Innervation of adult human laryngeal muscle fibers

Journal of Neurological Sciences 149 (1997) 81–86

Innervation of adult human laryngeal muscle fibers ´ ´ a , c , Jean Lacau St Guily a , Patrice Callard b , Alain Sebille c , * Sophie Perie a

ˆ Service d’ Otolarynologie et de chirurgie de la Face et du Cou, Hopital Tenon, 4 rue de la Chine, 75970 Paris Cedex 20, France b ˆ Service d’ Anatomopathologie, Hopital Tenon, 4 rue de la Chine, 75970 Paris Cedex 20, France c ´ ´ ´ ´ ´ Atelier de Regeneration Neuromusculaire, Departement de Physiologie, Faculte´ de Medecine Saint Antoine, 27 rue de Chaligny, 75571 Paris Cedex 12, France Received 16 October 1996; revised 3 January 1997; accepted 30 January 1997

Abstract The innervation of laryngeal muscle fibers was appraised in adult humans. Sixteen intrinsic laryngeal muscles were dissected during the autopsy of 4 adults (41–71 years old). Longitudinal serial frozen sections, 60 mm thick, of the whole muscles were double-stained for cholinesterase activity and axonal visualization. About 945 endplates per muscle were analysed using light microscopy. The neuromuscular junctions were always scattered throughout the whole muscles. Most of the muscle fibers showed a single neuromuscular junction, but multi-innervated fibers were found in all of the muscles. Their number was highest in interarytenoid muscles (21% of all the fibers). The distance between multiple neuromuscular junctions was most frequently less than 150 mm. Two neuromuscular junctions were frequently displayed, opposite one another, particularly in thyroarytenoid muscles, and this unusual feature seems specific for laryngeal muscles. The innervation of all of the muscle fibers was exclusively found to be unineuronal, with multi-innervated fibers being innervated by a single axon. Distal axonal degeneration occurred with aging, resulting in a loss in the number of multi-innervated muscle fibers.  1997 Elsevier Science B.V. Keywords: Aging; Cholinesterase; Human; Laryngeal muscles; Multi-innervation; Neuromuscular junction; Unineuronal innervation

1. Introduction Although the larynx plays a crucial role in the functions of respiration, swallowing and phonation in humans, little is known about the innervation pattern of the intrinsic laryngeal muscle fibers that move the pieces of cartilage dedicated to these functions. As a general rule, extrafusal fibers of mammalian skeletal muscles exhibit one neuromuscular junction (NMJ) that is innervated by a single motor axonal branch. Such a disposition is named focal unineuronal innervation (Salpeter, 1987). In the case of the thyroarytenoid laryngeal muscle, which supports phonation, previous reports pointed out the presence of fibers exhibiting more than one NMJ. This multi-innervation affected 20 to 80% of the total number of fibers, depending *Corresponding author: Tel: (33) 01 43 42 10 87; Fax: (33) 01 40 01 14 99; E-mail: [email protected] 0022-510X / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0022-510X( 97 )05395-1

of the report (Rudolph, 1962; Rossi and Cortesina, 1965a; Morales et al., 1980; Bendiksen et al., 1981; Lacau St Guily and Fardeau, 1983). The innervation of other laryngeal muscle fibers was thought to be multi-innervated also (Rossi and Cortesina, 1965b; Lacau St Guily and Fardeau, 1983). The posterior cricoarytenoid muscle, which is the single muscle opening the larynx during inspiration, was credited with the lower number of multiinnervated fibers (4%) (Rossi and Cortesina, 1965b; Lacau St Guily and Fardeau, 1983). However, it is not known if these NMJs are unineuronal or if the multi-innervated fibers are innervated by several axons or by branches of a single axon. Thus, due to the increasing use of botulinum toxin therapy to block NMJs in laryngeal dystonia (Blitzer et al., 1986), we thought that it would be of interest to delineate more accurately the pattern of innervation of the laryngeal muscles. For this purpose, we studied adult laryngeal muscles obtained from autopsies, frozen longi-

82

´ ´ et al. / Journal of Neurological Sciences 149 (1997) 81 – 86 S. Perie

tudinal sections of which were double-stained (cholinesterase activity and axons).

