EXPERIMENTAL
NEUROLOGY
Experimental Brachialis
K. TADA,
65, 301-314 (1979)
Study of Spinal Nerve Repair after Plexus Injury in Newborn Rats: A Horseradish Peroxidase Study
S. OHSHITA,
K. YONENOBU, N. SHIMIZU’
K. ONO, K. SATOH,
AND
Department of Orthopaedic Surgery, Osaka University Medical School, Fukushima, Osaka, Japan, 553* and Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Kitaku, Osaka, Japan Received
January
25, 1979
As an animal model of nerve injury at delivery, traction injury was made to the plexus brachialis nerves of newborn rats. By traction, spinal root avulsion or plexus brachialis injury was produced. Horseradish peroxidase (HRP) was injected into the denervated biceps or triceps brachii muscle at various intervals after the experimental nerve injury. Four weeks after the injury, HRP-labeled neurons were identified in the ventral horn of the injected side. In the experimental animals the labeled neurons were found in the ipsilateral ventral horn of the Cq through T, levels regardless of whether the injection was into the denervated biceps or triceps brachii muscle. In the control animals the distribution of the labeled neurons differed depending on the site of injection; labeled neurons were found in the ipsilateral ventral horn of C, through Ca after the injection of HRP into the biceps and in that of C, through C, after injection into the triceps. Within the same level of the cord, the labeled neurons in the experimental animal showed more widespread distribution in the vental horn than those in the control animal. Regeneration of the injured nerves by axonal sprouts from the proximal stumps with subsequent confusion of growth was supported by the present study. In a small number of experimental animals, the labeled neurons were identified not only in the ipsilateral but also in the contralateral ventral horns, suggesting the persistence of an immature mode of innervation of the forelimb muscle by bilateral ventral horn neurons. We also correlated the results of the study and the peculiar clinical findings that follow recovery of the nerve injury during delivery. avulsion, p.b.i.-plexus Abbreviations: HRP-horseradish peroxidase, r.a.-root brachialis injury. 1 Grateful acknowledgment should be given Professor Hideo Namiki, Hawaii University, for his valuable suggestions and kind revision of the manuscript. 301 0014-4886~79/080301-14!$02.00/0 Copyright 0 1979 by Academic Press. Inc. All tights of reproduction in any fom reserved
302
TADA
ET AL.
INTRODUCTION Birth palsy of the upper extremity resulting from a traction injury of the brachial plexus nerves at delivery has been the subject of fairly extensive studies basically from the standpoint of treatment. Precise analysis of degeneration and regeneration of these injured nerves, however, has not yet been made. Birth palsy has two distinct features from other types of nerve injury. First, the nerve is injured in the postnatal developing stage, and second, the cause of injury is traction force during delivery and the lesions consist of either spinal root avulsion or disruption of nerve trunks. The repair process of the nerve lesions in the early stage of life seems to be considerably different from that in adult life. To produce an animal model of human birth palsy, spinal root avulsion and brachial plexus injury were experimentally created in newborn albino rats. After this procedure, we used the horseradish peroxidase (HRP) method (7, 9) to investigate the process of regeneration of the injured nerves at various stages after injury. MATERIALS
AND METHODS
Animal Model ofBirth Palsy. Newborn albino rats, 1 day after delivery, were used for this experiment. A longitudinal incision was made in the left axillary region and the left plexus brachialis was exposed under a dissection microscope and pulled distally with a hook. All nerves innervating the muscles of the left forelimb were severed at various levels according to the direction and strength of the traction force. The severed nerves were left in the wound without an attempt to place them in the original anatomic position. The animals were divided into two groups, root avulsion (r. a.) and plexus brachialis injury (p. b. i.) groups according to the sites of the injury. Avulsion of the spinal root was confirmed by the presence of the dorsal root ganglion at the distal stump and leakage of cerebrospinal fluid (Fig. 1). Observation of voluntary movement of the left forelimb was periodically carried out to evaluate the recovery. The right forelimb was left intact as control. Horseradish Peroxidase Method. Under ether anesthesia, 33% horseradish peroxidase (Type II, Sigma Chemical Co., St. Louis, Missouri) was injected (10 to 15 mg/lOO g body weight) into the left biceps brachii muscle or the left triceps brachii muscle. HRP injected into the muscle spread throughout the muscle belly, which had a brown appearance beneath its fascia. Absence of leakage at the injection site was confirmed under a dissection microscope. Twenty-four hours after the injection the rats were killed by perfusion through the heart with a solution containing 0.5% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer
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(PH 7.2 to 7.4,4”C). The spinal cords from C, through T, were immediately removed and immersed 12 h in the same fixative. The specimens were then rinsed and stored overnight in 0.2 M phosphate buffer with 30% sucrose at 4°C. The spinal cords were frozen and sectioned serially in the frontal plane at 40-pm thickness using a freezing microtome. Sections were reacted according to the procedure of Graham and Karnovsky (5) and examined after staining with cresyl echt violet under a dark-field microscope. Control Study. The topographical localization and quantity of motoneurons innervating the biceps and triceps brachii muscles of the first group of control animals were studied. Ten albino rats (about 200 g body weight) were used. After the injection of HRP in the biceps or triceps brachii, serial frontal sections of the spinal cord were examined after treatment with the horseradish peroxidase technique