Delayed muscle fiber transformation after foreign-reinnervation of excessive muscle tissue.код для вставкиСкачать
THE ANATOMICAL RECORD 223:347-355 (1989) Delayed Muscle Fiber Transformation After ForeignReinnervation of Excessive Muscle Tissue LORENZO KASER AND MARKUS MUNTENER Institute of Anatomy, University of Zurich-Irchel, CH-8057 Zurich, Switzerland ABSTRACT Following partial denervation motor units can increase (by selfreinnervation) as much as four to five times their normal size. To investigate the still unknown quantitative reinnervation capacity of a motor nerve in the case of foreign-reinnervation, in adult male rats the denervated sternomastoid muscle was either self-reinnervated by its original nerve or foreign-reinnervation by the omohyoid nerve, which had to reinnervate the three times the amount of muscle fibers and six times the amount of muscle mass. After survival times of 7, 8, 9, or 10 months, nerves and muscles were investigated histochemically and immunohistochemically. The omohyoid nerve could fully reinnervate the sternomastoid muscle, but at 7 and 8 months this muscle still revealed nearly the same proportions of IIA and IIB fibers as were seen in the self-reinnervated sternomastoid at all stages. However, in the following 2 months a shift of the fiber pattern toward that of the normal omohyoid was observed, as evidenced by a strong increase in type IIB fibers (from 24% to 62%),at the expense of type IIA fibers. These findings are in contrast to those after foreign (cross) reinnervation of leg muscles where the fiber transformation (according t o the foreign motor input) occurs in parallel with the reinnervation process during the first 2-3 months. The delayed fiber transformation observed could be the consequence of the highly enlarged peripheral field of the omohyoid motoneuron pool or could merely reflect a general difference between limb and neck muscles. Whatever the case may be, in the present experiments the afferent input, which is not fully restored until 9 months, could play a crucial role in contrast to limb muscles, where the successful motor reinnervation takes place in the absence of sensory activity. The morphological, physiological, and metabolic characteristics of muscles are largely determined by the innervating motoneurons (Buller et al., 1960; Close, 1965; Guth, 1968; Buchthal and Schmalbruch, 1980; Jolesz and Sreter, 1981; Pette and Vrbova, 1985). Within most muscles the proportions of the different fiber types are rather constant over long periods, the content of transitional fibers being very low. However, the fiber spectrum of a given muscle may be altered by a variety of stimuli, such as training (Salmons and Henriksson, 1981; Howald, 1982; Schantz, 1986; Baumann et al., 1987), hormones (mainly thyroid hormones) (Ianuzzo et al., 1977; Fitts et al., 1980; Nicol and Bruce, 1981; Nwoye and Mommaerts, 1981; Nwoye et al., 1982; Johnson et al., 1983; Florini, 1987; Muntener et al., 1987),artificial chronic stimulation, or foreign-reinnervation (Guth, 1968; Jolesz and Sreter, 1981; Pette and Vrbova, 1985). A special kind of neuromuscular plasticity is seen in the aging muscle (Campbell et al., 1973; Kugelberg, 1976; Caccia et al., 1979; Jenny et al., 1980; Larsson, 1982; Edstrom and Larsson, 1987). Beside an altered fiber type composition (mainly in slow muscles), a gradual loss of motoneurons is observed (Larsson, 1982; Edstrom and Larsson, 1987), leading to a partial denervation of the muscle. A considerable amount of these muscle fibers are reinnervated by collateral 0 1989 ALAN R. LISS, INC. sprouting from neighboring axons (Harriman et al., 1970; Kugelberg et al., 1970; Grinnell and Herrera, 1981; Wernig and Herrera, 1986; Edstrom and Larsson, 1987), resulting in an enlargement of the remaining motor units. This denervation-reinnervation process leads to a consequent reorganization of fiber types in which type grouping (Karpati and Engel, 1968) replaces the usual checkerboard pattern of fiber types. These observations lead to the question of the amount of muscle fibers that can additionally be innervated by a single axon. Neurophysiological experiments with partially denervated muscles have shown that motor units can increase by as much as four to five times their normal size (Thompson and Jansen, 1977; Brown and Ironton, 1978; Brown et al., 1981; Grinnell and Herrera, 1981; Hatcher et al., 