2. Materials and methods

2.1. Material Sixteen laryngeal intrinsic muscles (4 thyroarytenoid, 4 posterior cricoarytenoid, 4 interarytenoid and 4 cricothyroid) were dissected out within 30 h of the death of 4 adults (41, 54, 68, 71 years old), according to current French law. These patients did not suffer from neuromuscular or respiratory disorders and were not infected with HIV or hepatitis C. The whole frozen muscles were sectioned longitudinally. The serial sections (20 or 60 mm thick) were mounted on polysilane precoated glass slides. The 20 mm thick sections were stained with hematoxylin– eosin (H and E). The 60 mm thick sections were incubated with 5-bromoindoxyl acetate for cholinesterase (ChE) activity and axons were gold–silver impregnated, according to the method of Pestronk and Drachman (1978).

2.2. Quantitative histology H and E sections were observed at magnifications of 3100 or 3250 to calculate the diameters of the fibers. In the double-stained sections (3250 or 3400), myofibers that were longer than 300 mm and showed the presence of at least one NMJ were investigated. The length (mm) of each NMJ was measured and its aspect was classified as ‘‘platelike’’ (i.e. a large compact or lobulated end-plate) (Engel, 1986; Oda, 1986), ‘‘grapelike’’ (i.e. a group of several small ChE spots) (Engel, 1986; Oda, 1986) or ‘‘complex’’ (scattered nerve endings) (Tuffery, 1971). Multiple NMJs were recorded and the distances between them were measured. The number of axonal branches innervating each NMJ was also recorded.

3. Results

Fig. 1. Three esterase activities detected in human laryngeal muscles by 5-bromo-indoxyl oxidation. (A) Neuromuscular junction (interarytenoid muscle, 41 year old); (B) myotendinous junction (thyroarytenoid muscle, 54 year old) and (C) Cross-section of a sensory organ with encapsulated nerve terminal (interarytenoid muscle, 54 year old). ABC: magnification 3400.

3.1. General features Each muscle provided about 40 serial sections, in which the fibers were constantly parallel, giving longitudinal aspects that were favourable for observing multiple NMJs. No fascicles were observed. Connective tissue was abundant between muscle fibers in all of the muscles. The diameter of the fibres ranged from 20–35 mm, regardless of the muscle. As shown in Fig. 1, esterase activity was detected at the myotendinous junctions, without nerve endings being present, as was previously described for skeletal muscles (Couteaux, 1953; Teravainen, 1969). In connective tissue, some nerve-ending terminals ensheathed in a conjunctive capsule exhibited esterase activity. We

assume that these structures were sensory receptors (but not spindles) as no myofibers were observable in their vicinity. NMJs were characterized by esterase activity (stained blue) coexisting with axon terminals (which stained black). They were scattered without definite endplate zones throughout all of the studied laryngeal muscles.

3.2. Patterns of innervation An average of 851 myofibers were observed in each of the muscles, in which an average of 945 NMJs were recorded. The 3 aspects of ‘‘platelike’’, ‘‘grapelike’’ or ‘‘complex’’ nerve endings were observed in all of the

´ ´ et al. / Journal of Neurological Sciences 149 (1997) 81 – 86 S. Perie

83

Fig. 3. Three aspects of multi-innervation of a single myofiber by sprouting of a single motor axon. (A) Interarytenoid muscle, 68 year old; (B) interarytenoid muscle, 41 year old and (C) thyroarytenoid muscle, 68 year old. ABC: magnification 3400. Fig. 2. Three aspects of a double-stained neuromuscular junction in human laryngeal muscles. (A) ‘‘Platelike’’ (posterior cricoarytenoid muscle, 68 year old); (B) ‘‘grapelike’’ (interarytenoid muscle, 68 year old) and (C) ‘‘complex’’ (thyroarytenoid muscle, 54 year old). ABC: magnification 3400.

muscles, however, ‘‘complex’’ nerve endings were found principally in thyroarytenoid muscles (Fig. 2). At the post-synaptic zone, 2 or more ChE spots were constantly observed. The mean length of the NMJs was 47 mm at

