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Reinnervation of the soleus muscle by its own or by an alien nerve
lVewoscience Vol. 10, No. Printed in Great Britain
0306-4522/83
4, pp. 1463 to 1469, 1983
$3.00 + 0.00
Pergamon Press Ltd IBRO
REINNERVATION OF THE SOLEUS MUSCLE ITS OWN OR BY AN ALIEN NERVE
BY
M. C. IP* and G. VRBOVA Dept of Anatomy & Embryology, Centre for Neuroscience, University College London, Gower Street, London, WCIE 6BT, U.K.
Abstract-The rates of reinnervation of the rat soleus muscle by its own or an alien nerve were compared using physiological and morphological criteria. The muscle’s own nerve was more effective than the alien nerve in establishing functional connections with the original endplates. The relative ineffectiveness of the alien nerve during early stages of reinne~ation was more apparent using physiolo~cal rather than mo~holo~~l criteria. Five to six weeks after nerve injury there was no longer any difference between the muscles supplied by either nerve.
It is accepted that mammalian slow twitch muscle fibres can be reinnervated by either their own or an alien nerve, This conclusion is based on a number of studies showing recovery of tension and weight of soleus muscles from various mammals when they were reinnervated by an alien nerve.‘.2’4’7”s At the turn of the century Langley and Anderson’l provided convincing evidence that cholinergic nerves are interchangeable, and that functional reinnervation of skeletal muscles by the vagus or by preganglionic s~patheti~ fibres can be achieved if these nerves are connected to denervated skeletal muscles. However, sensory nerves and postganglionic sympathetic nerves that do not release a~tylchoiine (ACh) are unabfe to establish functional connections with skeletal muscle fibres.” Thus, successful muscle reinnervation requires that the transmitter released by the ingrowing nerve be ACh. In the situation where autonomic nerves are in competition with somatic nerves the muscles are preferentially reinnervated by the somatic nerves.’ An indication that skeletal muscle fibres are preferentially reinnervated by nerves that previously supplied muscle fibres of similar characteristics was provided by results of Lewis, Rowlerson and Webb.” These authors noticed that if a soleus muscle was reinnervated by a nerve from a mixed muscle the size of the slow units was abnormally enlarged, while the size of fast units was much reduced. This changed the relationship between the conduction velocity of the axon and the size of motor unit; normally, fast conducting axons supply the largest and fastest motor units, but after crossinnervation the fast conducting axons supplied the smallest motor units, and the slow conducting ones the larger units. It may be that the change of this -*Present address: Dept of Anatomy, University of Hong Kong. ~~~~~i~~i~~: ACh, acetylcholine. hSC
IO/‘&”
relationship in the cross-innervated soleus is due to a selective reinnervation of the slow muscle fibres by nerves originally supplying slow tibres in the mixed muscle. If indeed soleus was selectively reinnervated by axons to slow muscle fibres, then it could be expected that reinnervation of the soleus muscle by its own nerve may proceed at a different rate from reinnervation by a nerve from predominantly fast muscle. This possibility was explored in the present study. EXPERIMENTAL PROCEDURES Wistar rats weighing 1%2OOg were anaesthetized with intrape~toneal injection of chloral hydrate (4.59{ solution, 1ml/l00 g of weight). Using sterile precautions the soleus muscles were exposed and on one side its own nerve was sectioned and resutured to the muscle at its place of entry. On the other side the soleus nerve was sectioned close to its entry to the muscle, bent proximally and sutured into the lateral head of gastrocnemius in a manner that would prevent it from reinnervating the soleus muscle. The deep branch of the lateral popliteal nerve was then dissected and cut and the proximal stump was sutured to the soleus muscle at the place of entry of the original nerve. The animals were left to recover and the final experiment was performed two to eight weeks later. The rats were again anaesthetized with chloral-hydrate and the soleus muscles prepared for tension recording. Contractions were elicited on the self-reinnervated side by stimulating the muscle’s own nerve. On the cross-innervated side, contractions were elicited by stimulating the lateral popliteal nerve as well as the muscle’s own nerve. In some cases, in spite of all the precautions, the muscle’s own nerve also grew back and contributed to reinnervation. When more than IO:/ of the cross-innervated muscle was reinnervated by its own nerve, the animal was excluded from the experiment. In addition to eliciting contractions by nerve stipulation, the muscles were also stimulated directly to establish the freauency at which maxima1 tetanic tension was produced. The ratio of d~rectiy~indirectly elicited contractions was taken as an index of reinnervation. When the tension recording was completed both soleus muscles were excised and processed for histological visualization of the endplates and axons, sections were stained by
1463
2464
M. C. ip and G. V&ova
a modification of the Namba, Nakamura and Grab” method described bv O’Brien. Ostberp: and Vrbova” to assess the proportion of innervated endplates as well as their morphology and the distribution of axons within the muscle.
