Radiation induced changes in profibrotic markers in the submental muscles and their correlation with tongue movement

Abstract

Swallowing impairment is a major complication of radiation treatment for oropharyngeal cancers. Developing targeted therapies that improve swallowing outcomes relies on an understanding of the mechanisms that influence motor function after radiation treatment. The purpose of this study was to determine whether there is a correlation between radiation induced changes in tongue movement and structural changes in irradiated submental muscles, as well as assess other possible causes for dysfunction. We hypothesized that a clinically relevant total radiation dose to the submental muscles would result in: a) quantifiable changes in tongue strength and displacement during drinking two months post treatment; and b) a profibrotic response and/or fiber type transition in the irradiated tissue. Sprague-Dawley adult male rats received radiation to the submental muscles at total dose-volumes known to provoke dysphagia in humans. A clinical linear accelerator administered 8 fractions of 8Gy for a total of 64Gy. Comparisons were made to sham-treated rats that received anesthesia only. Swallowing function was assessed using videofluoroscopy and tongue strength was analyzed via force lickometer. TGFβ1 expression was analyzed via ELISA. The amount of total collagen was analyzed by picrosirius red staining. Immunofluorescence was used to assess fiber type composition and size. Significant changes in licking function during drinking were observed at two months post treatment, including a slower lick rate and reduced tongue protrusion during licking. In the mylohyoid muscle, significant increases in TGFβ1 protein expression were found post radiation. Significant increases in the percentage of collagen content were observed in the irradiated geniohyoid muscle. No changes in fiber type expression were observed. Results indicate a profibrotic transition within the irradiated swallowing muscles that contributes to tongue dysfunction post-radiation treatment.

Fig 1. Changes in lick and swallow metrics in rodents 2-months post treatment.

Results demonstrate the mean and standard deviations for both groups. (A-E) Lick rate (Hz), swallow rate (Hz), inter-swallow interval (ISI; seconds), pharyngeal transit time (PTT; ms), and tongue protrusion (mm) were obtained from videofluoroscopic swallow studies. (F) The maximum voluntary lick force was obtained from force lickometer. The boundaries of each box depict the interquartile range and the line within the box marks the median. Whiskers above and below the box indicate the 90^th and 10^th percentiles. Each circle represents the mean value for an individual animal. Statistically significant differences (p < 0.05) between groups are indicated by an asterisk.

Fig 2. Changes in eating behavior in rodents 2-months post treatment.

Results demonstrate the mean and standard deviations for both groups. The mastication time (seconds) was measured by analyzing the length of time it took each rat to eat each individual pellet. The boundaries of each box depict the interquartile range and the line within the box marks the median. Whiskers above and below the box indicate the 90^th and 10^th percentiles. Each circle represents the mean value for an individual animal. Statistically significant differences (p < 0.05) between groups are indicated by an asterisk.

Fig 3. Changes in profibrotic response post radiation.

64Gy of radiation increases TGFβ1 expression in the mylohyoid muscle compared to sham treatment as determined by ELISA. TGFβ1 levels in the tissue were normalized to the protein concentration from the BCA assay and are expressed as TGFβ1/pg of protein. Radiation and sham treated controls were compared by t-test. Statistical significance of p< 0.05 is indicated by an asterisk.

Fig 4. Changes in collagen content post radiation in the geniohyoid and anterior digastric muscles.

Significant increases in the % of collagen content were observed post-radiation in geniohyoid muscle compared to sham treatment (p<0.05 is shown as *). The boundaries of each box depict the interquartile range and the line within the box marks the median. Whiskers above and below the box indicate the 90^th and 10^th percentiles. Each circle represents the mean value for an individual animal. Images show the cross section of the geniohyoid (A & B) and digastric (C & D) muscles from sham (A, C) and radiated (B, D) treated rats. Tissue was stained with Picrosirius red. Images are at 20x (Bar, 100μm). Red staining denotes collagen fibers and yellow indicates muscle bundles.

Fig 5. MyHC fiber type composition and size in the geniohyoid and digastric muscles after radiation or sham treatment.

Representative images of MyHC expression in the digastric muscle. (A) Section was stained for expression of MyHC IIa (red), IIb (green), and laminin (blue). (B) Section was stained for expression of MyHC IIa (red), IIx (green), and laminin (blue). (C) Section was stained for expression of MyHC I (red) and laminin (blue). (D) The percentage of fibers positive for MyHC type IIa, IIb, IIx, and I in the sham (light grey) and radiated (dark grey) treated animals. (E) The fibers cross sectional area (CSA) for each muscle fiber. The boundaries of each box depict the interquartile range and the line within the box marks the median. Whiskers above and below the box indicate the 90^th and 10^th percentiles. Each circle represents the mean value for an individual animal.