Impact of recurrent laryngeal nerve lesion on oropharyngeal muscle activity and sensorimotor integration in an infant pig model

Abstract

The successful performance of a swallow requires dynamic integration between a wide range of sensory inputs and muscle activities to produce the coordinated kinematics of oropharyngeal structures. Damage to the recurrent laryngeal nerve (RLN) produces dysphagia in infants, with food or liquid entering the airway despite this nerve having minimal direct sensory or motor connections to the act of swallowing, apart from vocal fold closure. Previous results have demonstrated that a complete RLN lesion disrupts both performance and kinematics before initiation of the pharyngeal swallow in infants. We tested the hypothesis that a RLN lesion produces changes in the normal activity of oral floor, tongue, and infrahyoid muscles during a swallow. We recorded swallowing in our validated infant pig model, with synchronous high-speed imaging and fine-wire, chronic electromyography. We found changes in the timing, duration, and amplitude of the motor pattern in an array of muscles that are supplied by several different cranial and cervical nerves. Some of these changes in muscle activity are associated with the preparatory aspects of bolus aggregation or movement and so occur before the pharyngeal swallow. Taken with previous biomechanical results, these patterns suggest an intricate brain stem sensorimotor integration that occurs as part of a swallow. In particular, the execution of oral motor function is changed as a result of this simple lesion.

NEW & NOTEWORTHY Damage to the recurrent laryngeal nerve compromises swallowing despite an absent or minimal contribution to either the motor or sensory aspects of this function. This study documents EMG changes, following RLN lesion, to non-RLN innervated muscles that are active during swallowing in an infant model. Some of these muscles fire before the pharyngeal swallow and are associated with the preparatory aspects of bolus aggregation and movement, suggesting important sensorimotor integration at a brain stem level.

INTRODUCTION

In mammals, safe swallows are produced when the bolus of food or liquid moves from the oral cavity through the oropharynx and into the esophagus, avoiding entry into the airway. The successful performance of a swallow requires dynamic integration between a wide range of sensory inputs and muscle activities to produce the coordinated kinematics of cheeks, tongue, palate, pharynx, hyoid, and larynx. The naturally occurring neural activity of the “single sequential motor performance” (36) is triggered primarily by sensory stimuli from the valleculae, the pharyngeal spaces at the base of the tongue, innervated by a branch of the superior laryngeal nerve (SLN) (36, 55). These stimuli trigger a series of motor events, in which pharyngeal muscles fire in a set sequence (52) that in general, although not completely, reflect the order of craniocervical nerves (CN) V3, VII, IX, X, and XII and C1/C2 via ansa cervicalis.

A lesion of the recurrent laryngeal nerve (RLN), a particular branch of CN X, is associated with dysphagia, a swallow where liquid enters the airway, either above or below the vocal folds (35, 42, 48). However, neither the sensory nor the motor components of the RLN directly contribute to bolus propulsion or to a safe swallow. For the sensory components, the RLN provides general sensation of the larynx below the vocal folds. A bolus must already have passed into the airway before a response from this field will be triggered. The RLN supplies all but one of the intrinsic muscles of the larynx and possibly some muscle of the upper esophagus. These muscles are not the primary movers of the bolus in the oral cavity or pharynx but do close the airway. An open airway presents the potential for boluses in the upper airway (penetration) to transit into the lower airway (aspiration). However, previous work indicates that the increase in aspirations following an RLN lesion is not paired with a reduction, or conversion, of penetrations into aspiration. However, in controlled animal studies, a consistent experimentally created RLN lesion produces change in both the early performance and the kinematics of the swallow (21).

