Acta Oto-Laryngologica. 2014; 134: 318–325
ORIGINAL ARTICLE
Changes in electrical response function and myosin heavy chain isoforms following denervation and reinnervation of bilateral posterior cricoarytenoid muscles in dogs
JINGYUAN LI1*, SHAOFENG LIU2*, QIUHUI CHENG1, MINGRONG NIE2, SIYI ZHANG1, XIAOLI SHENG1, SHAOHUA CHEN1 & PINGJIANG GE1 1
Department of Otolaryngology, Guangdong General Hospital & Guangdong Academy of Medical Sciences, Guangzhou city and and 2Department of Otolaryngology, Guangzhou Women and Children’s Medical Center, Guangzhou, China
Abstract Conclusions: Both electrical response function and mRNA expression of myosin heavy chain (MyHC) types 2X, 1, and Neonatal of bilateral posterior cricoarytenoid (PCA) muscle changed after denervation or reinnervation in canines. Objectives: There is a need to investigate the electrical response function MyHC alteration of denervation or reinnervation in the bilateral PCA muscle of large animals. Methods: MyHC isoforms expression profile and PCA muscle function outcome were detected by real-time reverse transcribed-polymerase chain reaction and muscle response to functional electrical stimulation, 9 weeks after denervation and reinnervation with ansa–recurrent laryngeal nerve anastomosis in dogs. Results: Denervation produced up-regulation of MyHC-1 and MyHC-Neonatal messenger ribonucleic acid (mRNA) expression. Reinnervation caused a decrease of MyHC-2X mRNA expression. The electrical voltage threshold of vocal fold movement and maximum abduction of denervation were greater than that of the reinnervated or control group. The denervated vocal abduction maximum of response to electrical stimulation was less than that in reinnervation or control groups.
Keywords: Electrical stimulation, regeneration, recurrent laryngeal nerve, canine, anastomosis, abduction
Introduction Laryngeal dysfunction occurs after denervation of the recurrent laryngeal nerve (RLN). Unilateral paralysis may result in transient or permanent dysphonia. For bilateral vocal folds paralysis, the loss of abductor function of bilateral posterior cricoarytenoid (PCA) muscles results in a life-threatening condition that often requires an emergency intubation tube or tracheotomy. Current treatment options for laryngeal paralysis are far from ideal. Although direct nerve anastomosis techniques result in nonselective reinnervation of adductor or abductor muscle and produce an uncoordinated, synkinetic movement of vocal folds, the nerve anastomosis technique can regain the bulk and tension of the vocal fold. This technique appears to be appropriate for
clinical applications, such as ansa cervicalis to the RLN in patients with unilateral paralysis [1]. Because the denervated PCA muscle was still 10–25% as responsive to stimulation as the innervated unilateral PCA muscle, the application of paced muscle electrical stimulation results has been favorable in animals and preliminary clinical trials using an implantable commercial stimulator [2]. Bilateral PCA muscle reinnervation may be more effective than unilateral PCA muscle denervation on vocal fold movement under the pace stimulation. A denervated muscle undergoes a rapid decline in mass and a change in muscle fiber phenotype [3,4]. Following reinnervation, there was a trend for fast-type fiber diameter and fiber phenotype expression to normalize [5]. Most of the investigator’s interest has been focused on myosin because of its central role in the
Correspondence: Pingjiang Ge and Siyi Zhang, Department of Otolaryngology, Guangdong General Hospital, 106, Zhongshan 2 Road, Guangzhou city 510080, China. Tel: +86 13751753465. Fax: +86 20 34242560. E-mail: [email protected] *These authors contributed equally to this study.
(Received 24 September 2013; accepted 24 October 2013) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2013.860657
PCA muscle electrical response and MyHC following reinnervation contractile process and because different isoforms of myosin give rise to variations in contractile properties and fiber types [6]. Skeletal muscles are composed of different types of myofibers. Six major myosin heavy chain (MyHC) phenotype genes have been identified in the skeletal muscle encoding 1, 2A, 2B, 2X, Neonatal, and extraocular protein of adult mammals. Electrophoresis in laryngeal muscles revealed the presence of the isoforms expected on the basis of reverse transcription polymerase chain reaction (RT-PCR). Five bands were detected in PCA muscle (1, 2A, 2X, 2B, and Neonatal) [7]. In the denervated condition, the changes of MyHC composition in laryngeal muscle include an increase and decrease in type 2A, 2X, and 2B, and a minimal change in type 1 MyHC. The reinnervation of muscle changes the MyHC phenotype of denervated muscle. The expression of normal MyHC composition is dependent on the condition of appropriate neural contact and functional reinnervation in rats [3,8]. The nerve transaction and repair also results in persistent alteration of MyHC composition and vocal fold dysfunction. Muscle reinnervation demonstrated by electromyography rarely achieves normal functional activity in rats [3,8]. There has been no study of the relationship between electrophysiological function and myosin in canines, which have a larger laryngeal muscle. The present study had two primary objectives. The first was to test the hypothesis that MyHC phenotype transition in canine bilateral PCA muscle denervation and reinnervation was similar to the results observed in unilateral PCA muscle paralysis studies, or in studies of other mammalian species. The second objective was to characterize the effects of denervation or reinnervation on the functional outcome of canine bilateral PCA muscles. Material and methods Animal care and model Approval was obtained from the Guangdong General Hospital Review Board before conducting this study. Twenty-four mongrel dogs (weight 14.4 ± 1.0 kg) were used in this study. They were randomly assigned to one of three groups: (1) control, (2) denervated, or (3) reinnervated. Animals were humanely euthanized 9 weeks after denervation or reinnervation. Induction of anesthesia was achieved using ketamine (35 mg/kg) administered intramuscularly. Heart rate, temperature, and oxygen saturation levels were monitored throughout the experiment to monitor the animal’s level of anesthesia and general well-being. Following laryngoscopy, an endotracheal tube was put into place. Subsequent intravenous drips of diazepam