as described above. For a topographical study, localization of HRP-labeled neurons in the ventral horn of the spinal cord was determined by dark-field microscopy. For the quantitative study, the number of HRP-labeled neurons in 20 randomly selected sections, 800 pm thick, was counted in each spinal segment. In addition, as the second control group a total of 33 albino rats of various ages (10 or 11 days, 2,3,5,8,12,20, or 50 weeks of age) were used for the study of the developmental change in spinal motoneuron innervation to the biceps and triceps brachii muscles. Serial frontal or horizontal sections were examined after treatment with the same methods as the first control group. Nerve Injury Group. Forty-two rats were used for the study of regeneration of axotomized motoneurons. HRP was injected into the biceps or triceps muscle of rats in the p. b. i. and r. a. groups at various intervals after nerve injury (4, 6, 8, or 10 weeks). The spinal cords were treated in the same manner as the controls using the HRP method. The localization of HRP-labeled motoneurons in the spinal cord was studied in each group. For a quantitative study of regenerated motoneurons, the total number of HRP-labeled neurons was counted in 20 sections 800 pm thick in each spinal segment. RESULTS Clinical Findings in Animal Model of Birth Palsy. Three types of clinical manifestations are known in human birth palsy: proximal paralysis (involvement of the 5th and 6th cervical nerves), distal paralysis (involvement of the 8th cervical and 1st thoracic nerves), and total paralysis (15). In the present experiment, all nerves innervating the forelimb muscles were damaged, giving rise to manifestations comparable
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to those of human total paralysis. Voluntary movement of the left forelimb reappeared 4 weeks after the injury. The pattern of recovery was not uniform; recovery of motor function predominated in the proximal part of the limb in some animals and in the wrist and fingers in others. As recovery proceeded, we observed that it was far better in rats with p. b. i. than in r. a. rats. Details of this type of bilateral innervation of the forelimb muscles detected in only newborn rats (less than 2 weeks old) were reported elsewhere (20). Retrograde Transport of HRP in the Control Groups. After injection of HRP into the biceps or triceps brachii muscles, HRP-labeled granules were found in the perikarya of the ipsilateral ventral horn (Fig. 2). In animals with the injection into the biceps brachii, HRP-labeled perikarya were located from the Cq to Cs segments. HRP-labeled motoneurons were distributed predominantly to the dorsolateral, dorsomedial, and ventrolatera1 nuclei at the level of Cq, Cg, and C, respectively (Fig. 3a). HRP-labled motoneurons innervating the triceps brachii muscle were located from the C5 to C8 segments in the ipsilateral ventral horn (Fig. 3b). They were in the dorsolateral nucleus at the Cg, Cc, and C, segments and in the ventrolateral nucleus at the C, segment. The total number of labeled motoneurons seen in the 800-pm-thick sections of each segment is shown in Table 1. HRP-marked neurons in animals with the injection into the biceps brachii were estimated to be 36 at Cd, 54 at Cg, 80 at Cg, 86 at CT, and 31 at the C, level. Those in animals with the injection into the triceps brachii were 6 at Cg, 81 at Cg, 117 at C7, and 20 at the C, level. No labeled neuron was observed in the contralateral ventral horn in animals of the first control group. In summary, the cell column innervating the biceps brachii was found to occupy the dorsolateral nucleus at C, and the ventrolateral nucleus at C, in the ipsilateral ventral horn, and to expand laterally at C, and C,. The cell column innervating the triceps brachii was located from C, to C, with its largest expansion in C,. Whereas in the second control group the same distribution of the labeled motoneurons was observed in the ipsilateral ventral horn as in the first, a marked difference was found in the contralateral ventral horn in this group. In eight of 10 rats 10 and 11 days of age and two of two rats 2 weeks of age, HRP-labeled perikarya were also present in the contralateral ventral horn. No perikarya were labeled in the contralateral ventral horn in animals more than 2 weeks of age. Retrograde Transport of HRP in the Traction Injury Group. HRPlabeled perikarya were found in the ventral horn of the injected side in both p. b. i. and r. a. groups after injection of HRP into the biceps or triceps brachii 4 weeks after injury (Figs. 4a, b). Similar findings were obtained
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FIG. 2. Photomicrograph of the spinal cord under dark-field microscope, x 100. This frontal section is froin the C, level of a control animal with injection of HRP into the biceps brachii muscle (animal 9).
from the 6-, 8-, and IO-week interval cases. The segmental distribution and the localization of the labeled neurons in the nerve injury groups differed from those of the control group (Fig. 4a, b). The labeled neurons were distributed to Cq through T, after HRP injection into both biceps brachii (Table 2a) (animals 74,110, and 210) and triceps brachii (Table 2b) (animals 57 and 61). The topographical study of both types of nerve injury showed HRP-labeled neurons scattered widely in the ventral horn beyond the
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NERVE REPAIR
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c6 CS
c7
C8
C8
FIG. 3. A diagrammatic representation of HRP-labeled neurons in the ventral horn of the control group. a-distribution of the labeled motoneurons is from C, to Ca in the animal with injection of HRP into the biceps brachii muscle. The labeled motoneurons are in the dorsolateral nucleus at Cq, in the dorsomedial nucleus at C, and C6, in the ventromedial nucleus at C,, and in the ventrolateral nucleus at Ce. b-HRP-labeled neurons innervating the triceps bra&ii muscle are present from the C5 to C, segments. They are localized in the dorsolateral nucelus at C,, C,, and C,, and in the ventrolateral nucleus at the C,.
