DOI: 10.5061/dryad.f7m0cfz5h
title: Data from: Effects of chemoradiation and tongue exercise on swallow biomechanics and bolus kinematics

description: Background: Common treatments for head and neck cancer (radiation and
 chemotherapy) can lead to dysphagia; tongue exercise is a common
 intervention. This study aimed to assess swallow biomechanics and bolus
 kinematics using a well-established rat model of radiation or
 chemoradiation treatment to the tongue base, with or without tongue
 exercise intervention. Methods: Pre- and post-treatment videofluoroscopy
 was conducted on thirty-two male Sprague-Dawley rats treated with
 radiation/chemoradiation and exercise/no exercise. Rats in the exercise
 groups completed a progressive resistance tongue training paradigm.
 Swallow biomechanics, bolus kinematics, jaw opening, and post-swallow
 respiration were assessed. Results: Both treatments impacted outcome
 measures; the addition of exercise intervention showed benefit for some
 measures, particularly in rats treated with radiation, vs. chemoradiation.
 Conclusions: Radiation and chemoradiation can significantly affect aspects
 of deglutition; combined treatment may result in worse outcomes. Tongue
 exercise intervention can mitigate deficits; more intensive intervention
 may be warranted in proportion to combined treatment.
description: Thirty-two male Sprague-Dawley rats were randomized into four
 groups: concurrent chemoradiotherapy without exercise (CCRT), radiotherapy
 without exercise (RT), CCRT+ tongue exercise (CCRT+TE), and radiotherapy
 (RT+TE), with 8 animals per group based on a priori power calculations).
 All rats underwent videofluoroscopic swallow studies (VFSS) at baseline
 (prior to treatment administration/exercise intervention) and 12 weeks
 post-treatment +/- exercise intervention, thus serving as their own
 controls. Treatment consisted of RT delivered to an 8X12
 mm<sup>2</sup> area of the tongue base over the course of ten
 days using methods previously described (Xstrahl Small Animal Radiation
 Research Platform (SARRP)
 Irradiator).<sup> </sup>Administration of radiotherapy to this
 area targets a structure crucial for swallowing and allows exploration of
 how treatment may impact swallow function. Each rat
 received 4.5 Gy/day, totaling 45 Gy – thus the biological effective dose
 was approximately 112 Gy. This fraction size was calculated using methods
 previously described, and allowed for maintenance of clinical relevance in
 the protocol of treatment delivery. In addition, rats in the CCRT groups
 received two doses of cisplatin (3mg/kg) in addition to RT – one dose
 prior to the first fraction of RT, and another prior to the sixth
 fraction. Cisplatin is a common chemotherapy drug and is often considered
 the standard of care in this patient
 population.<sup>49,50</sup> Rats in the TE groups underwent an
 8-week progressive resistance tongue training paradigm following treatment
 administration and a recovery period. Baseline and final VFSS recordings
 were used for data analysis via ImageJ. Rats were pair-housed in standard
 polycarbonate cages in the vivarium on a reversed 12:12 light/dark cycle.
 All animal work was done in accordance with NIH and university animal-use
 guidelines and approved by the University of Wisconsin School of Medicine
 and Public Health Animal Care and Use Committee (IACUC<strong>
 </strong>Protocol M005828). Progressive
 Resistance Tongue Training Paradigm:   The
 progressive resistance tongue training paradigm is a well-established
 tool, analogous to lingual strengthening exercises employed in the
 clinical setting, in translational models such as aging, Parkinson
 disease, and stroke. Intervention involves training the rat to press a
 disc connected to a force transducer with a specified amount of force to
 obtain a water reward (Figure 1). To avoid adding undue stress during
 treatment administration, rats were not water restricted until after
 treatment. Following a 1-week (7 days) gradual water restriction, rats
 were acclimated to the apparatus and underwent an 8-week training
 paradigm. Maximum force (g) was calculated at baseline, and individualized
 force requirements were initiated at 20% and increased by 20% every two
 weeks, ending with 80% maximum force required at eight weeks. Rats were
 water restricted over the training period to motivate their participation
 in the exercise, and given access to water <em>ad libitum</em>
 for 3 hours/day, 7 days/week.