1985). However, the studies mentioned above only examined the motoneurons that were self-reinnervating their original muscle. Nothing is known about the quantitative and qualitative reinnervation capacity in the case of foreign-reinnervation. Received May 4,1988, accepted August 8,1988. Address reprint requests t o Markus Miintener, Anatomisches Institut der Universitat Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. L. U S E R AND M. MUNTENER 348 In the present investigation the omohyoid nerve was brought to reinnervate the denervated sternomastoid muscle. The sternomastoid muscle has six times the amount of muscle mass and three times the amount of muscle fibers, including type I fibers, which are lacking in the omohyoid muscle (Gottschall et al., 198Ob; Muntener et al., 1980). Histo- and immunohistochemical investigation of nerve and muscle revealed a successful foreign-reinnervation. However, in this quantitatively asymmetric type of cross-reinnervation, the foreign motor input led to a delayed fiber transformation starting 7 months after reinnervation, in contrast to the classical cross-reinnervation experiments in limb muscles (with comparable fiber ratios) in which the fiber transformation roughly parallels the reinnervation process. MATERIALS AND METHODS Young adult male rats (2.5 months old; 250-300 g; strain Zur:SIV) supplied by the Institute of Laboratory Animal Science, University of Ziirich, were used. The animals were anesthetized with 0.25 m l k g body weight Innovar-Vet (Pitman-Moore) IM combined with 2.5 mg Valium (Roche) and 7.5 mg Nembutal (Abbott) IP. Surgical Procedures Anatomy As in the human, the sternomastoid muscle in rat arises from the manubrium of the sternum and is inserted into the mastoid. The sternomastoid nerve (entering laterally into the muscle) originates from two separate roots: a motor root from the accessory nerve and a sensory root from the cervical plexus (Gottschall et al., 1980b). The two omohyoid muscles (inferior and superior), which are connected by a n intermediate tendon, extend from the anterior border of the scapula to the hyoid bone, passing behind the sternomastoid. The omohyoid superior muscle lies close to the lateral border of the sternohyoid. It is supplied by a branch of the superior radix of the ansa cervicalis, which enters the muscle medially a t the point where it crosses the sternomastoid (Muntener et al., 1980). Foreign-reinnervation In 13 animals the right sternomastoid muscle was denervated by resecting the sternomastoid nerve between the point of entry into the muscle and the fusion of its motor and sensory roots. For additional prevention of self-reinnervation, the accessory nerve was transected and the proximal stump sutured into the posterior belly of the digastric muscle. The omohyoid superior nerve was exposed and cut near its entrance into the muscle. After excision of a short segment (for the first determination of the number of alpha-motor axons), the nerve was sutured medially into the sternomastoid muscle, opposite to the point of entry of the original nerve. To avoid a growth of omohyoid nerve fibers toward the denervated omohyoid superior muscle, this muscle was excised. Self-reinnervation In 11animals the accessory and sternomastoid nerves were transected and loosely adapted (without suture). Fourteen age-matched animals served as controls. Histochemical Procedures After survival times of 7,8, 9, and 10 months, respectively, the animals were deeply anesthetized with Nembutal, and the functional reinnervation was evidenced (see below and under Results). Muscles The experimental sternomastoid and contralateral sternomastoid and omohyoid superior muscles were either frozen in toto in melting isopentane (- 160°C) or divided transversely into three parts. The cranial third was frozen, the middle third was fixed with 4% formaldehyde in 0.2 M phosphate buffer @H 7.4), and the caudal third was fixed with Bouin’s fluid and embedded in paraffin. Control muscles were processed in the same fashion. Cryocut cross sections (12 pm) were reacted for myofibrillar ATPase according to Guth and Samaha (19701, with certain modifications as described elsewhere (Miintener, 1979,1982). For the demonstration of alkali-stable ATPase, preincubations at pH 10.4 and 10.5 were combined with incubation at pH 9.5 or 9.6. For the demonstration of acid-stable ATPase, preincubation at pH 4.3 