Animals and study design
Wild-type C57BL/6 mice (n = 120, 8 to 9-week-old female mice; Charles River Laboratories, Hollister, CA, USA) were maintained under standard vivarium conditions in a BioZone VentiRack™ (BioZone, Inc., Fort Mill, SC, USA). There were six groups: (1) 0 Gy; (2) Photons; (3) Protons; (4) LDR; (5) LDR + Photons; and (6) LDR + Protons. Subsets/groups were euthanized in 100% CO2 on days 21 and 56 post-irradiation. The protocol was approved by the Loma Linda University (LLU) Institutional Animal Care and Use Committee prior to initiation and followed all recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The mice were monitored daily by expert LLU animal care personnel and the investigators. All efforts were made to ensure little or no suffering.
Irradiation procedures
For LDR priming, mice were irradiated using 57Co plates (AEA Technology, Burlington, MA, USA) placed immediately beneath their cages (one plate/two cages in BioZone rack). A total dose of 0.01 Gy was delivered at 0.03 cGy/h to a maximum of five mice/large cage. Since γ-rays easily penetrate for long distances through many materials (major exceptions being lead and concrete), the dose for each individual animal was not determined. Dose confirmation, however, was performed using several thermoluminescent dosimeters (TLD) at various locations per cage. The data showed that dose uniformity was ± 5%.
For acute photon and proton irradiation, mice were placed individually into polystyrene aerated cubicles and irradiated to 2 Gy once within a very short period of time. Photon radiation was delivered using 60Co γ-rays (0.8 Gy/min; Eldorado Model G, Atomic Energy of Canada Ltd, Ottawa, Canada). Proton irradiation was done in the Proton Treatment and Research Center at Loma Linda University Medical Center; an average dose rate of 0.9 Gy/min and 230 MeV energy was used. Different groups received acute photon or proton radiation alone or within 1–2 h after protracted exposure to LDR γ-rays. Additional details on set-up and dosimetry have been previously published [20].
Analysis of gene expression by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
Immediately after sacrifice, the right lungs were frozen in liquid nitrogen (n = 5 mice/group/time point) and kept at −80°C until analysis. The 84 relevant genes were assessed using RT2 Profiler™ PCR Array PAMM-013 (SuperArray BioSciences, Frederick, MD, USA) to compare mRNA levels of ECM and CAM in lung tissue between irradiated groups and the 0 Gy control. To determine the effect of LDR γ-rays on response to acute radiation, gene expression levels in the LDR + Photon and LDR + Proton groups were compared to the levels obtained for their counterparts that received acute radiation alone. RNA extraction, RT-PCR, and its data analyses were performed by SuperArray BioSciences. Genes with ≥1.5-fold difference in expression and P <0.05 in the various group comparisons are emphasized. The ΔΔCt method used in PCR array of this study is widely accepted and known as a valid method for relative quantification of real-time PCR data.
Histopathology
After harvest, the left lungs were formalin-fixed, paraffin-embedded, and sections (5-μm thick) were prepared for standard hematoxylin/eosin (H&E) and Masson trichrome collagen staining. The changes in histology were assessed under a light microscope.
Masson Trichrome collagen staining
Briefly, deparaffinzed sections were pretreated in Bouin’s fluid at 56°C for 1 h before they were stained with Weigert’s Iron Hematoxylin, Biebrich Scarlet-Acid Fuchsin solution and Aniline Blue Stain Solution. Finally, 1% Acetic Acid solution was used to remove non-specific staining. After this procedure, the nuclei are stained black, cytoplasm and muscle fibers are displayed as red, and blue color represents collagen. The lung consists of a great number of alveoli and its expansion is not uniform throughout. Although, in this study, whole-mount sections of the lungs were not scanned for quantification of collagen, we could identify the difference in relative amounts of collagen in lung tissue from the Trichrome stained sections under a microscope.
Western blotting
A total of 50 mg of lung tissue from each mouse was homogenized in 50 μL of RIPA lysis buffer (Sigma, St. Louis, MO, USA) containing protease inhibitors (Roche, Indianapolis, IN, USA), and then pelleted via centrifugation at 13,000 g at 4°C. The protein concentration in supernatants was measured using the Coomassie Plus assay (Pierce, Rockford, IL, USA). Then, 25 mg of protein was loaded in each well of the gel for electrophoresis prior to electroblotting proteins onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA). In order to compare marker expression under the same condition, we loaded two of five samples from each group on the same gel with 12 wells. Six gels were used for a total of 60 specimens (five mice/group/time point). All membranes were incubated overnight with primary antibody at 4°C following blocking non-specific binding sites in 5% non-fat milk in Tris Buffered Saline (TBS) containing 0.1% Tween 20. The same membranes after electroblotting were incubated separately with different primary antibodies that were used to detect the proteins with different size prior to treatment with the secondary antibody conjugated with horseradish peroxidase (HRP, Millipore, Temecula, CA, USA). However, when detecting different proteins with similar size, stripping buffer (2% SDS, 62.5 mM Tris–HCl pH 6.7, 100 mM 2-mercaptoethanol) was used to remove primary and secondary antibodies on the PVDF membrane before proceeding to detect another target. This stripping buffer removes antibodies from membranes very effectively. Nonetheless, every time the stripped membranes were incubated with second antibody to confirm that no previous primary antibody remained on the membranes prior to incubating them with a new primary antibody. Four rabbit anti-mouse primary polyclonal antibodies were used to identify transforming growth factor (TGF)-β1 (recombinant full length inactive form), E-cadherin, Slug, and α-SMA. β-actin, utilized as a housekeeping protein, was detected with polyclonal antibody. All antibodies were purchased from Abcam. The enhanced chemiluminescence (ECL) and film exposure method (GE Healthcare-Bio-Sciences Corp., Piscataway, NJ, USA) was used to display the bands on the membranes. We used the Biospectrum 310 MultiSpectral System (Ultra-Violet Products, Ltd., Upland, CA, USA) and the appropriate software to measure band intensity on the films. This device is able to select real size of individual target bands, thus eliminating as much background as possible.
Statistical analysis
Gene expression data were evaluated using Student’s t-test, a well-accepted method for data obtained using the RT-PCR method described above. The ratios of target bands to β-actin obtained from Western blotting between each of the irradiated groups and 0 Gy at both time points was analyzed using two-way analysis of variance (ANOVA) followed by the Tukey test (Systat Software, Richmond, CA, USA).