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. 2001 Apr;82(2):123–128. doi: 10.1111/j.1365-2613.2001.iep0082-0123-x

Morphological aspects of rabbit masseter muscle after cervical sympathectomy

Maeli Dal Pai-Silva 1, Anete Kinumi Ueda 2, LuiZ A L Resende 3, Vitalino DaL Pai 1, Armando Alves 1, Antônio Tadeu Souza Faleiros 3, Antonio De Castro Rodrigues 4
PMCID: PMC2517706  PMID: 11454103

Abstract

The objective of this paper was to study the effect of sympathetic innervation on morphological and histochemical aspects of skeletal muscle tissue. Rabbit masseter muscle was studied using histochemical and immunohistochemical methods for periods of up to 18 months post-sympathectomy. The morphological and enzymatic characteristics of control masseter muscles were similar on both the left and right sides. The main features were muscle fibres with a mosaic pattern and a predominance of type IIa fibres, followed by type I. Type IIb fibres showed very low frequency. Sympathectomized animals showed varying degrees of metabolic and morphological alterations, especially 18 months after sympathectomy. The first five groups showed a higher frequency of type I fibres, whilst the oldest group showed a higher frequency of type IIb fibres. In the oldest group, a significant variation in fibre diameter was observed. Many fibres showed small diameter, atrophy, hypertrophy, splitting, and necrosis. Areas with fibrosis were observed. Thus cervical sympathectomy induced morphological alterations in the masseter muscles. These alterations were, in part, similar to both denervation and myopathy. These findings indicate that sympathetic innervation contributes to the maintenance of the morphological and metabolic features of masseter muscle fibres.

Keywords: sympathectomy, morphology, histochemistry, immunohistochemistry, masseter muscle, rabbit

Introduction

Striated muscles are composed of different fibre types, with variable morphological, enzymatic, and physiological characteristics, which are strongly related to motor innervation. In addition, some authors have demonstrated that sympathetic activity seems to be significant to fibre type composition in different muscles (Karlsson & Smith 1983). Lumbar sympathectomy in dogs revealed alterations in fibre type distribution in the gracilis muscle, with an increase in the fast fibre associated with changes in the composition of light-chain myosin isoforms. These findings, in some way, affect muscle contraction (Karlsson et al. 1988).

The absence of sympathetic activity was correlated with Romberg syndrome (progressive facial hemiatrophy) in humans. This is a serious disease which can cause major aesthetic facial deformities (Rees 1976; Rischbieth 1976; Freidel & Breton 1984). Its pathogenicity is unknown, but it has been hypothesized that the sympathetic nervous system is affected (Archambault & From 1932; Blatt et al. 1960; Tebloev et al. 1976; Miller et al. 1987).

In an experimental study using cats, dogs and rabbits, after ablation of the superior cervical sympathetic ganglion, Resende et al. (1991) were able to detect clinical alterations similar to progressive facial hemiatrophy. Skeletal muscle fibre innervation by sympathetic axons after motor denervation, has been demonstrated by several authors (Barker & Saito 1980, 1981; Nomoto et al. 1991).

The participation of sympathetic innervation in the morphological and histochemical aspects of the masseter muscle was studied to gain a better understanding of Romberg syndrome.

Materials and methods

Two groups of rabbits were used: 30 were submitted to ablation of the right superior cervical sympathetic ganglion, and 10 were used as control. Sympathectomy was confirmed by the presence of Claude Bernard–Horner Syndrome after ablation of the sympathetic ganglion, myosis, ptosis, enophthalmos, and morphological analysis of the sympathetic ganglion. The control rabbits were sacrificed 6, 12, and 18 months into the experiment and the experimental rabbits at 3, 6, 9, 12, 15, and 18 months. For the histochemical study, small segments from the superficial region of both the left and right masseter muscles were frozen in n-hexane, cooled in liquid nitrogen. Transverse cryostat sections were submitted to haematoxylin-eosin (HE), Gomori-trichrome, nonspecific Esterase (Chayen et al. 1969), Nicotinamide Adenine Dinucleotide-Tetrazolium Reductase (NADH-TR) (Peter et al. 1972), and myofibrillar ATPase (m-ATPase), after acid (pH 4.6) and alkaline (pH 10.4) preincubations (Dubowitz & Brooke 1973).