RESULTS
Morphological
evidence of reinnervation
When the central stump of either the soleus or lateral popliteal nerve was sutured to the soleus muscle at the place of entry of its original nerve, axons crossed the anastomosis and, 16 days after the operation, were seen in abundance within the muscle (Figs la,b and c). In both self- and alien-reinnervated muscles, the axons formed endplates near their site of entry (Figs lb,d,e,f), but reinnervation of the originaf endplates was much preferred by both nerves (Figs la,e and g). Twenty-one days after the operation, muscles reinnervated by their original nerves often had ultraterminal sprouts; these frequently terminated on adjacent endplates so that some of the endplates were contacted by more than one axon (Figs 2b and c). The morphology of endplates reinnervated by alien axons was not very different from those innervated by their original nerves. Figure 2(k) illustrates that 3 weeks after the initial operation the original endplates were contacted by the alien axons and ultraterminal sprouts were commonly seen. In these muscles also, the number of ultraterminal sprouts greatly varied but there was no consistent difference between muscles reinnervated by their own or alien nerves. However, when the proportion of reinnervated endplates was assessed at different times, it became apparent that until about 22 days after surgery, the proportion of reinnervated end-
plates in the muscles supplied by the alien nerve was smaller than in the self-reinnervated muscles (see Table 1). Figure 2g illustrates a group of “empty” endplates in a muscle reinnervated by an alien nerve 3 weeks after the initial operation, Some endplates within the same muscle were reinnervated hq very slender axons (Figs 2h and i) and this indicates that reinnervation was still very incomplete. Recovery of’flmction
of the self- and alien-rrinnrwated
soleus tnuscle
The contractions elicited by stimulating the corresponding nerves or the muscles directly were recorded at different times after surgery. The ratio of the tension developed by direct and indirect stimulation was then calculated as a measure of the reinnervation. After S-10 weeks, both nerves reinnervated the muscles completely, and at this late stage there was no detectable difference between the ability of the muscle’s own or alien nerve to reinnervate the muscle. During earlier stages of reinnervation, however. the muscle’s own nerve was more effective than the alien nerve (see Table 1 and Fig. 3). Thus, initially, there is some degree of incompatibility between the alien nerve and muscle. An indication of the slower recovery of function of those neuromuscular junctions supplied by the alien nerve is given by the responses of the muscles to indirect stimulation at relatively high frequencies. The records of isometric tetanic contraptions, elicited by direct and indirect stimulation at 80 Hz of a self-reinnervated soleus (A and B) and a muscle supplied by an alien nerve (C and D) from the same animal 21 days after the operation (Fig. 4), show that the muscle supplied by the alien nerve was unable to maintain tension. This inability of the muscle to maintain tension was apparent only when contractions were elicited by stimulating the
Fig. I. Photographs of frozen sections of self-reinnervated (a and d) and cross-reinnervated (b,c,e,f, and g) rat solei stained by a modified technique of Namba ef al.rs (a) Twenty-one days after self-reinnervation by own nerve, shows that the stitch (S, lower left corner) is very close to, or almost on the original endplate zone, the reinnervated endplates appear nearly normal and reinnervation is almost complete (94.77;). (h) Twenty-one days after cross-innervation of the contralateral muscle from the same animal, shows that extra junctional endplates (asterisk) can be seen not far from the stitch (S, top left corner). (c) One of the endplates shown in (b) at a higher magnification. (d) Twenty-four days after self-reinnervation, some extra junctional endplates (asterisk) near the stitch (S, lower left corner) are seen. (e) Twenty-four days after cross-innervation of the contralateral muscle, a montage shows some extra junctional endplates on the left hand side 4-5 mm away from the original endplate zone to the right, some endplates of both zones are also shown at a higher magnification in (f) and (g), respectively. As clearly seen from (g) the reinnervated endplates have normal appearance and their terminal axons and arborizations are apparently normal. Scale: 1OO~m. Fig. 2. Photographs of frozen sections of self-reinnervated (a-f, on left-hand column) and cross-innervated (g-k, on right-hand column) rat sotei treated as in Fig. 1. (a-c) 21 days after self-reinnervation by own nerve, show reinnervated endplates some with distinct ultrate~nals (top most in a and b, lowest in b and c) which innervate another endplate (top two in b, and right two in c). In contrast 21 days after cross-innervation the contralateral soieus of the same rat (g-i), shows rather poor reinnervation from none (g) to thin reinnervating axons (h and j). At 23 days after surgery, the cross-innervated soleus has some endplates that are well reinnervated (k) and similar to self-reinnervated endplates (d) from an animal 2 I days after surgery. Note some ultraterminal sprouts in d and e. (e and f) are taken from a self-reinnervated soleus 23 days after the operation. Scale: SOpm.
