This protocol facilitates the preparation of the rat and vocal fold for optimal longitudinal and cross-sectional analysis. The protocol utilizes laryngeal tracking during cryosectioning to ensure an accurate identification of the intrinsic laryngeal muscles and to reduce the chance of tissue loss. The rant larynx is a well-established model for investigating structural and functional neuromuscular laryngeal adaptations in response to development, aging, disease, and pharmacological agents.

Although this demonstration focuses on the thyroarytenoid muscle of the vocal fold, the methodology is also applicable to other intrinsic laryngeal muscles. For dissection of the rat larynx, shave the ventral neck from the mandible to the manubrium and swab the exposed area with alcohol to prevent fur contamination of the tissue specimens. Place the rat under a dissecting microscope with a 10 times magnification and use a scalpel to make a midline neck incision until the trachea is exposed.

Use forceps and a sharp instrument to separate the ventral extrinsic laryngeal muscles at the midline and sever the trachea caudal to the third tracheal ring. To excise the larynx, use dissecting scissors to make an incision caudal to the third tracheal ring and rostral to the hyoid bone. Use tweezers, pins, and microscissors to remove the esophagus, thyroid gland, and extrinsic laryngeal muscles from the larynx.

Using the midline between the posterior cricoarytenoid muscles as a landmark, use the microscissors to bisect the larynx dorsally between the arytenoids. Pin the lateral walls of the larynx to expose the vocal folds and use the microscissors to bisect the tissue ventrally through the midline of the thyroid cartilage between the anterior commissure of the vocal folds. Then rinse each hemilarynx in PBS for 10 seconds before delicately drying the tissue with a task wiper to reduce ice crystal formation during freezing.

To fix the hemilarynx tissues, place the samples in a centrifuge tube containing 4%formaldehyde and PBS for one hour at room temperature on an orbital shaker at 70 revolutions per minute. At the end of the incubation, transfer the tissues into a new centrifuge tube and rinse the samples three times for 20 minutes in fresh PBS per wash. After the last wash, transfer the tissues into a new centrifuge tube containing a 20%sucrose 5%glycerol solution at four degrees Celsius for up to 18 hours.

When the tissue has sunk to the bottom of the tube, place the hemilarynges into a cryomold filled with optimal cutting temperature medium with the medial surface of the vocal fold facing the bottom of the cryomold in the longitudinal aspect of the vocal fold parallel to the lower edge of the cryomold opening. Flash freeze the submerged tissues in isopentane chilled in a steel beaker surrounded by liquid nitrogen and wrap each mold in pre-labeled foil for minus 80 degrees Celsius storage. To acquire cross-sectional plane sections of the frozen hemilarynx tissue samples, set the cryostat chamber to minus 20 degrees Celsius and set the section thickness to 10 microns.

Transfer the tissue samples to the cryostat chamber and use additional optimal temperature cutting medium to attach one embedded tissue sample to the cryostat specimen disk with the ventral thyroid cartilage facing the cryostat blade and the arytenoid cartilage facing the specimen disk. To trim the cutting medium from the frozen tissue block, advance the specimen head by 100 microns until the ventral portion of the thyroid cartilage appears. Then trim and track 30 micrometer sections from the onset of the thyroid cartilage until the lamina propria, medial thyroid arytenoid muscles, and lateral thyroarytenoid muscle can be observed by light microscopy.

An accurate identification of the transparent tissues during cryosectioning is the most difficult and critical component of this protocol. Once the target muscle has been reached, collect 10 micron sections of the tissue on positively charged slides and store the sections at four degrees Celsius in PBS until they are ready to be stained. To obtain longitudinal vocal fold sections for neuromuscular junction analysis of the thyroarytenoid muscle, affix the specimens to the disks so that the epiglottitis is oriented toward the cryostat blade and the tracheal lumen faces down toward the specimen disk and trim the cutting medium as just demonstrated.

Trim and track 30 micrometer sections from the onset of the thyroid until the lamina propria, and medial and lateral divisions of the thyroarytenoid muscle can be observed by light microscopy. Once the target muscle has been reached, collect 30 micrometer sections onto positively charged slides and store the sections at four degrees Celsius in PBS until they are ready to be stained. In this analysis, the distance between laryngeal landmarks was tracked in both longitudinal and cross-sectional planes using laryngeal muscles in the surrounding cartilages to determine their progression during cryosectioning.

Here, the appearance of laryngeal landmarks during cross-sectional cryosectioning in temporal order with the thyroid can be observed, appearing prior to the medial thyroarytenoid muscle and the lamina propria. Here, the appearance of laryngeal landmarks during longitudinal cryosectioning in temporal order with the alar muscle appearing prior to the medial thyroarytenoid muscle and the lamina propria. Weight and laryngeal landmark appearances had weak to moderate correlations for young rats and weak correlations for aged rats.

The distances among landmarks within each plane were moderately to strongly correlated for both age groups, but weakly correlated between the two dissection planes. The most important thing to remember is that small variations in the tissue embedding and mounting will result in large variations during cryosectioning. These detailed procedures allow reliable neuromuscular histological investigations of the rat vocal fold.