Synopsis

Hyperpolarized ¹²⁹Xe diffusion morphometry allows the microstructural dimensions of the lungs to be measured noninvasively, providing a means to quantify normal alveolar growth and disease progression in disorders such as emphysema. In the preclinical setting, ¹²⁹Xe diffusion morphometry holds the potential to provide insights into fundamental pulmonary biology and to assess the efficacy of potential therapies. However, to have the greatest impact on pulmonary biomedicine, this approach must be applied reliably to mouse models, where tools to investigate disease mechanisms are most highly developed. Here, we demonstrate the feasibility of high-resolution, ¹²⁹Xe diffusion morphometry in living mice.

Abstract

Introduction: Hyperpolarized (HP) ¹²⁹Xe MRI has emerged a powerful tool to quantify regional lung function (e.g., ventilation and gas exchange) in a wide range of diseases including asthma, cystic fibrosis and chronic obstructive pulmonary disease ^1-3. In disorders characterized by alveolar destruction (e.g., emphysema), disease severity can also be assessed by measuring the ¹²⁹Xe apparent diffusion coefficient (ADC) ^{4, 5}. That is, on the millisecond timescale gas atoms diffuse a smaller distance after inhalation because of collisions with alveolar walls, but restricted diffusion decreases as airspace size increases. Further, by imaging HP gases with multiple diffusion-weightings (i.e., b-values), it is possible to quantify the dimensions of the pulmonary microstructure and extract physiologically relevant, morphological parameters, including alveolar surface-to-volume ratio and number density (Na) ^{6, 7} . Because these parameters are altered by disease, “diffusion morphometry” can non-invasively characterize pathology progression in patients. In the preclinical setting, diffusion morphometry provides direct information about lung development and pulmonary disease pathogenesis and could be used to preclinically assess the efficacy of potential therapies. Here, we demonstrate for the first time the feasibility of high-resolution, in vivo ¹²⁹Xe diffusion morphometry of the mouse lung.

Methods: Diffusion-weighted MR images were acquired using a multi-slice, gradient-echo sequence (Figure 1) on a 7 T Bruker BioSpin scanner operated with ParaVision 6.0. Acquisition parameters included: matrix = 64², FOV = 32×32 mm², axial slice thickness = 1.5 mm, slices = 12, bandwidth = 50 kHz, dummy pulses = 8, α = 45º, TE = 7.95 ms, TR = 1 s, δ = 2.575 ms, Δ = 2.585 ms, b = [0, 6.25, 12.5, 18.75, 25, 31.25, 37.5] s/cm². ¹²⁹Xe was polarized to ~30% using a commercial polarizer (Model 9810, Polarean Inc., Durham, NC). Seven C57BL/6 mice were ventilated at an inspiratory pressure of 10 cm H₂O using a customized-built, HP-gas-compatible ventilator ⁸, and data were acquired at full-inspiration using a mixture of 70% HP ¹²⁹Xe and 30% O₂. Maps of were extracted from multi-b-value ¹²⁹Xe images using Bayesian probability theory, the acinar model of Weibel (Figure 2), and the ¹²⁹Xe-specfic theory of Sukstanskii and Yablonskiy ^9-14. Following imaging, mice were euthanized by exsanguination. The lungs were inflation-fixed in situ at a pressure of 10 cm H₂O to match in vivo conditions, and the lungs were then removed, paraffin embedded, and processed for histology.

Results: Diffusion-weighted images with sufficient signal (signal-to-noise ratio ~20 on b=0 images) for diffusion morphometry were obtained from all mice (Figure 3). The mean, whole lung ADC of all mice was 0.0165 ± 0.0005 cm²/s. Population means for alveolar sleeve depth, h, and acinar duct radius, R, were 44 ± 1 µm and 99 ± 2 µm, respectively. From regional h and R measurements, additional morphometric parameters including mean linear intercept (Lm = 74 ± 3 µm,) surface-to-volume ratio (58 ± 2 mm^-1), and alveolar number density (Na = 4600 ± 200 mm^-3) were calculated. The parameters h, R, and Lm extracted from these data were found to be in good agreement with histological measurements (Figure 4).

Discussion and Conclusion: The mean ADC of ~0.016 cm²/s is substantially less than the ~0.04 cm²/s observed in adult humans⁴, consistent with highly restricted diffusion in the small airspaces of the mouse lung. Bland-Altman analysis showed that values of h and R obtained using diffusion morphometry were in good agreement with those obtained using conventional histology, and the mean difference between MRI-derived and histology-derived metrics were not significantly different than 0 µm (p>0.05) for both metrics. MRI-derived Lm values skewed to somewhat lower values (by ~10 µm) than those obtained from histology. This pattern is similar to that previously reported for HP ³He diffusion morphometry in mice¹⁰. Whether these small discrepancies reflect limitations of the geometric model (Figure 2) or non-physiological distortion of lung microstructure during inflation/fixation remains to be determined. The alveolar number density (Na) of ~4300 mm^-3 was consistent with expectations, but was somewhat larger that the previously reported value of 3200 mm^-3 obtained using HP ³He ¹⁰. While not investigated here, this discrepancy likely reflects differences in animal age or ventilation strategy. Together these results suggest that ¹²⁹Xe diffusion morphometry provides physiologically relevant measures of lung microstructure in mice and will allow changes such as fibrotic alveolar wall thickening, emphysematous tissue destruction, and alveolar growth to be quantified noninvasively, paving the way to assess normal lung development and disease mechanisms in mouse models of human disease.

Acknowledgements

References

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