The goal of this paper was to determine whether and what disruption there is in the pattern of electromyographic activities following a controlled RLN lesion. The null hypothesis is that an RLN lesion will not impact any muscle function beyond those directly supplied by the RLN. However, our previous results demonstrated changes in oropharyngeal performance and kinematics before the swallow initiation (21–23), while the bolus is still in the valleculae before it is moved over the laryngeal opening and into the esophagus. This suggests an alternative hypothesis that muscles important to bolus control and movement, including those that are active at the beginning of the swallow, before the bolus moves out of the valleculae, will be affected by this lesion. In particular, we hypothesized that changes would be seen in the activity of muscles innervated by CN V3 (mylohyoid), CN VII (stylohyoid), CN X (cricothyroid), CN XII (genioglossus), and ansa cervicalis (geniohyoid, sternothyroid, and thyrohyoid). These muscles have been shown to be important and active both in a normal swallow and in a decerebrate swallow in this infant pig model (14, 46). The muscles included in this study represent three groups of muscles critical to the kinematics and biomechanics of swallowing: 1) oral floor/suprahyoid, 2) tongue, and (3) infrahyoid/strap muscles (18, 52–54). Because electromyographic (EMG) output in general is a measurement of the signal from the central nervous system (CNS) to peripheral nerves to target muscles, specific changes in efferent output can be tested as changes in EMG signals.

The design of the experiments, paired, prelesion (control) vs. post-lesion (treatment), used to generate the results reported here permit testing of the null hypothesis that this lesion has no impact on non RLN innervated muscles involved in swallowing. Because it is a unilateral lesion, we propose an additional null hypothesis: that there will be no change in any left-right EMG signal differences following the lesion. The alternative hypothesis, that a left-right change occurs, suggests that the lesion will impact one side (lesioned or nonlesioned) differentially.

MATERIALS AND METHODS

Animals and surgical procedures.

In accordance with the Northeast Ohio Medical School (NEOMED) Institutional Animal Care and Use Committee (13-011), experimental procedures were followed as outlined in previous studies (21, 22, 54). Seven infant pigs (Sus scrofia Yorkshire; Michael Fanning Farm, Howe, IN) were obtained at ages 3–5 days and trained to drink pig formulated barium milk (Solustart Pig Milk Replacement; Land o’ Lakes, Arden Mills, MN) from a nipple bottle (NASCO Farm & Ranch, Fort Atkinson, WI). These infants ranged in weight from 2.0 to 2.8 kg at the start of the study; weight was monitored throughout the study. In an initial surgery under general anesthesia (2–5% isofluorane) and aseptic conditions, oral markers and duplicate bipolar electrode wires were placed, via a midline incision bilaterally under direct vision in each of the following muscles: mylohyoid, stylohyoid, cricothyroid, genioglossus, geniohyoid, sternothyroid, and thyrohyoid. The intralaryngeal muscles were excluded because of the difficulty in reaching them with this surgical approach and the subsequent impossibility of verifying functional (vocal) loss in swallowing in this animal model. The standard human procedures involve an awake human with subject confirmation of sensations or functions (5, 41); this is not possible in an animal model. The right recurrent laryngeal nerve (RLN) was identified immediately below the larynx and marked with a loose suture (21). Reported variations in RLN anatomy and configuration, including a nonrecurrent nerve, or branches between the RLN and SLN, were not seen (12), and their absences were confirmed post mortem.

As previously described (31, 54), each electrode was connected to a microconnector, and all collectively exited through a midline incision. Microconnectors were connected to standard 25-pin D-connectors outside the body. The cables were secured with Vetwrap to prevent disconnection and animal injury. Shortly after surgical recovery (pre-lesion), the animals were fed unrestrained while electromyographic signals (EMG) were amplified and recorded at 10 kHz using an MA-300 EMG System (Motion Laboratory Systems) with a band pass of 20 Hz–2 kHz and a 60-Hz notch filter. The signals were subsequently recorded on a 16-channel Powerlab 30/16 (AD Instruments, Colorado Springs, CO). Simultaneously, animals were filmed, while feeding until satiation, in front of biplanar C-arm fluoroscopes fitted with high-speed video cameras. Video data were collected for another study (21, 22) but were used to confirm the occurrence of swallows in the EMG data. Two to six feedings were recorded before the second surgery. After 2–3 days, each animal underwent a second surgery, where the right RLN was ligated. The nerve was located as it ascended the trachea, before entering the larynx. A 1- to 3-mm section was removed, and the free ends were displaced. The ends of the nerve were capped with two microhematological clips to prevent any potential regrowth. In some cases, before the animal awakened from anesthesia, the larynx was observed with a flexible veterinary laryngoscope. In all observed cases, the right vocal fold was paralyzed. However, this proved to be a difficult procedure, given the complex anatomy of the pig larynx (9, 10).