normal range (Figs. 4a, b). The above findings indicated redistribution of motor neurons innervating both biceps and triceps muscles as the result of regeneration of the axotomized nerves. In four experimental rats (animal 57 with p. b. i., and 27,61, and 158 with r. a.), HRP-labeled neurons were found in the bilateral ventral horns. The labeled neurons in both ipsilateral and contralateral sides were large and multipolar. They were located in the anterolateral nucleus (Fig. 5). The number of labeled neurons varied considerably from one animal to another regardless of the type of injury (Table 2), reflecting the fact that
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TADA ET AL. TABLE
1
Neuron Counts in 800~Pm-Thick Spinal Cord Sections of the Control Groups Number of labeled neurons HRP-injected muscle
Animal number
C3
C4
C5
C6
C7
C8
Tl
Biceps brachii
1 5 9 12 13 Average
0 0 14 4 0 4
31 34 49 39 29 36
16 38 85 100 30 54
74 95 105 85 41 80
92 73 120 70 73 86
42 63 22 15 13 31
0 0 10 0 0 2
Triceps brachii
7 11 14 18 19 Average
0 0 0 0 0 0
2 0 2 0 0 1
6 8 2 8 6 6
65 97 96 59 89 81
111 192 123 86 74 117
45 0 16 37 0 20
0 0 0 0 0 0
regeneration of the axotomized neurons was not uniform in the traction injury. Whereas labeled motoneurons were distributed at all segments from C4 to C, in the majority of animals in the p. b. i. group, they were restricted to certain segments in the r. a. group (animals 25, 80,87, 183, and 203). The limitation of labeled neurons in the latter group to particular segments was believed to reflect an incomplete recovery from injury. In both types of nerve injuries, there was a tendency for the labeled neurons to decrease in number proportional to the time interval after the injury. DISCUSSION Traction injury of the brachial plexus nerves at delivery presents a clinical course which is different from that of the adult (6, 15). Although nerve lesions in birth palsy have not been thoroughly investigated, it is well known that better recovery is expected in this type of lesion than in the adult type of similar lesions. It remains unknown whether this is due to the difference in the level of injury, in the neuronal capacity to recover, or other causes. At the junction with the spinal cord, the spinal root is partly composed of glial tissue projecting from the cord and devoid of perineurium. On the other hand, the peripheral nerve is ensheathed by an epineurium and a perineurium composed of resilient collagenous fibrous tissue, and supported by collagenous matrix, an endoneurium. In our experimental
309
SPINAL NERVE REPAIR
CS c6
L!J 0 :* ’ ‘4 w
c7 w 0
C8 CS
Dl a
.a
w
Dl b
cd
ul0 .*
FIG. 4. A diagrammatic representation of HRP-labeled neurons in the ventral horn of the plexus brachiahs injury group. a-the left biceps brachii was injected with HRP 8 weeks after nerve injury (animal 110). HRP-labeled neurons are found from the Cq to T, in the ipsilateral ventral horn and show widespread distribution within the ventral horn of each segment. b-the left triceps brachii was injected with HRP 4 weeks after the nerve injury (animal 57). HRP-labeled neurons were found in the segments identical to those of animals with injection into the biceps brachii. Their distribution within the ventral horn was also widespread.
model of the r. a. group, the spinal roots were disrupted at the junctional zone (Fig. 1) as already stated by Sunderland and Bradley (17,18) in human spinal root injury, whereas nerve lesions were in the brachial plexus in the p. b. i. group.
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TABLE
2
Neuron Counts in the Ipsilateral Horn of Nerve-injured
Group
Number of labeled neurons Animal number
Type of nerve injury”
163 168 198 210 63 65 72 73 74 80 81 110 171 172 182 183 25 26 27c 93 186
p.b.i. r.a. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. r.a. r.a. r.a. r.a. r.a. r.a. p.b.i.
a. Biceps Brachii-injected Group b 4 0 2 12 b 0 0000 * 0 12 1 b 2 1 0 6 b 10 27 8 0 5 8 22 4 30 0 3 0 3 6 51 b 23 100 217 3 4 * 0 8 b 80 200 * b 84 153 208 b 1 2 11 b 6 9 32 tl 1 5 2 b 2 0020 10 0 0 0 9 b 3 0 62 b 65 48 6 b 5 8 41 b 3 6 38
p.b.i. r.a. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. r.a. r.a. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. r.a.
b. Triceps Brachii-injected Group 4 4 32 143 46 0 61 b 28 15 5 b 29 * 19 13 6 35 b 2 27 b 10 b 4 18 b 4 22 8 b 7 10 4 * 4 B 6 17 12 b 0 b 12 5 0 0 0 10 2 0 b 0 0 0
57’ 61’ 199 211 64 75 149 150 158’ 87 111 117 122 123 23 24 28
Weeks after nerve injury
C3
C4
C5
C6
270 208 13 12 84 57 152 59 108 0 18 12 43 1 5 22 0
C7
C8
Tl
10
0
b
49 83 9 20 71 54 54 b 126 110 18 41 36
22 0 2 3 30 14 0 b 26 55 7 2 33
33 40 1 53 74
8 2 1 13 9
234 37 6 72 102 70 133 123 162 2 48 0 16 8 0 38 0
80 16 1 37 9 0 38 6 0 0 11 2 42 0 0 7 0
b
0 45 0 0 0 0 2 b b 12 b b * b
38 20 0 0 0 b 0 0 0 b 0 0 1 0 0 0 0
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SPINAL NERVE REPAIR TABLE
2 (Continued) Number of labeled neurons
Animal number
Type of nerve injury”
92 187 203 204
r.a. p.b.i. r.a. r.a.
Weeks after nerve injury
C3
C4
CS
C6
C7
C8
b b b b
0 2 5 0
I5 16 6 0
0 78 0 0
0 75 0 0
0 7 0 0
Tl 0 b b lJ
a p.b.i.-plexus brachialis injury, r.a.-root avulsion. ’ Labeled neurons could not be counted because of technical errors. c HRP-labeled neurons were found within bilateral ventral horns.