41–54 years of age and decreased significantly in the elderly, to reach 38 mm (Table 1). In interarytenoid muscles, some NMJs measured 200 mm in length. Most of the muscle fibers exhibited a single NMJ, but some fibers in all of the muscles exhibited multiple NMJs (Fig. 3). Most of the multi-innervated fibers displayed 2 end-plates. The number of these as a percentage of the total number of fibers was higher in interarytenoid muscles. It decreased with age, as in all the muscles (Table 2). The distance

Table 1 Mean length (mm) of the neuromuscular junctions (6one standard error) Age (years)

TA

PCA

IA

CT

Middle-aged adults

41 54

52 (0.82) 53 (1.02)

51 (0.69) 51 (0.70)

47 (0.63) 42 (0.49)

41 (0.42) 36 (0.38)

Elderly adults

68 71

47 (0.49) 40 (0.76)

46 (0.54) 46 (0.66)

41 (0.46) 34 (0.41)

37 (0.36) 36 (0.46)

TA (thyroarytenoid muscle), PCA (posterior cricoarytenoid muscle), IA (interarytenoid muscle) and CT (cricothyroid muscle).

´ ´ et al. / Journal of Neurological Sciences 149 (1997) 81 – 86 S. Perie

84

Table 2 Percentage of multi-innervated myofibers to the total number of fibers recorded (in brackets) Age (years)

TA (%)

PCA (%)

IA (%)

CT (%)

Middle-aged adults

41 54

14 (668) 14 (727)

12 (635) 14 (723)

21 (550) 21 (1077)

7 (915) 8 (1139)

Elderly adults

68 71

9 (1537) 10 (386)

7 (899) 7 (619)

10 (1176) 9 (877)

6 (1022) 5 (670)

Abbreviations as in Table 1.

between two NMJs seen in the same muscle fiber varied from 10 to 700 mm, but was generally less than 150 mm. Two usual aspects were observed in these multi-innervated fibers: (i) Multiple small NMJs, side by side, with a distance of 10–20 mm between them; (ii) double NMJs displayed opposite one another on the extremities of one diameter of the muscle fiber (Fig. 4). This particular feature occurred more frequently in the thyroarytenoid muscles (Table 3). Irrespective of the laryngeal muscle, the innervation was found to be exclusively unineuronal, with

Fig. 4. Two neuromuscular junctions of the same myofiber displayed opposite one another. A and B, longitudinal aspect (A5thyroarytenoid muscle, 54 year old; B5interarytenoid muscle, 41 year old). C, Crosssection at the level of the neuromuscular junctions (thyroarytenoid muscle, 68 year old). ABC: magnification 3400.

sprouts at the terminal part of a single axon innervating the multiple NMJs of the same myofiber. In the muscles of the oldest patients, most of the axons innervating an end-plate were shown to sprout at the level of the end plate (ultraterminal sprouting), innervating the end-plates of the adjacent muscle fibers, without a proper innervating axon (denervated myofibers).

4. Discussion Three striking features emerge from this study about the innervation of human laryngeal muscles: First, the NMJs were not restricted to an end-plate zone; second, two or more end-plates innervating the same fiber were seen in all of the laryngeal muscles and their disposition (opposite one another of most of these end-plates) seems specific for these muscles; third, the innervation of adult laryngeal muscle NMJs was found to be unineuronal, as is the rule in skeletal muscles. NMJs were randomly distributed along the muscle fibers, resulting in scattered distribution in all of the muscles, as was previously observed in the thyroarytenoid muscle (Rudolph, 1962; Rossi and Cortesina, 1965a; Rosen et al., 1983). No end-plate zones were observable in laryngeal muscles, in contrast with previous findings (Sonesson, 1960; Morales et al., 1980; De Vito et al., 1985; Gambino et al., 1985; Freije et al., 1986, 1987). This could be due rather to the spread insertion of the myofibers on the cartilages resulting in an irregular arrangement (contrasting with the pennate disposition due to a tendon in skeletal muscle), than to the presence of multi-innervated fibers. This peculiar insertion of myofibers in the laryngeal pieces of cartilage results in a lack of muscle fascicles and in the organisation of the myofibers in layers. All of these differences from skeletal muscles suggest that laryngeal