After lesion surgery, animals were again fed as previously described during EMG recording. Due to the loss of function of some electrodes, each animal had a variable number of post-lesion recordings, but all had a minimum of two post-lesion recordings. At the conclusion of the experiment, the animals were euthanized. Lesion and electrode placement were confirmed post mortem by a person different from the surgeon who implanted the electrodes.

Choice of muscles.

The muscles selected for this study were dictated by several different factors. First, we were technically limited to eight muscles (left and right) per animal, given a 16-channel EMG recording system. Second, given the nature of the surgery to implant electrodes, and concern for the animals, plus the length of surgery and associated damage, we selected muscles that were accessible without damaging any cartilaginous structures, neurovascular structures, or other muscles (50, 52, 54). This ruled out placing electrodes into any intrinsic muscles of the larynx, except for cricothyroid. The other factors involving choice of muscles was a function of the hypotheses we proposed to test. Muscle activity can be interpreted in two ways. It is a measure of the muscle activity that generates the kinematics and movements of the oropharyngeal structures. But, EMG is also a “read-out” of the central nervous system’s signals to the periphery. For the latter, we wished to see if muscles involved in swallowing, as determined by previous work (50, 52), and supplied by a variety of different cranial and spinal nerves, are impacted by the disruption to the RLN. Thus, we chose both supra- and subhyoid muscles, as well as a muscle of the tongue.

Experimental design.

The sample comprised seven infant pigs (4 female, 3 male). Previous work using similar EMG techniques in infant pigs of similar ages found that even three animals, with sufficient sampling of swallows nested within sequences nested within individual, provided a sufficient sample to detect differences among muscles and treatments (17, 18, 52–54). The unit of analysis for this study was the sequence, with swallows nested within sequences. A sequence was a single feeding bout by an animal, containing a variable number of swallows, ranging from 30 to 200. Sequences were selected that contained swallows that were free of EMG artifacts and obvious noise. Each animal had independent bipolar electrodes placed in up to eight muscles, duplicated on right and left. However, because some electrodes failed over time, and others proved to be noisy, the number of sequences of acceptable data varied per muscle, ranging from 6 to 68, with an average of 45.2. Muscle recordings with inadequate EMG signal were excluded from analysis. Data from the beginning (high swallow frequency) and the end (low swallow frequency) of each feeding sequence were also avoided (20). A set of 10–25 swallows, from muscles showing EMG activity, were selected from each feeding sequence and used for analysis. An average burst of rectified, constant time (10 ms) reset integrated EMG signals, using the set of swallows from a sequence, was calculated, so that sequence, nested within individual, was the unit of analysis for all statistical analyses. The use of an “average” burst is standard in fine-wire EMG studies because it produces more normally distributed data and is more reflective of the underlying physiology (17, 43). The sample included a total of 54 sequences, 19 pre-lesion and 35 post-lesion. Because not all sequences contained valid signals for all muscles, the sample size for each response/dependent variable for each muscle varied, but the total is reported with the results. Multiple sequences were recorded pre- and post-lesion in each animal. The design was a paired/repeated-measures design, with each individual serving as its own control (pre-lesion data) with subsequent recordings made post-lesion (27, 28, 56). Methods for analysis were modified from the previous EMG swallowing studies used to create this model system (51–54). Using a custom-designed computational program, Sequencer, available from the senior author (51), amplified and bandpass filtered EMG signals were rectified and integrated and then assessed for integrity of signal, e.g., suggestive of broken electrode, etc. Additionally, background noise was eliminated with a sequential, statistically defined threshold (49). These data were exported to a text file, and a separate Matlab (Mathworks) script was then used to determine the peak muscle activation for each swallow within a 400-ms window of analysis (timed relative to the consistent left thyrohyoid EMG signal, which was the indicator of swallowing). If a sequence did not contain the same active left thyrohyoid channel throughout all feeding terms, the sequence was eliminated from analysis. Three response variables were extracted from each EMG signal, as defined below.