Immunohistochemical tests were performed, as described by Hsu et al. (1981), using the Avidin-Biotin-Peroxidase method (ABC). Transverse cryostat sections were incubated for 1 h at room temperature in a humid chamber with antibody against myosin heavy chain (MHC) for slow and fast myosin (Gorza 1990) diluted 1 : 1000 in 1% bovine serum albumin (BSA, Sigma Chemical). Unbound antibodies were removed by PBS, pH 7.4. The sections were incubated with a biotinylated antimouse IgG (Vector Laboratories Inc., CA, USA) diluted 1 : 60 in 1% BSA for 30 min at room temperature. After rinsing in PBS, an avidin-biotin-horseradish peroxidase complex was added and incubation continued for 30 min. Peroxidase was located using 0.038% 3–3′-diaminobenzidine tetrahydrocloride (DAB, Sigma Chemical) and 0.025% hydrogen peroxide in 0.1 m Tris-HCl, pH 7.4, for 4 min

For the frequency calculation (%) of fibre types in the studied regions, we used a camera lucida coupled to the microscope. Ten areas in each region could be seen using 400× magnification. We used histological sections submitted to m-ATPase reactions. The frequency calculation was based on the average number of types of fibre in each region. In the statistical analysis we used Goodman's coefficient Y2 for multinomial comparisons (Curi & Moraes 1981).

Results

The control rabbits showed similar morphological and enzymatic characteristics in the superficial masseter muscle on both the left and right sides. The muscle fibres revealed the following normal features: polygonal shape, mosaic pattern, predominance of type IIa fibres, and low frequency of type IIb fibres (Table 1). A small variation in fibre diameter was observed (Figure 1).

Table 1.

Frequency (%) and total number of fibre types on both left (L) and right (R) sides of the superior (S), medium (M) and inferior (I) portions of the masseter muscle in the control rabbits

Age Fibres SR MR IR SL ML IL
3 months I 26.6 (120) 27.9 (118) 25.9 (114) 32.8 (144) 31.2 (136) 27.8 (115)
IIa 55.4 (249) 57.6 (245) 52.9 (233) 59.4 (260) 53.7 (232) 50.0 (206)
IIb 18.0 (81) 14.5 (62) 21.2 (93) 7.8 (34) 15.1 (65) 22.2 (92)
Total 100 (450) 100 (425) 100 (440) 100 (438) 100 (432) 100 (413)
12 months I 43.3 (171) 38.7 (160) 32.5 (129) 35.9 (140) 27.5 (114) 22.4 (92)
II 51.7 (204) 52.2 (217) 56.3 (224) 56.9 (221) 60.3 (250) 58.8 (243)
IIb 5.0 (20) 9.1 (38) 11.2 (45) 7.2 (28) 12.2 (50) 18.8 (78)
Total 100 (395) 100 (415) 100 (398) 100 (389) 100 (414) 100 (413)
18 months I 35.0 (107) 31.5 (94) 24.3 (76) 34.9 (99) 30.0 (87) 29.5 (70)
IIa 58.3 (178) 58.0 (173) 56.5 (178) 60.3 (170) 60.0 (174) 53.9 (128)
IIb 6.7 (20) 10.5 (31) 19.2 (60) 4.8 (14) 10.0 (29) 16.6 (40)
Total 100 (305) 100 (298) 100 (315) 100 (283) 100 (290) 100 (238)
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Figure 1.

Figure 1

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Control masseter muscle. a – Normal fibres. HE, × 80. b and c – Three types of muscle fibre (I, IIa, IIb) with mosaic pattern. NADH-TR, × 100 and Non-specific Esterase, × 80, respectively. d – Predominance of type IIa fibre. m-ATPase, pH 4.6, × 60. e and f – Immunohistochemical reaction with monoclonal antibody for slow myosin, × 80, and fast IIa myosin, respectively, × 80.

The sympathectomized animals showed a varying degree of metabolic and morphological alteration in the masseter muscle on both sides, more pronounced on the right. Eighteen months after sympathectomy, the alterations were more evident. The animals in the oldest group showed a significant variation in fibre diameter, with clusters of atrophic and hypertrophic fibres (Figure 2a), focus of necrosis with phagocytosis (Figure 2b), fibrosis, and an increase in adipose tissue (Figure 2c). Fibres with sarcoplasm splitting were observed (Figure 1a). Alterations in oxidative metabolism enzyme distribution, fibres lacking oxidative metabolism enzymes, or fibres with subsarcolemal patches of formazan were observed (Figure 2d). Non-specific esterase showed angulated atrophic fibres (Figure 2e). The first five groups (3,6,9,12, and 15 months) showed a higher frequency of type I fibres (Figure 2f), while in the oldest group (18 months) a higher frequency of type IIb fibres was observed (Table 2).

Figure 2.

Figure 2

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Masseter muscle after sympathectomy. a – Atrophic (A) and hypertrophic (H) fibres. Fibrosis (F). Splitting (arrow). HE, × 80. b – Necrotic fibres (N). Internal nucleus (Nu). HE, × 80. c – Atrophic (A) fibres. Adipose tissue (Ad). Gomori Stain, × 60. d – Fibre clusters with intense oxidative enzyme activity (*), and fibres with alteration in oxidative enzyme activity (arrow head). Atrophic fibre (f). NADH-TR. X60. e – Angulated atrophic fibre (f). Non-specific esterase, × 60. f – Clusters of type I (I) fibre. m-ATPase, pH 4.6. × 40.

Table 2.