The earlier work on the localization of the motoneurons innervating the forelimb muscles in the rat were based on retrograde degeneration following axotomy of the peripheral nerves. It is, however, generally accepted that the HRP method introduced by Kristenssen et al. (7) is the most accurate technique available today for tracing the cellular origin of the axon terminal networks. When HRP is injected into a muscle, it is taken by coated vesicles of the end plates (25) and transported by motor axons to their perikarya (8- 10). Many authors reported the regeneration of denervated neurons after the sequential degenerative period (2, 19, 23, 24). Regeneration of disrupted nerve fibers after axotomy depends on two major mechanisms: (i) a regenerative process within the soma, namely, reproduction of cellular protein, and (ii) formation of sprouts from the proximal stumps of the disrupted axons with final bridging between sprouts and distal stumps. The latter mechanism is often achieved by collateral sprouting by the neighboring nerve fibers. Many collateral sproutings in regenerating peripheral nerves were reported by Ehrlich and Mark (4) in axolotls, by van Essen and Jansen (22) in leeches, by Bennett and Pettigrew (1) in developing albino rats, and Chen (3) in adult cats. Our data indicated that HRP-labeled neurons can be detected within the ipsilateral ventral horn from the 4th week after nerve injury. The result substantiated the reestablishment of axonal continuity by means of sprouting and recovery of retrograde axonal flow from the nerve ending to the perikarya in the ventral horn. Furthermore, confused reinnervation with subsequent misdirection was confirmed in our experimental study. There was a widely spread distribution of the labeled neurons in the segmental as well as the topographical aspect within the ipsilateral ventral horn. These results indicate that functionally different neurons as well as proper neurons participate in the regeneration of the disrupted nerves which innervate the affected biceps or triceps brachii muscles. Two mechanisms may play a
FIG. 5. Photomicrograph under dark-field microscope, x 50. This frontal section of C, was from an animal with HRP injection into the biceps brachii muscle 10 weeks after the plexus brachialis injury (animal 27). The neurons were not only in the ipsilateral (arrow) but also in the contralateral ventral horn.
SPINAL NERVE REPAIR
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role in the disordered recovery: (i) misdirected sprouting from the proximal end of the injured axon (2) and (ii) collateral sprouting within the spinal cord. The latter mechanism remains to be proven. An interesting finding in the present study was that HRP-labeled neurons were recognized in the contralateral ventral horn in four experimental animals. This fact was interpreted as showing the monosynaptic innervation of the forelimb muscles by both the ipsilateral and contralateral ventral horns subsequent to injury. Innervation of the forelimb muscles by the bilateral ventral horn motor neurons cannot be justified on the basis of a conventional neuroanatomic concept. Our previous paper, however, showed that the forelimb muscles in developing rats were innervated by motoneurons in the bilateral ventral horns (20). The present study seemed to indicate the persistence of an immature mode of bilateral innervation seen in the early stage of life. The route of the cross-innervating motoneuron networks has not yet been fully analyzed. An alternative explanation of the bilateral innervation is that, after injury of the spinal roots and brachial plexus nerves, undamaged motoneurons in the contralateral ventral horn sprout axons via the ventral spinal roots ipsilateral to the injured roots and nerves to the degenerated neuromuscular junction to form new synapses. Raisman (12, 13) demonstrated collateral reinnervation after partial deafferentation of the septal nuclei in the albino rat. Tsukahara (21) and Nakamura et al. (11) reported synaptic reorganization of the red nucleus in the cat. Steward et al. (16) discovered a new projection growing from the contralateral entorhinal cortex to reinnervate the denervated regions in the stratum moleculare of the dentate gyrus after experimental injury of a unilateral entorhinal cortex. Those studies suggest that at least some nerve cells in the central nervous system retain enough plasticity to repair disrupted neuronal connections. The result of our study concerning reinnervation of the denervated forelimb muscles by both ipsilateral and contralateral ventral horn cells might provide an additional example of the plasticity of the spinal neurons in the recovery of nerve injury. REFERENCES M. R., AND A. G. PETTIGREW. 1974. The formation of synapses in reinnervated and cross-reinnervated striated muscle during development. J. Physiol.
1. BENNETT,
(London)
2.
241: 547-573.
H., AND J. Z. YOUNG. 1940. Treatment of gunshot wounds of peripheral nerves. Lancer 27: 123-125. 3. CHEN, D. H. 1978. Qualitative and quantitative study of synaptic displacement in chromatolyzed spinal motoneurons of the cat. J. Camp. Neural. 177: 635-664. 4. EHRLICH, D., AND R. F. MARK. 1977. Fiber counts of regenerating peripheral nerves in axolotls and the effect of metamorphosis. J. Comp. Neurol. 174: 307-316. CAIRUS,
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5. GRAHAM, R. C., AND M. J. KARNOVSKY. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14: 291-302. 6. HARA, T. 1966. Clinical study of the brachial plexus injury. J. Jup. Orthop. Assoc. 39: 959-985. 7. K~USTENSSON,K., Y. OLSSON, AND I. SJ~STRAND. 1971. Axonal uptake and retrograde transport of exogeneous proteins in the hypoglossal nerve. Brain Res. 32: 399-406. 8. LAVAIL, J. H., AND M. M. LAVAIL. 1972. Retrograde axonal transport in the central nervous system. Science 176: 1416-1417. 9. LITCHY, W. J. 1973. Uptake and retrograde transport of horseradish peroxidase in frog sartorius nerve in vitro. Brain Res. 56: 377-381. 10. MIZUNO, N., A. KONISHI, AND M. SATO. 1975. Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase. J. Comp. Neurol. 164: 105-116. 11. NAKAMURA, Y., N. MIZUNO, A. KONISHI, AND M. SATO. 1974. Synaptic reorganization of the red nucleus after chronic deafferentation from cerebellorubral fibers: an electron microscope study in the cat. Brain Res. 82: 298-301. 12. RAISMAN, G. 1969. Neuronal plasticity in the septal nuclei of the adult rat. Brain Res. 14: 25-48. 13. RAISMAN, G., AND M. FIELD. 1973. A quantitative investigation of the development of collateral reinnervation after peripheral deafferentation of the septal nuclei. Bruin Res. 50: 241-264. 14. RAM~N Y CAJAL, S. 1928. Degeneration and Regeneration of the Nervous System. London, Oxford. 15. SEDDON, H. 1972. Surgical Disorders of rhe Peripheral Nerves. Churchill Livingstone, Edinburgh and London. 16. STEWARD, O., C. W. COTMAN, ANDG. S. LYNCH. 1974. Growth of a new fiber projection in the brain of adult rats: reinnervation of the dentate gyrus by the contralateral entorhical cortex following ipsilateral entorhinal lesions. Exp. Brain Res. 20: 45-66. 17. SUNDERLAND, S., AND K. C. BRADLEY. 1961. Stress-strain phenomena in human peripheral nerve trunks. Brain 84: 102- 119. 18. SUNDERLAND, S., AND K. C. BRADLEY. 1961. Stress-strain phenomena in human spinal nerve roots. Brain 84: 120-124. 19. SUNDERLAND, S. 1968. Nerves and Nerve Injuries. Livingstone, London. 20. TADA, K., S. OSHITA, K. YONENOBU, K. ONO, K. SATOH, AND N. SHIMIZU. 1979. Development of spinal motoneuron innervation of the upper limb muscle in the rat. Exp.