Statistical analysis.

We calculated and tested three response or dependent variables: 1) relative timing of muscle activation (in ms), 2) duration of muscle activation (in ms), and 3) value of peak amplitude of activation (7, 54). Relative timing was onset of muscle activity relative to the peak activity of the left (non-lesion side) thyrohyoid signal. The thyrohyoid was selected as the timing indicator because it is the most consistent signal with the smallest within-electrode variation of all muscles in previous studies of intact animals (7, 18, 54). Alternatives to this included hyoglossus or mylohyoid, both of which are part of the “leading complex” in both intact and decerebrate models (18). However, the hyoglossus is difficult to record from reliably, and, as is also true of the mylohyoid, it has a variable signal over a sequence (18).

Duration of activation was the length of time that muscle activation was at least 20% of peak activity. Peak amplitude was a percentage of the highest burst of that electrode (muscle-side-animal) recorded, so that all muscles were scaled to their maximum EMG burst levels. Three variables were calculated for each of the seven pairs of muscles. Values of EMG signal duration, timing, and amplitude were averaged over the 10–25 swallows per sequence. The unit of analysis was sequence-day-animal. Thus, sequences were nested within days that were nested within individual animals. This is a more statistically conservative analysis than using an individual swallow as unit of analysis (17, 39, 43). Because there were over 1,000 swallows, divided among seven animals, the results could be liable to pseudo-replication and type I error. Although the model leaves us open to type-II errors and a lack of power (17), it biases the results against the hypothesis of differences between pre- and post-lesion, which we determined to be an acceptable bias.

We used a standard variance components mixed model (39), including 1) individual as a random factor, 2) treatment (pre-lesion/post-lesion), and 3) side (left/right) as fixed factors, with a side × treatment interaction [SYSTAT (57)]. Frequently, side and the interaction were not significant, and the model was rerun excluding those terms.

RESULTS

Timing of peak activation.

The timing of peak muscle activation (Fig. 1A and Table 1) relative to the left thyrohyoid peak was significantly different post-lesion for those muscles innervated by CN VII (stylohyoid), CN XII (genioglossus), and C1, C2 (sternothyroid) (P < 0.01, Table 2 specific test values). Significant differences between left and right side muscle timings occurred in genioglossus (CN XII) and sternothyroid (C1, C2). Sternothyroid showed significant timing differences between sides and also between lesion/control. The variation among individuals (Table 1) was accounted for as a random factor; Fig. 1B shows the variation in timing specifically of genioglossus in different individuals because this muscle had a large amount of variation.

Fig. 1.

A: timing of peak muscle activation relative to thyrohyoid activation. RLN, recurrent laryngeal nerve. There were significant differences between pre-lesion electromyographic (EMG) signals (gray) and post-lesion (open) in genioglossus (innervated by CN XII), stylohyoid (innervated by CN VII), and sternothyroid (innervated by ansa cervicalis). B: variation in timing of peak genioglossus activity in 4 individual animals. Relative to other muscles (A), the timing of genioglossus does not appear different between control and lesion. However, there is significant variation among individuals. The complete linear model, which includes this variation, using individual as a random factor, indicates that the differences between control and lesion were significant.

Table 1.

Means ± SE for timing of muscle activity by muscle and side

		Pre-Lesion Right Side, ms		Pre-Lesion Left Side, ms		Post-Lesion Right Side, ms		Post-Lesion Left Side, ms
Muscle	Number of Sequences	Mean	SE	Mean	SE	Mean	SE	Mean	SE
Stylohyoid	25	−16.25	0.47	−13.91	3.05	−20.34	4.57	−29.64	2.78
Thyrohyoid	54	23.77	8.30			14.31	3.89
Geniohyoid	58	−78.60	14.47	−80.11	9.59	−87.82	11.97	−81.44	9.76
Genioglossus	34	−76.42	20.59	−99.31	9.39	−92.16	13.13	−94.85	9.55
Mylohyoid	14	−44.32	10.91	−44.71	6.77	−47.67	2.46	−45.35	3.63
Cricothyroid	39	82.76	10.44	80.99	7.79	92.93	8.57	83.68	5.90
Sternothyroid		70.63	18.34	50.29	3.62	106.87	5.85	79.15	9.34

Table 2.

