Isolated muscle spindles from lower lumbrical muscles of rats were used to study the 3-dimensional organization of intrafusal structures by scanning electron microscopy following (a) complete denervation, (b) reinnervation after a single crush lesion of the sciatic nerve, or (c) reinnervation after transection and immediate suture of this nerve. One week after complete denervation, previous sites of intrafusal motor endplates were transformed into sarcolemmal ovoid bulges. These bulges persisted in denervated muscle spindles up to 12 weeks. Regenerated motor nerve endings were detected on intrafusal muscle fibers 1 month, and thereafter following sciatic nerve crush injuries, and 3 months and later following transection and suture of the nerve. Furthermore, 3 different types of subsynaptic areas of motor nerve terminals were observed. The scanning electron microscopic technique also allowed visualization of splitting and fusion of intrafusal muscle fibers. The findings are discussed in view of their possible functional implications. Key words: neuromuscular spindle denervation reinnervation nerve endings scanning electron microscopy MUSCLE & NERVE 15:433-441 1992
SCANNING ELECTRON MICROSCOPIC STUDY OF DENERVATED AND REINNERVATED INTRAFUSAL MUSCLE FIBERS IN RATS RALF DIELER, MD, ANJE VOLKER, MD, and J. MICHAEL SCHRODER, MD
T h e scanning electron microscope can resolve topographical surface details of 5 to 10 nm, with a depth of focus 500 times that of an optical microscope at equivalent magnification.8 Application of this technique to mammalian extrafusal muscle fibers (EMFs) has iven new insights into their overall morphology 1',18,28,29,37 as well as convincing morphological evidence for dynamic changes at
From the lnstitut fur Neuropathologie, Rheinisch-Westfalische Technische Hochschule, Aachen, Germany. Preliminary results were presented at the French-German Joint Meeting of Neuropathology in Frankfurt, West Germany, October 19-21, 1989. Dr. Dieler's present address is Klinik and Poliklinik fur Hals-, Nasen- und Ohrenkranke der Universitat Wurzburg, Josef-Schneider-Str 11, 8700 Wurzburg, Germany. Acknowledgments: The editorial comments of Michael Zidanic, PhD, The Johns Hopkins University School of Medicine, and the photographical assistance of Ms. Bettina Loschner are gratefully acknowledged. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Bonn, Germany), Di 38611.1. The micromanipulator was donated by the Deutsche Gesellschaft zur Bekampfung der Muskelkrankheiten Address reprint requests to Prof Dr. J.M Schroder, lnstitut fur Neuropathologie, Klinikum der RWTH, Pauwelsstrasse 30, D-5100 Aachen. Germany. Accepted for publication March 25, 1991
CCC 01 48-639x1921040433-09$04.00 0 1992 John Wiley & Sons, Inc.
SEM of Muscle Spindles
neuromuscular junctions during maturation,'" ageing,17 or after severance of inner~ation.'~''" The evaluation of surface structures in muscle spindles is limited by the difficulty of separating the inner and outer spindle capsule from the underlying intrafusal muscle fibers (IMFs) and their nerve terminals. In a preceding study,36 scanning electron microscopic (SEM) investigation of normal rat muscle spindles gave new information about their 3-dimensional organization. We now applied the SEM technique to denervated and reinnervated muscle spindles. Knowledge of structural changes in muscle spindles should help to understand the functional recovery of these proprioceptive receptors following peripheral nerve lesions.
MATERIALS AND METHODS
Muscle spindles from the lower lumbrical muscles of female Han:Sprague- Dawley rats weighing 200 to 250 g were used. All animals were handled according to the law of Germany to protect animals used for experimental purposes (Tierschutzgesetz, 1986). T h e experiments were licensed by the local authority. T h e animals were anesthetized with ether fol-
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lowed by intra-abdominal injection of 30 mg/mL chloral hydrate (1 mL per 100 g body weight), and the left sciatic nerves were exposed by a dorsal skin incision. For the denervation experiments, the exposed nerves of 15 rats were sectioned at the mid-thigh level and the proximal and distal stumps were tightly ligated to prevent spontaneous axonal outgrowth. For reinnervation studies, the sciatic nerve was either crushed unilaterally with a watchmaker forceps for 15 minutes (12 rats), or transected at the mid-thigh level and immediately approximated by two oblique 9-0 epineural sutures. Survival times were 3 days; 1, 4, or 12 weeks for denervatiodligation; or 1, 3, 6, or 12 months for both crush injury and transection/ suture. After survival times of 3 days to 1 year, the lower extremities were fixed by perfusing the abdominal aorta of the anesthetized rat with a solution containing 3.9% glutaraldehyde in Sorensen's phosphate buffer (pH 7.4). T h e lumbrical muscles were excised and kept for 1 to 3 hours in the same fixative, rinsed in phosphate buffer, postfixed in phosphate-buffered 2% osmium tetroxide for 3 hours, glycerinated for 48 hours in 66% glycerine, and subsequently stored in 100% glycerine. The muscle spindles were isolated (teased) from the lumbrical muscles in glycerine using a dissecting microscope ( x 40) and fine preparatory needles. In approximately one-half of the teased muscle spindles, w e attempted to mechanically remove the outer spindle capsule at the equatorial region using a micromanipulator (Leitz, Wetzlar, Germany) or fine injection needles. A total of 507 muscle spindles were isolated. The isolated muscle spindles were rinsed with distilled water, treated with 8N HCl for 20 to 60 minutes at 60°C to remove connective tissue elements," washed in distilled water, and dehydrated through a graded series of acetone. After drying at the critical point with liquid GO, for eluting acetone in a CPD 010 (Balzers Union, Liechtenstein), the spindles were sputter-coated with gold in a Balzers Union SCD 030, and examined with a Jeol JSM 50 A scanning electron microscope at 25 kV. Only 265 of the 507 isolated muscle spindles were available for SEM evaluation after the preparatory procedures. Considerable loss of muscle spindles was caused by HC1 treatment, because the length of treatment had to be determined by trial and error. During critical-point drying, some muscle spindles were lost from the enclosing envelopes by flushing the chamber with CO,. Repro-
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ducibility of results was further restricted by the difficulty of attaching the dried and brittle specimen to the specimen holders. Therefore, comparable positions of interesting regions could not intentionally be achieved. Equatorial and juxtaequatorial regions frequently remained concealed within the spindle capsule, or were damaged due to the attempts to mechanically remove the outer and inner spindle capsule. One lumbrical muscle of each animal from each side was used for transmission electron microscopic (TEM) evaluations. The processing of muscle s indles for TEM followed standard proceduresg These results, as well as T E M data available from denervated or reinnervated rat muscle s indles analyzed before in our laboratory,33-5' were used to classify the findings. The results of a previous scanning electron microscopic investigation of normal rat muscle spindles that had been carried out under the same conditions as the present study,36 served as controls. RESULTS
The diameters of IMFs ranged from 5 to 19 p,m. According to values given in revious studies for normal rat muscle spindles,3x33,36 muscle fibers with diameters 5 8 pm were regarded as nuclear chain fibers, and those with diameters > 8 pm as nuclear bag fibers. DENERVATED MUSCLE SPINDLES
In the early stages of denervation the observations in degenerated sensory and motor nerve endings corresponded to what has been reported in the literature. 14217,4'942 Sensory or motor nerve terminals were no longer seen following 1 week of denervation. Sites of previous sensory endings were identified by shallow depressions in the IMF associated with basal lamina duplications and remnants of nerve endings.
