家犬双侧环杓后肌失神经及颈袢神经再支配后肌卫星细胞活性变化

目的 探讨家犬双侧环杓后肌失神经支配及用颈袢神经再吻合支配后肌卫星细胞活性的变化.方法 随机数字表法将24只家犬分成3组,每组8只,分别为切断双侧喉返神经组,切断喉返神经后即刻颈袢神经再吻合组,不切断喉返神经的对照组.3组动物手术后再饲养9周后再次暴露喉返神经及环杓后肌,使用喉返神经诱发环杓后肌电位来验证喉返神经的再通情况;双盲法提取环杓后肌肌肉组织中总RNA,反转录后做实时定量聚合酶链反应(PCR)检测肌卫星细胞增殖分化标记物Myogenin、Myf5和Pax7的mRNA表达.结果 手术后3组动物各有1只死亡或感染退出实验.术后9周诱发神经环杓后肌肌电图提示对照组动物喉返神经功能均正常,切断组7只动物电刺激都没有反应,吻合组均有神经再通.Myogenin的mRNA相对表达量切断组较对照组和吻合组均有明显升高(Z值为1.42和1.38,P值均＜0.05),Myf5的mRNA表达切断组也明显高于对照组和吻合组(Z值为1.66和1.69,P值均＜0.01);切断组Pax7的mRNA表达明显高于对照组(Z=1.66,P＜0.01),也高于吻合组(Z=1.42,P＜0.05);而吻合组与对照组Myogenin、Myf5和Pax7的mRNA表达差异均无统计学意义(P值均＞0.05).结论 双侧喉返神经切断后,环杓后肌的肌卫星细胞增殖分化的mRNA表达增强,而同颈袢神经再吻合后肌卫星细胞的增殖分化表达下降.

Abstract：

Objective To investigate the activity of bilateral posterior cricoarytenoid muscle satellite cell after denervation or reinnervation with ansa cervicalis. Methods Twenty four dogs were randomly divided into 3 groups. The bilateral laryngeal recurrent nerves were cut in group one in all dogs. The bilateral laryngeal recurrent nerves were anastomosed with ansa cervicalis after incision in group two in all dogs. The dogs in group three were used as control. Nine weeks after surgery, the electromyography was used to test the regeneration of the nerve. The posterior cricoarytenoid muscles biopsy were collected. The expression of mRNA of Myogenin, Myf5, and Pax7 was assayed by realtime RT-PCR after total RNA isolation. Results Two dogs died after surgery in incision and anastomose group. The electromyography suggested that the RLN of all dogs had denervated in the incision group and had reinnervated in the anastomose group after 9 weeks. Myogenin mRNA from RLN incision dogs PCA muscles had greater expression versus controls ( Z = 1.42, P ＜ 0. 01 ) or anastomosed dogs ( Z = 1.38, P ＜ 0. 01 ). Myf5 mRNA expression from RLN incision dogs PCA muscles had significant increase versus control dogs ( Z = 1.66, P ＜0. 01 ) or anastomosed dogs ( Z = 1.69, P ＜ 0. 01 ). Pax7 mRNA expression from RNL incision dogs had significant increase compared with control ( Z = 1.66, P ＜0. 01 ) or anastomosed animals ( Z = 1.42, P ＜0. 05 ). There was no significant difference in Myogenin ( Z = 1.34, P ＞ 0. 05 ), Myf5 ( Z = 0. 54, P ＞0. 05) and Pax ( Z = 0. 54, P ＞ 0. 05 ) mRNA expression between controls and anastomosed animals.Conclusions The bilateral denervation of RLN cause significantly increasing in dog PCA muscle satellite cell proliferation and differentiation. The bilateral reinnervation of RLN cause PCA muscle satellite cell come back nonproliferative, quiescent state in dog.     