or 4.25 was followed by incubation at pH 9.5. In half of the muscles the cryocut sections were processed for the combined demonstration of myofibrillar ATPase (after alkali and acid preincubation) and acetylcholinesterase (AChE, E.C. 188.8.131.52.), according to Ashmore et al. (1981) and Toop (1976). Every eighth cryosection (12 pm) of the muscles frozen in toto was treated according to the alpha-naphthylacetate method for nonspecific esterases with fast red as a coupling agent (Pearse, 1972). Fibers were classified into the following types: IA (equivalent to the classical “slow-twitch oxidative,” i.e., type I fiber); IB and IIC (both transitional fibers) (Brooke and Kaiser, 1970; Karpati et al., 1975; Jansson et al., 1978); and IIA (“fast-twitch oxidative glycolytic”) and IIB (“fast-twitch glycolytic”). Fiber typing and counting of types IA, IB, and IIC was performed as described elsewhere (Muntener, 1982). For the determination of the number of IIA and IIB fibers (as well as the total number of fibers), the slides were directly enlarged on photographic paper and the fibers counted. In the normal sternomastoid muscle a deep “red” portion (consisting of types I and I1 fibers and containing all the muscle spindles) and a superficial “white” portion (lacking type I fibers) are clearly distinguishable. Such a grouping is no longer the case in the self- and foreign-reinnervated muscles. Thus mean percentages of different fiber types were also calculated for normal muscles. Nerves Omohyoid superior and sternomastoid nerves were either fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.6) and processed according to the method of Karnovsky and Roots (1964), or fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.61, postfixed with 0 ~ 0 4 and , embedded in epon (for 1 pm semithin sections). Single Fiber Preparation The formaldehyde-fixed middle portions of the muscles containing the majority of the end plates (EPs) and additional entire control muscles were divided into thin 349 FOREIGN-REINNERVATION cranial bundles, incubated in the medium of Koelle and Friedenwald (19491, and further processed for single fiber preparations as described elsewhere (Miintener and Zenker, 1986). On orthographically projected EPs, the AChE-stained areas were measured using a MOP-AM02 image analyzing system. Morphometric Procedures Determination of the mean diameters of the different muscle fiber types, mean diameter of axons, and number of alpha-motor axons was performed as described elsewhere (Miintener et al., 1980; Gottschall et al., 1980a,b). Briefly, the cross-sectional areas of a representative number of each muscle fiber type were measured with the MOP-AM02 image analyzing system and the fiber diameters calculated by assuming a cylindric form of the muscle fibers. Analogously, the diameters of all myelinated motor and sensory axons of the nerves investigated were determined in semithin sections. For differentiation between motor and sensory nerve fibers, histochemical demonstration of axonal AChE was used (Karnovsky and Roots, 1964) and the diameters of the AChE-positive(motor)axons evaluated. The populations of alpha- and gamma-motor axons were arbitrarily separated by dividing the smallest class (5.0-6.0 pm) between the two peaks of the diameter histogram. Number and localization of EPs were determined semiquantitatively. The sections (every eighth, stained for nonspecific esterases) were projected on to a screen on which 20 vertical test lines were superimposed. The muscle was aligned so that the lines passed parallel to its deep to superficial axis. The EPs within each strip were counted and each amount symbolized on a horizontal line with a dot of corresponding size (diameter of the dot = 1mm per EP). By assembling these dot diagrams along the longitudinal axis of the muscle, a two-dimensional representation of the extension and density of the area containing EPs (in the following called “end plate zone” [EZ]) was obtained. Superimposed on this representation of the EZ was a reconstruction of the intramuscular branching pattern of the nerve (Fig. 1). This reconstruction also served for the evaluation of a successful foreign-reinnervation. RESULTS Three months after reinnervation, the animals (selfand foreign-reinnervated) showed no asymmetry in head posture or movement in running and swimming tests. Self-reinnervation was successful in all