TEM Findings.
After 3 days of complete denervation, IMFs revealed slightly elevated subneural apparatuses of motor nerve terminals in polar regions with small and irregularly configurated, shallow impressions. At 1 week survival time, previous sites of motor nerve terminals were trarisformed into sarcolemmal bulges about 9 x 1 1 km in size, irregularly shaped and occasionally with clearly delineated sarcolemmal pits (Fig. I ). A t survival times of 4 and 12 weeks, these sarcolemma1 bulges were still visible. Sites of sensory nerve
SEM Findings.
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FIGURE 1. One week after complete denervation without reinnervation, a sarcolemmal bulge with an uneven shape and with irregular elevations is seen in the distal polar region of an IMF. It contains 2 sharply delineated sarcolemmal pits (arrowheads). Scale bar: 2 pm. FIGURE 2. A nuclear bag fiber in the midpolar region of a muscle spindle, 3 months afler transection and immediate suture of the sciatic nerve. It shows an oval sarcolemmal bulge crossed by a small nerve fascicle that branches off a preterminal axon (arrowheads). The fusiform enlargements along the nerve fiber are thought to represent Schwann cell perikarya (S). Scale bar: 2 pm. FIGURE 3. Plan view of a regenerated motor nerve terminal, 1 month after crushing the sciatic nerve. It is located on a nuclear chain fiber. The supplying preterminal axon reaches the nerve ending from the equatorial region (arrowheads). Scale bar: 3 pm, FIGURE 4. Lateral view of a similar plate-like ending as shown in Figure 3, at 3 months after crushing the sciatic nerve. It lies in a sarcolemmal depression. The supplying preterminal axon (arrowhead) reaches the nerve ending from the equatorial region. Scale bar: 2 pm.
seen. They were similar to those in denervated IMFs, and measured up to 8 x 1 3 p m in size. These sarcolemmal bulges were sometimes delinREINNERVATED MUSCLE SPINDLES eated from the IMF by small slits of' approximately 0.2 pm, and were traversed by unmyelinTEM Findings. After 3 months, regenerated moated axons 1 pm in thickness (Fig. 2). tor (see Fig. 8) or sensory nerve endings were enRegenerated motor nerve endings were decountered in all muscle spindles after crush letected at 1 month and thereafter following sciatic sions, whereas, in the transection/suture group, nerve crush injury, and 3 months and thereafter regenerated nerve endings were only occasionally in the transection/suture group. They were classiseen before 6 months of recovery. Their general fied into: (a) plate-like endings and (b) multitermimorphology and fine structural abnormalities have been reported in preceding s t ~ d i e s . ' ~ , ~nal ~ fusimotor endings. The plate-like motor nerve terminals usually had an oval or fusiform outline Some muscle spindles showed increased numbers measuring between 10 to 16 pm in length and 4 to of IMFs. 1 0 pm in width. They were situated on the surface of the underlying I M F not indenting the sarcoSEM Findings. One month after crushing the scilemma (Fig. 3 ) , or they caused slight sarcolemmal atic nerve, and at 1 and 3 months after transectroughs where the nerve endings were superfitiodsuture of the sciatic nerve, sarcolemmal cially embedded into the muscle fiber surface (Fig. bulges with various outlines and contours were endings at the equatorial or juxtaequatorial region could not be convincingly identified.
SEM of Muscle Spindles
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SEM of Muscle Spindles
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FIGURE 5. Normally appearing subsynaptic folds and grooves underlying a motor end-plate on an IMF in the distal polar region 6 months after crushing the sciatic nerve. The flat trough with synaptic grooves, partitioned by sarcoplasmic ridges (arrows), is indicated by arrowheads. Scale bar: 2 pm. FIGURE 6. Transmission electron microscopic aspect of subneural apparatus on intrafusal muscle fiber (IMF). Motor nerve ending on an IMF in the polar region from a normal rat muscle spindle. The nerve ending (E) is filled with small clear synaptic vesicles and mitochondria. A nonmyelinated preterminal axon (A) lies in close proximity to the nerve ending. A basal lamina separates the motor nerve ending from the underlying IMF (arrowhead). The subneural apparatus is formed by small sarcoplasmic ridges and grooves (arrows). Scale bar: 1 pm. FIGURE 7. The reinnervated IMF, 12 months after crushing the sciatic nerve, shows a subsynaptic trough in the midpolar region that is surrounded by a sarcolemmal rim, and shows some wide ridges and grooves. Scale bar: 2 pm. FIGURE 8. Transmission electron microscopic aspect of subneural apparatus on intrafusal muscle fiber (IMF). Regenerated motor nerve ending (E) in the polar region, 3 months after nerve crushing. The muscle fiber underlyingthis nerve ending shows a slight trough that is delineated by a sarcolemmal rim (arrowheads). Scale bar: 1 pm. FIGURE 9. Three months after transection and immediate suture of the sciatic nerve, the distal polar region of a nuclear bag fiber with a slight, flat, plate-like elevation (arrows) is shown. The sarcomeric pattern is flattened near the equatorial side of the elevation (arrowheads). Scale bar: 3 prn. FIGURE 10. A plan view of a similar plate-like elevation (large arrows), as in Figure 9, is shown in the polar region of an IMF, 1 month after nerve crush. It possesses small, irregular surface elevations. The sarcomeric pattern of the IMF (small arrows) does not continue into the elevation. Scale bar: 2 wm.