家犬双侧环杓后肌失神经及颈袢神经再支配后肌卫星细胞活性变化

Objective To investigate the activity of bilateral posterior cricoarytenoid muscle satellite cell after denervation or reinnervation with ansa cervicalis. Methods Twenty four dogs were randomly divided into 3 groups. The bilateral laryngeal recurrent nerves were cut in group one in all dogs. The bilateral laryngeal recurrent nerves were anastomosed with ansa cervicalis after incision in group two in all dogs. The dogs in group three were used as control. Nine weeks after surgery, the electromyography was used to test the regeneration of the nerve. The posterior cricoarytenoid muscles biopsy were collected. The expression of mRNA of Myogenin, Myf5, and Pax7 was assayed by realtime RT-PCR after total RNA isolation. Results Two dogs died after surgery in incision and anastomose group. The electromyography suggested that the RLN of all dogs had denervated in the incision group and had reinnervated in the anastomose group after 9 weeks. Myogenin mRNA from RLN incision dogs PCA muscles had greater expression versus controls ( Z = 1.42, P ＜ 0. 01 ) or anastomosed dogs ( Z = 1.38, P ＜ 0. 01 ). Myf5 mRNA expression from RLN incision dogs PCA muscles had significant increase versus control dogs ( Z = 1.66, P ＜0. 01 ) or anastomosed dogs ( Z = 1.69, P ＜ 0. 01 ). Pax7 mRNA expression from RNL incision dogs had significant increase compared with control ( Z = 1.66, P ＜0. 01 ) or anastomosed animals ( Z = 1.42, P ＜0. 05 ). There was no significant difference in Myogenin ( Z = 1.34, P ＞ 0. 05 ), Myf5 ( Z = 0. 54, P ＞0. 05) and Pax ( Z = 0. 54, P ＞ 0. 05 ) mRNA expression between controls and anastomosed animals.Conclusions The bilateral denervation of RLN cause significantly increasing in dog PCA muscle satellite cell proliferation and differentiation. The bilateral reinnervation of RLN cause PCA muscle satellite cell come back nonproliferative, quiescent state in dog.     

Study on contractile properties of the posterior cricoarytenoid muscle after delayed reinnervation]

To study date on the contractile properties of posterior cricoarytenoid muscle after delayed reinnervation of different reinnervated methods. Twenty four dogs were reinnervated at 0,4,5,6,10 and 12 month interval following recurrent laryngeal nerve via the phrenic nerve anastomosed to the recurrent laryngeal nerve after cutting the adductor branch and ansa cervicalis-sternothyroid muscle pedicle implanted into the posterior cricoarytenoid muscle. After 6 months, a series of contractions were recorded from each side in twenty living dogs. The results showed that contractile force of reinnervated muscle decreased gradually with the time of denervation, but contractile force of muscle was no significantly difference between reinnervated side of nerve anastomosed group in 4 months after denervated and normal side, and it was significantly difference between nerve anastomosed group and nerve-muscle pedicle implanted group at some time of delayed reinnervation. The contractile time of reinnervated side of two operated groups was similar to that of normal side. The conclusion demonstrated that the contractile properties can indicate exactly reinnervated degree of muscle, and the earlier reinnervation was performed, the better curative effect was.     

Experimental laryngeal reinnervation by phrenic nerve implantation into the posterior cricoarytenoid muscle

Under general anaesthesia, 5 dogs underwent sectioning of the right recurrent nerve followed by implantation of the phrenic nerve into the posterior cricoarytenoid (PCA) muscle. Some 6-7 months later the dogs were sacrificed after registration of vocal cord motility. Still photographs and movie film of the larynx were taken during quiet and forced respiration and at electrical stimulation of the implanted phrenic nerve. The PCA and vocal muscles were removed for histochemical studies. We found practically no abductory movement of the vocal cord on the reinnervated side, either during quiet or forced respiration. During forced inspiration there was, however, a slight medial bowing of the right vocal cord. At electrical stimulation there was a sphincteric movement of the entire larynx. Histochemistry showed a reinnervation picture of both the PCA and the vocal muscles on the experimental side. The conclusion drawn from this study is that axonal escape, probably from the implantation site, results in an unwanted reinnervation of laryngeal adductor muscles, which neutralize the abducting effect of the PCA muscle during inspiration. This method therefore does not seem to be suitable as a treatment alternative for bilateral recurrent nerve paralysis.     