Brain
Res. 35: 287-293.
21. TSUKAHARA, N., H. HULTBORN, F. MURAKAMI, AND Y. FUJITO. 1975. Electrophysiological study of formation of new synapses and collateral sprouting in red nucleus neurons after partial denervation. J. Neurophysiol. 38: 1359- 1372. 22. VAN ESSEN, D. C., AND J. K. S. JANSEN. 1977. The specificity of re-innervation by identified sensory and motor neurons in the leech. J. Comp. Neural. 171: 433-454. 23. WEISS, P. 1934. In vitro experiments on the factors determining the outgrowing nerve fibers. J. Exp. Zool. 68: 393-448. 24. WEISS, P., AND A. C. TAYLOR. 1944. Further experimental evidence against “neurotropin” in nerve regeneration. J. Exp. Zoo/. 95: 233-257. 25. ZACKS, S. I., AND A. SAITO. 1969. Uptake of exogenous horseradish peroxidase by coated vesicles in mouse neuromuscular junctions. J. Histochem. Cytochem. II: 161- 170.
NEUROLOGY
Experimental Brachialis
K. TADA,
65, 301-314 (1979)
Study of Spinal Nerve Repair after Plexus Injury in Newborn Rats: A Horseradish Peroxidase Study
S. OHSHITA,
K. YONENOBU, N. SHIMIZU’
K. ONO, K. SATOH,
AND
Department of Orthopaedic Surgery, Osaka University Medical School, Fukushima, Osaka, Japan, 553* and Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Kitaku, Osaka, Japan Received
January
25, 1979
As an animal model of nerve injury at delivery, traction injury was made to the plexus brachialis nerves of newborn rats. By traction, spinal root avulsion or plexus brachialis injury was produced. Horseradish peroxidase (HRP) was injected into the denervated biceps or triceps brachii muscle at various intervals after the experimental nerve injury. Four weeks after the injury, HRP-labeled neurons were identified in the ventral horn of the injected side. In the experimental animals the labeled neurons were found in the ipsilateral ventral horn of the Cq through T, levels regardless of whether the injection was into the denervated biceps or triceps brachii muscle. In the control animals the distribution of the labeled neurons differed depending on the site of injection; labeled neurons were found in the ipsilateral ventral horn of C, through Ca after the injection of HRP into the biceps and in that of C, through C, after injection into the triceps. Within the same level of the cord, the labeled neurons in the experimental animal showed more widespread distribution in the vental horn than those in the control animal. Regeneration of the injured nerves by axonal sprouts from the proximal stumps with subsequent confusion of growth was supported by the present study. In a small number of experimental animals, the labeled neurons were identified not only in the ipsilateral but also in the contralateral ventral horns, suggesting the persistence of an immature mode of innervation of the forelimb muscle by bilateral ventral horn neurons. We also correlated the results of the study and the peculiar clinical findings that follow recovery of the nerve injury during delivery. avulsion, p.b.i.-plexus Abbreviations: HRP-horseradish peroxidase, r.a.-root brachialis injury. 1 Grateful acknowledgment should be given Professor Hideo Namiki, Hawaii University, for his valuable suggestions and kind revision of the manuscript. 301 0014-4886~79/080301-14!$02.00/0 Copyright 0 1979 by Academic Press. Inc. All tights of reproduction in any fom reserved
302
TADA
ET AL.
INTRODUCTION Birth palsy of the upper extremity resulting from a traction injury of the brachial plexus nerves at delivery has been the subject of fairly extensive studies basically from the standpoint of treatment. Precise analysis of degeneration and regeneration of these injured nerves, however, has not yet been made. Birth palsy has two distinct features from other types of nerve injury. First, the nerve is injured in the postnatal developing stage, and second, the cause of injury is traction force during delivery and the lesions consist of either spinal root avulsion or disruption of nerve trunks. The repair process of the nerve lesions in the early stage of life seems to be considerably different from that in adult life. To produce an animal model of human birth palsy, spinal root avulsion and brachial plexus injury were experimentally created in newborn albino rats. After this procedure, we used the horseradish peroxidase (HRP) method (7, 9) to investigate the process of regeneration of the injured nerves at various stages after injury. MATERIALS
AND METHODS
Animal Model ofBirth Palsy. Newborn albino rats, 1 day after delivery, were used for this experiment. A longitudinal incision was made in the left axillary region and the left plexus brachialis was exposed under a dissection microscope and pulled distally with a hook. All nerves innervating the muscles of the left forelimb were severed at various levels according to the direction and strength of the traction force. The severed nerves were left in the wound without an attempt to place them in the original anatomic position. The animals were divided into two groups, root avulsion (r. a.) and plexus brachialis injury (p. b. i.) groups according to the sites of the injury. Avulsion of the spinal root was confirmed by the presence of the dorsal root ganglion at the distal stump and leakage of cerebrospinal fluid (Fig. 1). Observation of voluntary movement of the left forelimb was periodically carried out to evaluate the recovery. The right forelimb was left intact as control. Horseradish Peroxidase Method. Under ether anesthesia, 33% horseradish peroxidase (Type II, Sigma Chemical Co., St. Louis, Missouri) was injected (10 to 15 mg/lOO g body weight) into the left biceps brachii muscle or the left triceps brachii muscle. HRP injected into the muscle spread throughout the muscle belly, which had a brown appearance beneath its fascia. Absence of leakage at the injection site was confirmed under a dissection microscope. Twenty-four hours after the injection the rats were killed by perfusion through the heart with a solution containing 0.5% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer
