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The Regenerative Capacity of Nerves Affected by Fowl Paralysis
Brit. vet. J. (1965),121,278
THE REGENERATIVE CAPACITY OF NERVES AFFECTED BY FOWL PARALYSIS By P. A. L. Wight Agricultural Research Council, Poultry Research Centre, Edinburgh
=============s U M MAR Y ============= Regenerating neurites occurred in both normal and fowl-paralysis-affected sciatic nerves of the domestic fowl 2 I and 3 I days after experimental neurotmesis. It is concluded that many neurites in the diseased nerves possess the capacity to regenerate. INTRODUCTION
Degeneration ofaxons in peripheral nerves affected by fowl paralysis (Marek, 1907) has been recorded by several workers (Pappenheimer, Dunn & Cone, 1929; Seagar, 1933; Lerche & Fritzsche, 1933; Wight, I962a). However, Wight (1962b) found that, despite severe involvement of their associated peripheral nerves, routine histological methods rarely revealed morphological changes in the central neurones. Accordingly, an analysis of the numbers of axons in peripheral nerves was undertaken (Wight, 1964) by which it was confirmed that a reduction in the number ofaxons may occur in the disease. If, however, the central neurones remain intact, the question arises whether their axons in the affected nerve have the capacity to regenerate. The feasibility of spontaneous or therapeutically assisted recovery is also dependent on this regenerative capacity. The present communication records an investigation of this problem. MATERIAL AND METHODS
Brown Leghorns affected by unilateral hind-limb paralysis which clinically resembled spontaneous fowl paralysis were obtained from the closed flock at the Poultry Research Centre. Fowl paralysis is endemic in this flock (Wight, 1963). All were females aged between three and five months. Control birds were either sibs of the same sex and age, or, if this was not possible, of the same sex, age and genetic line. The birds can be divided into three experimental groups: Group 1. Six fowl-paralysis-affected birds and six controls were anaesthetized with intravenous Nembutal (sodium pentobarbitone) and the sciatic nerve of one limb was exposed by incising the skin on the inside of the thigh and retracting the adductor muscle. A piece of sciatic nerve 2 mm. in length was excised just distal to the transit of the deep femoral vein. The skin incision was then sutured. The extracted piece of nerve was examined histologically to find out whether
REGENERATION OF NERVES IN FOWL PARALYSIS
279
it was affected with fowl paralysis and to assess the severity of the involvement. Twenty~one days later the birds were killed and their sciatic nerves removed, spread on card and fixed in IO per cent formol-saline. After IO days the nerve was cut into three pieces, each I cm. in length, one piece being the neurectomized region and the other two being proximal and distal to this. These pieces were then embedded in paraffin and successive serial sections, 7 f.J, thick, were tained with haematoxylin and eosin, trichrome, and Holmes' silver method. Group II. A further four affected and four control birds were treated as above but were not killed until 3 I days after the operation. Their transected nerves were removed and processed exactly as in the preceding cases. Group III. Four affected and two control birds were anaesthetized and a segment of sciatic nerve removed as previously described . The cut ends of the nerve were then sutured into a sterile, 1'5 cm. long, non-toxic surgical vinyl tube of 3 mm. bore, in the centre of which was a plug of polyvinyl alcohol sponge (Prosthex; Ramer Chemical Co., Ltd., England) 0 '5 cm. wide. This tubing was held in place by two sutures passing through the underlying muscles. The adductor muscle was pulled over the tubing and the skin sutured. After 3 I days the birds were killed, the nerve removed and the vinyl tube cut off. The nerve, together with the central sponge, was processed and sections cut and stained as previously. RESULTS