4). These endings were seen on nuclear bag and on nuclear chain fibers at midpolar or distal polar sites. The regenerated multiterminal fusimotor nerve endings usually occupied proximal polar regions extending over areas of 15 x 50 pm. They were situated on IMFs measuring more than 12 km in diameter, i.e., on nuclear bag fibers, and emanated from dichotomously branching preterminal axons measuring 0.4 to 1.5 pm in diameter. These axons and their tapering endings were oriented toward the spindle pole and laid above the muscle fiber surface. Some nerve endings were removed from the underlying IMF due to mechanical manipulation and/or HCl hydrolysis. Thus, 3 different images of subneural apparatuses (SNAs) from motor nerve terminals were seen: They (1) either covered areas of approximately 3 x 11 pm, slightly indenting the IMF surface and showing rather small subsynaptic sarcolemmal ridges and grooves (Fig. 5); or (2) they formed deeper subsynaptic troughs of about 5 x 12 pm in size. These surfaces showed only few, wide sarcolemmal ridges and some broad and shallow grooves (Fig. 7). (3) The third type of subneural apparatuses represented flat, plate-like sarcolemmal elevations measuring up to 9 x 15 pm that were clearly delineated from the regular sarcomeric pattern (Figs. 9 and 10). These areas had only a few inconspicuous surface elevations. When equatorial regions were visualized in reinnervated muscle spindles, a variety of intrafusal components were encountered. Closely spaced spirals were observed in the equatorial region on nuclear chain as well as on nuclear bag fi-
SEM of Muscle Spindles
bers in one muscle spindle 12 months after crush lesion, and in two muscle spindles 1 and 6 months after transection/suture. Each segment measured 2 to 4 pm in diameter. 'These structures were regarded as regenerated primary sensory endings. Single spirals or cuff-like rings were either embedded into the IMF surface or were raised above the surface level for up to 1 pm. Irregularly formed ring-like structures were seen in a few muscle spindles in the juxta-equatorial region, with each segment measuring 1.5 to 3 pm in diameter. These rings only occurred on nuclear chain fibers and were thought to represent regenerated secondary sensory endings. IMFs were often covered by fibroblastic cell processes from the inner spindle capsule. Numerous small nerve fascicles or single nerve fibers with focal enlargements, presumably representing Schwann cell perikarya, were seen closely adjacent to the IMFs. Splitting of IMFs was evident in denervated as well as in reinnervated muscle spindles (Figs. 11 and 12). Because of their small diameter (6 pm) these IMFs were regarded as nuclear chain fibers. Both parts of the split IMFs were separated for variable distances. The longer fiber sometimes showed further splitting along its course inside the spindle. In SEM preparations, where the basal lamina was sufficiently removed, satellite cells appeared as flat oval or fusiform structures (Fig. 13). They were clearly delineated from the IMF by a small cleft, and were not or only slightly elevated above the muscle fiber surface. In T E M images (Fig. 14) satellite cells can be seen closely attached to IMFs and covered by a common basal lamina.
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FIGURE 11. Splitting of an IMF in the juxtaequatorial region of a reinnervated muscle spindle, 12 months after crushing the sciatic nerve. Scale bar: 2 pm. The branching of the IMF leads to 3 segments (a-c). Part a and b of the original IMF fuse again after a distance of about 15 pm. FIGURE 12. Splitting of an IMF in the juxtaequatorial region of a reinnervated muscle spindle, 12 months after crushing the sciatic nerve. Scale bar: 2 pm. The IMF, a presumed nuclear chain fiber, shows a thin branch (arrowheads) that is broken during the preparatory manipulations. FIGURE 13. An elliptical structure, a presumed satellite cell, is clearly delineated by a small cleft (arrowheads), and is completely embedded into a reinnervated IMF (12 months after crush lesion). The IMF is separated from a neighboring IMF by a fibroblast (F) and its cell processes. Scale bar: 3 pm. FIGURE 14. The TEM image shows the typical appearance of a satellite cell (S): It is closely attached to an IMF, and both are covered by a common basal lamina (arrowheads). Scale bar: 1 pm.
The results are summarized in Table 1 according to the frequency of occurrence in the different experimental groups.
DISCUSSION
This study presents, for the first time, SEM aspects of a variety of muscle spindle components following crush lesion or transection of the rat sciatic nerve with or without reinnervation. Four to 5 muscle spindles were found per lumbrical muscle in denervated and reinnervated muscles. These numbers correspond to the amount of muscle spindles in normal rat lumbrical muscles counted by Porayko and Smith3' and Schroder et al.3FArendt and Asmussen,' by contrast, reported that the number of muscle spindles was reduced in denervated and reinnervated muscles after repeated crush lesions or severance of the supplying nerve. I p et al." found only two-thirds of the normal number of muscle spindles in muscles after severance and suture of the nerve. After complete denervation, 1 month after crushing, and 1 and 3 months after transection and immediate suture of the sciatic nerve, the subneural apparatuses of previous motor nerve terminals in polar regions of muscle spindles showed an elevation of the subsynaptic membrane above the surface of the IMF or a loss of junctional folds. These changes resemble those reported by Matsuda et aLZ6in denervated and reinnervated neuromuscular junctions of extrafusal muscle fibers of the hamster, and may be caused by the greater resistance of the cytoskeletal network underneath the plasma membrane. The apparently less severe changes of the subneural apparatuses on IMFs are obviously due to the less pronounced denervation atrophy in IMFs comprising 17% to 34% of their cross-sectional area in rat muscle spindles following 12 months of complete dener-
SEM of Muscle Spindles
~ a t i o n whereas ,~~ EMF diameters in soleus muscles were found to be reduced by 80% after only 21 days of denervation.16 The subneural apparatuses of EMFs recovered their normal structural organization 20 weeks after nerve transection and suture.26 Likewise, the subsynaptic changes on intrafusal muscle fibers were no longer seen after 6 months survival time. This may indicate that areas of former endplates get reinnervated; fusimotor reinnervation of IMFs may resemble motor reinnervation of EMFs with respect to the known preference of old endplate regions for reestablishing neuromuscular j ~ n c t i o n s . However, ~~,~~~~~ complete disappearance of subneural apparatuses, as a result of reinnervation at ectopic sites, cannot be excluded as an alternative explanation. In some spindles, sarcolemmal bulges on reinnervated IMFs were crossed by thin, unmyelinnated axons. These changes were observed more frequently after nerve section and suture than after a crush lesion. Abnormal contact relationships between terminal axons and intrafusal muscle fibers, between terminal axons and Schwann cells, and between Schwann cells and intrafusal muscle fibers were also reported in TEM ~tudies.'"'~It is not known, whether these abnormalities due to reinnervation could cause clinical symptoms (e.g., hypertonia, hypotonia, tremor, ataxia, or dysesthesia). Regenerated motor nerve terminals were classified into (a) plate-like endings, and (b) into multiterminal endings that correspond in size and location to the P1-plates and multiterminal nerve endings as described by Barker et a1.' Mechanical manipulations or HC 1 hydrolysis can lead to detachment of motor nerve endings from the IMF Thus, 3 different types of subneural apparatuses (SNAs) could be distinguished according to the classification of Arbuthnott et al.': (a) SNAs with slight impressions in the IMF surface and a few sarcolemmal ridges and
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Table 1. Frequency of SEM findings after denervation and reinnervation of intrafusal muscle fibers Reinnervation Denervation 3d
Findings Sarcolemmal bulges Abnormally regenerated motor nerve endings Plate-like motor terminals Multiterminal endings Subneural apparatuses without nerve endings Primary sensory nerve endings Secondary sensory nerve endings Fiber splitting Satellite cells Abbreviafions: d
=
I w 4w
1 -
Crush lesion
12 w Total
2
3
6
-
-
-
1 m 31-17 6 m 1 2 m Total 1 m 3 1
3
1
-
-
-
_
-
-
3
-
1
_ 1
-
7
1
1
-
5
-
_
_
12 m Total 1
2
-
1
1
-
I
1 3 4
1
3 4 5
1
-
1
2 1 1
-
-
31-17 6 m
5
-
1 3
-
1
-
3
-
1
-
1
-
days, w = weeks, m = months, - = not observed.
