Minimally Invasive Murine Laryngoscopy for Close-Up Imaging of Laryngeal Motion During Breathing and Swallowing

My research investigates abnormal swallowing in neurological diseases across species, including humans, dogs, horses, and rodents. We focus on laryngeal dysfunction, which can lead to aspiration pneumonia and respiratory failure. However, effective prevention and treatment options are yet to be established.

The research gap we are addressing with this protocol is objective measurement of laryngeal function during swallowing in translational rodent models of normal versus impaired laryngeal airway protection. This protocol offers the advantage of longitudinal and highly-detailed examination of laryngeal function and airway protection in the same rodent over the course of disease with or without treatment to enhance our understanding of the pathophysiology of various neurologic diseases and treatment mechanisms. Begin by injecting glycopyrrolate into the anesthetized rodent.

Place it in the preheated induction cage on the warming station and cover the cage with a surgical drape for 10 minutes to minimize visual stimulation. Shave the submental and hip regions of the anesthetized rodent. Transfer it to a custom endoscopy platform.

Then, secure the rodent in dorsal recumbency on a heated platform and stabilize the head in ear bars. Using a surgical tape, secure a respiratory sensor to the rodent's abdomen, immediately coddled to the xiphoid process. Cover the rodent's torso with a transparent film to observe abdominal motion during respiration and facilitate thermo regulation.

Keep the hind limbs and lower abdomen exposed. Disinfect the shaved area with an alcohol wipe. Then, use a 22-gauge needle to create a small opening in the submental skin.

Insert a sterile, concentric EMG needle electrode into the tongue base through the submental skin at the midline. Place a ground electrode subcutaneously at the hip. Now, adjust the respiratory sensor location and DMG needle electrode depth for clean electrophysiology signals in both channels.

Attach the endoscope to a micro manipulator. Place a tapered, cotton-tipped applicator behind the central incisors, perpendicular to the jaw, to open the rodent's mouth. Rotate the cotton swab on the tongue's dorsal surface to slightly protrude the tongue.

Using a light finger grip, gently pull the tongue slightly out of the mouth to one side of the central incisors. Insert the endoscope lateral to the incisors on the same side as the retracted tongue. Then, turn on the light source to avoid harming the rodent size.

Use a microswab to remove visible food particles and excess saliva to reduce aspiration risk. Continue adjusting the endoscope until the hypopharynx is centered on the monitor and key anatomical structures are identifiable. Regularly check the rodent's tongue for signs of ischemia, such as darkened discoloration, every five minutes.

Now, apply light pressure to the velum with a microprobe inserted alongside the endoscope to visualize the larynx from a distance. Advance the endoscope slowly between the velum and epiglottis, keeping the larynx centered in the field of view. Use microlevel adjustments of the endoscope tip to avoid mucosal injury and airway obstruction.

Observe for evoked swallows identifiable as abrupt glottic closure events synchronized with visible jaw depression and laryngeal breathing. Evoke 5 to 10 swallows applying pressure to the laryngeal mucosa to observe the tongue EMG bursting activity and brief apnea visible in the respiratory trace. Carefully retract the endoscope into the oropharynx without removing it, and center the hypopharynx in the field of view to visualize the epiglottis and velum.

To resume nasal breathing, use a microswab to apply light pressure to the tongue base to trigger swallowing and recouple the velum and epiglottis trapping the epiglottis beneath the velar membrane. Afterward, moisten the tongue and central incisors with a saline-soaked cotton swab, and return the tongue to its normal position within the oral cavity. Administer atipamezole to the rodent, followed by manual stimulation along its back and stomach to hasten recovery.

Then, place the animal in a pre-warmed recovery cage on the warming station to recover from anesthesia. Return rodents to their warmed home cage once they can move around spontaneously in the recovery cage. To quantify laryngeal motion, identify at least one representative episode of spontaneous breathing lasting 10 to 20 seconds per animal.

Identify three to five representative swallow events per animal. Endoscopic images from an adult Sprague-Dawley rat showed normal laryngeal motion before and altered motion after transection of the right recurrent laryngeal nerve. Post surgery, the larynx's resting position remained unchanged, but laryngeal asymmetry was noticeable during inspiration and swallowing.

In a healthy rat, electrophysiological recordings showed rhythmic respiratory and EMG patterns during breathing, which were disrupted by swallowing. An expanded analysis of the recordings revealed various outcome measures, including inspiratory phase duration and inter-respiratory interval. Notably, during inspiration, the respiratory trace is delayed for approximately 150 milliseconds compared to the EMG bursting activity, which highlights the temporal differences between the two electrophysiological methods.