Group I. Histological examination of sections of control nerves from the proximal side of the transection revealed moderate oedema separating the neurites into bundles. Schwann cells were proliferated and Holmes' method showed that axons of both small and large diameter were present. Near the site of transection, the oedema increased and contained shreds of fibrin. The number of neurites per histological section appeared reduced and each was separated from its neighbour by oedema. Schwann cells were proliferated forming distinct Bungner's bands. Some digestion chambers containing debris and phagocytes were present. Holmes' method now showed that some neurites contained argentophilic debris, fragmented or swollen, vacuolated and jagged-edged axons, while others contained one or several fine, regenerating axon sprouts. Close to the transection there were no digestion chambers, only fine regenerating axons being present both in and outside Bungner's bands. The piece of nerve removed at the original operation had been replaced by fibrous granulation tissue. Adjacent to the proximal end of the nerve the fibrous tissue formed a densely cross-woven wall, but in the greater part of the granulation tissue band fibroblasts were aligned in the direction of the long axis of the nerve. The epineurium was thickened in this region and it blended into the fibrous granulation tissue to form a swollen cap fitting over the oedematous end of the nerve. Biingner's bands approached the fibrous wall, some being deflected parallel
BRITISH VETERINARY JOURNAL,
121 ,6
to it, and only a few actually entering the scar tissue. However, Holmes' method showed that, although some fine regenerating axon sprouts were also deflected by the granulation tissue, others penetrated between the connective tissue strands and made their way distally for varying distances. None was seen to traverse the entire granulation tissue bridge. Distal to the neurectomy, the nerve trunk was of reduced diameter and consisted essentially of shrunken neurite sheaths with very numerous proliferated Schwann cells. There were some small vacuolations of these neurites, probably representing old digestion chambers. Axons were absent. Essentially the same regenerative processes appeared to be taking place in the fowl-paralysis-affected nerves after the same post-neurectomy interval (Fig. I), the main morphological difference being the presence of infiltrating cells of haematogenous origin. In the proximal piece of nerve, these cells were similar in number and type to those in the piece removed at the original ·neurectomy. Near the transection they became less numerous, and they were infrequent distal to the neurectomy. In examples of those fowl-paralysis-affected nerves classed by Wight (Ig62a) as Type III and severe Type I, both of which are characterized by very great numbers of infiltrating cells, the number of Biingner's bands and regenerating axons was less than in the control nerves. However, even in these severe cases, fine, regenerating axons were seen. Group II. Thirty-one days after neurectomy, regeneration was more advanced in both control and fowl-paralysis-affected nerves. Just proximal to the severed ends regenerative activity was intense, numerous bundles of fine axon sprouts, tangled skeins and whirls of fine axons and end bulbs being seen (Fig. 2). In both control and fowl-paralysis-affected birds Biingner's bands, often containing several fine axons, had penetrated a considerable way across the granulation tissue bridge, while some axons had traversed the entire distance and were beginning to neurotize the chains of Schwann cells of the distal nerve. There was a noticeable increase in the amount of collagen in the endoneurium. Group III. Macroscopically, the vinyl alcohol sponge appeared incorporated in the granulation tissue uniting the severed ends of the nerves, but in fact only its periphery was invaded by fibrovascular tissue. In consequence, it had proved a barrier to the regenerating axons, and in both control and affected birds Biingner's bands and regenerating axons had penetrated only the interstices at the proximal face of the sponge.
DISCUSSION
These results show that fowl-paralysis-affected nerves possess the capacity for regeneration. Both axon and neurilemmal regeneration occurred and in the distal section the Schwann cells proliferated and preserved the architecture of the nerve for the regenerating fibres. Morphologically there was little difference between regeneration in the diseased and apparently healthy control nerves; classical regenerative phenomena such as fine axon sprouting,
PLATE [
Fig. I. Fine regene rating axons approaching th e connective tissue scar from th e proximal sid e o f a fowl-paral ysis-a ffected nerve (at the left ). They a re at first defl ected but subsequently adopl a course parallel lo th e long a xis of the ne rve. 2 I days afler n eurectomy. Holm es' m ethod. x 550.
Wight, Br. vet. J.,
I:U,
6
PLATE II
.~~
.~~~
Fig.
2.
.-:J
Fin e axon sprouts forming end bulbs and tangled whirls in th e proximal piece of a fowl-paral ysisaffected n erve . 3 1 days after neurectomy. Holmes' m ethod. x 250.