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(PH 7.2 to 7.4,4”C). The spinal cords from C, through T, were immediately removed and immersed 12 h in the same fixative. The specimens were then rinsed and stored overnight in 0.2 M phosphate buffer with 30% sucrose at 4°C. The spinal cords were frozen and sectioned serially in the frontal plane at 40-pm thickness using a freezing microtome. Sections were reacted according to the procedure of Graham and Karnovsky (5) and examined after staining with cresyl echt violet under a dark-field microscope. Control Study. The topographical localization and quantity of motoneurons innervating the biceps and triceps brachii muscles of the first group of control animals were studied. Ten albino rats (about 200 g body weight) were used. After the injection of HRP in the biceps or triceps brachii, serial frontal sections of the spinal cord were examined after treatment with the horseradish peroxidase technique as described above. For a topographical study, localization of HRP-labeled neurons in the ventral horn of the spinal cord was determined by dark-field microscopy. For the quantitative study, the number of HRP-labeled neurons in 20 randomly selected sections, 800 pm thick, was counted in each spinal segment. In addition, as the second control group a total of 33 albino rats of various ages (10 or 11 days, 2,3,5,8,12,20, or 50 weeks of age) were used for the study of the developmental change in spinal motoneuron innervation to the biceps and triceps brachii muscles. Serial frontal or horizontal sections were examined after treatment with the same methods as the first control group. Nerve Injury Group. Forty-two rats were used for the study of regeneration of axotomized motoneurons. HRP was injected into the biceps or triceps muscle of rats in the p. b. i. and r. a. groups at various intervals after nerve injury (4, 6, 8, or 10 weeks). The spinal cords were treated in the same manner as the controls using the HRP method. The localization of HRP-labeled motoneurons in the spinal cord was studied in each group. For a quantitative study of regenerated motoneurons, the total number of HRP-labeled neurons was counted in 20 sections 800 pm thick in each spinal segment. RESULTS Clinical Findings in Animal Model of Birth Palsy. Three types of clinical manifestations are known in human birth palsy: proximal paralysis (involvement of the 5th and 6th cervical nerves), distal paralysis (involvement of the 8th cervical and 1st thoracic nerves), and total paralysis (15). In the present experiment, all nerves innervating the forelimb muscles were damaged, giving rise to manifestations comparable
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to those of human total paralysis. Voluntary movement of the left forelimb reappeared 4 weeks after the injury. The pattern of recovery was not uniform; recovery of motor function predominated in the proximal part of the limb in some animals and in the wrist and fingers in others. As recovery proceeded, we observed that it was far better in rats with p. b. i. than in r. a. rats. Details of this type of bilateral innervation of the forelimb muscles detected in only newborn rats (less than 2 weeks old) were reported elsewhere (20). Retrograde Transport of HRP in the Control Groups. After injection of HRP into the biceps or triceps brachii muscles, HRP-labeled granules were found in the perikarya of the ipsilateral ventral horn (Fig. 2). In animals with the injection into the biceps brachii, HRP-labeled perikarya were located from the Cq to Cs segments. HRP-labeled motoneurons were distributed predominantly to the dorsolateral, dorsomedial, and ventrolatera1 nuclei at the level of Cq, Cg, and C, respectively (Fig. 3a). HRP-labled motoneurons innervating the triceps brachii muscle were located from the C5 to C8 segments in the ipsilateral ventral horn (Fig. 3b). They were in the dorsolateral nucleus at the Cg, Cc, and C, segments and in the ventrolateral nucleus at the C, segment. The total number of labeled motoneurons seen in the 800-pm-thick sections of each segment is shown in Table 1. HRP-marked neurons in animals with the injection into the biceps brachii were estimated to be 36 at Cd, 54 at Cg, 80 at Cg, 86 at CT, and 31 at the C, level. Those in animals with the injection into the triceps brachii were 6 at Cg, 81 at Cg, 117 at C7, and 20 at the C, level. No labeled neuron was observed in the contralateral ventral horn in animals of the first control group. In summary, the cell column innervating the biceps brachii was found to occupy the dorsolateral nucleus at C, and the ventrolateral nucleus at C, in the ipsilateral ventral horn, and to expand laterally at C, and C,. The cell column innervating the triceps brachii was located from C, to C, with its largest expansion in C,. Whereas in the second control group the same distribution of the labeled motoneurons was observed in the ipsilateral ventral horn as in the first, a marked difference was found in the contralateral ventral horn in this group. In eight of 10 rats 10 and 11 days of age and two of two rats 2 weeks of age, HRP-labeled perikarya were also present in the contralateral ventral horn. No perikarya were labeled in the contralateral ventral horn in animals more than 2 weeks of age. Retrograde Transport of HRP in the Traction Injury Group. HRPlabeled perikarya were found in the ventral horn of the injected side in both p. b. i. and r. a. groups after injection of HRP into the biceps or triceps brachii 4 weeks after injury (Figs. 4a, b). Similar findings were obtained
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FIG. 2. Photomicrograph of the spinal cord under dark-field microscope, x 100. This frontal section is froin the C, level of a control animal with injection of HRP into the biceps brachii muscle (animal 9).