 Videofluoroscopy:
 Videofluoroscopic swallow study (VFSS) allows for radiographic
 imagining of bolus movement and swallowing physiology from the oral cavity
 to the esophagus. Quantitative measures of deglutition including bolus
 speed, bolus area, and mastication rate have been validated in prior
 animal studies as clinically-relevant measures of oral phase efficiency.
 Such outcomes provide information on potential physiological changes, thus
 enabling translation to human studies given anatomical similarities
 present between this model and humans. Further, assessment of swallow
 biomechanics and post-swallow respiration using VFSS recordings provide
 critical information regarding deglutition.  Analysis of these outcomes
 can provide clinically-relevant insight into aspects of swallow physiology
 in both the oral and pharyngeal phases of swallowing.
 All rats underwent baseline and post-treatment/intervention
 recordings to compare potential changes both within and between groups.
 Rats were food restricted prior to recording and acclimated to smooth
 peanut butter placed on the side of the home cage prior to recording day.
 To conduct VFSS, rats were individually placed in their home cage on an
 L-shaped platform perpendicular to the C-arm fluoroscope (Genoray ZEN-7000
 C-Arm 3<sup>rd</sup> Gen Fluoroscope; Origin Republic of
 Korea, Vendor Mulit Inc., Ontario, CA). A mixture of peanut butter and
 barium was placed on a stage affixed to the side of the home cage; rats
 were allowed to consume the mixture <em>ad libitum</em> for
 approximately five minutes (Figure 2). X-ray was only initiated when the
 rat began to consume the mixture, as to avoid unnecessary radiation
 exposure. Swallows were recorded at 30 frames/second and analyzed offline,
 using MorphoJ for swallowing biomechanics, and ImageJ for all other
 measures. Computational Analysis of Swallow
 Mechanics adapted for the Rodent Model (CASM-R): 
 Computational Analysis of Swallowing Mechanics (CASM) is a
 morphometric analysis software that has been used for studying swallowing
 biomechanics in humans with dysphagia, and has been adapted for use in
 rodents (CASM-R). CASM-R tracks markers at skeletal levers formed by the
 hard palate, 1st and 4th cervical vertebrae, and mandible (Figure 3A) at
 each frame, in order to describe body and jaw movements during the
 swallow. Skeletal lever tracking also serves to re-axis / “anchor” the
 position of soft tissue structures relative to the skeletal landmarks,
 allowing for more specific analysis of the isolated movement of swallowing
 structures, including the upper esophageal sphincter (UES) and base of
 tongue (BOT), without interference of gross body/jaw movements.
 Assessment of swallow biomechanics can provide information on
 potential musculoskeletal dysfunction through analysis of key muscular and
 skeletal landmarks affiliated with swallowing, thereby guiding
 investigation of the underlying mechanisms treatment may have on swallow
 physiology. An initial group of sixteen male Sprague-Dawley rats (n =
 4/group) were included for a preliminary pilot analysis, following methods
 previously described in Kletzien <em>et al.,</em> 2017 and in
 alignment with published clinical work supporting significant outcomes
 with limited sample size, to determine the impacts of treatment and
 exercise intervention on swallow biomechanics. Briefly, three VFSS
 recorded swallows were selected for analysis. Eight muscular and skeletal
 landmarks were identified – the hard palate, C1, C4, the UES, three
 mandibular points (anterior, mid, and posterior), and tongue base – and
 mapped in each frame of the individual swallow for multivariate
 morphometric analysis. The pharyngeal phase was defined as the frame
 immediately preceding swallow onset until the bolus head made contact with
 the UES. Swallows in which the rat’s positioning was suboptimal for data
 analysis (<em>i.e.,</em> obliqued, extraneous movement, or
 upper extremities blocking structures) were identified as outliers by
 MorphoJ and were subsequently excluded from final analysis, as inadequate
 positioning could provide incorrect data related to the positioning and
 movement of the key anatomical landmarks. In these cases, said excluded
 swallow was replaced with another from the same rat, selected at random.