Wight, Br. vet. ]. ,
121,
6
REGENERATION OF NERVES IN FOWL PARALYSIS
spirals of Perroncito, proliferation of neurilemmal cells, and Bungner's band formation, were seen in both. The results do not t ell us whether individual diseased neurites can regenerate, but merely that, in the nerve trunk as a whole, some fibres can do so. It would be difficult to devise a method which would resolve this question: the number of n eurites in regenerating nerves of mammals may be increased by 200 per cent (Thomas & Davenport, 1949), and maturation is not obtained until connection is made with the periphery (Sanders & Young, 1946) so that quantitative estimates would be technically complex. However, reinnervation by axon branching can compensate for loss of a proportion of netirones and their fibres in mammals. In practice, therefore, the results of these experiments suggest that, if the progress of the disease could be terminated spontaneously or by therapeutic measures, the regenerative capacity displayed in all but the severest cases of fowl paralysis may be sufficient to restore function. These findings also signify, in confirmation of previous histological investigations (Wight, 1962b), that at least some neurones in the central nervous system must be viable. REFEREN CES
LERCHE, & FRITZSCHE, K. (1933). Z. InfektKrankh. parasit. Krankh. Hyg. Haustiere, 45. 89· MAREK, J. (1907). Dt. tieriir:<;tl. Wschr., 15. 417. PAPPENHEIMER, A. M., DUNN, L. C. & CONE, V. (1929). J. expo Med., 49. 63. SANDERS, F. K. & YOUNG, J. Z. (1946). J. expo Biol., 22. 203 . SEAGAR, E. A. (1933). Vet. J., 89. 454· THOMAS, R. W. & DAVENPORT, H. A. (1949). Q.. Bull. NWest. Univ . med. Sch., 23. 170. WIGHT, P. A. L. (1962a) J. compo Path. Ther., 72. 40. WIGHT, P. A. L. (1962b). J . compo Path. Ther., 72. 348. WIGHT, P. A. L. (1963)' Vet. Rec., 75. 685. WIGHT, P. A. L. (1964). Res. vet. Sci. , 5. 46. (R eceived for publication 28 J anuary, 1965 )
THE REGENERATIVE CAPACITY OF NERVES AFFECTED BY FOWL PARALYSIS By P. A. L. Wight Agricultural Research Council, Poultry Research Centre, Edinburgh
=============s U M MAR Y ============= Regenerating neurites occurred in both normal and fowl-paralysis-affected sciatic nerves of the domestic fowl 2 I and 3 I days after experimental neurotmesis. It is concluded that many neurites in the diseased nerves possess the capacity to regenerate. INTRODUCTION
Degeneration ofaxons in peripheral nerves affected by fowl paralysis (Marek, 1907) has been recorded by several workers (Pappenheimer, Dunn & Cone, 1929; Seagar, 1933; Lerche & Fritzsche, 1933; Wight, I962a). However, Wight (1962b) found that, despite severe involvement of their associated peripheral nerves, routine histological methods rarely revealed morphological changes in the central neurones. Accordingly, an analysis of the numbers of axons in peripheral nerves was undertaken (Wight, 1964) by which it was confirmed that a reduction in the number ofaxons may occur in the disease. If, however, the central neurones remain intact, the question arises whether their axons in the affected nerve have the capacity to regenerate. The feasibility of spontaneous or therapeutically assisted recovery is also dependent on this regenerative capacity. The present communication records an investigation of this problem. MATERIAL AND METHODS
Brown Leghorns affected by unilateral hind-limb paralysis which clinically resembled spontaneous fowl paralysis were obtained from the closed flock at the Poultry Research Centre. Fowl paralysis is endemic in this flock (Wight, 1963). All were females aged between three and five months. Control birds were either sibs of the same sex and age, or, if this was not possible, of the same sex, age and genetic line. The birds can be divided into three experimental groups: Group 1. Six fowl-paralysis-affected birds and six controls were anaesthetized with intravenous Nembutal (sodium pentobarbitone) and the sciatic nerve of one limb was exposed by incising the skin on the inside of the thigh and retracting the adductor muscle. A piece of sciatic nerve 2 mm. in length was excised just distal to the transit of the deep femoral vein. The skin incision was then sutured. The extracted piece of nerve was examined histologically to find out whether
REGENERATION OF NERVES IN FOWL PARALYSIS
279
