Morphometric analysis of neuronal soma size within the motor nucleus of a transplanted murine skeletal muscle.код для вставкиСкачать
THE ANATOMICAL RECORD 217:402-406 (1987) Morphometric Analysis of Neuronal Soma Size Within the Motor Nucleus of a Transplanted Murine Skeletal Muscle KATHLEEN M. KLUEBER Medical Sciences ProgradAnatorny Section, Indiana University School of Medicine, Bloomington, IN 47405 ABSTRACT Since the number of motor neurons supplying a muscle graft is reduced, the peripheral field for the surviving motor neurons would be enlarged. This possible change in motor unit size may result in morphologic changes in the size of motor neurons within the motor neuron pool of the graft. The extensor digitorum longus (EDL) muscle from 19 transplanted and 17 age-matched normal 129 ReJ female mice were injected with horseradish peroxidase in order to examine the cell size distributions of the motor neuron pools supplying these muscles. Computer-assisted morphometric analysis of cell sizes within the motor neuron pool to the transplants indicated a significant shift in cell size, with the largest areas ranging between 630 and 1250 pm2. A number of the alpha neurons supplying the grafts were twice the average cell size for the control population = 592 pm2). The increase in the number of large motor neurons indicates a n hypertrophy of neurons reinnervating the grafts. The long-term graft is reinnervated by a decreased population of motor neurons = 71, which vary in size from small to very large, reflecting both changes in the possible source of the nerve reinnervating the graft as well as alteration in the size of the motor unit, respectively. (x (x Subsequent to whole muscle transplantation, the myofibers of the denervated, devascularized graft undergo necrosis. The necrotic myofibers are phagocytosed, leaving only the empty basal lamina1 tubes. Finally, regenerating myofibers are formed de novo within these tubes, and most, if not all, of the myofibers receive motor innervation. This secondary myogenesis occurs without the presence of nerves (Carlson et al., 1979) with reinnervation occurring at two weeks posttransplantation in the mouse (Ontell et al., 1982). Although the reinnervating neurons of the murine extensor digitorium long u s (EDL) are located within the same spinal cord level and Rexed's lamina as those to nontransplanted EDL muscles, the motor neuron pool of murine grafts is significantly smaller in size than those innervating the nonoperated muscle (Klueber et al., 1984). Since the murine EDL regenerate contains a third less myofibers than unoperated controls (Bourke and Ontell, 1984) and the number of motor neurons supplying the grafts is reduced (Klueber et al., 1984), the motor units to the graft are modified as indicated by the myofiber type grouping in the murine model of secondary myogenesis (Thomas et al., 1984).Thus, the reinnervation pattern of the EDL graft is not the same as the innervation pattern seen prior to transplantation. The question arises: Does the altered peripheral field of the transplant influence the soma size of the reinnervating motor neurons supplying the graft? It was the purpose of this study to examine the motor neuron soma sizes within the motor neuron pool of transplanted murine EDL muscles. 0 1987 ALAN R. LISS. INC MATERIALS AND METHODS Under methoxyflurane anesthesia (Metafane, PitmanMoore, Inc., Washington Crossing, NJ) administered via inhalation, the extensor digitorum longus (EDL) muscles of 19 four-week-old 129 ReJ female mice were surgically removed and then soaked in 0.75% bupivacaine HCl (Marcaine, Sterling Drug Inc., New York, NY), a known myotoxic agent (Benoit and Belt, 1970; Libelius et al., 1970). The muscle was then reattached to the tendonous ends (Carlson and Gutman, 19741, but no attempt was made for neurovascular anastornosis. At 100 days posttransplantation, the grafts as well as EDL muscles from 17 age-matched normal 129 ReJ female mice were surgically exposed. Using a n endodontic file technique (Klueber and Ontell, 1984), a semisolid paste of horseradish peroxidase (HRP, Sigma Type VD was placed intramuscularly. After a n 18-24-hour survival time, the animals were anesthetized with methoxyflurane via inhalation, intravenously injected with heparin, then transventricularly perfused with physiological saline followed by 1% paraformaldehyde and 1% glutaraldehyde in 0.1M phosphate b d e r (pH 7.2). The spinal cords were removed and fixed a n additional 2 hours in fresh fixative (4"C), processed through a graded series of sucrose solutions to be stored in 30% sucrose (4°C) overnight. Spinal cord segments L2 through L4 were mounted and cut in serial transverse Received July 28, 1986; accepted November 19, 1986 MOTOR NEURON SOMA SIZE INNERVATING MUSCLE GRAFTS section (42 pm) on a cryostat (Damon/IEC Division, model CTD) and subsequently processed for HRP reaction product with the tetramethylbenzidine method of Mesulam (1978). Selection of spinal cord segments L2 to L4 was made based on prior work in this laboratory as well as the description of the location of the motor neurons to the deep and superficial peroneal nerves, which supply the muscles of the shank in the mouse (McHanwell and Biscoe, 1981a). The number of cells within the motor neuron pool for both the control and grafted EDL was evaluated using a A 0 microstar microscope equipped with dark-field and phase optics. Composite camera lucida drawings from each spinal cord were made to record the position and size of each motor neuron within the motor nucleus. No correction factor was necessary since the motor neuron pool was small and