Differences in timing of muscle activity

	Random Effect	Treatment Differences		Side Differences		Treatment and Side Differences
Muscle	A$, Error	F ratio	P value	F ratio	P value	F ratio	P value	Degrees of Freedom
Stylohyoid	314.18 39.09	9.42	0.01	1.02	0.32	2.42	0.13	1,24
Thyrohyoid	343.20 816.09	2.71	0.10	Right side only				1,53
Geniohyoid	2,027.22 598.91	0.210	0.64	0.773	0.38	0.18	0.66	1,57
Genioglossus	0.00 122.03	4.31	0.04	4.34	0.04	0.27	0.60	1,33
Mylohyoid	152.75 24.55	0.099	0.75	1.81	0.20	2.07	0.17	1,13
Cricothyroid	1,356.26 3,907.09	0.083	0.77	3.88	0.05	1.19	0.28	1,38
Sternothyroid	1,446.80 167.65	27.93	0.00	14.89	0.00	17.61	0.00	1,13

Duration of muscle activation.

The duration of muscle activation (Fig. 2 and Tables 3 and 4) during a swallow was significantly (P < 0.02) different post-lesion in muscles innervated by CN V3 (mylohyoid), CN VII (stylohyoid), and ansa cervicalis (thyrohyoid). The only significant difference between the duration of left and right muscle activation was in geniohyoid. There were no significant interactions.

Fig. 2.

Duration of muscle activation during a swallow cycle. Comparison of control data (gray) to post-lesion data (open) in the same individuals indicates statistically significant differences observed in mylohyoid (innervated by CN V3), stylohyoid (innervated by CN VII), and thyrohyoid (innervated by C1 and C2 of ansa cervicalis).

Table 3.

Means ± SE for duration of muscle activity by muscle and side

		Pre-Lesion Left Side, ms		Post-Lesion Left Side, ms		Pre-Lesion Right Side, ms		Post-Lesion Right Side, ms
Muscle	Number of Sequences	Mean	SE	Mean	SE	Mean	SE	Mean	SE
Stylohyoid	27	141.08	6.93	171.68	3.23	146.47	5.48	156.05	7.80
Thyrohyoid	43	118.07	4.99	130.70	4.56	126.38	5.82	139.43	5.06
Geniohyoid	47	93.31	6.74	108.33	9.39	122.99	12.37	120.49	12.08
Genioglossus	62	95.00	5.63	109.17	7.79	102.19	6.62	105.85	5.47
Mylohyoid	24	113.17	7.54	128.00	7.05	111.35	8.80	142.71	7.60
Cricothyroid	52	159.33	12.50	161.96	8.27	119.60	5.07	150.10	8.00
Sternothyroid	14	125.56	12.70	117.01	6.36	126.18	13.40	133.50	8.66

Table 4.

Differences in duration of muscle activity

		Treatment Differences		Side Differences		Treatment and Side Differences
Muscle	Random Effect Error Variance	F ratio	P value	F ratio	P value	F ratio	P value	Degrees of Freedom
Stylohyoid	63.07	8.33	0.01	0.04	0.84	1.85	0.18	1,26
	250.73
Thyrohyoid	956.72	5.13	0.02	0.73	0.39	0.044	0.83	1,42
	385.60
Geniohyoid	1,259.16	0.23	0.63	7.69	0.01	0.546	0.46	1,46
	781.77
Genioglossus	560.29	1.33	0.25	0.33	0.56	0.06	0.79	1,61
	338.40
Mylohyoid	0.0001	6.80	0.02	0.52	0.47	0.870	0.36	1,23
	489.44
Cricothyroid	826.54	2.56	0.11	2.76	0.10	2.33	0.13	1,51
	779.29
Sternothyroid	214.53	0.26	0.61	0.14	0.71	0.03	0.87	1,13
	369.89

Peak amplitude.

