Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

The overall goal of this procedure is to study swallowing function in freely behaving rodents, referred to as a video fluoroscopic swallow study or VFSS first. A feeding chamber is constructed to confine rodents in a small space during testing. The second step is to perform behavioral conditioning with a test solution to ensure maximal participation.

The next step is to conduct VFSS testing with the preconditioned mice. Finally, videos are subjected to frame by frame analysis to quantify swallow parameters. Ultimately, this novel assessment tool can be used to detect and characterize dysphagia in mirroring disease models and provide functional biomarkers for use in preclinical trials.

We first had the idea for using observation chambers because VFSS testing of humans is voluntary and unrestrained, and we wanted to closely mimic the human clinical procedure. The main advantage of this technique over similar existing methods is that it quickly elicits voluntary feeding behaviors in rodents to permit rapid testing of all stages of swallowing. Generally, individuals new to this method will struggle with video analysis because the small and rapid movements of swallowing and mice are difficult to discern.

Begin by assembling the observation chambers next, assemble a syringe delivery system to deliver solution to peg bowls during testing. Once assembled, place the observation chamber onto a motorized scissor lift for remote positioning. The main advantage of the remote controlled scissor lift table is improved radiation safety for researchers while maintaining freely behaving rodents in the fluoroscopy field of view.

Prepare the mice by habituating them to the observation chamber and subjecting them to an overnight water regulation period. When ready for testing, remove the chamber from the cage and clean it thoroughly. Use a dry erase marker to label each cleaned tube before placing it back in the home cage.

Next, prepare the chocolate flavored iohexol solution to set up the fluoroscopy environment. Use a spare observation chamber and peg bowl to determine the optimal height and position within the fluoroscope beam that permits visualization of drinking in the lateral plane. Set the fluoroscopy frame rate to 30 frames per second.

Higher frame rates can be used if available. Ensure that a radiopaque calibration marker is appropriately placed on the fluoroscope camera so that it is visible on the display monitor during the entire test. This step is necessary to permit calibration of length measurements to reduce stress due to handling.

Observe the cage until a mouse freely enters the observation chamber. Once entered, lift the chamber out of the cage and gently attach the second end cap. With repeated testing, mice can be easily coaxed to enter the chamber when it is placed in front of them inside the cage, or when suspended by the tail over the chamber opening.

Position the observation chamber within the fluoroscopy machine to begin VFSS testing in the lateral plane. Next, use a syringe delivery system to fill the peg bowl with chocolate flavored iohexol solution. Start the video fluoroscopy recording.

When the mouse starts drinking during testing, use the remote controlled lift table to adjust the position of the observation chamber so that the swallowing mechanism is visible in the field of view. Pause recording each time the mouse turns away from the peg bowl to minimize the duration of radiation exposure. Resume recording when the mouse returns to the peg bowl, refill the peg bowl as needed.

The goal is to record several long bouts of continuous drinking, which is typical for most mice. Within the first two minutes of testing, stop testing if the mouse does not drink within five minutes, return non-compliant mice to the home cage for retesting at a later time the same day. Do not exceed a 24 hour water regulation.

When testing multiple mice from the same home cage. Clean the peg bowl and tubing with a dry paper towel between mice. Trim the tip of the PE tubing as needed.

If chewed during testing, clean the observation chamber as needed to remove any splattered iohexol on the chamber walls. Rinse the chamber with tap water and dry with a paper towel. Use a video editing software program that permits frame by frame analysis of the video fluoroscopy recordings.

To quantify the swallow parameters of interest, at least two trained reviewers should analyze each video in a blinded fashion. The primary reviewer views each video to identify and analyze three to five long drinking bouts. This criterion is based on published non-radiographic swallow studies showing that this parameter is sufficient for statistical analysis.

The secondary reviewer then independently analyzes the three to five measures per swallow parameter for each mouse that were initially identified and analyzed by the primary reviewer. Reviewer discrepancies are then identified for each mouse and reanalyzed until 100%consensus is reached. The undisputed values for each swallow parameter are then averaged to obtain the mean value for each mouse.

When fewer than three measures are obtained for a swallow parameter, enter a missing value in the statistical database for that measure. These examples were obtained using a low energy fluoroscopy system position. One permits visualization of the entire head and proximal thoracic region.

The swallow trigger point is centered within the field of view. Position two permits visualization from the swallow trigger point to the gastroesophageal junction. Note, the bolus passing through the distal esophagus even at the lowest magnification setting.

Bony structures of the head and neck are readily visible using the low energy fluoroscopy system. Anatomical structures are also examined at higher magnification to permit quantification of several additional swallow parameters. This figure shows representative preliminary findings for two VFSS swallow parameters, swallow rate, and inter swallow interval swallow rate was significantly slower for SOD one mice compared to aged C 57.

Mice and controls inters swallow interval was not significantly different between groups. Once mastered video capture can be completed in just under two minutes per mass if it is performed properly. While attempting this procedure, it's important to remember to utilize the chocolate flavored test solution within a few hours of preparation to prevent changes in viscosity and palatability that can result in avoidance behaviors.

This new technique is paving the way for researchers in the field of neuroscience to explore natural feeding and swallowing behaviors in healthy rodents and disease models. After watching this video, you should have a good understanding of how to successfully perform VFSS testing of freely behaving rodents To quantify typical feeding behaviors and swallowing function, Don't forget that working with radiation emitting equipment can be extremely hazardous, and radiation precautions for time, distance, and shielding should always be followed while performing this procedure.