Fig. I465
1
Fig. 2. 1466
1467
Reinnervation of soleus muscle Table I. The Table compares self-reinnervated (self) and cross-reinnervated ._ (days) 14 14 16 19 20 21 21 22 22 23 23 23 27 35 41 80 120
(x-inn.) soleus muscles
No. extra j. endpl. (x-inn) (self) (x-inn) ._~ __._..~._~_ “_~_ 15(386) 24(534) 90 12(2465) 21 22(1065) ,461) 82(468) 98( 1256) 32 51 100(1022) 14(38?1
Reinn. endpl. I/D Ratio x 100 (self) (x-inn) _~~~~~-.~~-~- Wf) 416332) 1.5 34 1.5 351429) 21 18(1564) IS 15 18(1145) 17 37 9.5 62 95(632) 22 66 89(363) 15 100 94(674) 100 100 96f 1080) 16 100 83(478) 12 54 24 70 98(610) 41 80 100(973) 100 100 961642) 100 100 Iit: -_ 100 100 100 100
-
95(606) l~(lOS9) 92(673)
70 33
29 4 70
-
-
-
Results obtained using physioiogical techniques, i.e. the ratios of indirectly- to directly-elicited maximal tetanic contraction (I/D Ratio x 100) are compared to those obtained using rno~holo~~l techniques where the ‘/* of reinnervated endplates (Reinn. endpi.) was calculated. Numbers in brackets indicate the total number of endplates counted. The time after the operation is indicated in days.
nerve; when the muscle was stimulated directly it could maintain its tension even at high rates of stimulation (Fig. 4~). Thus, neuromuscular transmission between. the alien nerve and soleus muscle was less effective than transmission between the muscle’s own nerve and soleus. This point is further supported by the finding that in the self-reinnervated muscle the proportion of reinnne~ated endplates assessed histolo~cally correlated well with the ratio of the directly to indirectly
elicited contractions, whereas, in the muscles supplied by the alien nerve, the proportion of endplates contacted by axons was usually higher than the ratio of directly to indirectly elicited contractions. Figure 3 shows that in the cross-innervated muscles even 3 to 4 weeks after surgery, the tension developed in response to nerve stimulation was considerably less than that produced by stimulating the muscle directly. Thus, the results show that functional reinner-
IOO-
50-
S-in
-2-3
X-in
S-in weeks
X-m
-...--J
S-m
-3-4
X-in
S-in
X-m
weeks------’
Fig. 3. The block diagram shows the degree of ~inne~ation assessed by histological means (reinne~ated endplates) and by recording the ratio of direct/ indirect tetanic tension (tetanic tension) of self and cross-innervated soleus muscles.
146X
M. C. Ip and G. Vrbova A
c
-
1.
Fig. 4. Records of isometric contractions of the selfreinnervated (A + B) and cross-innervated (C + D) soleus muscles 21 days after the operation are shown. The contractions were elicited by trains of stimuli at 80 Hz applied directly to the muscle (A and C) or to the respective motor nerves (B + D). Note that the x-innervated muscle was unable to maintain tension when stimulated via its motor nerve.
vation of soleus muscles proceeds faster if the muscle’s own nerve is connected to the muscle, than if an alien nerve is made to reinnervate it.