grooves corresponding to the SNAs of In,,-plates; (b) SNAs with sarcolemmal rims and a few shallow grooves corresponding to the SNAs of rn,-plates; and (c) SNAs showing plaque-like elevations above the IMF surface resembling SNAs of ma-plates. I t was suggested that ma-plates correspond to multiterminal nerve endings, whereas m,-plates represent pl- or p,-nerve terminals.' Regenerated sensory nerve endings were encountered 6 and 12 months after a crush lesion and 1 to 6 months after transection and suture of the sciatic nerve. T h e endings were, however, often covered by remnants of basal lamina and connective tissue elements, or they were partly destroyed while removing the connective tissue. Some evidence for structural abnormalities of reinnervated sensory endings (e.g., incomplete contact relationship between sensory endings and IMFs, intervening basal lamina between sensory nerve ending and IMF, covering of sensory terminals by Schwann cells and axonal sprouts, hyperinnervation, etc.) were more clearly identified in preceding TEM s t u ~ i i e s . ' ~ , ~ ~ Intrafusal muscle fiber splitting and fusion of intrafusal muscle fibers was unequivocally visualized in the present SEM study. Although this phenomenon may also occur in normal muscle spindles,52"." it has been observed more fre uently after denervation and Data on the frequency of muscle fiber splitting following denervation and reinnervation are rare in the literature. Kucera," mentioned splitting in 67% of all reinnervated IMFs after crush lesion of the supplying nerve, whereas Walro et al.40 observed fiber splitting in 14.3% of regenerated IMFs in
re innervation.",",^
440
Transection and suture
SEM of Muscle Spindles
muscle grafts of rats. Muscle fiber splitting may be one cause of a numerical increase of IMFs after nerve lesions. Another mechanism leading to an increased number of IMFs may be the stimulation of satellite cells. They may become motile and mitotic, forming myoblasts and myotubes that fuse incompletely with the parent muscle fiber, or with newly formed myoblasts and m y o t ~ b e s . * ~ T h e occurrence of regenerated motor and sensory nerve terminals confirms that reinnervation of IMFs does take place, even after transection and immediate suture of the nerve. T h e necessary period of time for reinnervation, however, appears to be longer after nerve transection than after crush lesions, where axonal guidance is more easily accomplished by intact endoneurial and perineurial connective tissue elements. Regenerated nerve terminals were noted to be functional to various degrees in electrophysiological ~tudies.~,~."'".'" These studies also revealed effective, though incomplete, restoration of muscle spindle discharge patterns, particularly for sensory nerve responses. However, following peripheral nerve suture or transplantation in man, Struppler et ai.,38were not able to confirm muscle spindle reinnervation using electromyography or microneurography. Because discharge patterns often correlate to the morphology of the nerve terminals,4x20x3q the normal appearance of at least some regenerated intrafusal nerve endings suggests that muscle spindle function could have been, in fact, re-established. Abnormal reinnervation of pre-existing, split, or newly formed IMFs, however, may cause functional disturbances that would have to be analyzed using electrophysiological techniques.
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REFERENCES 1. Arbuthnott ER, Sutherland FI, Boyd IA, Gladden MH: Axon terminal indentation and postsynaptic folding as criteria for classifying fusimotor nerve endings, in Boyd IA, Gladden MH (eds): The Miiscle Spindle. Basingstoke, Macmillan, 1985, p p 57-62. 2. Arendt KW, Asmussen G: Die Muskelspindeln in1 denervierten und reinnervierten M. soleus d e r Ratte: I. Veranderungen d e r Anzahl und d e r Lange d e r Muskelspindeln. Anat An,z 1976;140:241-253. 3. l h n k s RW, Barker 1). Brown HG: Sensory reinnervation of muscles following nerve section and suture in tars. ] Hand Surg 1985; 10-B:340-344. 4. Banks KW, Barker D, Stacey M,J: On the classification of motor endings in mammalian muscle spindles, in Boyd IA, Gladden MH (eds): The Muscle Spindle. Rasingstoke, Macmillan, 1985, p p 69-74. 5. Barker D, Gidumal JL: T h e morphology of intrafusal muscle libers in the cat.J Physiol 1961;157:513-528. 6. Barker D, Banks RW: 'I'he muscle spindle, in Engel AG, Ranker BQ (eds): Myology. New York, McGraw-Hill, 1986, p p S09- 34 1. 7. Barker D, Scott JJA, Stacey MJ: Reinnervation and recovery of cat muscle receptors after long-term denervation. Exp Neurol 1986;94: 184-202. 8. Black J ' I : T h e scanning electron microscope: operating principles, in Hayat MA (ed): Princzpks and Techniques of Scanning Electron Microscopy. New York, van Nostrand Keinhold, 1974, p p 1-43. 9. Brown MC, Butler RG: Regeneration of afferent and efferent fibers to muscle spindles after nerve injury in adult cats. J Physiol 1976;260:253-266. 10. Collins WF 111, Mendell LM, Munson JB: O n the specilicity of sensory reinnervation of cat skeletal muscle. J Physiol 1986;375:587-609. 11. Desaki J, Uehara Y: Evidence of branching of intrafusal muscle fibers.] Electron Microsc 1979;28:128- 130. 12. Desaki J , Uehara Y: T h e overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. J Nrmvcytol 108 1 ;10: 10 1 - 1 10. 13. Desaki J , Uehara Y: Formation and maturation of subneural apparatuses at neuomuscular junctions in postnatal rats: a scanning and transmission electron microscopical study. Dev Biol 1987;119:390-401. 14. DeSantis M, Norman WP: An ultrastructural study of nerve terminal degeneration in muscle spindles of the tenuissimus muscle of the cat. J Neurocytol 1979;8:67-80. 15. Dieler R, Schrijder JM: Abnormal sensory and motor reinnervation of rat muscle spindle following nerve transection and suture. Acta Neuropathol (Berlin) 1990;80:163- 17 1. 16. Engel AC;, Stonnington H H : 'I'rophic functions of the neuron. 11. Denervation and regulation of muscle. Morphological effects of denervation of muscle. A quantitative ultrastructural study. Ann NY Acad Sci 1974;228:68-88. 17. Fahim MA, Holley J A , Robbins N: Scanning and light inicroscopic study of age changes at a neuromuscular ,junction in the mouse. J Neurocytol l983;12: 13-25. 18. Fahim MA, Holley J A , Robbins N : Topographic comparison of neuromuscular junctions in mouse slow and fast twitch muscle. Neuroscience 1984;13:227-235. 19 Gregory JE, Luff AR, Proske U: Muscle receptors in the cross-reinnervated soleus niuscle of the cat. ] Physiol 1982;331:367-383. 20 I p MC, Vrbova G , Westbury DR: T h e sensory reinnervation of. the hind limb muscles of the cat following denervation and de-efferentiation. Neuroscience 1977;2:423-434. 21 I p MC:, Luff AR, Proske U: Innervation of muscle receptors in the cross-reinnervated soleus muscle of the cat. Anat Rec 1988;220:2 12-2 18. 22. Iwayama T: Relation of regenerating nerve terminals to original endplates. Nature 1969;224:81-82.