Table 3 Percentage of neuromuscular junctions opposite one to another to the number of multi-innervated myofibers recorded (in brackets) Age (years)

TA (%)

PCA (%)

IA (%)

CT (%)

Middle-aged adults

41 54

36 (91) 40 (105)

24 (74) 19 (99)

10 (114) 17 (228)

15 (66) 11 (92)

Elderly adults

68 71

18 (140) 34 (41)

24 (61) 25 (43)

18 (113) 15 (83)

10 (61) 9 (34)

Abbreviations as in Table 1.

´ ´ et al. / Journal of Neurological Sciences 149 (1997) 81 – 86 S. Perie

muscles should be defined as cartilaginous muscles and not as skeletal ones. In three previous studies performed on laryngectomy material (Rossi and Cortesina, 1965a; Bendiksen et al., 1981; Lacau St Guily and Fardeau, 1983), a high frequency of multiple innervation in the thyroarytenoid muscle was reported. This high percentage of multi-innervation was proposed on the basis of multiple sites of ChE activity along the same fiber without visualisation of the motor axon terminals. Due to the laryngectomy origin of the specimens, one can argue that some of the multiinnervated fibers resulted from denervation, with a spread of acetylcholine receptors. In a preliminary study, we observed the large extent of neurogenic atrophy in muscles dissected from the healthy side of a neoplastic larynx (not shown). In view of this result, we avoided the use of laryngectomy muscles in the present study and we decided to use simultaneous staining of end-plates and nerve terminals to delineate the pattern of innervation of the laryngeal muscle fibers. We observed multiple end-plates in fibers from all of the laryngeal muscles from autopsy material and the percentage of these was about the same as that previously reported in extraocular muscles (Teravainen, 1968; Salpeter et al., 1974). Interestingly, the interarytenoid muscle, which has not been studied to our knowledge, showed the highest percentage of multi-innervated fibers of all of the laryngeal muscles, except in aging muscle, where it decreases rapidly (more than 20% until 54 years of age and 10% or less from 68 years of age). In thyroarytenoid muscles, a high frequency of double NMJs that were opposite each other were seen. This arrangement seems specific to human laryngeal muscles and was never observed in any of the NMJs described in vertebrates to date. Transverse sections, as shown in Fig. 4, confirmed their arrangement on each extremity of one diameter of the muscle fiber. They were constantly innervated by two sprouts of the same axon. We suggest that this feature could result from the segmentation of a single NMJ when the muscle fiber increased in size, as this aspect was not reported in child laryngeal muscles (Konig and Von Leden, 1961). During the embryonic development of skeletal muscles, each NMJ is transiently innervated by multiple axons and the myofiber is refractory to further innervation at other sites along its length (Bennett and Pettigrew, 1974). The poly-axonal innervation progressively disappears until the early postnatal period and this regression is thought to be due to the withdrawal of axon terminals from NMJs without neuronal degeneration (Redfern, 1970; Brown et al., 1976; Jansen and Fladby, 1990). A single axon at least persists, which innervates the single NMJ of each myofiber (focal innervation) and the terminal part of this axon further arborizes to extend the area of the NMJ (Tuffery, 1971; Juntunen and Teravainen, 1972). There is a controversy about the uni- or polyneuronal innervation in multi-innervated muscle fibers from adults. In extraocular