it was affected with fowl paralysis and to assess the severity of the involvement. Twenty~one days later the birds were killed and their sciatic nerves removed, spread on card and fixed in IO per cent formol-saline. After IO days the nerve was cut into three pieces, each I cm. in length, one piece being the neurectomized region and the other two being proximal and distal to this. These pieces were then embedded in paraffin and successive serial sections, 7 f.J, thick, were tained with haematoxylin and eosin, trichrome, and Holmes' silver method. Group II. A further four affected and four control birds were treated as above but were not killed until 3 I days after the operation. Their transected nerves were removed and processed exactly as in the preceding cases. Group III. Four affected and two control birds were anaesthetized and a segment of sciatic nerve removed as previously described . The cut ends of the nerve were then sutured into a sterile, 1'5 cm. long, non-toxic surgical vinyl tube of 3 mm. bore, in the centre of which was a plug of polyvinyl alcohol sponge (Prosthex; Ramer Chemical Co., Ltd., England) 0 '5 cm. wide. This tubing was held in place by two sutures passing through the underlying muscles. The adductor muscle was pulled over the tubing and the skin sutured. After 3 I days the birds were killed, the nerve removed and the vinyl tube cut off. The nerve, together with the central sponge, was processed and sections cut and stained as previously. RESULTS
Group I. Histological examination of sections of control nerves from the proximal side of the transection revealed moderate oedema separating the neurites into bundles. Schwann cells were proliferated and Holmes' method showed that axons of both small and large diameter were present. Near the site of transection, the oedema increased and contained shreds of fibrin. The number of neurites per histological section appeared reduced and each was separated from its neighbour by oedema. Schwann cells were proliferated forming distinct Bungner's bands. Some digestion chambers containing debris and phagocytes were present. Holmes' method now showed that some neurites contained argentophilic debris, fragmented or swollen, vacuolated and jagged-edged axons, while others contained one or several fine, regenerating axon sprouts. Close to the transection there were no digestion chambers, only fine regenerating axons being present both in and outside Bungner's bands. The piece of nerve removed at the original operation had been replaced by fibrous granulation tissue. Adjacent to the proximal end of the nerve the fibrous tissue formed a densely cross-woven wall, but in the greater part of the granulation tissue band fibroblasts were aligned in the direction of the long axis of the nerve. The epineurium was thickened in this region and it blended into the fibrous granulation tissue to form a swollen cap fitting over the oedematous end of the nerve. Biingner's bands approached the fibrous wall, some being deflected parallel
BRITISH VETERINARY JOURNAL,
121 ,6
to it, and only a few actually entering the scar tissue. However, Holmes' method showed that, although some fine regenerating axon sprouts were also deflected by the granulation tissue, others penetrated between the connective tissue strands and made their way distally for varying distances. None was seen to traverse the entire granulation tissue bridge. Distal to the neurectomy, the nerve trunk was of reduced diameter and consisted essentially of shrunken neurite sheaths with very numerous proliferated Schwann cells. There were some small vacuolations of these neurites, probably representing old digestion chambers. Axons were absent. Essentially the same regenerative processes appeared to be taking place in the fowl-paralysis-affected nerves after the same post-neurectomy interval (Fig. I), the main morphological difference being the presence of infiltrating cells of haematogenous origin. In the proximal piece of nerve, these cells were similar in number and type to those in the piece removed at the original ·neurectomy. Near the transection they became less numerous, and they were infrequent distal to the neurectomy. In examples of those fowl-paralysis-affected nerves classed by Wight (Ig62a) as Type III and severe Type I, both of which are characterized by very great numbers of infiltrating cells, the number of Biingner's bands and regenerating axons was less than in the control nerves. However, even in these severe cases, fine, regenerating axons were seen. Group II. Thirty-one days after neurectomy, regeneration was more advanced in both control and fowl-paralysis-affected nerves. Just proximal to the severed ends regenerative activity was intense, numerous bundles of fine axon sprouts, tangled skeins and whirls of fine axons and end bulbs being seen (Fig. 2). In both control and fowl-paralysis-affected birds Biingner's bands, often containing several fine axons, had penetrated a considerable way across the granulation tissue bridge, while some axons had traversed the entire distance and were beginning to neurotize the chains of Schwann cells of the distal nerve. There was a noticeable increase in the amount of collagen in the endoneurium. Group III. Macroscopically, the vinyl alcohol sponge appeared incorporated in the granulation tissue uniting the severed ends of the nerves, but in fact only its periphery was invaded by fibrovascular tissue. In consequence, it had proved a barrier to the regenerating axons, and in both control and affected birds Biingner's bands and regenerating axons had penetrated only the interstices at the proximal face of the sponge.