animals; type I fibers were regularly present in these muscles. The foreign-reinnervation of the sternomastoid was successful in ten animals, as evidenced by physiologic and morphologic criteria, i.e., there was a twitch response when cutting the omohyoid nerve, and an absence of such a response when cutting the sternomastoid nerve stump; the reinnervated muscle completely lacked type I fibers; the intramuscular nerves were branches of the omohyoid superior nerve. Since the short omohyoid nerve Fig. 1. Diagrammatic representation of the end plate zone (shadowed) of a normal sternomastoid muscle. The density of the end plates (EPs) is indicated hy dots of graded size (one to ten EPs per unit area) or squares (more than ten EPs; see text for details). For graphical reasons the end plate zone is elongated by a factor of 4. The dark lines represent the intramuscular main branches of the sternomastoid nerve. med lat caudal 351 FOREIGN-REINNERVATION N-STM S-STM S-STM S-STM S-STM 7 8 9 10 F-STM F-STM F-STM F-STM N-OM0 7 8 9 10 7-10 7-10 N-STM 7-10 IIA IA Fig. 3. Relative fiber type proportions in self-reinnervated (S-STM) (A) and foreign-reinnervated (F-STM)(B) sternomastoid muscle. Numbers indicate months after surgery; each bar represents the mean values of two to three animals (for clarity the indication of the SD has been omitted). For comparison the fiber type ratios of normal sterno- 1- ( IIB mastoid (N-STM)and normal omohyoid (N-OMO)are shown on the left and on the right side in A and B, respectively. Note the strong increase of the proportion of IIB fibers in the F-STM between 8 and 9 months, leading to a fiber pattern similar to that of N-OMO. ran through scar tissue adhesions (to adjacent muscles), 2B,C). The former deep (red) portion was only identifiathe sternomastoid muscle could not be stimulated ble according to the presence of the persistent muscle spindles. One foreign-reinnervated muscle exhibited a indirectly. narrow area with very small denervated fibers ( - 5% of Muscles total fiber number). Seven months postoperatively both the self- and forThe regained muscle mass (in percentage of agematched control muscles) was comparable in all stages. eign-reinnervated sternomastoid muscles revealed a low The following percentages (mean t- SD) were calculated: percentage of type IIB fibers (25% and 24%, respectively). While in the self-reinnervated muscles the ratio 91.5 15.5% after foreign-reinnervation and 93.6 12.4%after self-reinnervation. In all experimental mus- of the fiber types remained the same during the whole cles the total fiber number as well as the diameters of period investigated, in the foreign-reinnervated muscles the different fiber types (myosin-based classification) the proportion of IIB fibers strongly increased, from 24% varied within the normal range. Transitional fibers (type to 60%, at the expense of IIA fibers, until month 9 (Fig. IB and IIC) were only sporadically found. Fiber splitting 3). Thereafter the fiber type distribution remained eswas not observed. Groups of fiber types were dispersed sentially constant in the foreign-reinnervated muscles over the entire cross-sectional area of the muscles (Fig. also (Fig. 3). * + Fig. 2. Transverse sections of normal (A), self-reinnervated (B; 9 fibers (present only in normal and self-reinnervated sternomastoid) months postoperatively), and foreign-reinnervated (C; 10 months post- appear white, as they are completely lacking in activity. Type IIA operatively) sternomastoid muscle and of normal omohyoid muscle 0) fibers are black to dark gray, and IIB are intermediate-stained in stained for myofibrillar ATPase (preincubation at pH 10.5, incubation different degrees of intensity (see left and right side in A). Note “type at pH 9.5 or 9.6). In the sternomastoid muscles (A-C), the deep muscle grouping” in the experimental muscles (B,C). x40. portion is on the left and the superficial one on the right. Type IA L. KASER AND M. MUNTENER 352 Fig. 4. Cross section of the same omohyoid superior nerve before (A) and 10 months after (B,C)reinnervation of the sternomastoid muscle. The reinnervating nerve consists of one large (B) and two small (C) branches embedded within scar tissue. Before implantation of the nerve (A), 21 alpha-axons and after 10 months of survival (B,C), 23 alpha-axons were determined (see text for details). Epon-embedded semithin sections, phase contrast. x 1,150. The