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(20 mg/kg), atropine (1 mg/kg), isopropylphenol (15 mg/kg), and fentanyl (20 mg/kg) were provided as needed to maintain a surgical anesthetic plane. Animals were placed in a supine position on an operating platform. The neck was shaved and prepared for surgery from the level of the submentum down to the chest. The larynx and trachea were exposed using a midline neck incision extending from the hyoid bone to the sternal notch. A 2 cm section of each RLN was removed from the trachea region. The resected ends of the RLN were folded back on the remainder of the nerve and tied with a 3-0 suture in the denervated group [9]. The wound was closed in layers after the trachea and the RLN were exposed in the control group. The surgical technique of reinnervation was as follows. The sternocleidomastoid muscle was reflected posteriorly to expose the omohyoid muscle. Mobilization of the omohyoid muscle was used to identify the ansa cervicalis overlying the internal jugular vein. The ansa cervicalis branches to the sternohyoid and sternothyroid muscles were identified and transected on the lateral aspect of these strap muscles. They were freely mobilized in preparation for anastomosis. The resected laryngeal ends of the RLN were anastomosed to the ansa cervicalis branches with four or five epineurial stitches of nylon 11-0 thread. The wound was then closed in layers [10]. PCA muscle stimulus-response activation The PCA muscle activation was measured 9 weeks after denervation or reinnervation. Seven animals were assigned to each group. With each animal in a supine position, a midline neck incision was made. After thyrohyoid membrane incision, the posterior surface of the bilateral PCA muscle and vocal folds were exposed through the pharyngeal cavity. The RLN trunk was then stimulated with hook electrodes (2 ms pulse duration, 5 V; BL-420s, TaiMeng, Chendu, China) to check that no abductor branches remained intact. Reinnervation was confirmed at the termination of the experiment by the presence of vocal fold motion evoked by RLN stimulation. Vocal fold abductions elicited by PCA muscle stimulation were measured by superimposing a grid, which was calibrated with a 4 mm ruler, on the vocal fold. Both stimulation needle electrodes were positioned into the upper and lower end of the PCA muscle for electrical stimulation. The distance between anode and cathode stimulation electrodes was 1 cm. Identity of RLN reinnervation was confirmed by the presence of vocal fold movement associated with electrical stimulation. The pattern of electrical pulse delivery (biphase, 10 ms pulse duration, 40 pps, 5 s train interval time, 3 s on and 2 s off) was used to study the characteristics of PCA muscle
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stimulus-response [2]. The amplitudes of the pulses were increased in a stepwise fashion (0.1 V) using the pulse generator (BL-420s, TaiMeng). The pulse amplitudes were obtained from thresholds of movement and complete dynamic PCA muscle response. The distance of vocal movement from complete dynamic PCA muscle responses was also measured in each animal [2]. The tissue specimens of muscle were collected using 2 mm diameter cup forceps after the measurement of PCA muscle stimulus-response activation. Total RNA isolation A 1 mm tissue specimen of muscle was obtained from the middle portion of the PCA muscle bilaterally from denervation, reinnervation or control animals, immediately following data collection. The specimens were quickly submerged in RNAlater Stabilization Reagent (Qiagen Inc., Valencia, CA, USA), incubated at 4 C overnight, and stored at –80 C until extraction. Animals were euthanized without recovery from anesthesia after collection of specimens. Tissue specimens were placed in 120 mg zirconia/ silica beads (1 mm diameter) and homogenized at 4800 rpm for 90 s with the use of a Mini-Beadbeater homogenizer (BioSpec Products, Inc., Bartlesville, OK, USA). We used the RNeasy Fibrous Tissue Mini Kit (Qiagen Inc.) to extract the mRNA from tissue specimens. The total mRNA was stored at –80 C. Real-time RT-PCR Reverse transcription was performed with the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). The real-time PCR was
performed using iQTM SyBR Green Supermix Kit (Invitrogen Corp., Carlsbad, CA, USA) in a 50 ml volume reaction mixture composed of 500 nmol/l primer one, 500 nmol/l primer two, 25 ml iQ SyBR Green Supermix, and 1.2 ml template complementary DNA ribonuclease-free water. Canine-specific primers were used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MyHC-1, MyHC-2A, MyHC-2B, and MyHC-2X. The detailed forward and reverse primer sequences and the sizes of the products are summarized in Table I. The PCR was performed under the following conditions: 1 cycle at 94 C for 5 min, followed by 36 cycles at 94 C for 45 s, 60 C for 45 s, 72 C for 45 s, and 1 cycle at 50 C, increasing to 95 C in 0.5 C increments to make a melting curve [11]. The PCR measurement was repeated twice per specimen. We used the real-time PCR detection system (Option Tm2; Bio-Rad Laboratories) to detect the PCR products. Relative quantitative gene expression was determined by the ratio of target gene concentration to the internal control GAPDH. For PCR-negative control samples, primers were not added during PCR. We used gel electrophoresis to verify the PCR products according to fragment size in 2.5% agarose gels containing 0.5 mg/ml ethidium bromide. Statistical analysis All data are reported as mean ± SD. Gene expression ratios and stimulus-response activation values from denervation, reinnervation, and normal groups were assessed with the one-way analysis of variance (ANOVA) test for three grouping variables. The threshold for significance was defined as p < 0.05.
Table I. Primer sequences. Phenotype
Product length (bp)
Sequences
MyHC-1
221
F: 5’-ATGCCAACCGCATGGCTGCT-3’ R: 5’-CTGCTCCGCCAGTTTCCGGG-3’
MyHC-2A
241
F: 5’-GGTTGCAGGCGGCTGAGGAG-3’ R: 5’-GAGCGGGCCTCCTTCTGGGA-3’
MyHC-2B
349
F: 5’-CGCCAGGCTGCAGAGGCAAT-3’ R: 5’-TGCGGGCTTCCTGGACGATG-3’
MyHC-2X
82
F: 5’-CCCTGCAATCAGCCCGCCAC-3’ R: 5’-CTCTGCAGCTCGGCCTTGCC-3’
MyHC-Neonatal
319
F:5’-GCAGCTGGCCCTGAAGGGTG-3’
GAPDH
429
F: 5’-AACCATGAGAAGTATGACAAC-3’
R:5’-CTCGGCCTCCTCCAGCTCGT-3’
R: 5’-CTCAGTGTAGCCCAGGATGC-3’ GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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PCA muscle electrical response and MyHC following reinnervation Table II. Posterior cricoarytenoid (PCA) muscle stimulus-response activation values. Vocal abduction distance maximum (mm)
Threshold of vocal max abduction (V)
Threshold of vocal movement (V)
Denervation (n = 8)
2.5 ± 1.1*
6.1 ± 0.5*
1.4 ± 0.6*†
Reinnervation (n = 7)
3.0 ± 0.8
4.9 ± 1.5
0.5 ± 0.1
Normal control (n = 7)
3.8 ± 1.1
3.4 ± 1.2
0.4 ± 0.2
Group
*Significant difference between the denervation and normal control groups (p < 0.05). †Significant difference between the denervation and reinnervation groups (p < 0.05).
Two animals died from aspiration; one dog was in the reinnervation group and the other was in the control group.
vocal movement from maximum generated glottal opening (Table II). There was a significant difference between the denervation and control groups as regards the stimulus pulse amplitudes threshold value. The maximum threshold voltage of the denervation group was less than that of the control group (p < 0.05; Table II). The stimulus pulse amplitude threshold value of vocal movement of the denervation group was more than the threshold value of the control or reinnervation groups (p < 0.05; Table II).