The peak EMG amplitude relative to the maximal values in the sequence data was significantly different in two muscles, sternothyroid and thyrohyoid (Fig. 3 and Tables 5 and 6). Stylohyoid (innervated by CN VII) showed significant differences in the side and treatment interactions. The only difference between left and right side amplitude was in genioglossus. However, significant differences were observed between right side control values and right side lesion values. Sternothyroid, innervated by ansa cervicalis, displayed significant differences between side and treatment interactions and overall treatment effect. Again, no differences were seen between left side control and lesion values, but significant differences were observed between right side control values and lesion values. No differences were found between left and right side control samples for stylohyoid and sternothyroid. Significant differences were, however, seen between left and right sides post-lesion.

Fig. 3.

Scaled peak amplitude relative to maximal sequence burst. Comparison of control data (gray) to post-lesion data (open) from the same individuals indicates statistically significant differences in stylohyoid (innervated by CN VII) and sternothyroid (innervated by ansa cervicalis).

Table 5.

Means ± SE for scaled amplitude of muscle activity by muscle and side

		Pre-Lesion Left Side		Post-Lesion Left Side		Pre-Lesion Right Side		Post-Lesion Right Side
Muscle	Number of Sequences	Mean	SE	Mean	SE	Mean	SE	Mean	SE
Stylohyoid	28	0.66	0.04	0.62	0.03	0.57	0.05	0.68	0.04
Thyrohyoid	46	0.50	0.05	0.50	0.04			0.50	0.04
Geniohyoid	46	0.39	0.05	0.43	0.03	0.31	0.04	0.39	0.05
Genioglossus	53	0.53	0.04	0.45	0.05	0.35	0.04	0.33	0.04
Mylohyoid	24	0.51	0.03	0.38	0.05	0.51	0.06	0.49	0.05
Cricothyroid	50	0.59	0.03	0.57	0.04	0.50	0.08	0.63	0.03
Sternothyroid	13	0.52	0.00	0.53	0.05	0.73	0.03	0.28	0.02

Table 6.

Differences in scaled amplitude of muscle activity

		Treatment Differences		Side Differences		Treatment and Side Differences
Muscle	Random Effect (Error Variance)?	F ratio	P value	F ratio	P value	F ratio	P value	Degrees of Freedom
Stylohyoid	0.009	0.62	0.440	2.74	0.11	7.54	0.01	1,27
	0.009
Thyrohyoid	0.037	4.67	0.03					1,45
	0.016
Geniohyoid	0.001	1.71	0.19	2.30	0.13	0.18	0.66	1,45
	0.023
Genioglossus	0.018	0.58	0.44	5.95	0.02	0.24	0.62	1,52
	0.018
Mylohyoid	0.007	2.13	0.15	1.47	0.23	1.19	0.28	1,23
	0.013
Cricothyroid	0.001	1.59	0.21	0.13	0.71	3.51	0.06	1,49
	0.025
Sternothyroid	0.001	12.73	0.00	0.15	0.70	13.99	0.00	1,12
	0.014

DISCUSSION

Testing the effect of a lesion of the RLN on swallowing.

Unilateral damage to the RLN impacts the behavior and performance of swallowing, both in human pediatric patients who have sustained injury (35, 42) and in controlled animal models with a complete lesion (21–23). The results from these animal studies detail the impacts of such a lesion on performance and kinematics in a pediatric model (21–23). The changes in penetration and aspiration are not a simple result of vocal fold paralysis or an open airway. Following RLN lesion, there is an increase in the frequency of aspiration. If the increase in aspiration occurred passively, this was due to a passive conversion of liquid that penetrated the upper airway merely transiting through, that is, liquid that had penetrated the upper airway merely through the open folds into the lower airway, then we would see a concomitant drop in the number of penetration events. On the other hand, what is observed is an increase in aspiration, no change in frequency of penetration, but a decrease in the frequency safe or normal swallows. There are also changes in how the bolus is handled at different times during the swallow (21, 23). A swallow consists of several integrated kinematic events, as well as preparatory intraoral transport occurring before the swallow stages (24, 29, 30, 34). The initiation of the pharyngeal stage of the swallow is the propulsion of the bolus out of the valleculae, through the oropharynx and piriform sinuses, and into the esophagus, and is concurrent with the onset of thyrohyoid EMG activity (18, 24, 52). Changes following RLN lesion in performance, timing, and kinematics of oropharyngeal behavior occur not only during the swallow but also before this initiation. Gould et al. (23) found differences in tongue shape, tongue movement, and shape and size of the bolus. Finally, the relative timing of tongue movements changes with this lesion (19).