 Following this preliminary pilot multivariate morphometric analysis,
 analysis of bolus kinematics and bolus area was completed to study the
 dynamic interaction on swallow function in a larger sample size. Given
 interpretation of CASM-R findings is limited, the subsequent inclusion of
 kinematic analysis allowed for more robust assessment and subsequent
 conclusions of the relationship between HNC treatment, tongue exercise
 intervention, and physiologic outcomes. Bolus
 Kinematics and Bolus Area: Prior to analysis,
 three swallows were selected per rat. Selection was based on the following
 criteria: unobstructed sagittal view, adequate positioning during
 recording (ideally little to no movement, rat positioned straight without
 curvature of body/movement of limbs), and visualization of bolus from oral
 cavity (mastication) through esophagus. To measure bolus speed (mm/sec), a
 marker was placed at the leading edge of the bolus (referred to as the
 head of the bolus), beginning at the frame immediately preceding swallow
 onset, and at each subsequent frame until the head of the bolus made
 contact with the UES. A marker was placed on the skull (rostral and
 posterior to the eye) at the frame before swallow onset, and again at the
 final frame of the swallow, in order to account for whole body movement.
 Bolus area was measured at the final frame of the swallow (when bolus head
 first encounters UES) as the slowed transit caused by impedance of the UES
 permits greater visualization of the bolus, with less interference of
 movement artifact on bolus edges. To further reduce measurement
 variability, the same bolus frame was measured 3 times, with the average
 used for final analysis. Pixel area was converted to
 mm<sup>2</sup> using a calibration factor derived from a
 17.9cm metal disc. Mastication rate (cycles/sec) was
 calculated from the frame of minimum jaw opening (jaw closure) following
 bolus acquisition, through 5 complete cycles of jaw opening-closing, until
 the closure of the 5<sup>th</sup> cycle. All videos were
 analyzed by rater 1 (NSH), and a random subset of videos (10%) was
 analyzed by a second rater (AFP) to calculate inter-rater reliability.
 Agreement amongst the raters was excellent (ICC=0.99).
 Jaw Opening:  Following
 results obtained from the assessment of mastication rate, the length of
 jaw opening (aperture) was measured to investigate the effects of
 treatment and intervention on the mandibular range of motion. 3
 mastication cycles were selected, analyzed, and averaged per rat. The
 point of maximum jaw opening in a cycle was identified, and a line was
 drawn from the lowest point of the mandible (mid-anterior) to the highest
 point of the hard palate. Measurements were taken pre-and
 post-treatment/tongue exercise intervention for all groups.
 Respiratory-Swallow Coordination:
  The typical process of deglutition involves a post-swallow
 exhalation immediately following the swallow apnea period. HNC treatments
 can impact the coordination of breathing and swallowing, resulting in an
 unsafe post-swallow inhale that elevates the risk of aspiration. A
 previous study in our lab confirmed the presence of this aberrant
 post-swallow inhale pattern in a rat model of combined chemoradiation
 treatment, using a validated diaphragm tracking measure as a proxy for
 respiration. In the present study, an abbreviated version of this assay
 was used to determine the differential effects of CCRT, RT, and tongue
 exercise on respiratory-swallow coordination. The position of the
 diaphragm was visually tracked during the breathing cycle associated with
 each analyzed swallow, and a binary code was assigned for statistical
 analysis: post-swallow exhale (0; Ex) or post-swallow inhale (1; In).
 Raters, blinded to rat ID, group assignment, and time point, identified
 the onset frame and directionality of each phase: pre-swallow respiration
 (In/Ex), swallow apnea (cessation of movement), and post-swallow
 respiration (In/Ex). All files were analyzed by NSH, and 10% of all videos
 were analyzed by a second rater (LMR) for inter-rater reliability (IRR =
 90%). <strong>STATISTICAL
 ANALYSIS</strong> Swallow
 Biomechanics: Procrustes fit was generated to
 evaluate the distribution of shape change among rats and highlight
 potential outliers. Following identification and removal of outliers, a
 canonical variate analysis was conducted to determine biomechanical
 differences between the groups. A discriminant function <em>post
 hoc</em> analysis was completed to visualize said changes through
 corrected eigenvectors. Mahalanobis distance (D) was used to indicate
 significance. To account for multiple comparisons across 8 coordinates, a
 <em>post hoc</em> Bonferroni correction was performed with a
 corresponding set critical value of α&lt;0.006.