DISCUSSION
These results show that fowl-paralysis-affected nerves possess the capacity for regeneration. Both axon and neurilemmal regeneration occurred and in the distal section the Schwann cells proliferated and preserved the architecture of the nerve for the regenerating fibres. Morphologically there was little difference between regeneration in the diseased and apparently healthy control nerves; classical regenerative phenomena such as fine axon sprouting,
PLATE [
Fig. I. Fine regene rating axons approaching th e connective tissue scar from th e proximal sid e o f a fowl-paral ysis-a ffected nerve (at the left ). They a re at first defl ected but subsequently adopl a course parallel lo th e long a xis of the ne rve. 2 I days afler n eurectomy. Holm es' m ethod. x 550.
Wight, Br. vet. J.,
I:U,
6
PLATE II
.~~
.~~~
Fig.
2.
.-:J
Fin e axon sprouts forming end bulbs and tangled whirls in th e proximal piece of a fowl-paral ysisaffected n erve . 3 1 days after neurectomy. Holmes' m ethod. x 250.
Wight, Br. vet. ]. ,
121,
6
REGENERATION OF NERVES IN FOWL PARALYSIS
spirals of Perroncito, proliferation of neurilemmal cells, and Bungner's band formation, were seen in both. The results do not t ell us whether individual diseased neurites can regenerate, but merely that, in the nerve trunk as a whole, some fibres can do so. It would be difficult to devise a method which would resolve this question: the number of n eurites in regenerating nerves of mammals may be increased by 200 per cent (Thomas & Davenport, 1949), and maturation is not obtained until connection is made with the periphery (Sanders & Young, 1946) so that quantitative estimates would be technically complex. However, reinnervation by axon branching can compensate for loss of a proportion of netirones and their fibres in mammals. In practice, therefore, the results of these experiments suggest that, if the progress of the disease could be terminated spontaneously or by therapeutic measures, the regenerative capacity displayed in all but the severest cases of fowl paralysis may be sufficient to restore function. These findings also signify, in confirmation of previous histological investigations (Wight, 1962b), that at least some neurones in the central nervous system must be viable. REFEREN CES
LERCHE, & FRITZSCHE, K. (1933). Z. InfektKrankh. parasit. Krankh. Hyg. Haustiere, 45. 89· MAREK, J. (1907). Dt. tieriir:<;tl. Wschr., 15. 417. PAPPENHEIMER, A. M., DUNN, L. C. & CONE, V. (1929). J. expo Med., 49. 63. SANDERS, F. K. & YOUNG, J. Z. (1946). J. expo Biol., 22. 203 . SEAGAR, E. A. (1933). Vet. J., 89. 454· THOMAS, R. W. & DAVENPORT, H. A. (1949). Q.. Bull. NWest. Univ . med. Sch., 23. 170. WIGHT, P. A. L. (1962a) J. compo Path. Ther., 72. 40. WIGHT, P. A. L. (1962b). J . compo Path. Ther., 72. 348. WIGHT, P. A. L. (1963)' Vet. Rec., 75. 685. WIGHT, P. A. L. (1964). Res. vet. Sci. , 5. 46. (R eceived for publication 28 J anuary, 1965 )