The results presented in this paper reject the initial null hypothesis that the RLN lesion will only impact those immediate structures and fields that it supplies directly. In supporting the alternative hypothesis, these EMG results extend the implications of the changes that occur in performance and kinematics following RLN damage. As was true of kinematics, the changes in EMG activity from pre-lesion to post-lesion were evident both before swallow initiation and during the time of bolus transit. Furthermore, these changes occurred in muscles that are not directly controlled by the RLN, although they did occur in the cricothyroid muscle supplied by the superior laryngeal nerve (SLN).

Potential underlying mechanisms for dysfunction caused by RLN lesion.

The results herein extend our understanding of the underlying mechanisms of the dysfunction caused by RLN lesion. Our previous work documented the performance deficits (21) and changes in kinematics (22). The value of the results in this paper lies first in understanding the muscle activity that generates kinematics as well as in assessing the changes in CNS activity that alter the motor signals (19). If the changes in EMG signals were only in the infrahyoid musculature, which is active as or after the bolus passes over the laryngeal opening, then it is possible that the changes could have resulted from sensory feedback from the kinematics of the swallow. However, the altered motor output in the lesion model includes even earlier changes in the activity of muscles that are important for propelling the bolus out of the oral cavity or out of the valleculae at the base of the tongue. These actions occur before the pharyngeal portion of the swallow, before any aspiration might occur. This is consistent with other data indicating that changes in tongue function and bolus shape associated with the lesion occur before the beginning of the pharyngeal swallow (23).

Another potential mechanism for these changes lies in feed-forward models of neural control of swallowing (32, 33). Wong et al. (58) suggest that in adult humans laryngeal elevation, an important component of swallowing, is responsive to changes in airway opening before the swallow. They conclude that internal sensorimotor adaptation occurs in response to changing demands for airway protection. The work of Gross and colleagues (25, 26) also demonstrates the impact of subglottal air pressure on swallowing, suggesting that these sensory changes could impact the safety of swallowing in adult humans.

The classical effect of simple lesion or section of a mixed nerve is the loss of sensory input to the CNS and paralysis of the striated muscle supplied by that nerve. However, more recent research indicates that a much more complicated situation can arise in the CNS after peripheral nerve damage. A peripheral lesion can have multiple upstream impacts. These include degenerative atrophy of substance P sensory input and of met-enkephalin interneurons in the dorsal horns (11), increased density of microglia, which contributes to the abnormal sensations arising from nerve damage (3, 4), local increases in the permeability of the blood-brain barrier to inflammatory mediators independently of the microglia (16), synapse stripping from motoneurons (47), and, in the adjacent territories of the central terminations of uninjured nerves, both up- and downregulation of neuroactive peptides in the dorsal horn of the spinal cord (44, 45). Any of these mechanisms, operating at a brain stem level, has the potential to affect multiple peripheral nerves. These results suggest numerous central locations that could be impacted by the RLN lesion.

Within- and among-individual variation in swallowing.

The results presented here, together with the earlier results on kinematics and behavior, contain a significant amount of interindividual variation. Many of these individuals were siblings, and significant efforts were made for consistency in the surgical procedures. The approach, the insertion of electrodes, and the specific site of lesion were the same in all animals. The changes following RLN lesion, however, were variable across muscles. These changes included different responses to lesion in timing, duration, and amplitude of the EMG signal as well as changes in left-right differences among muscles. Even the polarity of change varied, in that within one muscle some individuals showed a longer duration of activity but others had a shorter duration. These differences most likely resulted from one of several potential biological explanations.