DISCUSSION
The results show that the muscle’s own nerve reinnervates the rat soleus muscle more rapidly than an alien nerve. Although the alien nerve makes morphological contacts with the muscle fibres, initially these contacts are functionally less effective than connections between the muscle’s own nerve and its muscle fibres. It could be that the postsynaptic specialisations of the endplate region are adjusted in some way so as to respond best to the original nerve ending. Soleus nerve endings have different fimctional characteristics from those of fast nerves, in that they are smaller, release less transmitter” and fire at low rates3 and so the postsynaptic specialisation may be better suited to their function. The inability of the muscle to maintain tension in response to stimulation
of the alien nerve during early stages of reinnervation could be due to a mis-matching of the postsynaptic specialisation to the activity of the nerve ending. It may be that the activity of cholinesterase. or its particular molecular form, is different in slow and fast muscles,‘4 and so the greater release of ACh from the fast endings could cause transient depolarisation block at high rates of stimulation. It is known that an alien nerve is able to change the structure of neuromuscular junctions’ and so it can be expected that with time the synapse between the alien nerve and soleus muscle fibres will become transformed and more efficient; this is indeed brought out by the present results. The finding that functional reinnervation of the soleus muscle proceeds more rapidly when the muscle is connected to its own nerve has implications for the recovery of function and redistribution of motor units within a mixed muscle after nerve injury. It is generally assumed that after nerve injury muscle fibres of the same type are segregated. This grouping was attributed to random, non-selective reinnervation of muscle fibres and their subsequent conversion into a similar fibre type. Since the motoneurone determines the histochemical properties of the muscle fibres5,16~19this process can be expected to take place. If, however, the rate of reinnervation is not entirely random, as the present results and those of Lewis et ~1.‘~ suggest, then axons would preferentially reinnervate the type of muscle fibre they were originally connected to. This could lead to a more rapid recovery and could explain results showing that the distribution of motor unit properties and sizes returns to normal relatively quickly after nerve injury.’ The fibre grouping may then occur in situations when the muscle was deprived of its innervation for long periods of time, during which the endplate could have lost its specialisation. The more rapid reinnervation of muscle fibres by their own nerve fibres may then represent an important mechanism for ensuring that the distribution of motor unit sizes and types is not drastically altered after nerve injury, and subsequent reinnervation by the muscles’ own nerve. Acknowledgements-We are grateful to the Muscular Dystrophy Group of G.B. and the M.R.C. for their support. We thank MS K. Ward for her help.
REFERENCES
1. Buller A. J., Eccles J. C. and Eccles R. M. (1960) Interactions between motoneurones and muscles in respect to the characteristic speeds of their responses. J. Physiol., Land. 150, 417-439. 2. Close R. (1969) Dynamic properties of fast and slow skeletal muscles of the rat after nerve cross-union. J. fhysiol., Land. 204, 331-346. 3. Denny-Brown D. (1929) On the nature of postural reflexes. Proc. R. Sot. B. 104, 252-301. 4. Dhoot G. K., Perry S. V. and Vrbova G. (1981) Changes in the distribution of the components of the troponin complex in muscle fibres after cross-innervation. Expl Neurol. 72, 513-530.
5. Edstrom L. and Kugelberg E. (1968) Histochemical composition, distribution of fibres and fatiguability of single motor units. J. Neurol. Neurosurg. Psych&f. 31, 424433. 6. Hnik P., Jirmanova J., Vyklickjl L. and ZclenP J. (1,967) Fast and slow muscles of the chick after nerve cross-union. J. Physiol., Land. 193, 309-335.
Reinnervation of soleus muscle
1469
7. Hoh J. F. Y. (1975) Selective and non-selective reinnervation of fast twitch and slow twitch rat skeletal muscle. J. Physiol., Lond. 251, 791801. 8. Gordon T. and Stein R. B. (1980) Rematching of nerve and muscle properties in cat motor units after reinnervation. In Plasticity of Muscle (eds Pette D. and de Guyter W.) 9. Grinnell A. D., Letinsky M. S. and Rheuben M. B. (1979) Competitive interaction between foreign nerves innervating frog skeletal muscle. J. Physiol., Lond. 289, 241-262. 10. Gutmann E. and Young J. 2. (1944) The reinnervation of muscle after various periods of atrophy. J. Anaf. 78, 15-43. 11. Kuno M., Turkanis S. A. and Weakly J. N. (1971) Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog. J. Physiol., Lond. 213, 545-556. 12. Langley J. N. and Anderson H. K. (1904) The union of different kinds of nerve fibres. J. Physiol., Lond. 31, 365-391. 13. Lewis D. M., Rowlerson A. and Webb S. (1982) Motor units and immunohistochemistry of cat soieus muscle after long periods of cross-reinnervation. J. Physiol., Lond. 325, 395-403. 14. Massoulie J. and Bon S. (1982) The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. A. Rev. Neurosci. 5, 57-106. 15. Namba T., Nakamura T. and Grob D. (1967) Staining for nerve fibre and cholinesterase activity in fresh frozen sections, Am. J. clin. Path. 47, 74-77. 16. Nemeth P. M., Pette D. and Vrbova G. (1981) Comparison of enzyme activities among single muscle fibres within defined motor units. J. Physiol., Lond. 311, 489-495.