85

muscles, the innervation of fibers was found to be unineuronal (Bach-y-Rita and Lennerstrand, 1975), but also multi-neuronal (Pilar, 1967). In laryngeal muscles, no evidence of polyneuronal innervation was shown in dogs (Martensson, 1966). In our study, unineuronal innervation was constantly observed in all human laryngeal muscles, including the interarytenoid muscle, which is unpaired and receives motor nerve fibers coming from the two inferior laryngeal nerves. Spontaneous retrograde axonal degeneration occurs with age in human peripheral nerves (Toghgi et al., 1977). Age-related changes have been also reported in clinical and pathological series concerned with laryngeal sensory and motor function (Ward et al., 1981; Malmgren and Ringwood, 1988). The superior laryngeal nerve in old people exhibits a loss of number and a decrease in axonal diameter in myelinated nerve fibers (Mortelliti et al., 1990). As a result of this spontaneous axonal degeneration, we observed a decrease both in the number of multiinnervated fibers and in the mean length of NMJs in old laryngeal muscles. Simultaneously, several muscle fibers previously denervated were reinnervated by ultraterminal sprouts of intact axons, as occurs in skeletal muscle (Brown et al., 1981). In summary, we found multi-innervated fibers in all of the human laryngeal muscles and their innervation was always unineuronal. In many cases, two neuromuscular junctions were found opposite one another, an unusual feature that seems specific to laryngeal muscles. Finally, no defined end-plate zone was delineated, regardless of the laryngeal muscle recorded in this series, suggesting (from a therapeutic point of view) that a diffuse spreading of botulinum toxin might be warranted to ensure a durable effect rather than targeting the same spot with repeated injections.

References Bach-y-Rita, P. and Lennerstrand, G. (1975) Absence of polyneuronal innervation in cat extraocular muscles. J. Physiol. (Lond.), 244: 613– 624. Bendiksen, F.S., Dahl, H.A. and Teig, E. (1981) Innervation pattern of different type of muscle fibres in the human thyroarytenoid muscle. Acta Oto-Laryngol., 91: 391–397. Bennett, M.R. and Pettigrew, A.G. (1974) The formation of synapses in striated muscle during development. J. Physiol. (Lond.), 241: 515– 545. Blitzer, A., Brin, M.F., Fahn, S. and Lovelace, R.E. (1986) Localized injections of botulinum toxin for the treatment of focal laryngeal dystonia. Laryngoscope, 98: 193–197. Brown, M.C., Holland, R.L. and Hopkins, W.G. (1981) Motor nerve sprouting. Ann. Rev. Neurosci., 4: 17–42. Brown, M.C., Jansen, J.K.S. and Van Essen, D. (1976) Polyneuronal innervation of skeletal muscle in newborn rats and its elimination during maturation. J. Physiol. (Lond.), 261: 387–424. ´ histochimiques des zones d’insertions Couteaux, R. (1953) Particularites ´ C.R. Soc. Biol. (Paris), 147: 1974–1976. du muscle strie. De Vito, M., Malmgren, L.T. and Gacek, R.R. (1985) Three-dimensional

86

´ ´ et al. / Journal of Neurological Sciences 149 (1997) 81 – 86 S. Perie

distribution of neuromuscular junctions in human cricothyroid. Arch. Otolaryngol. Head Neck Surg., 111: 110–113. Engel, A.G. (1996) The neuromuscular junction. In: A.G. Engel and B.Q. Banker (Eds.), Myology: Basic and Clinical, 2nd ed. McGraw-Hill, New York, NY, pp. 209–253. Freije, J., Malmgren, L.T. and Gacek, R.R. (1986) Motor end-plate distribution in the human lateral cricoarytenoid muscle. Arch. Otolaryngol. Head Neck Surg., 112: 176–179. Freije, J., Malmgren, L.T. and Gacek, R.R. (1987) Motor end-plate distribution in the human interarytenoid muscle. Arch. Otolaryngol. Head Neck Surg., 113: 63–68. Gambino, D.R., Malmgren, L.T. and Gacek, R.R. (1985) Three dimensional computer reconstruction of the neuromuscular junction distribution in the human posterior cricoarytenoid muscle. Laryngoscope, 95: 556–560. Jansen, J.K.S. and Fladby, T. (1990) The perinatal reorganization of the innervation of skeletal muscle in mammals. Prog. Neurobiol., 34: 39–90. Juntunen, J. and Teravainen, H. (1972) Structural development of myoneural junctions in the human embryo. Histochemistry, 32: 107– 112. Konig, W.F. and Von Leden, H. (1961) The peripheral nervous system of the human larynx. Part II: The thyroarytenoid (vocalis) muscle. Arch. Otolaryngol. Head Neck Surg., 74: 153–163. ` Lacau St Guily, J. and Fardeau, M. (1983) Muscles intrinseques du larynx ´ de l’homme: caracteristiques histoenzymologiques des fibres musculaires. Ann. Otolaryngol. (Paris), 100: 1–12. Malmgren, L.T. and Ringwood, M.A. (1988) Aging of the recurrent laryngeal nerve: an ultrastructural morphometric study. In: O. Fjimura (Ed.), Vocal Fold Physiology: Voice Production, Mechanisms and Functions, Raven Press, New York, NY, pp. 159–170. Martensson, A. (1966) Testing for doubly innervated fibers in the intrinsic laryngeal muscles of the dog. Acta Physiol. (Scand.), 67: 152–164. Morales, J., Rama, J. and Gayoso, M. (1980) Some aspects of the nerve endings and synapses in the vocalis muscle. J. Laryngol. Otol., 94: 1047–1063. Mortelliti, A., Malmgren, L.T. and Gacek, R.R. (1990) Ultrastructural changes with age in the human superior laryngeal nerve. Arch. Otolaryngol. Head Neck Surg., 116: 1062–1069. Oda, K. (1986) Motor innervation and acetylcholine receptor distribution of human extraocular muscle fibres. J. Neurol. Sci., 74: 125–133.