Experimental laryngeal reinnervation by phrenic nerve implantation into the posterior cricoarytenoid muscle

Under general anaesthesia, 5 dogs underwent sectioning of the right recurrent nerve followed by implantation of the phrenic nerve into the posterior cricoarytenoid (PCA) muscle. Some 6-7 months later the dogs were sacrificed after registration of vocal cord motility. Still photographs and movie film of the larynx were taken during quiet and forced respiration and at electrical stimulation of the implanted phrenic nerve. The PCA and vocal muscles were removed for histochemical studies. We found practically no abductory movement of the vocal cord on the reinnervated side, either during quiet or forced respiration. During forced inspiration there was, however, a slight medial bowing of the right vocal cord. At electrical stimulation there was a sphincteric movement of the entire larynx. Histochemistry showed a reinnervation picture of both the PCA and the vocal muscles on the experimental side. The conclusion drawn from this study is that axonal escape, probably from the implantation site, results in an unwanted reinnervation of laryngeal adductor muscles, which neutralize the abducting effect of the PCA muscle during inspiration. This method therefore does not seem to be suitable as a treatment alternative for bilateral recurrent nerve paralysis.     

Histochemical study of posterior cricoarytenoid muscle reinnervation by a nerve-muscle pedicle in the cat

Reinnervation of the posterior cricoarytenoid (PCA) muscle with a nerve-muscle pedicle (NMP) has been proposed for patients with bilateral abductor vocal cord paralysis. Since its success has been controversial, a glycogen depletion histochemical technique was used to examine reinnervation. An ansa cervicalis NMP was implanted into the denervated PCA in nine cats. Eight months later, vocal cord activity was evaluated. The NMP nerve was stimulated extensively in seven cats (experimental group). Optical densities of NMP-supplied PCA muscle fibers from experimental and control groups were compared to detect differences in glycogen content. The results demonstrated quantitative evidence of reinnervation in two experimental animals. Electrical stimulation of the NMP produced abduction in one of these two animals, but was never observed during spontaneous respiration or airway occlusion. These observations indicate that reinnervation can occur but abduction requires electrical stimulation. The NMP technique may be more successful with an electrical pacer.     

实验性声带麻痹神经吻合术后神经传导初步研究

目的研究喉返神经完全性损伤后颈袢神经支单纯支配环杓侧肌的电生理恢复情况。方法将实验组犬左喉返神经完全切断,3个月后,再将其内收肌支与同侧颈袢胸舌肌支延期吻合,吻合术后6个月检测环杓侧肌肌电活动情况。结果吻合术后6个月,左环杓侧肌可见肌电动作电位,其潜伏期为1.60±0.05ms,与术前相比较无显著差异,但神经传导速度为15.60±0.25m/s,较术前减慢,差异有显著性。结论喉返神经损伤后3月再行选择性环杓侧肌神经支配吻合术,能基本恢复肌肉电生理功能,但要达到完全正常水平,仍有待继续观察。     

Ganglion cells in the posterior cricoarytenoid muscle of the rat following experimental denervation

OBJECTIVE: To investigate morphological changes of the i.m. ganglion cells in the posterior cricoarytenoid (PCA) muscle of the rat following denervation of the recurrent laryngeal nerve. MATERIAL AND METHODS: The recurrent laryngeal nerve on the left side of the rat was resected. Three weeks after transection, the PCA muscle was removed for morphological study using light and electron microscopy. RESULTS: No morphological changes were found in the i.m. ganglion cells in the PCA muscle, even though the myelinated nerve fibers were destroyed and had disappeared in ramified i.m. bundles. Around the cell body, numerous non-myelinated nerve fibers were found; these contained a large number of clear, spherical synaptic vesicles approximately 50 nm in diameter and several dense-cored vesicles approximately 100 nm in diameter. In contrast, neuromuscular junctions in most muscle fibers with partially disoriented and/or disintegrated myofibrils showed degenerative figures. In some instances, however, multiple nerve terminals were detected in contact with the postsynaptic membrane. Like the varicose swellings of non-myelinated nerve fibers around the ganglion cell body, these nerve terminals contained, in addition to clear synaptic vesicles (50 nm in diameter), several dense-cored vesicles (100 nm in diameter). CONCLUSION: We suggest that i.m. ganglion cells in the rat PCA muscle may supply postganglionic nerve fibers to the denervated neuromuscular junctions after transection of the nerve.     