17. O’Brien R. A. D., Ostberg J. C. and Vrbova G. (1978) Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle. J. Physiol., Lond. 282, 571-582. 18. Romanul F. C. A. and van der Meulen J. P. (1967) Slow and fast muscle after cross-innervation: enzymatic and physiological changes. Archs Neural. Psychiat., Chicago 17, 387402. 19. Vrbova G., Gordon T. and Jones R. (1978) Nerve-Muscle Interaction. Chapman & Hall, London. (Accepted 7 July 1983)
0306-4522/83
4, pp. 1463 to 1469, 1983
$3.00 + 0.00
Pergamon Press Ltd IBRO
REINNERVATION OF THE SOLEUS MUSCLE ITS OWN OR BY AN ALIEN NERVE
BY
M. C. IP* and G. VRBOVA Dept of Anatomy & Embryology, Centre for Neuroscience, University College London, Gower Street, London, WCIE 6BT, U.K.
Abstract-The rates of reinnervation of the rat soleus muscle by its own or an alien nerve were compared using physiological and morphological criteria. The muscle’s own nerve was more effective than the alien nerve in establishing functional connections with the original endplates. The relative ineffectiveness of the alien nerve during early stages of reinne~ation was more apparent using physiolo~cal rather than mo~holo~~l criteria. Five to six weeks after nerve injury there was no longer any difference between the muscles supplied by either nerve.
It is accepted that mammalian slow twitch muscle fibres can be reinnervated by either their own or an alien nerve, This conclusion is based on a number of studies showing recovery of tension and weight of soleus muscles from various mammals when they were reinnervated by an alien nerve.‘.2’4’7”s At the turn of the century Langley and Anderson’l provided convincing evidence that cholinergic nerves are interchangeable, and that functional reinnervation of skeletal muscles by the vagus or by preganglionic s~patheti~ fibres can be achieved if these nerves are connected to denervated skeletal muscles. However, sensory nerves and postganglionic sympathetic nerves that do not release a~tylchoiine (ACh) are unabfe to establish functional connections with skeletal muscle fibres.” Thus, successful muscle reinnervation requires that the transmitter released by the ingrowing nerve be ACh. In the situation where autonomic nerves are in competition with somatic nerves the muscles are preferentially reinnervated by the somatic nerves.’ An indication that skeletal muscle fibres are preferentially reinnervated by nerves that previously supplied muscle fibres of similar characteristics was provided by results of Lewis, Rowlerson and Webb.” These authors noticed that if a soleus muscle was reinnervated by a nerve from a mixed muscle the size of the slow units was abnormally enlarged, while the size of fast units was much reduced. This changed the relationship between the conduction velocity of the axon and the size of motor unit; normally, fast conducting axons supply the largest and fastest motor units, but after crossinnervation the fast conducting axons supplied the smallest motor units, and the slow conducting ones the larger units. It may be that the change of this -*Present address: Dept of Anatomy, University of Hong Kong. ~~~~~i~~i~~: ACh, acetylcholine. hSC
IO/‘&”
relationship in the cross-innervated soleus is due to a selective reinnervation of the slow muscle fibres by nerves originally supplying slow tibres in the mixed muscle. If indeed soleus was selectively reinnervated by axons to slow muscle fibres, then it could be expected that reinnervation of the soleus muscle by its own nerve may proceed at a different rate from reinnervation by a nerve from predominantly fast muscle. This possibility was explored in the present study. EXPERIMENTAL PROCEDURES Wistar rats weighing 1%2OOg were anaesthetized with intrape~toneal injection of chloral hydrate (4.59{ solution, 1ml/l00 g of weight). Using sterile precautions the soleus muscles were exposed and on one side its own nerve was sectioned and resutured to the muscle at its place of entry. On the other side the soleus nerve was sectioned close to its entry to the muscle, bent proximally and sutured into the lateral head of gastrocnemius in a manner that would prevent it from reinnervating the soleus muscle. The deep branch of the lateral popliteal nerve was then dissected and cut and the proximal stump was sutured to the soleus muscle at the place of entry of the original nerve. The animals were left to recover and the final experiment was performed two to eight weeks later. The rats were again anaesthetized with chloral-hydrate and the soleus muscles prepared for tension recording. Contractions were elicited on the self-reinnervated side by stimulating the muscle’s own nerve. On the cross-innervated side, contractions were elicited by stimulating the lateral popliteal nerve as well as the muscle’s own nerve. In some cases, in spite of all the precautions, the muscle’s own nerve also grew back and contributed to reinnervation. When more than IO:/ of the cross-innervated muscle was reinnervated by its own nerve, the animal was excluded from the experiment. In addition to eliciting contractions by nerve stipulation, the muscles were also stimulated directly to establish the freauency at which maxima1 tetanic tension was produced. The ratio of d~rectiy~indirectly elicited contractions was taken as an index of reinnervation. When the tension recording was completed both soleus muscles were excised and processed for histological visualization of the endplates and axons, sections were stained by
1463
2464
M. C. ip and G. V&ova
a modification of the Namba, Nakamura and Grab” method described bv O’Brien. Ostberp: and Vrbova” to assess the proportion of innervated endplates as well as their morphology and the distribution of axons within the muscle.