 Bolus Kinematics, Bolus Area, Jaw Opening, and
 Post-Swallow Respiration: To compare the
 effects of treatment and tongue exercise on bolus speed, bolus area,
 mastication rate, and jaw aperture within (pre vs. post time points) and
 between groups, a two-way repeated measures analysis of variance (ANOVA;
 assumptions met) with pairwise comparisons and <em>post
 hoc</em> Bonferroni correction was conducted using SPSS (IBM,
 Chicago, IL). To assess potential changes in post-swallow respiration
 patterns, descriptive statistics were generated (frequency and percentage
 of post-swallow inhales across 3 trials per rat) to compare the two time
 points and four groups – RT without exercise, RT+TE, CCRT without
 exercise, and CCRT+TE. Given there were no aberrant post-swallow inhales
 during the pre-treatment and pre-intervention time point (as expected for
 healthy controls), McNemar’s test to assess repeated measures of a binary
 outcome could not be completed. As such, Fisher’s Exact Test was conducted
 to assess the differences between groups in the post-treatment and
 intervention time point. Further, to determine how bolus area and
 post-swallow respiration impacted post-bolus speed, an ANCOVA was
 conducted on post-bolus speed data, with the covariates being
 pre-treatment bolus speed, post-treatment bolus area, and post-swallow
 respiration using SAS. A critical value of
 <em>α</em>=0.05<em> </em>indicated
 significance.
description: # Updated Effects of Chemoradiation and Tongue Exercise on Swallow
 Biomechanics and Bolus Kinematics: Open Source Data
  Description_of_the_data,_software,_and_file_descriptions    We have
 submitted our swallow and respiration patterns for the animals
 (ChemRad_Breathing_Analysis.xlsx); rat jaw aperture (opening) data
 (ChemRad_Jaw_Averages.xlsx); final organized data for bolus area, bolus
 speed, and mastication rate for pre and post WHAT?
 (ChemRad_Master_Sheet_Final_Organized.xlsx); bolus speed, bolus area, and
 mastication for each animal (Fluro_Template_Copy.xlsx); analysis of
 swallow-respiratory patterns of the animals in which the rater is blinded
 to timepoint and group (Respiration_Binding_02092022.xlsx); images showing
 the process we used to analyze the fluorscopic data
 (Fluoro_Protocol_Copy.docx); a written walkthrough of how we used MorphoJ
 to use CASM-R (MorphoJ_Analysis_Protocol.docx); statistical breakdown of
 collected data (PB_VELOC_GLM_Glen stats.docx); Processing and analyzing
 software ImageJ; and software used to analyze swallow biomechanics.
 Abbreviations HNC - Head and Neck Cancer RAD - Radiation Associated
 Dysphagia LRAD - Late Radiation Associated Dysphagia VFSS -
 Videofluoroscopic Swallow Study RT - Radiotherapy CCRT - Concurrent
 Chemotherapy and Radiation Treatment TE - Tongue Exercise CASM-R -
 Computational Analysis of Swallow Mechanics adapted for the Rodent model
 Software_Descriptions 1\)Image J     This program was used to process and
 analyze video-recorded swallows (using java) both pre- and post-treatment
 and intervention for all animals included in the study to obtain the
 following outcomes: bolus speed, bolus area, mastication rate, jaw
 aperture, and post-swallow respiration. 2\)MorphoJ     This program was
 used to analyze swallow biomechanics via the video-recorded swallows
 identified for analysis and converted in Matlab for morphometric analysis.