膈神经替代喉返神经修复治疗双侧声带麻痹的应用解剖

目的　探讨膈神经干与喉返神经喉内段前支吻合治疗双侧声带麻痹的解剖学基础。方法　解剖并观察 12具 (2 4侧 )成年尸体、7只喉全切除术切除的喉体、12例 (2 4侧 )根治性颈淋巴结清扫术和 6例 (6侧 )膈神经替代修复喉返神经患者共 46侧膈神经的起源、走行、血供及毗邻关系 ,测量膈神经干相关的长度 ,图像分析仪观测 30侧膈神经、喉返神经前支相关的组织学参数。结果　颈段膈神经营养动脉均自膈神经根部进入 ,来自于颈升动脉的占 95 6 % (4 4/4 6 )。膈神经干位置较深 ,在颈根部位于颈总动脉、椎静脉外侧 ,颈内静脉及胸导管 (左 )深面 ;在胸腔入口处跨过锁骨下动脉在锁骨下静脉深面下行。膈神经起点至锁骨下静脉上缘平面及至环甲关节的距离平均 ( x±s)分别为(7 2± 1 6 )cm及 (5 5± 1 4)cm ,两者相差至少 1 5cm。膈神经干平均有髓纤维数及神经束截面积分别为喉返神经前支的 2 41及 2 15倍 ,膈神经颈段单个神经束约占 75 0 % (18/2 4)。结论　临床上在胸腔入口解剖膈神经干安全可行 ,在锁骨下静脉上缘平面切断膈神经与喉返神经前支吻合无张力     

The role of the posterior cricoarytenoid muscle in phonation: an electromyographic investigation in dogs

The activity of the posterior cricoarytenoid muscle during respiration has been well investigated electromyographically. Its activity during phonation, however, has not been studied systematically. We, therefore, focused our attention on the phonatory activity of the posterior cricoarytenoid muscle to confirm whether it also contracts during phonation, as has been reported by some researchers. In our series of 12 adult dogs, the posterior cricoarytenoid was active in 11 dogs and inactive in one dog during phonation. Our present study also showed that the posterior cricoarytenoid activity was stronger for phonation than for inspiration in 6, stronger for inspiration than for phonation in 3, and the same for phonation and inspiration in 2 dogs. The results obtained from the present electromyographic evaluation demonstrated that the posterior cricoarytenoid is activated during phonation. The authors believe that the posterior cricoarytenoid muscle has phonatory function and that the phonatory effect of this muscle on the vocal cord may play an important role in precise glottis control.     

Applied anatomy for the reinnervation of posterior cricoarytenoid muscle by phrenic nerve for bilateral vocal cord paralysis]

OBJECTIVE: To study the anatomic basis for the anastomosis of phrenic nerve (PN) to the anterior branch of recurrent laryngeal nerve(RLN) for the treatment of the injured bilateral RLN. METHODS: The origin and the nutritive arteries and the adjacent tissue construction of PNs in 46 cases were studied. The longest utilizable length of PNs and the distance from the root of PN to cricothyroid joint were measured. The sectional area and the number of myelinated fibers of PNs and the anterior branch of RLNs were measured by computer image processing system. RESULTS: PNs coming from C4 comprised of 93.5%, 95.6% (44/46) of the nutritive arteries came from the ascending carotid artery and got into the cervical segment of PN from its root. The common trunk of PN was very deep, to the external of the common carotid artery and the vertebral vein, and deep to the internal jugular vein and thoracic duct (left), and in the superficies of the subclavian artery and in the deep of the subclavian vein when it was crossing the thoracic entrance. The distance from the root of PN to the level of the subclavian vein and to cricothyroid joint were (7.2 +/- 1.6) cm and (5.5 +/- 1.4) cm, respectively. The former was at least 1.5 cm longer than the latter. The average number of myelinated fibers and the sectional area of the PNs were 2.41 times and 2.15 times as many as those of the anterior branch of RLNs, respectively. The single-fasciculated PNs comprised of about 75.0% (18/24)). CONCLUSION: Clinically, it may be safe and available for cutting PN off at the level of the subclavian vein. The length of PN is enough for the anastomosis of PN to the anterior branch of RLN.     