PCA muscle stimulus-response activation
MyHC mRNA isoforms
The BL-420s generator delivered a train of squarewave pulses (bipolar, 20 ms pulse duration, 40 pps, 5 s train interval time, 3 s on and 2 s off). With this experimental design, differences in the stimulus current amplitude threshold of vocal fold movement would reflect differences in the state of PCA muscle innervation, and differences from complete dynamic PCA muscle responses would reflect differences in the state of PCA muscle atrophy. The distance of vocal fold movement from the maximum generated glottal opening in denervation and reinnervation groups could be generated throughout the functional stimulus. The denervated vocal abduction maximum was less than the reinnervated or control vocal abduction maximum (p < 0.05). There was no significant difference between the reinnervation and control group as regards the distance of
Log-transformed expression ratios were compared among the reinnervated, denervated, and control dogs (Table III) with the one-way ANOVA test for the variables MyHC-1, 2A, 2B, 2X, and Neonatal. It was observed that the mRNA expression of PCA muscle had fast phenotype 2A, 2B, 2X, Neonatal MyHC, and slow phenotype 1. Denervation produced an up-regulation (9 weeks after denervation) of the MyHC-1 and MyHC-Neonatal mRNA expression. Reinnervation caused a decrease (9 weeks after denervation) of MyHC-2X mRNA expression (Table III). Figure 1 shows significantly increased MyHC-1 gene expression of PCA muscle in the RLN denervated dogs compared with reinnervated dogs or controls (mean ± SD: 0.880.26 vs 0.360.36 or 0.260.29, respectively, p < 0.01 and p < 0.01). There was no significant
All statistical analyses were performed using SPSS version 15.0 for Windows (SPSS, Inc., Chicago, IL, USA).
Results Animal model
Table III. Comparison of log transformed gene expression ratios for five MyHC phenotypes in denervated, reinnervated, and control groups. Group MyHC-1
Controls (n = 7)
Denervated (n = 8)
Reinnervated (n = 7)
F value
p value
0.36 ± 0.36
0.88 ± 0.26
0.26 ± 0.29
9.164
0.002*
MyHC-2A
0.01 ± 0.27
0.54 ± 0.26
–0.08 ± 0.93
2.680
0.094
MyHC-2B
–2.69 ± 0.55
–2.71 ± 0.52
–3.40 ± 1.03
2.198
0.138
MyHC-2X
0.34 ± 0.81
–0.04 ± 0.64
–0.93 ± 0.86
5.038
0.017*
–2.06 ± 0.40
–0.38 ± 0.32
–1.61 ± 0.60
28.665
0.000*
MyHC-Neonatal
*Significant difference among the reinnervated, denervated, and control groups according to the ANOVA analysis (p < 0.05).
J. Li et al. significant difference between the denervated dogs and the controls. There was no significant difference in mRNA expression of MyHC-2A and MyHC-2B among denervation, reinnervation, and control groups in the PCA muscle (Figures 4 and 5).
1.5
* *
1.0
0.5
Discussion
0.0
–0.5 Controls
Denervation group
Reinnervation
Figure 1. Log-transformed MyHC-1 gene expression ratios for control, denervation, and reinnervation groups. Results revealed significantly increased MyHC-1 gene expression of posterior cricoarytenoid (PCA) muscle in recurrent laryngeal nerve (RLN) denervated dogs compared with reinnervated dogs or controls. *p < 0.01.
mRNA, Log (expression) (Mean ± SD)
difference between the reinnvervated dogs and the controls. Figure 2 shows a significant increase in MyHCNeonatal gene expression of PCA muscle in RLN denervated dogs, compared with reinnervated dogs or controls (mean ± SD: –1.610.60 vs –0.040.64 or – 2.060.40, respectively, p < 0.001 and p < 0.001). There was no significant difference between the reinnervated dogs and the controls. Figure 3 shows significantly increased MyHC-2X gene expression of PCA muscle in RLN reinnervated dogs compared with denervated dogs or controls (mean ± SD: –0.930.86 vs –0.040.64 or 0.340.81, respectively, p < 0.05 and p < 0.01). There was no
The PCA muscle is the sole abductor of the vocal folds during inspiration and acts as a co-contractor antagonist to the adductors during phonation in mammals. In the case of bilateral nerve damage, both folds are paralyzed in the paramedian position, severely restricting ventilation. The loss of abduction often requires an emergency tracheotomy to create a functional airway in patients with bilateral vocal paralysis. Treatment of bilateral vocal fold paralysis is challenging because the clinician always needs to balance between airway and voice. A surgical approach to vocal fold paralysis is laryngeal muscle reinnervation to re-establish working motor endplates and produce muscular contraction and abduction of vocal fold during inspiration through reinnervation of the abductor muscle. Two types of laryngeal muscle reinnervation that have been reported are selective and unselective reinnervation. Selective reinnervation aims at re-establishing functional glottis mobility and is the solution to PCA muscle synkinesis by targeting reinnervation to muscles. Unselective reinnervation mainly provides tone and bulk to the laryngeal muscle, whereas re-establishment of vocal motion is secondary. The ansa cervicalis best fits the criteria for reinnervating the PCA muscle, and is anatomically adjacent to the
0.0 ## –0.5 –1.0 –1.5 –2.0 –2.5 Controls
Denervation group
Reinnervation
Figure 2. Log-transformed MyHC-Neonatal gene expression ratios for control, denervation, and reinnervation groups. Results revealed increased MyHC-Neonatal gene expression of posterior cricoarytenoid (PCA) muscle in recurrent laryngeal nerve (RLN) denervated dogs compared with reinnervated dogs or controls. # p < 0.001.
mRNA, Log (expression) (Mean ± SD)
mRNA, Log (expression) (Mean ± SD)
322
1
0 *
–1
–2 Control
Denervation group
Reinnervation
Figure 3. Log-transformed MyHC-2X gene expression ratios for control, denervation, and reinnervation groups. Results revealed increased MyHC-2X gene expression of posterior cricoarytenoid (PCA) muscle in recurrent laryngeal nerve (RLN) reinnervated dogs compared with denervated dogs or controls. *p < 0.01; †p < 0.05.
mRNA, Log (expression) (Mean ± SD)
PCA muscle electrical response and MyHC following reinnervation 1.0
0.5
0.0
–0.5
–1.0 Control
Denervation group
Reinnervation
Figure 4. Log-transformed MyHC-2A gene expression ratios for control, denervation, and reinnervation groups. No significant difference was observed among the three groups.
mRNA, Log (expression) (Mean ± SD)
larynx [12]. The ansa–RLN anastomosis also demonstrated significant PCA muscle functional improvement in clinical and animal studies of unilateral vocal paralysis [10,13]. Although PCA muscle reinnervation can cause greater vocal fold mobility than denervation under functional electrical stimulation, research into bilateral PCA muscle reinnervation was limited due to the variability and complexity of nerve supply, especially in ansa–RLN anastomosis studies [2,6]. Our study developed a large animal model of ansa–RLN anastomosis bilateral vocal fold paralysis to investigate the denervation and reinnervation of the PCA muscle, for better vocal abduction in the use of PCA muscle pacing. Satellite cells are normally mitotically quiescent in a niche beneath the basal lamina of muscle, but are activated in response to denervation. When activated, these cells proliferate rapidly to produce myoblasts that fuse with pre-existing fibers and to replace part or –2.0 –2.5 –3.0 –3.5 –4.0 –4.5 Control
Denervation group
Reinnervation
Figure 5. Log-transformed MyHC-2B gene expression ratios for control, denervation, and reinnervation groups. No significant difference was observed among the three groups.
