann-La Roche (unrelated to this work). LCD reports consultation with GlaxoSmithKline, AstraZeneca, Sanofi, and OM Pharma during the course of this study. MJM, KJC, SK, SE, MDE, RLS, MS and NNJ have no disclosures.

### DATA AVAILABILITY STATEMENT

Data measures and analysis results can be made available by the corresponding author by request.

### ETHICS STATEMENT

This human study was approved by University of Wisconsin Health Sciences Institutional Review Board - approval: (IRB H-2005-0070). All participants provided written informed consent to participate in this study.

### ORCID

Loren C. Denlinger https://orcid.org/0009-0008-1299-1186

Sean B. Fain https://orcid.org/0000-0001-5461-0646

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**How to cite this article:** Mummy, D. G., McIntosh, M. J., Carey, K. J., Kehoe, S., Esnault, S., Johansson, M. W., Evans, M. D., Sorkness, R. L., Schiebler, M., Jarjour, N. N., Denlinger, L. C., & Fain, S. B. (2025). A method for MRI-guided bronchoscopy to identify obstructed airway segments. *Physiological Reports, 13*, e70119. https://doi.org/10.14814/phy2.70119