dispersed, and the individual cells could be followed by visual inspection within the serial sections. However, only those cells exhibiting a nucleus or nucleolus (Strick et al., 1976), determined with phase optics, were considered as cell bodies for counting and morphometric analysis. Morphometric analysis of soma size for all neurons within each of the motor neuron pools was accomplished by tracing the outline of soma perimeter, including the roots of dendritic and axonal processes within the plane of focus (Fig. 1). The resulting camera lucida drawings (50x1 were used for morphometric analysis using the Bioquant I1 analysis programs (R&M Biometrics), which stores perimeter and area measurements in individual 403 files for control and experimental groups. The files for each group were combined to produce normalized histograms of cell areas of the transplants and controls. Statistical analysis of motor neuron sizes for the control and grafted muscles was completed using a two-tailed Student's T-test, and the level of significance was set at P < .05. RESULTS The average size of the motor neuron pool for the control EDL was 16 f 1.03 SEM cells (N = 17),whereas the average size of the motor neuron pool to the EDL grafts was 7 & 0.90 SEM cells (N = 19, range 2-14). Morphometric analysis of the motor neuron soma size of the normal EDL indicates that the motor nucleus consists of a trimodal population of cells (Fig. 2) whose soma area ranges from 250 to 1250 pm2. Within the motor nucleus for the normal EDL, there is a population of small motor neurons ranging in area from 250 to 420 pm2 representing 18.3% of the cells. The midsized group of neurons consisted of areas ranging from 420 to 630 pm2, while the large group of cells had soma areas from 630 to 1250 pm2. The latter groups represent 44.5% and 34.9% of the total cell population for the EDL motor nucleus, respectively (Table 1). The soma size distribution of the motor neurons of the transplanted EDL was not trimodal in nature but rather widely dispersed. When the ranges of soma size found in the control group are superimposed on the motor neuron Fig. 1. A phase micrograph of 6 HRP-filled neurons from the motor nucleus of the EDL. The black dashes enclose the area used for morphometric analysis of the soma cell size. In this plane of focus, the nuclei (n) of 4 cells was noted; thus, only those cells were measured. x 100. 404 K.M. KLUEBER CONTROL L [7 GRAFTS l 0 20 10 30 40 CELL AREA (pm’) Bin width = 4 2 p ’ Fig. 2. Normalized histogram of soma size distribution from the motor neuron pools of control and transplanted EDL muscles. Note that the control group has a distinct trimodal population of cell size. For the grafts, however, the soma size distribution has a wider range with no definitive pattern. TABLE 1. Percentages of motor neuron soma sizes for control and transplanted EDL muscles Range of soma area (Km2) <250 250-420 420-630 630-1250 > 1250 Control spinal cords (N= 17) cells measured (N= 277) 1.4 18.3 & 44.5 34.9 & 0.8 +_ 1.0 4.1 4.1* 5.1** 0.6 Transplant spinal cords (N = 19) cells measured (N = 135) 2.8 & 1.4 17.9 4.7 25.9 k 4.7* 51.1 f 7.3** 2.2 & 1.4 *P < ,005. **P c . 0 5 . size distribution of the grafts, it becomes apparent that there is a shift in soma size for those neurons reinnervating the graft (Table 1). The percentage of neurons falling within the control ranges decreased for the small and medium populations, while increasing in the largest group of neurons (17.9%, 25.9%, and 51.1%, respectively). However, the range of soma size for both the small and large neuron populations increased for the grafts (Table 1).For the transplant motor neurons, 2.8% of the small neuron population were < 250 pm2 and 2.2% of the large neurons were > 1250 pm2. Further evaluation of individual soma size distributions for both the transplants and the control group revealed that 21.2% of the motor nuclei of the grafts did not contain motor neurons smaller than 630 pm2, while none of the control motor nuclei had all their motor neurons greater than 630 pm2. Within the transplant group, 15.8% of the motor nuclei did not contain neurons above 630 pm2, while 11.8% of the control muscles fit into this category. The transplant group had 63.2% of the individual motor nuclei in which over half of the neurons were greater than 630 pm2, while only 29.4% of the control motor neuron pools had a similar composition. In general, there appears to be a tendency for the small-sized motor neuron pools of the transplants to consist of the large-sized neurons ( > 630 pm2). Comparison of the soma size distributions for the control and transplant motor nuclei (Fig. 1; Table 1)reveal a slight shift in the percentage of small motor neurons for the transplant. Similarly, there is a highly significant (P < .005) decrease in the percentage of medium motor neurons in the transplant motor nucleus, with a significant (P < .05) increase in the percentage of very large neurons. Overall, theaverage soma size for the transplant’s motor neurons (X = 642.2 pm2 f 22.5 SEM) was significantly larger than that of the control neurons (X = 592.2 pm2 k 12.5 SEMI indicating a small yet significant (P < .05) shift in cell soma size for those neurons reinnervating the grafts. DISCUSSION In a n HRP study of the motor neuron pool to normal feline muscle, Burke et al. (1982) described a bimodal population of neuron sizes for both the medial gastrocnemius and soleus muscles. The motor nuclei of these muscles consisted of a population of small neurons (gamma) and a population of large (alpha) neurons. Pelligrini et al. (1977) further subdivided the large motor neuron