RESULTS
Morphological
evidence of reinnervation
When the central stump of either the soleus or lateral popliteal nerve was sutured to the soleus muscle at the place of entry of its original nerve, axons crossed the anastomosis and, 16 days after the operation, were seen in abundance within the muscle (Figs la,b and c). In both self- and alien-reinnervated muscles, the axons formed endplates near their site of entry (Figs lb,d,e,f), but reinnervation of the originaf endplates was much preferred by both nerves (Figs la,e and g). Twenty-one days after the operation, muscles reinnervated by their original nerves often had ultraterminal sprouts; these frequently terminated on adjacent endplates so that some of the endplates were contacted by more than one axon (Figs 2b and c). The morphology of endplates reinnervated by alien axons was not very different from those innervated by their original nerves. Figure 2(k) illustrates that 3 weeks after the initial operation the original endplates were contacted by the alien axons and ultraterminal sprouts were commonly seen. In these muscles also, the number of ultraterminal sprouts greatly varied but there was no consistent difference between muscles reinnervated by their own or alien nerves. However, when the proportion of reinnervated endplates was assessed at different times, it became apparent that until about 22 days after surgery, the proportion of reinnervated end-
plates in the muscles supplied by the alien nerve was smaller than in the self-reinnervated muscles (see Table 1). Figure 2g illustrates a group of “empty” endplates in a muscle reinnervated by an alien nerve 3 weeks after the initial operation, Some endplates within the same muscle were reinnervated hq very slender axons (Figs 2h and i) and this indicates that reinnervation was still very incomplete. Recovery of’flmction
of the self- and alien-rrinnrwated
soleus tnuscle
The contractions elicited by stimulating the corresponding nerves or the muscles directly were recorded at different times after surgery. The ratio of the tension developed by direct and indirect stimulation was then calculated as a measure of the reinnervation. After S-10 weeks, both nerves reinnervated the muscles completely, and at this late stage there was no detectable difference between the ability of the muscle’s own or alien nerve to reinnervate the muscle. During earlier stages of reinnervation, however. the muscle’s own nerve was more effective than the alien nerve (see Table 1 and Fig. 3). Thus, initially, there is some degree of incompatibility between the alien nerve and muscle. An indication of the slower recovery of function of those neuromuscular junctions supplied by the alien nerve is given by the responses of the muscles to indirect stimulation at relatively high frequencies. The records of isometric tetanic contraptions, elicited by direct and indirect stimulation at 80 Hz of a self-reinnervated soleus (A and B) and a muscle supplied by an alien nerve (C and D) from the same animal 21 days after the operation (Fig. 4), show that the muscle supplied by the alien nerve was unable to maintain tension. This inability of the muscle to maintain tension was apparent only when contractions were elicited by stimulating the
Fig. I. Photographs of frozen sections of self-reinnervated (a and d) and cross-reinnervated (b,c,e,f, and g) rat solei stained by a modified technique of Namba ef al.rs (a) Twenty-one days after self-reinnervation by own nerve, shows that the stitch (S, lower left corner) is very close to, or almost on the original endplate zone, the reinnervated endplates appear nearly normal and reinnervation is almost complete (94.77;). (h) Twenty-one days after cross-innervation of the contralateral muscle from the same animal, shows that extra junctional endplates (asterisk) can be seen not far from the stitch (S, top left corner). (c) One of the endplates shown in (b) at a higher magnification. (d) Twenty-four days after self-reinnervation, some extra junctional endplates (asterisk) near the stitch (S, lower left corner) are seen. (e) Twenty-four days after cross-innervation of the contralateral muscle, a montage shows some extra junctional endplates on the left hand side 4-5 mm away from the original endplate zone to the right, some endplates of both zones are also shown at a higher magnification in (f) and (g), respectively. As clearly seen from (g) the reinnervated endplates have normal appearance and their terminal axons and arborizations are apparently normal. Scale: 1OO~m. Fig. 2. Photographs of frozen sections of self-reinnervated (a-f, on left-hand column) and cross-innervated (g-k, on right-hand column) rat sotei treated as in Fig. 1. (a-c) 21 days after self-reinnervation by own nerve, show reinnervated endplates some with distinct ultrate~nals (top most in a and b, lowest in b and c) which innervate another endplate (top two in b, and right two in c). In contrast 21 days after cross-innervation the contralateral soieus of the same rat (g-i), shows rather poor reinnervation from none (g) to thin reinnervating axons (h and j). At 23 days after surgery, the cross-innervated soleus has some endplates that are well reinnervated (k) and similar to self-reinnervated endplates (d) from an animal 2 I days after surgery. Note some ultraterminal sprouts in d and e. (e and f) are taken from a self-reinnervated soleus 23 days after the operation. Scale: SOpm.