 File_Descriptions 1\)ChemRad_Breathing_Analysis.xlsx     ID: Randomized
 animal identification number     Treatment: Experimental condition
 describing what treatment the animal underwent.         CRT = Chemo
 therapy and radiotherapy         RT = Radiotherapy     Condition:
 Experimental condition denoting if the animal underwent tongue exercise or
 not.         Ex: = Exercise         NonEx = Non-Exercise     Pre_(E_or_I):
 A value measured before the animal underwent treatment         E = Exhale
         I = Inhale     Post_(E_or_I): A value measured after the animal
 underwent treatment         E = Exhale         I = Inhale         NA =
 Diaphragm could not be visualized 2\)ChemRad_Jaw_Averages.xlsx     ID:
 Randomized animal identification number    
 Jaw_Aperature_with_Calibration_Pre: Pre-treatment measurement of jaw
 opening     Jaw_Aperature_with_Calibration_Post: Post-treatment
 measurement of jaw opening 3\)ChemRad_Master_Sheet_Final_Organized.xlsx  
   ID: Randomized animal identification number     Bolus_Area(Pre): The
 size of the swallowed amount post treatment     Bolus_Area(Post): The size
 of the swallowed amount measured post treatment     Bolus_Velocity(Pre):
 The velocity of the swallowed bolus measured before the animal underwent
 treatment     Bolus_Velocity(Post): The velocity of the swallowed bolus
 measured after the animal underwent treatment     Mastication_Rate(Pre):
 The amount of chews before the bolus was swallowed before the animal
 underwent treatment     Mastication_Rate(Post): The amount of chews before
 the bolus was swallowed after the animal underwent treatment
 4\)Fluro_Template_Copy.xlsx     Animal_ID: Randomized animal
 identification number     Swallow#: swallow 1-3 included for analysis    
 Frame_Start: The video frame number corresponding with the start of the
 animals swallow     Frame_End: The video frame number corresponding with
 the end of the animals swallow     Stable_Marker_Start: stable head marker
 used to account for movement during the swallow, prior to swallow
 initiation     Initation: intial movement of bolus/initiation of swallow  
   Stable_Marker_End: stable head marker used to account for movement
 during the swallow, post-swallow cessation     Point_Number: point number
 on frame during bolus tracking     Area: The area of the swallowed bolus  
   Mean: The mean value of     Min: The minimum value of     Max:The
 maximum value of     X: the value on the X axis of     Y: The value on the
 Y axis of     Slice: # of frames included in swallow (i.e. 1-6)    
 Correction: documenting if animal moved forward or backward during swallow
 - calculations adjusted accordingly per template     Point Number: point
 number re: bolus tracking (i.e. 1-6) as you click through frames of
 swallow     Initiation: frame and all related measurements from ImageJ
 when swallow was initiated     Multi-point_tool_number: same as point
 number above     X-axis: measurement provided from ImageJ
 "measure" function (control+m)     Y-axis: measurement provided
 from ImageJ "measure" function (control+m)     Distance(Pixels):
 distance in pixels re: bolus measurement     Distance(mm): distance in mm
 re: bolus measurement     Frames: video frames containing the current
 swallow being analyzed     Location: measure provided by ImageJ    
 Velocity: bolus speed     Calibration_Value: calibration value determined
 by dime measurement at beginning of video recording used in calculation
 for outcomes     All: overall measure of bolus speed     Average: average
 speed across frames     Moved_Forward: head moved forward between swallow
 initiation and end of swallow     Moved_Backward: head moved backward
 between swallow initiation and end of swallow
 5\)Respiration_Blinding_02092022.xlsx     ID:Randomized animal
 identification number     Frames_for_Swallow_1: The total number of frames
 it took for the animal to complete one full swallow    
 Pre_Swallow1_BlindCode: The code used in the blind evaluation process to
 replace the animals ID number, used to identify the corresponding video
 file of the animals swallow pre treatment     Frames_for_Swallow_2: The
 total number of frames it took for the animal to complete a second full
 swallow     Pre_Swallow2_BlindCode: The code used in the blind evaluation
 process to replace the animals ID number, used to identify the
 corresponding video file of the animals swallow pre treatment    
 Frames_for_Swallow_3: The total number of frames it took for the animal to
 complete a third full swallow     Pre_Swallow3_BlindCode: The code used in
 the blind evaluation process to replace the animals ID number, used to
 identify the corresponding video file of the animals swallow pre treatment
     Post_Swallow1_BlindCode: The code used in the blind evaluation process
 to replace the animals ID number, used to identify the corresponding video
 file of the animals swallow post treatment     Post_Swallow2_BlindCode:
 The code used in the blind evaluation process to replace the animals ID
 number, used to identify the corresponding video file of the animals
 swallow post treatment     Post_Swallow3_BlindCode: The code used in the
 blind evaluation process to replace the animals ID number, used to
 identify the corresponding video file of the animals swallow post
 treatment 6\)Fluoro_Protocol_Copy.docx     Word file (.docx) containing
 images showing the process of analyzing fluoroscopic data.
 7\)MorphoJ_Analysis_Protocol.docx     Word file (.docx) containing a
 written walkthrough of using MorphoJ to conduct CASM-R.
 8\)PB_VELOC_GLM_Glen_stats.docx     Word file (.docx) containing the
 statistical breakdown of the collected data.
 9\)Respiration_Glen_Stats.docx     Word file (.docx) containing the
 statistical breakdown of the animal's respiration data.