population of the same feline muscles into small and large alpha neurons. The latter two types of alpha motor neurons were correlated to the fiber type of the muscle fibers they innervated: the small and large alpha neurons innervate the fast twitch oxidative (Type IIa) and the fast twitch glycolytic fibers (Type Ilk), whereas the smallest cells (gamma) innervate the slow twitch fibers (Type I) (Strick et al., 1976; Burke et al., 1982). The normal EDL muscle consists of a mixed myofiber population that contains 36% of Type IIa and 63% of Type IIb with less than 1% Type I (Thomas et al., 1984). In contrast, great variability of fiber type percentages was seen in the transplanted EDL (Thomas et al., 1984); therefore, the variation of motor neuron size to the transplants could be the result of the myofiber types found in the regenerated muscle. The trimodal distribu- MOTOR NEURON SOMA SIZE INNERVATING MUSCLE GRAFTS tion of cell size was noted in the motor nucleus of the normal EDL muscle of this study, but not for the transplants. The size range for motor neurons for the control and transplanted muscles in the current study correspond to those described by McHanwell and Biscoe (1981b)for the deep peroneal nerve. The EDL motor nucleus would be included within this motor column. The morphometric analysis of motor neuron cell size distribution for the transplanted EDL indicates a change in the size and number of small motor neurons and the addition of a population of very large motor neurons. This is the first study evaluating cell size distribution of motor neurons supplying a muscle graft. Similar changes in cell size distributions have been described following nerve section (Brushart and Mesulam, 1980).The transplantation procedure used in the current study involved the cutting of the muscle nerve to the EDL without reanastomosis following the repositioning of the graft. The size of the motor neurons to the grafts is similar to the size of motor neurons supplying the soleus muscle in murine muscular dystrophy (Parry et al., 1982). Since both the transplant and the dystrophic muscle undergo secondary myogenesis, similar changes in reinnervation may be expected. The decrease in the number of small neurons (250420 pm2) reinnervating the graft may be due to the poor reinnervation potential of gamma motor neurons following nerve section (Brushart and Mesulam, 1980).Following nerve section, motor neurons have been shown to retract dendrites in response to the peripheral injury (Kreutzberg, 1986). This type of response could account for the presence of the population of small neurons (< 250 pm2) within the motor columns of the grafts since the peripheral nerve is cut at the time of transplantation. Another possibility is that the small neurons found reinnervating the grafts may belong to the motor column of a more distal muscle. McHanwell and Biscoe (1981) describe such a relationship of neuronal size to muscle placement in the hindlimb of the mouse with a decrease in neuronal cell size for the more distal muscles. The possibility of nerves from surrounding muscles sprouting to supply denervated myofibers has been described (see Brown et al., 19811, but this possibility of muscle neurotization has not been examined in the current study. The increase in size of some of the alpha motor neurons for the graft could be the result of hypertrophy normally seen during the chromatolytic response of a neuron following nerve section (Gutmann, 1961). Similar increases in the large motor neuron size were found in a study of the motor innervation of the dystrophic soleus muscle (Parry et al., 1982). Muscular dystrophy is thought to be a neuropathy resulting in denervation of dystrophic myofibers (Parry et al., 1982). Although the neuronal swelling remains only as long as the regeneration of the damaged axon persists, it has been reported to last up to two years in the rat (Ducker, 1972). Another explanation for the increased size of some of the alpha motor neurons to the graft is a true hypertrophy in response to a change in motor unit size following reinnervation. After nerve section, both the muscle and nerve undergo changes (Brown et al., 1981). Among the changes that can take place in the motor nucleus is a loss of neurons capable of reinnervation due to severe 405 damage and death (Turner, 1943), or diversion of the regenerating axons away from their target (Barron et al., 1981). In either case there would be a reduction of nerve supply to the graft. Thus the decrease in the number of neurons to the graft would result in a n increase in motor unit size (Lowrie et al., 1985). Although the number of myofibers found within the murine graft is reduced by 32% (Bourke and Ontell, 1984) and the motor neuron pool is reduced by 53% (Klueber et al., 1984), a change in the motor unit size is likely a s can be inferred in this study. The grafts with the smallest number of motor neurons exhibit the tendency to be reinnervated by the largest motor neurons. Since motor neurons have been noted to be capable of sustaining a much larger peripheral field when the normal number of neurons is unavailable (Betz et al., 1979), the expansion of the graft’s motor unit could be reflected in a n increase in motor neuron cell body size as noted by Lowrie et al. (1985) following nerve section in the rat. In summary, the long-term graft is reinnervated by a decreased population of motor neurons, which vary in size from small to very large, reflecting possible changes in the source of the nerve supply as well as alteration in the size of the motor unit, respectively. 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