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all of any damaged fibers [14]. It has been reported that denervation of skeletal muscle actually leads to an accelerated level of satellite cell activation and differentiation, and the skeletal muscle denervation or reinnervation may result in a change of MyHC isoform in PCA muscle [4,15]. In the rat unilateral PCA muscle, denervation resulted in a reduction in type 1 MyHC protein isoform and an increase in type 2A + 2X MyHC protein isoform from day 30 to day 180, after denervation [16]. The mRNA levels of MyHC isoforms also changed after denervation. There were significant elevations in the neonatal type and MyHC-2X mRNA isoform levels, and a decrease in the MyHC-2B mRNA isoform levels at day 30, after rat unilateral PCA muscle denervation [15]. In the present study, denervation produced an up-regulation of the MyHC-1 and MyHC-Neonatal mRNA expression in a dog at 9 weeks after bilateral PCA muscle denervation. There are some differences between bilateral denervation of PCA muscle in the dog and unilateral denervation of PCA muscle in the rat. Reinnervation resulted in a significant decrease in MyHC-2B and a significant increase in MyHC-2X from day 30 to day 180, following rat unilateral PCA muscle reinnervation. No significant change was observed in the type 1 or type 2B MyHC [3,8,16]. In a study using mini-pigs, there was a significantly reduced expression of all type 2 MyHC, and a significant rise in the expression of type 1 MyHC at 2 months after reinnervation of unilateral PCA muscles [5]. Our data showed that the reinnervation caused a significant increase in mRNA expression of MyHC-2X at 9 weeks after bilateral PCA muscle denervation in the dog. No significant change was observed in the type 1 MyHC mRNA expression. The results suggest that MyHC alterations are similar in the dog and the rat after RLN reinnervation, although different in bilateral and unilateral RNL reinnervation. There are still some variations of MyHC alteration among species after reinnervation, such as dog, rat, and mini-pig. Early studies have concentrated on small animal models, where it is impossible to correlate MyHC phenotype alteration with functional outcome or clinical application. The present study, which provides strong bilateral abductor denervation or reinnervation by ansa–RLN anastomosis in canines, allows a comprehensive assessment of muscles and observation of laryngeal function in response to RLN anastomosis. Additionally, we observed alteration of mRNA expression of MyHC in bilateral PCA muscles after denervation and reinnervation, which to the best of our knowledge has not been previously shown in canines. The variation in PCA muscle fiber types
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between canines and rodents or mini-pigs may lead to the difference in alterations in the PCA muscle MyHC isoform profile after denervation or reinnervation [6]. The expression of fast-type 2A/X and 2B MyHX proteins following RLN injury is consistent with the fact that their regulation is dependent on neural activity [8,17]. With this experimental design, differences in vocal fold locomotivity would reflect differences in the state of PCA muscle innervation, atrophy, and MyHC phenotype expression. With the use of the stimulus paradigm, the functional electrical stimulus produced a different response in innervated and chemical denervated muscle. Longer pulse width (>0.45 ms) was required for direct activation of muscle fibers after chemical denervation [2]. For complete PCA muscle denervation with RLN resection, we used the pulse width 10 ms. Zealear et al. suggested that the optimum pulse frequency was 30–40 Hz [2]. In this study we used a pulse frequency of 40 Hz. A pulse train of 3 s was chosen because it generated a sustained abduction of sufficient duration for the measurement of vocal fold displacement. In the present study, the electrical stimulus voltage thresholds that generated vocal fold primary movement and maximum abduction in denervated PCA muscle were higher than those in normal PCA muscle. The vocal maximum abduction distance of response to electrical stimulation in denervated PCA muscle was shorter than that in normal PCA muscle. These findings corroborate the findings of Zealear et al., who reported that the maximum response of denervated PCA muscle that could be produced throughout the useable stimulus range was only 10–25% of that of the innervated muscle in the canine [2]. In the present study, the bilateral RLN traumatic denervation had a similar effect on PCA muscle function to unilateral chemical denervation. Electrical stimulation of the denervated PCA muscle caused vocal abduction in patients with vocal fold paralysis by pace stimulator [18]. As in the previous study, PCA muscle contraction would occur primarily through the activation of nerve terminals by electrical stimulus because of their lower threshold for activation, while in the latter, muscle fibers would be activated directly by electrical stimulus [2]. Broniatowski et al. [19] used a nerve–muscle pedicle to reinnervate the one side PCA muscle fitted with a perineural electrode for pacemaker stimulation. Peak inspiratory flows were significantly higher (p < 0.001) after reinnervation. This suggested that nerve–muscle pedicle reinnervation could improve the PCA muscle contractile amplitude under the electrical stimulus [19]. In this study, there was no significant difference
between the reinnervated and control animals in the electrical stimulus voltage threshold that generated vocal fold primary movement and maximum abduction. The vocal abduction distance maximum of response to electrical stimulation in reinnervated PCA muscle also was not significantly different from that of normal PCA muscle. The ansa–RLN anastomosis can reinnervate the bilateral PCA muscle effectively and lead to the recovery of electrical stimulation response function. Additionally, we were interested in the relationship between muscle function and alteration of MyHC phenotype expression after denervation or reinnervation of bilateral PCA muscle. In the present study, upregulation of type 1 MyHC and MyHC-Neonatal mRNA expression in PCA muscle may be correlated with down-regulation of electrical response function after denervation of bilateral PCA muscle in canines. The up-regulation of mRNA expression levels of type 2X MyHC in PCA muscle also may be correlated with recovery of electrical response function after reinnervation of bilateral PCA muscle in canines. This study may lay the foundation for future work into the application of muscle pacing stimulators on reinnervated muscle, especially in bilateral laryngeal muscle paralysis.