Variation in peripheral innervation patterns, including a variable connection between SLN and RLN, which could be a pathway where some proprioception from the laryngeal muscles could be maintained despite the complete lesion of the RLN (37, 40, 46), may be relevant. Although we found no evidence of such connections in these animals, it could possibly exist more proximally within the vagus nerve or even within the brain stem. Another potential source of variation is simply developmental variation that exists at any single age in an infant or juvenile mammal. Because brain stem central pattern generator (CPG) networks and their neuromuscular targets attain functional status at different rates, this also influences cross-system interactions among individual CPGs (2, 6). Thus, there is likely an interaction between lesion effect and maturation that is not fully accounted for in this model. The comparison of these results with results from adult pigs would clarify the role of neurological development in the variable response of infant pigs to identical lesion.

Implications for central control of swallowing.

Considering the EMG signal as a proxy for motor nerve activity, these results support the finding that the RLN lesion has a more extensive impact than would be expected from the immediate and direct effects on its sensorimotor fields. The sensory axons in the RLN (and other craniocervical nerves) enter the CNS via a complex pathway, where the signals pass via groups of interneurons and premotor neurons to the motor neurons (36). These signals activate the complex of muscles that produce the various movements of the functional swallow, including a wide range of supra- and infrahyoid muscles, as well as tongue and palate (18, 52). Currently, information exists on both sensory and motor ends of this process with limited understanding of the central pathways and processes.

On the sensory input side, there are two routes by which the RLN might impact swallowing behavior. First, the general sensation below the vocal folds may be important for detecting aspirated fluid in the trachea, signaling that a previous swallow was defective. Second, proprioception from the intrinsic muscles of the larynx (which, except for cricothyroid, provide the last barrier against fluid entering the larynx) may provide critical information for any subsequent swallows. On the motor side, the intrinsic muscles of the larynx, supplied by the RLN are responsible for the closure of the airway.

However, it should be recognized that peripheral nerve damage does not cause only loss of afferent information and/or motor outflow. Initiated by the injury, there is also a cascade of events at the molecular, cellular, and system levels, which progresses through plastic changes at spinal cord, brain stem, thalamic, and cortical levels (38). Furthermore, axotomy does not simply equate to loss of central excitation, because axotomy may equally well reduce synaptic excitation of inhibitory neurons (8). This suggests that there could be further and less obvious sequelae of RLN damage yet to be identified, in part because both the complex interneuron connections in the brain stem and also the sequential nature of the waves of excitation and inhibition of successive interneuron groups that culminate in a swallow (23) are poorly understood.

The results presented here increase our understanding of the role of afferent RLN fibers in generating a normal swallow. Although these results do not pinpoint a specific mechanism for the increase in aspiration frequency following lesion, the wide range of muscle activities that are changed supports the hypothesis that central brain stem integration is involved. This is both similar to and different from the impact of a purely sensory lesion of the SLN, which functions as a trigger for the swallow (1, 55). With either a unilateral or a bilateral SLN lesion, the frequency of aspiration increased (13, 15). Some of the increase was likely due to a simple failure to appropriately initiate the swallow (13, 15). However, these results, as is true of the results from the RLN lesion, also suggest that there is a more complex integration among the timing of different oropharyngeal elements responsible for a successful swallow (13, 14). More work is necessary to determine the exact sensorimotor interactions that are required for a normal swallow and how the absence of specific sensory components impacts that integration.

GRANTS

This work supported by National Institutes of Health Grants DC-9880 and HD-88561 to R. Z. German.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.R.D., F.D.H.G., A.J.T., and R.Z.G. conceived and designed research; K.R.D., F.D.H.G., J.O., and R.Z.G. performed experiments; K.R.D., F.D.H.G., J.O., and R.Z.G. analyzed data; K.R.D., F.D.H.G., A.J.T., and R.Z.G. interpreted results of experiments; R.Z.G. prepared figures; K.R.D., F.D.H.G., and R.Z.G. drafted manuscript; K.R.D., F.D.H.G., A.J.T., and R.Z.G. edited and revised manuscript; K.R.D., F.D.H.G., J.O., A.J.T., and R.Z.G. approved final version of manuscript.