Fig. I465
1
Fig. 2. 1466
1467
Reinnervation of soleus muscle Table I. The Table compares self-reinnervated (self) and cross-reinnervated ._ (days) 14 14 16 19 20 21 21 22 22 23 23 23 27 35 41 80 120
(x-inn.) soleus muscles
No. extra j. endpl. (x-inn) (self) (x-inn) ._~ __._..~._~_ “_~_ 15(386) 24(534) 90 12(2465) 21 22(1065) ,461) 82(468) 98( 1256) 32 51 100(1022) 14(38?1
Reinn. endpl. I/D Ratio x 100 (self) (x-inn) _~~~~~-.~~-~- Wf) 416332) 1.5 34 1.5 351429) 21 18(1564) IS 15 18(1145) 17 37 9.5 62 95(632) 22 66 89(363) 15 100 94(674) 100 100 96f 1080) 16 100 83(478) 12 54 24 70 98(610) 41 80 100(973) 100 100 961642) 100 100 Iit: -_ 100 100 100 100
-
95(606) l~(lOS9) 92(673)
70 33
29 4 70
-
-
-
Results obtained using physioiogical techniques, i.e. the ratios of indirectly- to directly-elicited maximal tetanic contraction (I/D Ratio x 100) are compared to those obtained using rno~holo~~l techniques where the ‘/* of reinnervated endplates (Reinn. endpi.) was calculated. Numbers in brackets indicate the total number of endplates counted. The time after the operation is indicated in days.
nerve; when the muscle was stimulated directly it could maintain its tension even at high rates of stimulation (Fig. 4~). Thus, neuromuscular transmission between. the alien nerve and soleus muscle was less effective than transmission between the muscle’s own nerve and soleus. This point is further supported by the finding that in the self-reinnervated muscle the proportion of reinnne~ated endplates assessed histolo~cally correlated well with the ratio of the directly to indirectly
elicited contractions, whereas, in the muscles supplied by the alien nerve, the proportion of endplates contacted by axons was usually higher than the ratio of directly to indirectly elicited contractions. Figure 3 shows that in the cross-innervated muscles even 3 to 4 weeks after surgery, the tension developed in response to nerve stimulation was considerably less than that produced by stimulating the muscle directly. Thus, the results show that functional reinner-
IOO-
50-
S-in
-2-3
X-in
S-in weeks
X-m
-...--J
S-m
-3-4
X-in
S-in
X-m
weeks------’
Fig. 3. The block diagram shows the degree of ~inne~ation assessed by histological means (reinne~ated endplates) and by recording the ratio of direct/ indirect tetanic tension (tetanic tension) of self and cross-innervated soleus muscles.
146X
M. C. Ip and G. Vrbova A
c
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1.
Fig. 4. Records of isometric contractions of the selfreinnervated (A + B) and cross-innervated (C + D) soleus muscles 21 days after the operation are shown. The contractions were elicited by trains of stimuli at 80 Hz applied directly to the muscle (A and C) or to the respective motor nerves (B + D). Note that the x-innervated muscle was unable to maintain tension when stimulated via its motor nerve.
vation of soleus muscles proceeds faster if the muscle’s own nerve is connected to the muscle, than if an alien nerve is made to reinnervate it.