Acknowledgments The authors thank Liming Yao, ZhouCuo Qi, and Xianbing Wang for assistance with manuscript preparation and data interpretation. This work was supported by Guangzhou Government ZhiCheng Plan Funding (grant no. 11A56040659) and Guangdong Province Government Technology Research and Developing Funding. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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ORIGINAL ARTICLE
Changes in electrical response function and myosin heavy chain isoforms following denervation and reinnervation of bilateral posterior cricoarytenoid muscles in dogs
JINGYUAN LI1*, SHAOFENG LIU2*, QIUHUI CHENG1, MINGRONG NIE2, SIYI ZHANG1, XIAOLI SHENG1, SHAOHUA CHEN1 & PINGJIANG GE1 1
Department of Otolaryngology, Guangdong General Hospital & Guangdong Academy of Medical Sciences, Guangzhou city and and 2Department of Otolaryngology, Guangzhou Women and Children’s Medical Center, Guangzhou, China
Abstract Conclusions: Both electrical response function and mRNA expression of myosin heavy chain (MyHC) types 2X, 1, and Neonatal of bilateral posterior cricoarytenoid (PCA) muscle changed after denervation or reinnervation in canines. Objectives: There is a need to investigate the electrical response function MyHC alteration of denervation or reinnervation in the bilateral PCA muscle of large animals. Methods: MyHC isoforms expression profile and PCA muscle function outcome were detected by real-time reverse transcribed-polymerase chain reaction and muscle response to functional electrical stimulation, 9 weeks after denervation and reinnervation with ansa–recurrent laryngeal nerve anastomosis in dogs. Results: Denervation produced up-regulation of MyHC-1 and MyHC-Neonatal messenger ribonucleic acid (mRNA) expression. Reinnervation caused a decrease of MyHC-2X mRNA expression. The electrical voltage threshold of vocal fold movement and maximum abduction of denervation were greater than that of the reinnervated or control group. The denervated vocal abduction maximum of response to electrical stimulation was less than that in reinnervation or control groups.
Keywords: Electrical stimulation, regeneration, recurrent laryngeal nerve, canine, anastomosis, abduction
Introduction Laryngeal dysfunction occurs after denervation of the recurrent laryngeal nerve (RLN). Unilateral paralysis may result in transient or permanent dysphonia. For bilateral vocal folds paralysis, the loss of abductor function of bilateral posterior cricoarytenoid (PCA) muscles results in a life-threatening condition that often requires an emergency intubation tube or tracheotomy. Current treatment options for laryngeal paralysis are far from ideal. Although direct nerve anastomosis techniques result in nonselective reinnervation of adductor or abductor muscle and produce an uncoordinated, synkinetic movement of vocal folds, the nerve anastomosis technique can regain the bulk and tension of the vocal fold. This technique appears to be appropriate for
clinical applications, such as ansa cervicalis to the RLN in patients with unilateral paralysis [1]. Because the denervated PCA muscle was still 10–25% as responsive to stimulation as the innervated unilateral PCA muscle, the application of paced muscle electrical stimulation results has been favorable in animals and preliminary clinical trials using an implantable commercial stimulator [2]. Bilateral PCA muscle reinnervation may be more effective than unilateral PCA muscle denervation on vocal fold movement under the pace stimulation. A denervated muscle undergoes a rapid decline in mass and a change in muscle fiber phenotype [3,4]. Following reinnervation, there was a trend for fast-type fiber diameter and fiber phenotype expression to normalize [5]. Most of the investigator’s interest has been focused on myosin because of its central role in the
Correspondence: Pingjiang Ge and Siyi Zhang, Department of Otolaryngology, Guangdong General Hospital, 106, Zhongshan 2 Road, Guangzhou city 510080, China. Tel: +86 13751753465. Fax: +86 20 34242560. E-mail: [email protected] *These authors contributed equally to this study.
(Received 24 September 2013; accepted 24 October 2013) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2013.860657
PCA muscle electrical response and MyHC following reinnervation contractile process and because different isoforms of myosin give rise to variations in contractile properties and fiber types [6]. Skeletal muscles are composed of different types of myofibers. Six major myosin heavy chain (MyHC) phenotype genes have been identified in the skeletal muscle encoding 1, 2A, 2B, 2X, Neonatal, and extraocular protein of adult mammals. Electrophoresis in laryngeal muscles revealed the presence of the isoforms expected on the basis of reverse transcription polymerase chain reaction (RT-PCR). Five bands were detected in PCA muscle (1, 2A, 2X, 2B, and Neonatal) [7]. In the denervated condition, the changes of MyHC composition in laryngeal muscle include an increase and decrease in type 2A, 2X, and 2B, and a minimal change in type 1 MyHC. The reinnervation of muscle changes the MyHC phenotype of denervated muscle. The expression of normal MyHC composition is dependent on the condition of appropriate neural contact and functional reinnervation in rats [3,8]. The nerve transaction and repair also results in persistent alteration of MyHC composition and vocal fold dysfunction. Muscle reinnervation demonstrated by electromyography rarely achieves normal functional activity in rats [3,8]. There has been no study of the relationship between electrophysiological function and myosin in canines, which have a larger laryngeal muscle. The present study had two primary objectives. The first was to test the hypothesis that MyHC phenotype transition in canine bilateral PCA muscle denervation and reinnervation was similar to the results observed in unilateral PCA muscle paralysis studies, or in studies of other mammalian species. The second objective was to characterize the effects of denervation or reinnervation on the functional outcome of canine bilateral PCA muscles. Material and methods Animal care and model Approval was obtained from the Guangdong General Hospital Review Board before conducting this study. Twenty-four mongrel dogs (weight 14.4 ± 1.0 kg) were used in this study. They were randomly assigned to one of three groups: (1) control, (2) denervated, or (3) reinnervated. Animals were humanely euthanized 9 weeks after denervation or reinnervation. Induction of anesthesia was achieved using ketamine (35 mg/kg) administered intramuscularly. Heart rate, temperature, and oxygen saturation levels were monitored throughout the experiment to monitor the animal’s level of anesthesia and general well-being. Following laryngoscopy, an endotracheal tube was put into place. Subsequent intravenous drips of diazepam