SEM of Muscle Spindles
23. Kucera J : Splitting of the nuclear bag, fiber in the course of muscle spindle denervation and reinnervation. J Ifutochem Cytochem 1977;25:1102- 1104. 24. Labovitz SS, Robbins N, Fahim MA: Endplate topography of denervated and disused rat neuromuscular junctions: comparison by scanning and light microscopy. Neuro.\rirnce 1984;11:963-971. 25. Letinsky MS, Fischbeck KH, McMahan UJ: Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush.] Ne~.rocytd1976;5:691-718. 26. Matsuda Y, Oki S, Kitaoka K, Nagano Y, Nojinia M, Desaki J : Scanning electron microscopic study of denervatrd and reinnervated neuromuscular junction. Muscle NrnJe 1988;11:1266- 1271. 27. Mazanet R, Franzini-Armstrong c:The satellite cell, in Engel AG, Banker BQ (eds): M J ~ J New ~ o ~York, ~ . McGrawHill, 1986, p p 285-307. 28. Ogata T, Yamasaki Y: T h e three-dimensional structure of rnotor endplates in different fiber types of rat intercostal muscle. Cell Tissue Res 1985;241:465-472. 29. Ogata T, Yaniasaki Y: Scanning electron-microscopic study on the three-dimensional structure of motor endplates ot. the slow (tonic) muscle fibers in the frog, Rana 11. nigromaculata. Cell Tissue Re5 1988;252:211-213. 30. Porayko 0, Smith RS: Morphology of muscle spindles i n the rat. Expperientia (Hasrl) 1968;24:588-589. 3 1. Sahgal V, Morgan CA: Histochemical and morphological changes in human muscle spindle in upper and lower motor neuron lesion. Acta Neuropathol (Berlin) 1976;34:4 1-46. 32. Sanes J R , Marshall LM, McMahan UJ: Reinnervation of muscle fiber basal lamina after removal of niyofibers. Differentiation of regenerating axons at original synaptic sites.] Cell B i d 1978;78:176- 198. 33. SchrOder JM: T h e fine structure of de- and reinnervated muscle spindle. I. T h e increase, atrophy and "hypertrophy" of intrafusal muscle fibers. Arte Neurripath.o/ (BPrlin) 1974;30: 109- 128. 34. Schriider JM: T h e fine structure of de- and reinnervated muscle spindles. 11. Regenerated sensory and motor nerve terminals. Acla Npuroipathol (Berlin) 1974;30: 129- 144. 35. Schroder JM, Kemme PT, Scholz I.: T h e fine structure of denervated and reinnervated muscle spindles: morphometric study of intrafusal muscle hbers. Acta Nruropnthol (Berlin) 1979;46:95- 106. 36. Schrijder JM, Bodden H , Hamacher A, Verres C: Scanning electron microscopy of teased intrafusal muscle fibers from rat muscle spindles. Muscle Nerur 1989;12:22 1-232. 37. Shotton DM, Heuser J E , Reese BF, Reese TS: Postsynaptic membrane folds of the frog neuromuscular junction visualized by scanning electron microscopy. Neurosci~ucr 1979;4:427-435. 38. Struppler A, Mackel R, Resinger UA, Waldhiir F: Electromyography and microneurography following peripheral nerve transplantations, in Gorio A, Millesi I I , Mingrino S (eds): Postraumatic Peripheral Nrrur Rrgrnei-ation: Exl,erimoltal Basis and Clinical Implications. New York, Raven Press, 1981, p p 581-589. 39. Swash M, Fox KP: T h e pathology of the niuscle spindle: effect of denervation. J Neui-ol Sci 1974;22:1-24. 40. Walro JM, Kucera J , Cui F, Staffield regenerated spindles in muscle grafts of the rat. Histocheniistry 1989;W:1- 13. 41. Winlow W, Usherwood PNR: Ultrastructural studies of normal and degenerated mouse neuromuscular junctions. J Neurocytol 1975;4:377-394. 42. Yamamura Y, Schober R: Zur Feinstruktur der Nervenendigungen in den Muskelspindeln des M. lumbricalis der Ratte nach Durchschneidung des Nerven. Acta Neuropathol (Berlin) 1982;57:23-36. I
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SCANNING ELECTRON MICROSCOPIC STUDY OF DENERVATED AND REINNERVATED INTRAFUSAL MUSCLE FIBERS IN RATS RALF DIELER, MD, ANJE VOLKER, MD, and J. MICHAEL SCHRODER, MD
T h e scanning electron microscope can resolve topographical surface details of 5 to 10 nm, with a depth of focus 500 times that of an optical microscope at equivalent magnification.8 Application of this technique to mammalian extrafusal muscle fibers (EMFs) has iven new insights into their overall morphology 1',18,28,29,37 as well as convincing morphological evidence for dynamic changes at
From the lnstitut fur Neuropathologie, Rheinisch-Westfalische Technische Hochschule, Aachen, Germany. Preliminary results were presented at the French-German Joint Meeting of Neuropathology in Frankfurt, West Germany, October 19-21, 1989. Dr. Dieler's present address is Klinik and Poliklinik fur Hals-, Nasen- und Ohrenkranke der Universitat Wurzburg, Josef-Schneider-Str 11, 8700 Wurzburg, Germany. Acknowledgments: The editorial comments of Michael Zidanic, PhD, The Johns Hopkins University School of Medicine, and the photographical assistance of Ms. Bettina Loschner are gratefully acknowledged. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Bonn, Germany), Di 38611.1. The micromanipulator was donated by the Deutsche Gesellschaft zur Bekampfung der Muskelkrankheiten Address reprint requests to Prof Dr. J.M Schroder, lnstitut fur Neuropathologie, Klinikum der RWTH, Pauwelsstrasse 30, D-5100 Aachen. Germany. Accepted for publication March 25, 1991
CCC 01 48-639x1921040433-09$04.00 0 1992 John Wiley & Sons, Inc.
SEM of Muscle Spindles
neuromuscular junctions during maturation,'" ageing,17 or after severance of inner~ation.'~''" The evaluation of surface structures in muscle spindles is limited by the difficulty of separating the inner and outer spindle capsule from the underlying intrafusal muscle fibers (IMFs) and their nerve terminals. In a preceding study,36 scanning electron microscopic (SEM) investigation of normal rat muscle spindles gave new information about their 3-dimensional organization. We now applied the SEM technique to denervated and reinnervated muscle spindles. Knowledge of structural changes in muscle spindles should help to understand the functional recovery of these proprioceptive receptors following peripheral nerve lesions.