DISCUSSION
The results show that the muscle’s own nerve reinnervates the rat soleus muscle more rapidly than an alien nerve. Although the alien nerve makes morphological contacts with the muscle fibres, initially these contacts are functionally less effective than connections between the muscle’s own nerve and its muscle fibres. It could be that the postsynaptic specialisations of the endplate region are adjusted in some way so as to respond best to the original nerve ending. Soleus nerve endings have different fimctional characteristics from those of fast nerves, in that they are smaller, release less transmitter” and fire at low rates3 and so the postsynaptic specialisation may be better suited to their function. The inability of the muscle to maintain tension in response to stimulation
of the alien nerve during early stages of reinnervation could be due to a mis-matching of the postsynaptic specialisation to the activity of the nerve ending. It may be that the activity of cholinesterase. or its particular molecular form, is different in slow and fast muscles,‘4 and so the greater release of ACh from the fast endings could cause transient depolarisation block at high rates of stimulation. It is known that an alien nerve is able to change the structure of neuromuscular junctions’ and so it can be expected that with time the synapse between the alien nerve and soleus muscle fibres will become transformed and more efficient; this is indeed brought out by the present results. The finding that functional reinnervation of the soleus muscle proceeds more rapidly when the muscle is connected to its own nerve has implications for the recovery of function and redistribution of motor units within a mixed muscle after nerve injury. It is generally assumed that after nerve injury muscle fibres of the same type are segregated. This grouping was attributed to random, non-selective reinnervation of muscle fibres and their subsequent conversion into a similar fibre type. Since the motoneurone determines the histochemical properties of the muscle fibres5,16~19this process can be expected to take place. If, however, the rate of reinnervation is not entirely random, as the present results and those of Lewis et ~1.‘~ suggest, then axons would preferentially reinnervate the type of muscle fibre they were originally connected to. This could lead to a more rapid recovery and could explain results showing that the distribution of motor unit properties and sizes returns to normal relatively quickly after nerve injury.’ The fibre grouping may then occur in situations when the muscle was deprived of its innervation for long periods of time, during which the endplate could have lost its specialisation. The more rapid reinnervation of muscle fibres by their own nerve fibres may then represent an important mechanism for ensuring that the distribution of motor unit sizes and types is not drastically altered after nerve injury, and subsequent reinnervation by the muscles’ own nerve. Acknowledgements-We are grateful to the Muscular Dystrophy Group of G.B. and the M.R.C. for their support. We thank MS K. Ward for her help.
REFERENCES
1. Buller A. J., Eccles J. C. and Eccles R. M. (1960) Interactions between motoneurones and muscles in respect to the characteristic speeds of their responses. J. Physiol., Land. 150, 417-439. 2. Close R. (1969) Dynamic properties of fast and slow skeletal muscles of the rat after nerve cross-union. J. fhysiol., Land. 204, 331-346. 3. Denny-Brown D. (1929) On the nature of postural reflexes. Proc. R. Sot. B. 104, 252-301. 4. Dhoot G. K., Perry S. V. and Vrbova G. (1981) Changes in the distribution of the components of the troponin complex in muscle fibres after cross-innervation. Expl Neurol. 72, 513-530.
5. Edstrom L. and Kugelberg E. (1968) Histochemical composition, distribution of fibres and fatiguability of single motor units. J. Neurol. Neurosurg. Psych&f. 31, 424433. 6. Hnik P., Jirmanova J., Vyklickjl L. and ZclenP J. (1,967) Fast and slow muscles of the chick after nerve cross-union. J. Physiol., Land. 193, 309-335.
Reinnervation of soleus muscle
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7. Hoh J. F. Y. (1975) Selective and non-selective reinnervation of fast twitch and slow twitch rat skeletal muscle. J. Physiol., Lond. 251, 791801. 8. Gordon T. and Stein R. B. (1980) Rematching of nerve and muscle properties in cat motor units after reinnervation. In Plasticity of Muscle (eds Pette D. and de Guyter W.) 9. Grinnell A. D., Letinsky M. S. and Rheuben M. B. (1979) Competitive interaction between foreign nerves innervating frog skeletal muscle. J. Physiol., Lond. 289, 241-262. 10. Gutmann E. and Young J. 2. (1944) The reinnervation of muscle after various periods of atrophy. J. Anaf. 78, 15-43. 11. Kuno M., Turkanis S. A. and Weakly J. N. (1971) Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog. J. Physiol., Lond. 213, 545-556. 12. Langley J. N. and Anderson H. K. (1904) The union of different kinds of nerve fibres. J. Physiol., Lond. 31, 365-391. 13. Lewis D. M., Rowlerson A. and Webb S. (1982) Motor units and immunohistochemistry of cat soieus muscle after long periods of cross-reinnervation. J. Physiol., Lond. 325, 395-403. 14. Massoulie J. and Bon S. (1982) The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. A. Rev. Neurosci. 5, 57-106. 15. Namba T., Nakamura T. and Grob D. (1967) Staining for nerve fibre and cholinesterase activity in fresh frozen sections, Am. J. clin. Path. 47, 74-77. 16. Nemeth P. M., Pette D. and Vrbova G. (1981) Comparison of enzyme activities among single muscle fibres within defined motor units. J. Physiol., Lond. 311, 489-495.
17. O’Brien R. A. D., Ostberg J. C. and Vrbova G. (1978) Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle. J. Physiol., Lond. 282, 571-582. 18. Romanul F. C. A. and van der Meulen J. P. (1967) Slow and fast muscle after cross-innervation: enzymatic and physiological changes. Archs Neural. Psychiat., Chicago 17, 387402. 19. Vrbova G., Gordon T. and Jones R. (1978) Nerve-Muscle Interaction. Chapman & Hall, London. (Accepted 7 July 1983)