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(20 mg/kg), atropine (1 mg/kg), isopropylphenol (15 mg/kg), and fentanyl (20 mg/kg) were provided as needed to maintain a surgical anesthetic plane. Animals were placed in a supine position on an operating platform. The neck was shaved and prepared for surgery from the level of the submentum down to the chest. The larynx and trachea were exposed using a midline neck incision extending from the hyoid bone to the sternal notch. A 2 cm section of each RLN was removed from the trachea region. The resected ends of the RLN were folded back on the remainder of the nerve and tied with a 3-0 suture in the denervated group [9]. The wound was closed in layers after the trachea and the RLN were exposed in the control group. The surgical technique of reinnervation was as follows. The sternocleidomastoid muscle was reflected posteriorly to expose the omohyoid muscle. Mobilization of the omohyoid muscle was used to identify the ansa cervicalis overlying the internal jugular vein. The ansa cervicalis branches to the sternohyoid and sternothyroid muscles were identified and transected on the lateral aspect of these strap muscles. They were freely mobilized in preparation for anastomosis. The resected laryngeal ends of the RLN were anastomosed to the ansa cervicalis branches with four or five epineurial stitches of nylon 11-0 thread. The wound was then closed in layers [10]. PCA muscle stimulus-response activation The PCA muscle activation was measured 9 weeks after denervation or reinnervation. Seven animals were assigned to each group. With each animal in a supine position, a midline neck incision was made. After thyrohyoid membrane incision, the posterior surface of the bilateral PCA muscle and vocal folds were exposed through the pharyngeal cavity. The RLN trunk was then stimulated with hook electrodes (2 ms pulse duration, 5 V; BL-420s, TaiMeng, Chendu, China) to check that no abductor branches remained intact. Reinnervation was confirmed at the termination of the experiment by the presence of vocal fold motion evoked by RLN stimulation. Vocal fold abductions elicited by PCA muscle stimulation were measured by superimposing a grid, which was calibrated with a 4 mm ruler, on the vocal fold. Both stimulation needle electrodes were positioned into the upper and lower end of the PCA muscle for electrical stimulation. The distance between anode and cathode stimulation electrodes was 1 cm. Identity of RLN reinnervation was confirmed by the presence of vocal fold movement associated with electrical stimulation. The pattern of electrical pulse delivery (biphase, 10 ms pulse duration, 40 pps, 5 s train interval time, 3 s on and 2 s off) was used to study the characteristics of PCA muscle
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stimulus-response [2]. The amplitudes of the pulses were increased in a stepwise fashion (0.1 V) using the pulse generator (BL-420s, TaiMeng). The pulse amplitudes were obtained from thresholds of movement and complete dynamic PCA muscle response. The distance of vocal movement from complete dynamic PCA muscle responses was also measured in each animal [2]. The tissue specimens of muscle were collected using 2 mm diameter cup forceps after the measurement of PCA muscle stimulus-response activation. Total RNA isolation A 1 mm tissue specimen of muscle was obtained from the middle portion of the PCA muscle bilaterally from denervation, reinnervation or control animals, immediately following data collection. The specimens were quickly submerged in RNAlater Stabilization Reagent (Qiagen Inc., Valencia, CA, USA), incubated at 4 C overnight, and stored at –80 C until extraction. Animals were euthanized without recovery from anesthesia after collection of specimens. Tissue specimens were placed in 120 mg zirconia/ silica beads (1 mm diameter) and homogenized at 4800 rpm for 90 s with the use of a Mini-Beadbeater homogenizer (BioSpec Products, Inc., Bartlesville, OK, USA). We used the RNeasy Fibrous Tissue Mini Kit (Qiagen Inc.) to extract the mRNA from tissue specimens. The total mRNA was stored at –80 C. Real-time RT-PCR Reverse transcription was performed with the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). The real-time PCR was
performed using iQTM SyBR Green Supermix Kit (Invitrogen Corp., Carlsbad, CA, USA) in a 50 ml volume reaction mixture composed of 500 nmol/l primer one, 500 nmol/l primer two, 25 ml iQ SyBR Green Supermix, and 1.2 ml template complementary DNA ribonuclease-free water. Canine-specific primers were used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MyHC-1, MyHC-2A, MyHC-2B, and MyHC-2X. The detailed forward and reverse primer sequences and the sizes of the products are summarized in Table I. The PCR was performed under the following conditions: 1 cycle at 94 C for 5 min, followed by 36 cycles at 94 C for 45 s, 60 C for 45 s, 72 C for 45 s, and 1 cycle at 50 C, increasing to 95 C in 0.5 C increments to make a melting curve [11]. The PCR measurement was repeated twice per specimen. We used the real-time PCR detection system (Option Tm2; Bio-Rad Laboratories) to detect the PCR products. Relative quantitative gene expression was determined by the ratio of target gene concentration to the internal control GAPDH. For PCR-negative control samples, primers were not added during PCR. We used gel electrophoresis to verify the PCR products according to fragment size in 2.5% agarose gels containing 0.5 mg/ml ethidium bromide. Statistical analysis All data are reported as mean ± SD. Gene expression ratios and stimulus-response activation values from denervation, reinnervation, and normal groups were assessed with the one-way analysis of variance (ANOVA) test for three grouping variables. The threshold for significance was defined as p < 0.05.
Table I. Primer sequences. Phenotype
Product length (bp)
Sequences
MyHC-1
221
F: 5’-ATGCCAACCGCATGGCTGCT-3’ R: 5’-CTGCTCCGCCAGTTTCCGGG-3’
MyHC-2A
241
F: 5’-GGTTGCAGGCGGCTGAGGAG-3’ R: 5’-GAGCGGGCCTCCTTCTGGGA-3’
MyHC-2B
349
F: 5’-CGCCAGGCTGCAGAGGCAAT-3’ R: 5’-TGCGGGCTTCCTGGACGATG-3’
MyHC-2X
82
F: 5’-CCCTGCAATCAGCCCGCCAC-3’ R: 5’-CTCTGCAGCTCGGCCTTGCC-3’
MyHC-Neonatal
319
F:5’-GCAGCTGGCCCTGAAGGGTG-3’
GAPDH
429
F: 5’-AACCATGAGAAGTATGACAAC-3’
R:5’-CTCGGCCTCCTCCAGCTCGT-3’
R: 5’-CTCAGTGTAGCCCAGGATGC-3’ GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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PCA muscle electrical response and MyHC following reinnervation Table II. Posterior cricoarytenoid (PCA) muscle stimulus-response activation values. Vocal abduction distance maximum (mm)
Threshold of vocal max abduction (V)
Threshold of vocal movement (V)
Denervation (n = 8)
2.5 ± 1.1*
6.1 ± 0.5*
1.4 ± 0.6*†
Reinnervation (n = 7)
3.0 ± 0.8
4.9 ± 1.5
0.5 ± 0.1
Normal control (n = 7)
3.8 ± 1.1
3.4 ± 1.2
0.4 ± 0.2
Group
*Significant difference between the denervation and normal control groups (p < 0.05). †Significant difference between the denervation and reinnervation groups (p < 0.05).
Two animals died from aspiration; one dog was in the reinnervation group and the other was in the control group.
vocal movement from maximum generated glottal opening (Table II). There was a significant difference between the denervation and control groups as regards the stimulus pulse amplitudes threshold value. The maximum threshold voltage of the denervation group was less than that of the control group (p < 0.05; Table II). The stimulus pulse amplitude threshold value of vocal movement of the denervation group was more than the threshold value of the control or reinnervation groups (p < 0.05; Table II).