from the 6-, 8-, and IO-week interval cases. The segmental distribution and the localization of the labeled neurons in the nerve injury groups differed from those of the control group (Fig. 4a, b). The labeled neurons were distributed to Cq through T, after HRP injection into both biceps brachii (Table 2a) (animals 74,110, and 210) and triceps brachii (Table 2b) (animals 57 and 61). The topographical study of both types of nerve injury showed HRP-labeled neurons scattered widely in the ventral horn beyond the
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c6 CS
c7
C8
C8
FIG. 3. A diagrammatic representation of HRP-labeled neurons in the ventral horn of the control group. a-distribution of the labeled motoneurons is from C, to Ca in the animal with injection of HRP into the biceps brachii muscle. The labeled motoneurons are in the dorsolateral nucleus at Cq, in the dorsomedial nucleus at C, and C6, in the ventromedial nucleus at C,, and in the ventrolateral nucleus at Ce. b-HRP-labeled neurons innervating the triceps bra&ii muscle are present from the C5 to C, segments. They are localized in the dorsolateral nucelus at C,, C,, and C,, and in the ventrolateral nucleus at the C,.
normal range (Figs. 4a, b). The above findings indicated redistribution of motor neurons innervating both biceps and triceps muscles as the result of regeneration of the axotomized nerves. In four experimental rats (animal 57 with p. b. i., and 27,61, and 158 with r. a.), HRP-labeled neurons were found in the bilateral ventral horns. The labeled neurons in both ipsilateral and contralateral sides were large and multipolar. They were located in the anterolateral nucleus (Fig. 5). The number of labeled neurons varied considerably from one animal to another regardless of the type of injury (Table 2), reflecting the fact that
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1
Neuron Counts in 800~Pm-Thick Spinal Cord Sections of the Control Groups Number of labeled neurons HRP-injected muscle
Animal number
C3
C4
C5
C6
C7
C8
Tl
Biceps brachii
1 5 9 12 13 Average
0 0 14 4 0 4
31 34 49 39 29 36
16 38 85 100 30 54
74 95 105 85 41 80
92 73 120 70 73 86
42 63 22 15 13 31
0 0 10 0 0 2
Triceps brachii
7 11 14 18 19 Average
0 0 0 0 0 0
2 0 2 0 0 1
6 8 2 8 6 6
65 97 96 59 89 81
111 192 123 86 74 117
45 0 16 37 0 20
0 0 0 0 0 0
regeneration of the axotomized neurons was not uniform in the traction injury. Whereas labeled motoneurons were distributed at all segments from C4 to C, in the majority of animals in the p. b. i. group, they were restricted to certain segments in the r. a. group (animals 25, 80,87, 183, and 203). The limitation of labeled neurons in the latter group to particular segments was believed to reflect an incomplete recovery from injury. In both types of nerve injuries, there was a tendency for the labeled neurons to decrease in number proportional to the time interval after the injury. DISCUSSION Traction injury of the brachial plexus nerves at delivery presents a clinical course which is different from that of the adult (6, 15). Although nerve lesions in birth palsy have not been thoroughly investigated, it is well known that better recovery is expected in this type of lesion than in the adult type of similar lesions. It remains unknown whether this is due to the difference in the level of injury, in the neuronal capacity to recover, or other causes. At the junction with the spinal cord, the spinal root is partly composed of glial tissue projecting from the cord and devoid of perineurium. On the other hand, the peripheral nerve is ensheathed by an epineurium and a perineurium composed of resilient collagenous fibrous tissue, and supported by collagenous matrix, an endoneurium. In our experimental
309
SPINAL NERVE REPAIR
CS c6
L!J 0 :* ’ ‘4 w
c7 w 0
C8 CS
Dl a
.a
w
Dl b
cd
ul0 .*
FIG. 4. A diagrammatic representation of HRP-labeled neurons in the ventral horn of the plexus brachiahs injury group. a-the left biceps brachii was injected with HRP 8 weeks after nerve injury (animal 110). HRP-labeled neurons are found from the Cq to T, in the ipsilateral ventral horn and show widespread distribution within the ventral horn of each segment. b-the left triceps brachii was injected with HRP 4 weeks after the nerve injury (animal 57). HRP-labeled neurons were found in the segments identical to those of animals with injection into the biceps brachii. Their distribution within the ventral horn was also widespread.
model of the r. a. group, the spinal roots were disrupted at the junctional zone (Fig. 1) as already stated by Sunderland and Bradley (17,18) in human spinal root injury, whereas nerve lesions were in the brachial plexus in the p. b. i. group.
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TABLE
2
Neuron Counts in the Ipsilateral Horn of Nerve-injured
Group
Number of labeled neurons Animal number
Type of nerve injury”
163 168 198 210 63 65 72 73 74 80 81 110 171 172 182 183 25 26 27c 93 186
p.b.i. r.a. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. r.a. r.a. r.a. r.a. r.a. r.a. p.b.i.
a. Biceps Brachii-injected Group b 4 0 2 12 b 0 0000 * 0 12 1 b 2 1 0 6 b 10 27 8 0 5 8 22 4 30 0 3 0 3 6 51 b 23 100 217 3 4 * 0 8 b 80 200 * b 84 153 208 b 1 2 11 b 6 9 32 tl 1 5 2 b 2 0020 10 0 0 0 9 b 3 0 62 b 65 48 6 b 5 8 41 b 3 6 38
p.b.i. r.a. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. r.a. r.a. p.b.i. r.a. p.b.i. p.b.i. p.b.i. p.b.i. r.a.
b. Triceps Brachii-injected Group 4 4 32 143 46 0 61 b 28 15 5 b 29 * 19 13 6 35 b 2 27 b 10 b 4 18 b 4 22 8 b 7 10 4 * 4 B 6 17 12 b 0 b 12 5 0 0 0 10 2 0 b 0 0 0
57’ 61’ 199 211 64 75 149 150 158’ 87 111 117 122 123 23 24 28
Weeks after nerve injury
C3
C4
C5
C6
270 208 13 12 84 57 152 59 108 0 18 12 43 1 5 22 0
C7
C8
Tl
10
0
b
49 83 9 20 71 54 54 b 126 110 18 41 36
22 0 2 3 30 14 0 b 26 55 7 2 33
33 40 1 53 74
8 2 1 13 9
234 37 6 72 102 70 133 123 162 2 48 0 16 8 0 38 0
80 16 1 37 9 0 38 6 0 0 11 2 42 0 0 7 0
b
0 45 0 0 0 0 2 b b 12 b b * b
38 20 0 0 0 b 0 0 0 b 0 0 1 0 0 0 0
311
SPINAL NERVE REPAIR TABLE
2 (Continued) Number of labeled neurons
Animal number
Type of nerve injury”
92 187 203 204
r.a. p.b.i. r.a. r.a.