MATERIALS AND METHODS
Muscle spindles from the lower lumbrical muscles of female Han:Sprague- Dawley rats weighing 200 to 250 g were used. All animals were handled according to the law of Germany to protect animals used for experimental purposes (Tierschutzgesetz, 1986). T h e experiments were licensed by the local authority. T h e animals were anesthetized with ether fol-
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lowed by intra-abdominal injection of 30 mg/mL chloral hydrate (1 mL per 100 g body weight), and the left sciatic nerves were exposed by a dorsal skin incision. For the denervation experiments, the exposed nerves of 15 rats were sectioned at the mid-thigh level and the proximal and distal stumps were tightly ligated to prevent spontaneous axonal outgrowth. For reinnervation studies, the sciatic nerve was either crushed unilaterally with a watchmaker forceps for 15 minutes (12 rats), or transected at the mid-thigh level and immediately approximated by two oblique 9-0 epineural sutures. Survival times were 3 days; 1, 4, or 12 weeks for denervatiodligation; or 1, 3, 6, or 12 months for both crush injury and transection/ suture. After survival times of 3 days to 1 year, the lower extremities were fixed by perfusing the abdominal aorta of the anesthetized rat with a solution containing 3.9% glutaraldehyde in Sorensen's phosphate buffer (pH 7.4). T h e lumbrical muscles were excised and kept for 1 to 3 hours in the same fixative, rinsed in phosphate buffer, postfixed in phosphate-buffered 2% osmium tetroxide for 3 hours, glycerinated for 48 hours in 66% glycerine, and subsequently stored in 100% glycerine. The muscle spindles were isolated (teased) from the lumbrical muscles in glycerine using a dissecting microscope ( x 40) and fine preparatory needles. In approximately one-half of the teased muscle spindles, w e attempted to mechanically remove the outer spindle capsule at the equatorial region using a micromanipulator (Leitz, Wetzlar, Germany) or fine injection needles. A total of 507 muscle spindles were isolated. The isolated muscle spindles were rinsed with distilled water, treated with 8N HCl for 20 to 60 minutes at 60°C to remove connective tissue elements," washed in distilled water, and dehydrated through a graded series of acetone. After drying at the critical point with liquid GO, for eluting acetone in a CPD 010 (Balzers Union, Liechtenstein), the spindles were sputter-coated with gold in a Balzers Union SCD 030, and examined with a Jeol JSM 50 A scanning electron microscope at 25 kV. Only 265 of the 507 isolated muscle spindles were available for SEM evaluation after the preparatory procedures. Considerable loss of muscle spindles was caused by HC1 treatment, because the length of treatment had to be determined by trial and error. During critical-point drying, some muscle spindles were lost from the enclosing envelopes by flushing the chamber with CO,. Repro-
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ducibility of results was further restricted by the difficulty of attaching the dried and brittle specimen to the specimen holders. Therefore, comparable positions of interesting regions could not intentionally be achieved. Equatorial and juxtaequatorial regions frequently remained concealed within the spindle capsule, or were damaged due to the attempts to mechanically remove the outer and inner spindle capsule. One lumbrical muscle of each animal from each side was used for transmission electron microscopic (TEM) evaluations. The processing of muscle s indles for TEM followed standard proceduresg These results, as well as T E M data available from denervated or reinnervated rat muscle s indles analyzed before in our laboratory,33-5' were used to classify the findings. The results of a previous scanning electron microscopic investigation of normal rat muscle spindles that had been carried out under the same conditions as the present study,36 served as controls. RESULTS
The diameters of IMFs ranged from 5 to 19 p,m. According to values given in revious studies for normal rat muscle spindles,3x33,36 muscle fibers with diameters 5 8 pm were regarded as nuclear chain fibers, and those with diameters > 8 pm as nuclear bag fibers. DENERVATED MUSCLE SPINDLES
In the early stages of denervation the observations in degenerated sensory and motor nerve endings corresponded to what has been reported in the literature. 14217,4'942 Sensory or motor nerve terminals were no longer seen following 1 week of denervation. Sites of previous sensory endings were identified by shallow depressions in the IMF associated with basal lamina duplications and remnants of nerve endings.
TEM Findings.
After 3 days of complete denervation, IMFs revealed slightly elevated subneural apparatuses of motor nerve terminals in polar regions with small and irregularly configurated, shallow impressions. At 1 week survival time, previous sites of motor nerve terminals were trarisformed into sarcolemmal bulges about 9 x 1 1 km in size, irregularly shaped and occasionally with clearly delineated sarcolemmal pits (Fig. I ). A t survival times of 4 and 12 weeks, these sarcolemma1 bulges were still visible. Sites of sensory nerve
SEM Findings.
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FIGURE 1. One week after complete denervation without reinnervation, a sarcolemmal bulge with an uneven shape and with irregular elevations is seen in the distal polar region of an IMF. It contains 2 sharply delineated sarcolemmal pits (arrowheads). Scale bar: 2 pm. FIGURE 2. A nuclear bag fiber in the midpolar region of a muscle spindle, 3 months afler transection and immediate suture of the sciatic nerve. It shows an oval sarcolemmal bulge crossed by a small nerve fascicle that branches off a preterminal axon (arrowheads). The fusiform enlargements along the nerve fiber are thought to represent Schwann cell perikarya (S). Scale bar: 2 pm. FIGURE 3. Plan view of a regenerated motor nerve terminal, 1 month after crushing the sciatic nerve. It is located on a nuclear chain fiber. The supplying preterminal axon reaches the nerve ending from the equatorial region (arrowheads). Scale bar: 3 pm, FIGURE 4. Lateral view of a similar plate-like ending as shown in Figure 3, at 3 months after crushing the sciatic nerve. It lies in a sarcolemmal depression. The supplying preterminal axon (arrowhead) reaches the nerve ending from the equatorial region. Scale bar: 2 pm.
seen. They were similar to those in denervated IMFs, and measured up to 8 x 1 3 p m in size. These sarcolemmal bulges were sometimes delinREINNERVATED MUSCLE SPINDLES eated from the IMF by small slits of' approximately 0.2 pm, and were traversed by unmyelinTEM Findings. After 3 months, regenerated moated axons 1 pm in thickness (Fig. 2). tor (see Fig. 8) or sensory nerve endings were enRegenerated motor nerve endings were decountered in all muscle spindles after crush letected at 1 month and thereafter following sciatic sions, whereas, in the transection/suture group, nerve crush injury, and 3 months and thereafter regenerated nerve endings were only occasionally in the transection/suture group. They were classiseen before 6 months of recovery. Their general fied into: (a) plate-like endings and (b) multitermimorphology and fine structural abnormalities have been reported in preceding s t ~ d i e s . ' ~ , ~nal ~ fusimotor endings. The plate-like motor nerve terminals usually had an oval or fusiform outline Some muscle spindles showed increased numbers measuring between 10 to 16 pm in length and 4 to of IMFs. 1 0 pm in width. They were situated on the surface of the underlying I M F not indenting the sarcoSEM Findings. One month after crushing the scilemma (Fig. 3 ) , or they caused slight sarcolemmal atic nerve, and at 1 and 3 months after transectroughs where the nerve endings were superfitiodsuture of the sciatic nerve, sarcolemmal cially embedded into the muscle fiber surface (Fig. bulges with various outlines and contours were endings at the equatorial or juxtaequatorial region could not be convincingly identified.