PCA muscle stimulus-response activation
MyHC mRNA isoforms
The BL-420s generator delivered a train of squarewave pulses (bipolar, 20 ms pulse duration, 40 pps, 5 s train interval time, 3 s on and 2 s off). With this experimental design, differences in the stimulus current amplitude threshold of vocal fold movement would reflect differences in the state of PCA muscle innervation, and differences from complete dynamic PCA muscle responses would reflect differences in the state of PCA muscle atrophy. The distance of vocal fold movement from the maximum generated glottal opening in denervation and reinnervation groups could be generated throughout the functional stimulus. The denervated vocal abduction maximum was less than the reinnervated or control vocal abduction maximum (p < 0.05). There was no significant difference between the reinnervation and control group as regards the distance of
Log-transformed expression ratios were compared among the reinnervated, denervated, and control dogs (Table III) with the one-way ANOVA test for the variables MyHC-1, 2A, 2B, 2X, and Neonatal. It was observed that the mRNA expression of PCA muscle had fast phenotype 2A, 2B, 2X, Neonatal MyHC, and slow phenotype 1. Denervation produced an up-regulation (9 weeks after denervation) of the MyHC-1 and MyHC-Neonatal mRNA expression. Reinnervation caused a decrease (9 weeks after denervation) of MyHC-2X mRNA expression (Table III). Figure 1 shows significantly increased MyHC-1 gene expression of PCA muscle in the RLN denervated dogs compared with reinnervated dogs or controls (mean ± SD: 0.880.26 vs 0.360.36 or 0.260.29, respectively, p < 0.01 and p < 0.01). There was no significant
All statistical analyses were performed using SPSS version 15.0 for Windows (SPSS, Inc., Chicago, IL, USA).
Results Animal model
Table III. Comparison of log transformed gene expression ratios for five MyHC phenotypes in denervated, reinnervated, and control groups. Group MyHC-1
Controls (n = 7)
Denervated (n = 8)
Reinnervated (n = 7)
F value
p value
0.36 ± 0.36
0.88 ± 0.26
0.26 ± 0.29
9.164
0.002*
MyHC-2A
0.01 ± 0.27
0.54 ± 0.26
–0.08 ± 0.93
2.680
0.094
MyHC-2B
–2.69 ± 0.55
–2.71 ± 0.52
–3.40 ± 1.03
2.198
0.138
MyHC-2X
0.34 ± 0.81
–0.04 ± 0.64
–0.93 ± 0.86
5.038
0.017*
–2.06 ± 0.40
–0.38 ± 0.32
–1.61 ± 0.60
28.665
0.000*
MyHC-Neonatal
*Significant difference among the reinnervated, denervated, and control groups according to the ANOVA analysis (p < 0.05).
J. Li et al. significant difference between the denervated dogs and the controls. There was no significant difference in mRNA expression of MyHC-2A and MyHC-2B among denervation, reinnervation, and control groups in the PCA muscle (Figures 4 and 5).
1.5
* *
1.0
0.5
Discussion
0.0
–0.5 Controls
Denervation group
Reinnervation
Figure 1. Log-transformed MyHC-1 gene expression ratios for control, denervation, and reinnervation groups. Results revealed significantly increased MyHC-1 gene expression of posterior cricoarytenoid (PCA) muscle in recurrent laryngeal nerve (RLN) denervated dogs compared with reinnervated dogs or controls. *p < 0.01.
mRNA, Log (expression) (Mean ± SD)
difference between the reinnvervated dogs and the controls. Figure 2 shows a significant increase in MyHCNeonatal gene expression of PCA muscle in RLN denervated dogs, compared with reinnervated dogs or controls (mean ± SD: –1.610.60 vs –0.040.64 or – 2.060.40, respectively, p < 0.001 and p < 0.001). There was no significant difference between the reinnervated dogs and the controls. Figure 3 shows significantly increased MyHC-2X gene expression of PCA muscle in RLN reinnervated dogs compared with denervated dogs or controls (mean ± SD: –0.930.86 vs –0.040.64 or 0.340.81, respectively, p < 0.05 and p < 0.01). There was no
The PCA muscle is the sole abductor of the vocal folds during inspiration and acts as a co-contractor antagonist to the adductors during phonation in mammals. In the case of bilateral nerve damage, both folds are paralyzed in the paramedian position, severely restricting ventilation. The loss of abduction often requires an emergency tracheotomy to create a functional airway in patients with bilateral vocal paralysis. Treatment of bilateral vocal fold paralysis is challenging because the clinician always needs to balance between airway and voice. A surgical approach to vocal fold paralysis is laryngeal muscle reinnervation to re-establish working motor endplates and produce muscular contraction and abduction of vocal fold during inspiration through reinnervation of the abductor muscle. Two types of laryngeal muscle reinnervation that have been reported are selective and unselective reinnervation. Selective reinnervation aims at re-establishing functional glottis mobility and is the solution to PCA muscle synkinesis by targeting reinnervation to muscles. Unselective reinnervation mainly provides tone and bulk to the laryngeal muscle, whereas re-establishment of vocal motion is secondary. The ansa cervicalis best fits the criteria for reinnervating the PCA muscle, and is anatomically adjacent to the
0.0 ## –0.5 –1.0 –1.5 –2.0 –2.5 Controls
Denervation group
Reinnervation
Figure 2. Log-transformed MyHC-Neonatal gene expression ratios for control, denervation, and reinnervation groups. Results revealed increased MyHC-Neonatal gene expression of posterior cricoarytenoid (PCA) muscle in recurrent laryngeal nerve (RLN) denervated dogs compared with reinnervated dogs or controls. # p < 0.001.
mRNA, Log (expression) (Mean ± SD)
mRNA, Log (expression) (Mean ± SD)
322
1
0 *
–1
–2 Control
Denervation group
Reinnervation
Figure 3. Log-transformed MyHC-2X gene expression ratios for control, denervation, and reinnervation groups. Results revealed increased MyHC-2X gene expression of posterior cricoarytenoid (PCA) muscle in recurrent laryngeal nerve (RLN) reinnervated dogs compared with denervated dogs or controls. *p < 0.01; †p < 0.05.
mRNA, Log (expression) (Mean ± SD)
PCA muscle electrical response and MyHC following reinnervation 1.0
0.5
0.0
–0.5
–1.0 Control
Denervation group
Reinnervation
Figure 4. Log-transformed MyHC-2A gene expression ratios for control, denervation, and reinnervation groups. No significant difference was observed among the three groups.
mRNA, Log (expression) (Mean ± SD)
larynx [12]. The ansa–RLN anastomosis also demonstrated significant PCA muscle functional improvement in clinical and animal studies of unilateral vocal paralysis [10,13]. Although PCA muscle reinnervation can cause greater vocal fold mobility than denervation under functional electrical stimulation, research into bilateral PCA muscle reinnervation was limited due to the variability and complexity of nerve supply, especially in ansa–RLN anastomosis studies [2,6]. Our study developed a large animal model of ansa–RLN anastomosis bilateral vocal fold paralysis to investigate the denervation and reinnervation of the PCA muscle, for better vocal abduction in the use of PCA muscle pacing. Satellite cells are normally mitotically quiescent in a niche beneath the basal lamina of muscle, but are activated in response to denervation. When activated, these cells proliferate rapidly to produce myoblasts that fuse with pre-existing fibers and to replace part or –2.0 –2.5 –3.0 –3.5 –4.0 –4.5 Control
Denervation group
Reinnervation
Figure 5. Log-transformed MyHC-2B gene expression ratios for control, denervation, and reinnervation groups. No significant difference was observed among the three groups.