Weeks after nerve injury
C3
C4
CS
C6
C7
C8
b b b b
0 2 5 0
I5 16 6 0
0 78 0 0
0 75 0 0
0 7 0 0
Tl 0 b b lJ
a p.b.i.-plexus brachialis injury, r.a.-root avulsion. ’ Labeled neurons could not be counted because of technical errors. c HRP-labeled neurons were found within bilateral ventral horns.
The earlier work on the localization of the motoneurons innervating the forelimb muscles in the rat were based on retrograde degeneration following axotomy of the peripheral nerves. It is, however, generally accepted that the HRP method introduced by Kristenssen et al. (7) is the most accurate technique available today for tracing the cellular origin of the axon terminal networks. When HRP is injected into a muscle, it is taken by coated vesicles of the end plates (25) and transported by motor axons to their perikarya (8- 10). Many authors reported the regeneration of denervated neurons after the sequential degenerative period (2, 19, 23, 24). Regeneration of disrupted nerve fibers after axotomy depends on two major mechanisms: (i) a regenerative process within the soma, namely, reproduction of cellular protein, and (ii) formation of sprouts from the proximal stumps of the disrupted axons with final bridging between sprouts and distal stumps. The latter mechanism is often achieved by collateral sprouting by the neighboring nerve fibers. Many collateral sproutings in regenerating peripheral nerves were reported by Ehrlich and Mark (4) in axolotls, by van Essen and Jansen (22) in leeches, by Bennett and Pettigrew (1) in developing albino rats, and Chen (3) in adult cats. Our data indicated that HRP-labeled neurons can be detected within the ipsilateral ventral horn from the 4th week after nerve injury. The result substantiated the reestablishment of axonal continuity by means of sprouting and recovery of retrograde axonal flow from the nerve ending to the perikarya in the ventral horn. Furthermore, confused reinnervation with subsequent misdirection was confirmed in our experimental study. There was a widely spread distribution of the labeled neurons in the segmental as well as the topographical aspect within the ipsilateral ventral horn. These results indicate that functionally different neurons as well as proper neurons participate in the regeneration of the disrupted nerves which innervate the affected biceps or triceps brachii muscles. Two mechanisms may play a
FIG. 5. Photomicrograph under dark-field microscope, x 50. This frontal section of C, was from an animal with HRP injection into the biceps brachii muscle 10 weeks after the plexus brachialis injury (animal 27). The neurons were not only in the ipsilateral (arrow) but also in the contralateral ventral horn.
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role in the disordered recovery: (i) misdirected sprouting from the proximal end of the injured axon (2) and (ii) collateral sprouting within the spinal cord. The latter mechanism remains to be proven. An interesting finding in the present study was that HRP-labeled neurons were recognized in the contralateral ventral horn in four experimental animals. This fact was interpreted as showing the monosynaptic innervation of the forelimb muscles by both the ipsilateral and contralateral ventral horns subsequent to injury. Innervation of the forelimb muscles by the bilateral ventral horn motor neurons cannot be justified on the basis of a conventional neuroanatomic concept. Our previous paper, however, showed that the forelimb muscles in developing rats were innervated by motoneurons in the bilateral ventral horns (20). The present study seemed to indicate the persistence of an immature mode of bilateral innervation seen in the early stage of life. The route of the cross-innervating motoneuron networks has not yet been fully analyzed. An alternative explanation of the bilateral innervation is that, after injury of the spinal roots and brachial plexus nerves, undamaged motoneurons in the contralateral ventral horn sprout axons via the ventral spinal roots ipsilateral to the injured roots and nerves to the degenerated neuromuscular junction to form new synapses. Raisman (12, 13) demonstrated collateral reinnervation after partial deafferentation of the septal nuclei in the albino rat. Tsukahara (21) and Nakamura et al. (11) reported synaptic reorganization of the red nucleus in the cat. Steward et al. (16) discovered a new projection growing from the contralateral entorhinal cortex to reinnervate the denervated regions in the stratum moleculare of the dentate gyrus after experimental injury of a unilateral entorhinal cortex. Those studies suggest that at least some nerve cells in the central nervous system retain enough plasticity to repair disrupted neuronal connections. The result of our study concerning reinnervation of the denervated forelimb muscles by both ipsilateral and contralateral ventral horn cells might provide an additional example of the plasticity of the spinal neurons in the recovery of nerve injury. REFERENCES M. R., AND A. G. PETTIGREW. 1974. The formation of synapses in reinnervated and cross-reinnervated striated muscle during development. J. Physiol.
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21. TSUKAHARA, N., H. HULTBORN, F. MURAKAMI, AND Y. FUJITO. 1975. Electrophysiological study of formation of new synapses and collateral sprouting in red nucleus neurons after partial denervation. J. Neurophysiol. 38: 1359- 1372. 22. VAN ESSEN, D. C., AND J. K. S. JANSEN. 1977. The specificity of re-innervation by identified sensory and motor neurons in the leech. J. Comp. Neural. 171: 433-454. 23. WEISS, P. 1934. In vitro experiments on the factors determining the outgrowing nerve fibers. J. Exp. Zool. 68: 393-448. 24. WEISS, P., AND A. C. TAYLOR. 1944. Further experimental evidence against “neurotropin” in nerve regeneration. J. Exp. Zoo/. 95: 233-257. 25. ZACKS, S. I., AND A. SAITO. 1969. Uptake of exogenous horseradish peroxidase by coated vesicles in mouse neuromuscular junctions. J. Histochem. Cytochem. II: 161- 170.