Pacing parameters of the canine posterior cricoarytenoid muscle

The electrical pacing of laryngeal tissue consists of many uncharted parameters. We carried out several acute experiments in the canine model to narrow parametric ranges to serve future research and development in this field. Our findings were as follows. Stimulation intensities greater than 6 V caused tissue damage. Optimum stimulation frequency ranged from 60 to 90 Hz. Optimum pulse duration was 2.0 milliseconds. Biphasic stimulation reduced accumulation of electrical charge at the electrodes.     

环杓后肌延期神经再支配后收缩特性的定量分析

为观察延期神经再支配环杓后肌的收缩特性，比较不同神经再支配方法的疗效，我们选择２４只犬，在右喉返神经切断后，于即刻，４，６，８，１０和１２个月时，分别以２只犬行选择性膈神经与喉返神经吻合（切断内收肌支）支配右环杓后肌（神经吻合组）；另１２只犬分别以２只行颈袢胸骨甲状肌蒂植入右环杓后肌（神经植入组）。组后饲养６个月，测定环杓后肌收缩强度及时间。结果表明，两组术侧的环杓后肌收缩力恢复率随病程延长而下降     

The effect of tracheostomy on posterior cricoarytenoid muscle

It has been reported previously that the amount of electromyographic (EMG) potential of the posterior cricoarytenoid (PCA) decreases after prolonged tracheostomy. It is, therefore, reasonable to assume that a significant alteration of the biochemical characteristics of this muscle would also occur. In addition to histochemical analysis, endoscopic and EMG data were recorded to give a direct comparison in each subject. Seven male beagles were used for this study. Four were tracheostomized and three served as controls. They were examined immediately before and after surgery and again after 4 weeks by EMG and endoscopic techniques. Histochemical staining was performed on each subject. All three modalities failed to demonstrate a substantial difference between the controls and the experimental dogs.     

Newer technique of laryngeal reinnervation: superior laryngeal nerve (motor branch) as a driver of the posterior cricoarytenoid muscle

This report analyzes the experience gained using two different techniques to reinnervate the paralyzed vocal cord. In the neurotization group, the superior laryngeal nerve (SLN) motor branch-cricothyroid muscle pedicle was used to reinnervate the posterior cricoarytenoid muscle. In the direct nerve anastomosis group, the SLN was anastomosed to the abductor branch of the recurrent laryngeal nerve (RLN), and the ansa hypoglossi (AH) to the adductor branch of the RLN. A third group of animals (control) had the right RLN sectioned without any anastomosis. About 5 to 6 months postoperatively the animals were killed painlessly and evaluated. The neurotization group revealed vocal fold mobilization on the right side to have an average of about half of the mobility of the left, normal side. After the RLN and SLN on the left were severed as well as the AH bilaterally, the vocal cord mobility was reduced to about one fourth. The direct nerve anastomosis group showed about fourfold less vocal cord mobility than the neurotization group. After the SLN, RLN, and AH were severed bilaterally, the control group showed no vocal cord mobility. The neurotization technique has been selected for further experimentation in human adults.     

The human posterior cricoarytenoid (PCA) muscle and diaphragm. A histochemical comparison as a basis for reinnervation attempts

PCA (posterior cricoarytenoid) muscles and biopsies from the SCM (sterno-cleidomastoid) muscles as well as the diaphragm were serially sectioned and incubated for myofibrillar ATPase and selected metabolic enzymes. The three main fibre types were present in all muscles, although some PCA muscles seemed to lack IIB fibres. The mean fibre type pattern of the PCA muscle was 57% type I, 36% type IIA and 7% type IIB, as compared with 42% type I, 42% type IIA and 16% type IIB in the diaphragm. All fibre types of the PCA muscle and the diaphragm were significantly more oxidative and less glycolytic than the corresponding SCM muscle fibres. Most striking was the finding of high 3-HBDH activity in the PCA and diaphragm muscle fibres, especially in type I.