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all of any damaged fibers [14]. It has been reported that denervation of skeletal muscle actually leads to an accelerated level of satellite cell activation and differentiation, and the skeletal muscle denervation or reinnervation may result in a change of MyHC isoform in PCA muscle [4,15]. In the rat unilateral PCA muscle, denervation resulted in a reduction in type 1 MyHC protein isoform and an increase in type 2A + 2X MyHC protein isoform from day 30 to day 180, after denervation [16]. The mRNA levels of MyHC isoforms also changed after denervation. There were significant elevations in the neonatal type and MyHC-2X mRNA isoform levels, and a decrease in the MyHC-2B mRNA isoform levels at day 30, after rat unilateral PCA muscle denervation [15]. In the present study, denervation produced an up-regulation of the MyHC-1 and MyHC-Neonatal mRNA expression in a dog at 9 weeks after bilateral PCA muscle denervation. There are some differences between bilateral denervation of PCA muscle in the dog and unilateral denervation of PCA muscle in the rat. Reinnervation resulted in a significant decrease in MyHC-2B and a significant increase in MyHC-2X from day 30 to day 180, following rat unilateral PCA muscle reinnervation. No significant change was observed in the type 1 or type 2B MyHC [3,8,16]. In a study using mini-pigs, there was a significantly reduced expression of all type 2 MyHC, and a significant rise in the expression of type 1 MyHC at 2 months after reinnervation of unilateral PCA muscles [5]. Our data showed that the reinnervation caused a significant increase in mRNA expression of MyHC-2X at 9 weeks after bilateral PCA muscle denervation in the dog. No significant change was observed in the type 1 MyHC mRNA expression. The results suggest that MyHC alterations are similar in the dog and the rat after RLN reinnervation, although different in bilateral and unilateral RNL reinnervation. There are still some variations of MyHC alteration among species after reinnervation, such as dog, rat, and mini-pig. Early studies have concentrated on small animal models, where it is impossible to correlate MyHC phenotype alteration with functional outcome or clinical application. The present study, which provides strong bilateral abductor denervation or reinnervation by ansa–RLN anastomosis in canines, allows a comprehensive assessment of muscles and observation of laryngeal function in response to RLN anastomosis. Additionally, we observed alteration of mRNA expression of MyHC in bilateral PCA muscles after denervation and reinnervation, which to the best of our knowledge has not been previously shown in canines. The variation in PCA muscle fiber types
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between canines and rodents or mini-pigs may lead to the difference in alterations in the PCA muscle MyHC isoform profile after denervation or reinnervation [6]. The expression of fast-type 2A/X and 2B MyHX proteins following RLN injury is consistent with the fact that their regulation is dependent on neural activity [8,17]. With this experimental design, differences in vocal fold locomotivity would reflect differences in the state of PCA muscle innervation, atrophy, and MyHC phenotype expression. With the use of the stimulus paradigm, the functional electrical stimulus produced a different response in innervated and chemical denervated muscle. Longer pulse width (>0.45 ms) was required for direct activation of muscle fibers after chemical denervation [2]. For complete PCA muscle denervation with RLN resection, we used the pulse width 10 ms. Zealear et al. suggested that the optimum pulse frequency was 30–40 Hz [2]. In this study we used a pulse frequency of 40 Hz. A pulse train of 3 s was chosen because it generated a sustained abduction of sufficient duration for the measurement of vocal fold displacement. In the present study, the electrical stimulus voltage thresholds that generated vocal fold primary movement and maximum abduction in denervated PCA muscle were higher than those in normal PCA muscle. The vocal maximum abduction distance of response to electrical stimulation in denervated PCA muscle was shorter than that in normal PCA muscle. These findings corroborate the findings of Zealear et al., who reported that the maximum response of denervated PCA muscle that could be produced throughout the useable stimulus range was only 10–25% of that of the innervated muscle in the canine [2]. In the present study, the bilateral RLN traumatic denervation had a similar effect on PCA muscle function to unilateral chemical denervation. Electrical stimulation of the denervated PCA muscle caused vocal abduction in patients with vocal fold paralysis by pace stimulator [18]. As in the previous study, PCA muscle contraction would occur primarily through the activation of nerve terminals by electrical stimulus because of their lower threshold for activation, while in the latter, muscle fibers would be activated directly by electrical stimulus [2]. Broniatowski et al. [19] used a nerve–muscle pedicle to reinnervate the one side PCA muscle fitted with a perineural electrode for pacemaker stimulation. Peak inspiratory flows were significantly higher (p < 0.001) after reinnervation. This suggested that nerve–muscle pedicle reinnervation could improve the PCA muscle contractile amplitude under the electrical stimulus [19]. In this study, there was no significant difference
between the reinnervated and control animals in the electrical stimulus voltage threshold that generated vocal fold primary movement and maximum abduction. The vocal abduction distance maximum of response to electrical stimulation in reinnervated PCA muscle also was not significantly different from that of normal PCA muscle. The ansa–RLN anastomosis can reinnervate the bilateral PCA muscle effectively and lead to the recovery of electrical stimulation response function. Additionally, we were interested in the relationship between muscle function and alteration of MyHC phenotype expression after denervation or reinnervation of bilateral PCA muscle. In the present study, upregulation of type 1 MyHC and MyHC-Neonatal mRNA expression in PCA muscle may be correlated with down-regulation of electrical response function after denervation of bilateral PCA muscle in canines. The up-regulation of mRNA expression levels of type 2X MyHC in PCA muscle also may be correlated with recovery of electrical response function after reinnervation of bilateral PCA muscle in canines. This study may lay the foundation for future work into the application of muscle pacing stimulators on reinnervated muscle, especially in bilateral laryngeal muscle paralysis.
Acknowledgments The authors thank Liming Yao, ZhouCuo Qi, and Xianbing Wang for assistance with manuscript preparation and data interpretation. This work was supported by Guangzhou Government ZhiCheng Plan Funding (grant no. 11A56040659) and Guangdong Province Government Technology Research and Developing Funding. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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