SEM of Muscle Spindles
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FIGURE 5. Normally appearing subsynaptic folds and grooves underlying a motor end-plate on an IMF in the distal polar region 6 months after crushing the sciatic nerve. The flat trough with synaptic grooves, partitioned by sarcoplasmic ridges (arrows), is indicated by arrowheads. Scale bar: 2 pm. FIGURE 6. Transmission electron microscopic aspect of subneural apparatus on intrafusal muscle fiber (IMF). Motor nerve ending on an IMF in the polar region from a normal rat muscle spindle. The nerve ending (E) is filled with small clear synaptic vesicles and mitochondria. A nonmyelinated preterminal axon (A) lies in close proximity to the nerve ending. A basal lamina separates the motor nerve ending from the underlying IMF (arrowhead). The subneural apparatus is formed by small sarcoplasmic ridges and grooves (arrows). Scale bar: 1 pm. FIGURE 7. The reinnervated IMF, 12 months after crushing the sciatic nerve, shows a subsynaptic trough in the midpolar region that is surrounded by a sarcolemmal rim, and shows some wide ridges and grooves. Scale bar: 2 pm. FIGURE 8. Transmission electron microscopic aspect of subneural apparatus on intrafusal muscle fiber (IMF). Regenerated motor nerve ending (E) in the polar region, 3 months after nerve crushing. The muscle fiber underlyingthis nerve ending shows a slight trough that is delineated by a sarcolemmal rim (arrowheads). Scale bar: 1 pm. FIGURE 9. Three months after transection and immediate suture of the sciatic nerve, the distal polar region of a nuclear bag fiber with a slight, flat, plate-like elevation (arrows) is shown. The sarcomeric pattern is flattened near the equatorial side of the elevation (arrowheads). Scale bar: 3 prn. FIGURE 10. A plan view of a similar plate-like elevation (large arrows), as in Figure 9, is shown in the polar region of an IMF, 1 month after nerve crush. It possesses small, irregular surface elevations. The sarcomeric pattern of the IMF (small arrows) does not continue into the elevation. Scale bar: 2 wm.
4). These endings were seen on nuclear bag and on nuclear chain fibers at midpolar or distal polar sites. The regenerated multiterminal fusimotor nerve endings usually occupied proximal polar regions extending over areas of 15 x 50 pm. They were situated on IMFs measuring more than 12 km in diameter, i.e., on nuclear bag fibers, and emanated from dichotomously branching preterminal axons measuring 0.4 to 1.5 pm in diameter. These axons and their tapering endings were oriented toward the spindle pole and laid above the muscle fiber surface. Some nerve endings were removed from the underlying IMF due to mechanical manipulation and/or HCl hydrolysis. Thus, 3 different images of subneural apparatuses (SNAs) from motor nerve terminals were seen: They (1) either covered areas of approximately 3 x 11 pm, slightly indenting the IMF surface and showing rather small subsynaptic sarcolemmal ridges and grooves (Fig. 5); or (2) they formed deeper subsynaptic troughs of about 5 x 12 pm in size. These surfaces showed only few, wide sarcolemmal ridges and some broad and shallow grooves (Fig. 7). (3) The third type of subneural apparatuses represented flat, plate-like sarcolemmal elevations measuring up to 9 x 15 pm that were clearly delineated from the regular sarcomeric pattern (Figs. 9 and 10). These areas had only a few inconspicuous surface elevations. When equatorial regions were visualized in reinnervated muscle spindles, a variety of intrafusal components were encountered. Closely spaced spirals were observed in the equatorial region on nuclear chain as well as on nuclear bag fi-
SEM of Muscle Spindles
bers in one muscle spindle 12 months after crush lesion, and in two muscle spindles 1 and 6 months after transection/suture. Each segment measured 2 to 4 pm in diameter. 'These structures were regarded as regenerated primary sensory endings. Single spirals or cuff-like rings were either embedded into the IMF surface or were raised above the surface level for up to 1 pm. Irregularly formed ring-like structures were seen in a few muscle spindles in the juxta-equatorial region, with each segment measuring 1.5 to 3 pm in diameter. These rings only occurred on nuclear chain fibers and were thought to represent regenerated secondary sensory endings. IMFs were often covered by fibroblastic cell processes from the inner spindle capsule. Numerous small nerve fascicles or single nerve fibers with focal enlargements, presumably representing Schwann cell perikarya, were seen closely adjacent to the IMFs. Splitting of IMFs was evident in denervated as well as in reinnervated muscle spindles (Figs. 11 and 12). Because of their small diameter (6 pm) these IMFs were regarded as nuclear chain fibers. Both parts of the split IMFs were separated for variable distances. The longer fiber sometimes showed further splitting along its course inside the spindle. In SEM preparations, where the basal lamina was sufficiently removed, satellite cells appeared as flat oval or fusiform structures (Fig. 13). They were clearly delineated from the IMF by a small cleft, and were not or only slightly elevated above the muscle fiber surface. In T E M images (Fig. 14) satellite cells can be seen closely attached to IMFs and covered by a common basal lamina.
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FIGURE 11. Splitting of an IMF in the juxtaequatorial region of a reinnervated muscle spindle, 12 months after crushing the sciatic nerve. Scale bar: 2 pm. The branching of the IMF leads to 3 segments (a-c). Part a and b of the original IMF fuse again after a distance of about 15 pm. FIGURE 12. Splitting of an IMF in the juxtaequatorial region of a reinnervated muscle spindle, 12 months after crushing the sciatic nerve. Scale bar: 2 pm. The IMF, a presumed nuclear chain fiber, shows a thin branch (arrowheads) that is broken during the preparatory manipulations. FIGURE 13. An elliptical structure, a presumed satellite cell, is clearly delineated by a small cleft (arrowheads), and is completely embedded into a reinnervated IMF (12 months after crush lesion). The IMF is separated from a neighboring IMF by a fibroblast (F) and its cell processes. Scale bar: 3 pm. FIGURE 14. The TEM image shows the typical appearance of a satellite cell (S): It is closely attached to an IMF, and both are covered by a common basal lamina (arrowheads). Scale bar: 1 pm.
The results are summarized in Table 1 according to the frequency of occurrence in the different experimental groups.
