Radiation induced pulmonary fibrosis as a model of progressive fibrosis: Contributions of DNA damage, inflammatory response and cellular senescence genes

<p><b>Purpose/Aim of Study</b>: Studies of pulmonary fibrosis (PF) have resulted in DNA damage, inflammatory response, and cellular senescence being widely hypothesized to play a role in the progression of the disease. Utilizing these aforementioned terms, genomics databases were interrogated along with the term, “pulmonary fibrosis,” to identify genes common among all 4 search terms. Findings were compared to data derived from a model of radiation-induced progressive pulmonary fibrosis (RIPF) to verify that these genes are similarly expressed, supporting the use of radiation as a model for diseases involving PF, such as human idiopathic pulmonary fibrosis (IPF). <b>Materials and Methods</b>: In an established model of RIPF, C57BL/6J mice were exposed to 12.5 Gy thorax irradiation and sacrificed at 24 hours, 1, 4, 12, and 32 weeks following exposure, and lung tissue was compared to age-matched controls by RNA sequencing. <b>Results</b>: Of 176 PF associated gene transcripts identified by database interrogation, 146 (>82%) were present in our experimental model, throughout the progression of RIPF. Analysis revealed that nearly 85% of PF gene transcripts were associated with at least 1 other search term. Furthermore, of 22 genes common to all four terms, 16 were present experimentally in RIPF. <b>Conclusions</b>: This illustrates the validity of RIPF as a model of progressive PF/IPF based on the numbers of transcripts reported in both literature and observed experimentally. Well characterized genes and proteins are implicated in this model, supporting the hypotheses that DNA damage, inflammatory response and cellular senescence are associated with the pathogenesis of PF.</p

⁵⁶Fe particle radiation causes endothelial activation.

<p>(<i>A</i>) Representative images of ICAM-1 staining. Pictures are at 20x magnification and the scale bar is 10 µm. (<i>B</i>) ICAM-1 area was measured as percent total area in the entire cortex in 2 serial sections with the results being averaged together. Each dot represents a single animal. (<i>C</i>) Protein samples were analyzed for LRP1 using Western blot. LRP1 levels were standarized against α-tubulin as a loading control. Representative immunoblot image is present in <i>C’</i>. Data is presented as mean ± SD. Results were analysed with a Student’s t-test. <i>n</i> = 13–14 animals per dose.</p

Radiation increases select Aβ isoforms but has no effect on APP processing.

<p>Dot plot analysis of soluble Aβ40 (<i>A</i>), Aβ42 (<i>B</i>) and insoluble Aβ40 (<i>C</i>) and Aβ42 (<i>D</i>). Each dot represents one animal. Data was analyzed with Student’s t-test for the females and one-way ANOVA with a Bonferroni post test for the males. (<i>E, F</i>) Male 0 cGy and 100 cGy APP (<i>E</i>) and β-C terminal fragment (<i>F</i>) protein levels were measured via Western blot and standardized to α-tubulin. Representative images of blots are present in <i>E’</i> and <i>F’</i>. Results were analyzed with Student’s t-test. Data displayed as mean ± SD, <i>n</i> = 8–14 animals per dose. *<i>P<.05, **P<.01</i>.</p

Immunohistochemical staining for Congo red and 6E10 increases after ⁵⁶Fe particle irradiation.

<p>(<i>A, C</i>) Representative images of half male brains stained for Congo red (<i>A</i>) or 6E10 (<i>C</i>) 6 months after 0 cGy or 100 cGy ⁵⁶Fe particle radiation. Scale bar is 1 mm. (<i>B, D</i>) Quantitative measurement of percent plaque area assessed with Congo red (<i>B</i>) and 6E10 (<i>D</i>). In addition, total number of individual 6E10 positive plaques (<i>E</i>) and the average size of plaques (µm²) (<i>F</i>) was determined. Each dot represents a single animal measured as percent area of the cortex and hippocampus combined. Data was analyzed with Student’s t-test for the females and one-way ANOVA with a Bonferroni post test for the males. Data displayed as mean ± SD, <i>n</i> = 8–14 animals per dose. *<i>P<.05, **P<.01</i>.</p

Effect of ⁵⁶Fe particle radiation on memory and cognition using contextual fear conditioning and novel object recognition tests.

<p>(<i>A</i>) Fear conditioning results quantified as percent time freezing. (<i>B</i>) No significant difference was found between any groups in freezing to a novel context or a tone stimulus. (<i>C</i>) Novel object recognition test using the recognition index generated for time spent with the novel object. All data is compared within the respective gender. Data was analyzed with Student’s t-test for the females and one-way ANOVA with a Bonferroni post test for the males. Graphs show means ± SD, <i>n = </i>8–14 animals per condition at each dose. **<i>P</i><.01, ***<i>P</i><.001.</p