contralateral muscles did not differ from normal control muscles. mean sizes of the EPs in normal omohyoid and sternomastoid and in experimental sternomastoid muscles did not differ significantly during the period investigated. With the semiquantitative method used, 41.7 -t 6.8% (SD) of the EPs present in the muscles (one EP per muscle fiber) were statistically surveyed for the graphic representation of the EZ. In normal and in both self- and foreign-reinnervated sternomastoid, the pyriform EZ was constantly situated in the center, extending over 24-40 32% of the muscle length. In the foreign-reinnervated sternomastoid, the branches of the omohyoid superior nerve invaded the EZ medially according to the implantation site. Nerves The reinnervating omohyoid nerve was regularly found to branch before its entry into the muscle, leading mostly to a very short nerve stem (Fig. 4).However, in four animals the nerve was long enough to allow a clear determination of the number of alpha-axons (as determined by a series of consecutive sections lying 0.2 mm from one another); on an average, 61 & 5% of the myelinated axons were alpha-axons. In these cases the numbers of alpha-axons counted before and after reinnervation were comparable (& 10%).In the other animals the second determination yielded higher (4585%) values because of the proximal branching of some axons. The number of alpha-axons in the self-reinnervating sternomastoid nerve varied within the normal range. In this nerve between 34% and 39% of the myelinated axons were found to be alpha-motoric.Muscle fiber type ratio, fiber diameter, total fiber number, number of alpha-axons, and motor unit size of experimental and control animals after ten months’ survival time are summarized in Table 1. DISCUSSION It is well known from partial denervation experiments that motor axons can sustain a much larger peripheral field as long as motoneurons and muscle are well matched (Thompson and Jansen, 1977; Brown and Ironton, 1978; Brown et al., 1981; Grinnell and Herrera, 1981; Hatcher et al., 1985).The aim of the present study was to evaluate if motoneurons display the same capacity when reinnervating a much larger foreign muscle of different fiber composition and function. Fiber number and diameter were restored quantitatively throughout, End Rate Zone (€2’) following self- and foreign-reinnervation, reflecting an Analysis of the single fiber preparations of entire mus- undisturbed trophic situation of the reinnervated cles showed all fibers to have a single end plate. The muscles. 353 FOREIGN-REINNERVATION TABLE 1. Overall proportions and diameters of the different fiber types (myofibrillar ATPase), number of muscle fibers and alpha-axons, average size of motor units in normal (N-STM), self-reinnervated (S-STM), and foreign-reinnervated (F-STM) sternomastoid and normal omohyoid (N-OMO) muscle after 10 months of survival (mean f SD) Fiber type proportions (%) IA IIA IIB Fiber diameter (pm) IA IIA IIB Number of muscle fibers Number of alpha-axons Average size of N-STM S-STM F-STM N-OM0 (3F (2)1 (2Y (3)’ 4.2 f 1.0 49.3 f 6.2 46.5 f 7.1 5.6 5.0 71.5 k 4.9 22.9 f 0.2 -2 38.2 f 2.4 61.8 f 2.4 -2 26.1 f 7.8 73.9 7.8 43.1 f 7.1 66.8 f 16.2 86.5 f 10.0 44.5 & 8.2 70.7 f 12.1 87.1 f 12.8 65.8 15.6 83.4 f 9.1 * -2 -2 + 61.8 f 8.1 65.9 & 6.3 430 4,820 89 f 8 + 4,630 420 93 f 11 4,940 f 600 25 f 10 1,650 f 310 22 & 6 54 50 198 75 ‘Number of animals. ’Fiber type not present. Establishment of final fiber pattern IReinnervation I symmetric 0 1 3 2 4 5 6 7 8 9 10 months Establishment of intermediate Establishment of final fiber pattern IReinnervation I asymmetric 0 1 2 3 4 5 6 7 8 9 10 months Fig. 5. Time course of the establishment of the fiber pattern after foreign-reinnervation. If the fiber ratio of the original to the foreign muscle is comparable (“symmetric” reinnervation), the fiber transformation leading to the final fiber pattern occurs parallel to the reinnervation. In the case of a considerable excess of musculature to be reinnervated (“asymmetric” reinnervation), a fiber transformation in two steps is observed. The first reinnervation phase with an intermediate fiber pattern, precedes the establishment of the final fiber pattern, according to the foreign motor input. The cases in which the number of alpha-axons was the same before and after foreign-reinnervation demonstrated the capability of the omohyoid