Pestronk, A. and Drachman, D.B. (1978) A new stain for quantitative measurement of sprouting at neuromuscular junctions. Muscle Nerve, 1: 70–74. Pilar, G. (1967) Further study of the electrical and mechanical responses of slow fibers in cat extraocular muscles. J. Gen. Physiol., 50: 2289–2300. Redfern, P.A. (1970) Neuromuscular transmission in newborn rats. J. Physiol. (Lond.), 209: 701–709. Rosen, M., Malmgren, L.T. and Gacek, R.R. (1983) Three-dimensional computer reconstruction of the distribution of neuromuscular junctions in the thyroarytenoid muscle. Ann. Otol. Rhinol. Laryngol., 92: 424– 429. Rossi, G. and Cortesina, G. (1965a) Multi-motor end-plate muscle fibres in the human vocalis muscle. Nature, 206: 629–630. Rossi, G. and Cortesina, G. (1965b) Morphological study of the laryngeal muscles in man. Acta Otolaryngol., 59: 575–592. ´ des synapses neuromusculaires dans le Rudolph, G. (1962) Particularites muscle vocal de l’homme. Rev. Laryngol. (Bord.), 83: 569–583. Salpeter, M.M., McHenry, F.A. and Feng, H.H. (1974) Myoneural junctions in the extraocular muscles of the mouse. Anat. Rec., 179: 201–224. Salpeter, M.M. (1987) Vertebrate neuromuscular junctions: general morphology, molecular organization, and functional consequences. In: M.M. Salpeter, (Ed.), Neurology and Neurobiology: The Vertebrate Neuromuscular Junction, Vol. 23, Alan R. Liss, New York, NY, pp. 1–54. Sonesson, B. (1960) On the anatomy and vibratory pattern of the human vocal folds. Acta Oto-Laryngol., Suppl 156: 1–80. Teravainen, H. (1968) Electron microscopic and histochemical observations on different types of nerve endings in extraocular muscles of the rat. Z. Zellforsch., 90: 372–388. Teravainen, H. (1969) Localization of acetylcholinesterase activity in myotendinous and myomuous junctions of the striated skeletal muscles of the rat. Experientia, 25: 524–525. Toghgi, H., Tsukagoshi, H. and Toyokura, Y. (1977) Quantitative changes with age in normal sural nerves. Acta Neuropathol., 38: 213–220. Tuffery, A.R. (1971) Growth and degeneration of motor end-plates in normal cat hind limb muscles. J. Anat., 2: 221–247. Ward, P.H., Colton, R., McConnell, F., Malmgren, L., Kashima, H. and Woodson, G. (1981) Aging of the voice and swallowing. Otolaryngol. Head Neck Surg., 100: 283–286.