Frequency (%) of fibre types on both left (L) and right (R) sides of the superior (S), medium (M) and inferior (I) portions of the masseter muscle in sympathectomized rabbits

Age Fibres SR MR IR SL ML IL
3 months I 61.9 (286) 57.8 (264) 54.6 (220) 64.2 (277) 55.5 (252) 46.9 (188)
IIa 34.6 (160) 38.7 (177) 41.2 (166) 33.3 (144) 40.5 (184) 48.1 (193)
IIb 3.5 (016) 3.5 (016) 4.2 (017) 2.5 (011) 4.0 (018) 5.0 (020)
Total 100 (462) 100 (457) 100 (403) 100 (432) 100 (454) 100 (401)
12 months I 51.9 (230) 47.5 (181) 38.3 (193) 60.0 (222) 46.5 (180) 40.3 (157)
IIa 45.2 (200) 47.8 (182) 54.7 (276) 35.9 (133) 48.1 (186) 50.8 (198)
IIb 2.9 (013) 4.7 (018) 7.0 (035) 4.1 (015) 5.4 (021) 8.9 (035)
Total 100 (443) 100 (381) 100 (504) 100 (370) 100 (387) 100 (390)
18 months I 26.6 (075) 34.6 (082) 28.0 (061) 38.9 (094) 33.4 (077) 24.1 (047)
IIa 52.1 (147) 45.6 (108) 38.5 (084) 44.2 (107) 48.3 (111) 35.9 (070)
IIb 21.3 (060) 19.8 (047) 33.5 (073) 16.9 (041) 18.3 (042) 40.0(078)
Total 100 (282) 100 (237) 100 (218) 100 (242) 100 (230) 100 (195)
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Discussion

The masseter muscle, which plays an active role in masticatory activity, is composed of several anatomic compartments, with different compositions of muscle fibre types (Bredman et al. 1990). In this work, we studied three portions of the superficial region: superior, medium, and inferior. The control animals showed a predominance of type IIa fibres in all portions, followed by type I. The frequency of type IIb fibres was low, especially in the inferior portion. Regional variations in fibre distribution of the skeletal muscles are common findings (Hering et al. 1979; Clark & Luschei 1981; Eriksson & Thornell 1983). In rat limb muscles, slow fibres usually lie deep within the muscle and fast fibres are superficial (Armstrong & Phelps 1984). In the jaw muscles, fibre distribution within the compartments allows the muscle to perform different roles during mastication (Bredman et al. 1990).

In this study, the experimental groups showed a predominance of type I fibres in all superior portions of the masseter muscle. However, the 18-month group showed a high frequency of type IIb fibres. The variation in fibre type distribution in the masseter muscle is correlated with mastigatory physiology (Bredman et al. 1990). In our study, however, this variation was more evident in the sympathectomized animals. The predominance of type I fibres in the first five groups (3, 6, 9, 12, and 15), and the predominance of type IIb fibres in the superficial masseter muscle in the 18-month group presents an interesting modulation in fibre type. The observation is similar to results obtained in athlete muscles submitted to anaerobic (strength) exercises (Anderson & Henriksson 1977; Keul et al. 1982). According to these authors, the characteristics acquired by the muscle after training, could be explained by the reduction in muscle sensitivity to the autonomic nervous system and might also be observed after sympathetic denervation (Karlsson & Smith 1983).

The absence of sympathetic activity in the striated muscle may have changed the proportion of fibre types (Karlsson & Smith 1983). After sympathetic denervation, an alteration in fibre type distribution was seen in the gracilis muscle. There was a reduction in slow fibres and in the percentage of slow myosin isoforms, with an increase in fast fibres and fast myosin isoforms. This resulted in a reduced endurance capacity and an increase in muscle fatigability (Karlsson & Smith 1983).

Blood flow to different regions of the organism is dependent on autonomic nervous system activity. As proposed by Cronenwett & Lindenauer (1977) an alteration in normal blood flow occurs after sympathectomy, with redistribution and increased flow in the arteriovenous anastomoses. In our study, this redistribution of flow might have contributed significantly to the changes observed in the fibre types.

After ablation of the sympathetic ganglion, the masseter muscle showed atrophic and hypertrophic fibres, fibre splitting, necrosis and phagocytosis, fibrosis, and alterations in the distribution of oxidative enzymes, which were more evident 18 months after sympathectomy. Similar alterations were observed in various muscular disorders. Some alterations are common in muscles after denervation, others after myopathy (Cullen et al. 1992; Loughlin 1993).

In conclusion, therefore, this study shows that after interruption of sympathetic activity in the masseter muscle, a change in fibre composition is observed, with an increase in type IIb fibres and morphological alterations in muscle fibres. These findings may contribute to the understanding of the mechanism of hemiatrophy seen in Romberg syndrome.

Acknowledgments

This work was supported by grants from FAPESP, Proc. 94/2163–1.

We wish to thank Sueli Cruz Michelin and José Carlos Pedroso Lima for technical assistance.

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