Masaya Takahashi1, Keisuke Ishimatsu1, Shanrong Zhang1, Hua Lu2, and Connie C.W. Hsia2
1Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, United States, 2Pulmonary and Critical Care Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States
Synopsis
The purpose of this study was to investigate the ability of in vivo ultra-short echo time (UTE)-MRI for assessment of pulmonary microstructure and functions of ventilation-perfusion in an animal model of pulmonary fibrosis in comparison with high-resolution MRI, physiological global measures and histomorphology.Purpose
We have demonstrated in mice that renal fibrosis is suppressed by administration of soluble Klotho protein, which is the cleaved extracellular domain of the single-pass transmembrane Klotho that was initially identified as a putative aging-suppressor1. The objectives of the study were to investigate whether UTE-MRI could quantify tissue integrity, including microstructure, ventilation and perfusion, in pulmonary fibrosis and whether the method could detect the effects of upregulated Klotho expression in lung parenchyma.
Materials and Methods
Animal preparation: Twenty-seven mice (12-13 week-old) were divided into three groups (A-C), anesthetized, intubated, and underwent tracheal instillation with different substances as follows: A; control (2.5 μg empty vector, n=9), B; Bleomycin (3 U/kg bleomycin + 2.5 μg empty vector, n=9), and C; Bleomycin+Klotho (3 U/kg bleomycin + 2.5 μg Klotho cDNA, n=9). All substances were dissolved in 50 μl saline and instilled using a micro-sprayer (Penn Century, Inc. Wyndmoor, PA) in each mouse. All animals were subjected to the MRI session at 3 weeks after instillation.
MRI: UTE-MRI studies were conducted in a 3T human MRI (AchievaÔ, Philips, Best, Netherlands) with a small solenoid coil2. Under anesthesia with isoflurane mixed in either medical grade air or 100% oxygen (O2), each entire lung was imaged using a 3D radial UTE sequence at two different TEs of 110 μs and 1.15 ms. The other parameters were: TR=10 ms, FA=10°, FOV=403 mm, matrix size=683 (reconstructed to 1443 matrix), NEX=2, affording a total scan time of 3.1 min. Subsequently, FA was increased to 30° and a set of two UTE images was acquired under air (baseline) and 100% O2 inhalation for each mouse. An approximately 5-min interval was used between the scans after switching the gasses. Under air inhalation, post-Gd UTE image was obtained immediately after intravenous injection of Gd (0.2 mmol/kg). Further, high-resolution images were obtained on the entire lung in vivo with cardiac-respiratory dual gating3 in a 7T animal MRI (Varian, Palo Alto, CA). The imaging parameters were: TE=1.83 ms, FA=22°, FOV=322 mm, matrix size=2562 (125 μm2 in-plane resolution), thickness=1 mm and NEX=4, affording a total scan time of 6-7 min. Lungs were inflation-fixed at 20 cmH2O after the MRI session for 3D ex-vivo imaging (200 μm3 isotropic resolution) and histology. To calculate prevalence of apparent fibrotic lesions, two observers evaluated the presence or absence of fibrosis in the lung parenchyma on the high-resolution image and UTE images with long TE (1.15 ms) in a blind manner. For SI measurements, multiple regions of interest (ROIs) were selected avoiding main pulmonary vessels in the normal-appearing lung parenchyma (NALP) for a representative value for each mouse. SI was measured in the most obviously identified lesion in each mouse when it presented. Percent change in SI due to administration of O2 or Gd was defined as: (SIpost – SIpre)/SIpre × 100, where SIpre is SI of the baseline and SIpost is SI measured with 100% O2 inhalation or injection of Gd.
Results and discussion
The prevalence of pulmonary fibrosis in the A) control, B) bleomycin, and C) bleomycin+Klotho groups were 0% (0/6), 78% (7/9, P<.01 vs. control), 44% (4/9), respectively. Figure 1 demonstrates representative high-resolution image (a), UTE images (b-c), T2* map (d) and %change in SI maps due to 100% O2 inhalation (e) or Gd injection (f) in a mouse with fibrosis lesions. The fibrotic lesions were revealed as abnormal increase of SI in the lung parenchyma in both UTE and high-resolution images (Fig. 1a-c). The T2* (ca. 0.95 ms), O2 (ca. 6-7%) or Gd (ca. 230%) enhancement in the NALP did not differ among the groups, A-C. The lesions seen in both groups B and C showed increased T2* compared to the NALP (Fig. 2), which implied increased tissue density. These lesions exhibited decreased O2 enhancement (Fig. 3) and almost identical Gd enhancement (Fig.4). The lung volume measured by ex-vivo 3D imaging in the groups B (856±64 mm3) and C (849±71 mm3) were smaller than that in the control group (985±101 mm3), which suggested restriction of lung inflation due to bleomycin-induced deposition of connective tissue in the parenchyma. Thus, the UTE imaging allowed us to evaluate the parenchymal structure and ventilation-perfusion changes in patchy pulmonary fibrotic lesions. Results suggest that gas-exchange was impaired but perfusion was maintained in the fibrotic lesions. Klotho treatment reduced the prevalence of apparent fibrotic lesions on MRI; however, further analysis is needed to evaluate if Klotho alleviated the severity of fibrosis.
Acknowledgements
This research was supported by grants from the National Heart, Lung and Blood Institute (U01 HL111146 and HL110967).References
1. Doi S, Zou Y, Togao O et al. Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice. J Biol Chem. 2011;286(10):8655-8665.
2. Togao O, Ohno Y, Dimitrov I et al. Ventilation/perfusion imaging of the lung using ultra-short echo time (UTE) MRI in an animal model of pulmonary embolism. J Magn Reson Imaging. 2011;34(3):539-546.
3. Kubo S, Levantini E, Kobayashi S et al. Three-dimensional magnetic resonance microscopy of pulmonary solitary tumors in transgenic mice. Magn Reson Med. 2006;56(3):698-703.