DISCUSSION
This study presents, for the first time, SEM aspects of a variety of muscle spindle components following crush lesion or transection of the rat sciatic nerve with or without reinnervation. Four to 5 muscle spindles were found per lumbrical muscle in denervated and reinnervated muscles. These numbers correspond to the amount of muscle spindles in normal rat lumbrical muscles counted by Porayko and Smith3' and Schroder et al.3FArendt and Asmussen,' by contrast, reported that the number of muscle spindles was reduced in denervated and reinnervated muscles after repeated crush lesions or severance of the supplying nerve. I p et al." found only two-thirds of the normal number of muscle spindles in muscles after severance and suture of the nerve. After complete denervation, 1 month after crushing, and 1 and 3 months after transection and immediate suture of the sciatic nerve, the subneural apparatuses of previous motor nerve terminals in polar regions of muscle spindles showed an elevation of the subsynaptic membrane above the surface of the IMF or a loss of junctional folds. These changes resemble those reported by Matsuda et aLZ6in denervated and reinnervated neuromuscular junctions of extrafusal muscle fibers of the hamster, and may be caused by the greater resistance of the cytoskeletal network underneath the plasma membrane. The apparently less severe changes of the subneural apparatuses on IMFs are obviously due to the less pronounced denervation atrophy in IMFs comprising 17% to 34% of their cross-sectional area in rat muscle spindles following 12 months of complete dener-
SEM of Muscle Spindles
~ a t i o n whereas ,~~ EMF diameters in soleus muscles were found to be reduced by 80% after only 21 days of denervation.16 The subneural apparatuses of EMFs recovered their normal structural organization 20 weeks after nerve transection and suture.26 Likewise, the subsynaptic changes on intrafusal muscle fibers were no longer seen after 6 months survival time. This may indicate that areas of former endplates get reinnervated; fusimotor reinnervation of IMFs may resemble motor reinnervation of EMFs with respect to the known preference of old endplate regions for reestablishing neuromuscular j ~ n c t i o n s . However, ~~,~~~~~ complete disappearance of subneural apparatuses, as a result of reinnervation at ectopic sites, cannot be excluded as an alternative explanation. In some spindles, sarcolemmal bulges on reinnervated IMFs were crossed by thin, unmyelinnated axons. These changes were observed more frequently after nerve section and suture than after a crush lesion. Abnormal contact relationships between terminal axons and intrafusal muscle fibers, between terminal axons and Schwann cells, and between Schwann cells and intrafusal muscle fibers were also reported in TEM ~tudies.'"'~It is not known, whether these abnormalities due to reinnervation could cause clinical symptoms (e.g., hypertonia, hypotonia, tremor, ataxia, or dysesthesia). Regenerated motor nerve terminals were classified into (a) plate-like endings, and (b) into multiterminal endings that correspond in size and location to the P1-plates and multiterminal nerve endings as described by Barker et a1.' Mechanical manipulations or HC 1 hydrolysis can lead to detachment of motor nerve endings from the IMF Thus, 3 different types of subneural apparatuses (SNAs) could be distinguished according to the classification of Arbuthnott et al.': (a) SNAs with slight impressions in the IMF surface and a few sarcolemmal ridges and
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Table 1. Frequency of SEM findings after denervation and reinnervation of intrafusal muscle fibers Reinnervation Denervation 3d
Findings Sarcolemmal bulges Abnormally regenerated motor nerve endings Plate-like motor terminals Multiterminal endings Subneural apparatuses without nerve endings Primary sensory nerve endings Secondary sensory nerve endings Fiber splitting Satellite cells Abbreviafions: d
=
I w 4w
1 -
Crush lesion
12 w Total
2
3
6
-
-
-
1 m 31-17 6 m 1 2 m Total 1 m 3 1
3
1
-
-
-
_
-
-
3
-
1
_ 1
-
7
1
1
-
5
-
_
_
12 m Total 1
2
-
1
1
-
I
1 3 4
1
3 4 5
1
-
1
2 1 1
-
-
31-17 6 m
5
-
1 3
-
1
-
3
-
1
-
1
-
days, w = weeks, m = months, - = not observed.
grooves corresponding to the SNAs of In,,-plates; (b) SNAs with sarcolemmal rims and a few shallow grooves corresponding to the SNAs of rn,-plates; and (c) SNAs showing plaque-like elevations above the IMF surface resembling SNAs of ma-plates. I t was suggested that ma-plates correspond to multiterminal nerve endings, whereas m,-plates represent pl- or p,-nerve terminals.' Regenerated sensory nerve endings were encountered 6 and 12 months after a crush lesion and 1 to 6 months after transection and suture of the sciatic nerve. T h e endings were, however, often covered by remnants of basal lamina and connective tissue elements, or they were partly destroyed while removing the connective tissue. Some evidence for structural abnormalities of reinnervated sensory endings (e.g., incomplete contact relationship between sensory endings and IMFs, intervening basal lamina between sensory nerve ending and IMF, covering of sensory terminals by Schwann cells and axonal sprouts, hyperinnervation, etc.) were more clearly identified in preceding TEM s t u ~ i i e s . ' ~ , ~ ~ Intrafusal muscle fiber splitting and fusion of intrafusal muscle fibers was unequivocally visualized in the present SEM study. Although this phenomenon may also occur in normal muscle spindles,52"." it has been observed more fre uently after denervation and Data on the frequency of muscle fiber splitting following denervation and reinnervation are rare in the literature. Kucera," mentioned splitting in 67% of all reinnervated IMFs after crush lesion of the supplying nerve, whereas Walro et al.40 observed fiber splitting in 14.3% of regenerated IMFs in
re innervation.",",^
440
Transection and suture
SEM of Muscle Spindles
muscle grafts of rats. Muscle fiber splitting may be one cause of a numerical increase of IMFs after nerve lesions. Another mechanism leading to an increased number of IMFs may be the stimulation of satellite cells. They may become motile and mitotic, forming myoblasts and myotubes that fuse incompletely with the parent muscle fiber, or with newly formed myoblasts and m y o t ~ b e s . * ~ T h e occurrence of regenerated motor and sensory nerve terminals confirms that reinnervation of IMFs does take place, even after transection and immediate suture of the nerve. T h e necessary period of time for reinnervation, however, appears to be longer after nerve transection than after crush lesions, where axonal guidance is more easily accomplished by intact endoneurial and perineurial connective tissue elements. Regenerated nerve terminals were noted to be functional to various degrees in electrophysiological ~tudies.~,~."'".'" These studies also revealed effective, though incomplete, restoration of muscle spindle discharge patterns, particularly for sensory nerve responses. However, following peripheral nerve suture or transplantation in man, Struppler et ai.,38were not able to confirm muscle spindle reinnervation using electromyography or microneurography. Because discharge patterns often correlate to the morphology of the nerve terminals,4x20x3q the normal appearance of at least some regenerated intrafusal nerve endings suggests that muscle spindle function could have been, in fact, re-established. Abnormal reinnervation of pre-existing, split, or newly formed IMFs, however, may cause functional disturbances that would have to be analyzed using electrophysiological techniques.
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SEM of Muscle Spindles
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