nerve to reinnervate a considerable excess of fiber number (threefold) and muscle mass (sixfold), also proving that it is not the nerve that determines the number of muscle fibers to be reinnervated. There is strong evidence that in the other cases, with higher axon numbers after reinnervation, the sternomastoid muscle was also reinnervated exclusively by the omohyoid nerve (and not by branches of axons to other infrahyoid muscles). Branching of regenerating myelinated axons leading to the same increased number of axons (distally to the regeneration site) as in the present study (average 60%) has also been found in other nerves (Carter and Lisney, 1987). However, two remarkable observations were made: a strong predominance of oxidative fibers (types IA and IIA) in the self-reinnervated muscles on the one hand and a highly delayed fiber transformation in the foreignreinnervated muscles on the other hand. Compared with controls, in the self-reinnervated sternomastoid muscles at all stages increased percentages of types IA and IIA fibers were found at the expense of IIB fibers. This finding is in marked contrast to hindlimb muscles (cat 354 L. KASER AND M. MUNTENER and rat) and infrahyoid muscles (rat) where after selfreinnervation mainly a normal fiber distribution is restored (Chan et al., 1982; Dum et al., 1985a,b; authors’ unpublished observation). Probably in the sternomastoid muscle IIA alpha-axons are more aggressive and vigorous in reinnervating than IIB alpha-axons. The same phenomenon has been observed in partially denervated hindlimb muscles (Hatcher et al., 1985). As expected, the foreign-reinnervated sternomastoid muscles exhibited qualitatively the fiber type distribution of the omohyoid muscle, i.e., only type I1 (A and B) fibers. However, the quantitative fiber type ratios were most striking. At 7 and 8 months postoperatively the proportions of IIA and IIB fibers were mostly the same as in the self-reinnervated sternomastoid muscles. But in the following 2 months the proportion of IIB fibers strongly increased, from 24% to 62%, changing the fiber pattern of the foreign-reinnervated muscle toward that of the normal omohyoid muscle (Fig. 3). This shift in fiber composition was also evidenced histochemically by the reaction for cytochrome c oxidase and immunohistochemically using antibodies against the Ca2+-binding protein parvalbumin (data not shown). To our knowledge this kind of delayed muscle transformation in response to cross- or foreign-reinnervation has not yet been observed. On the one hand, this could be the consequence of the highly enlarged peripheral field of the omohyoid motoneuron pool, meaning that a n excess of muscle mass is trophically reinnervated in a first consolidating phase before it is transformed according to the new motor input (Fig. 5). On the other hand, the delayed transformation could also reflect a general difference between limb and neck muscles, which are primarily controlled from supraspinal centers (Rapoport, 1979). In leg muscles with comparable fiber ratios the establishment of the final fiber pattern occurs parallel to the reinnervation, and the motoneuron activation patterns remain largely unaltered when their axons reinnervate foreign muscles (O’Donovan et al., 1985). The motor reinnervation of the extrafusal fibers is complete after 2-3 months (depending on the age of the animal), while the sensory reinnervation of the muscle spindles takes around 9 months (Collins et al., 1986). Thus the omohyoid motoneuron pool reinnervating the sternomastoid muscle receives the full afferent input long after the re-establishment of the motor innervation. Additionally, the sternomastoid muscle contains 10 to 15 times more muscle spindles than the omohyoid muscle (Gottschall et al., 1980b; Muntener et al., 1980). It can be hypothesized that the afferent feedback modifies and/or increases the activity pattern of the motoneurons, which would not be able to induce a corresponding muscle fiber pattern until afferent reinnervation is largely completed. If so, this could explain why a second fiber transformation occurred at such a late period (between 8 and 9 months) in the present experiments. The transformation was most probably brought forth by a n increased synaptic remodeling, leading to larger IIB and smaller IIA motor units (Wernig and Herrera, 1986). 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