- Email: [email protected]
Peripheral nerve fibres regenerate through myenteric plexus
.¥euro.wiem'e l.elter.s'. 76 (1987) 129 132 Elsevier Scientific Publishers Ireland Ltd.
129
NSL 04534
Peripheral nerve fibres regenerate through myenteric plexus P . N . A n d e r s o n a n d M. T u r m a i n e I)~7~arttn('nl ol'.411atonly and Biolog, y as Applied to Medieim'. The MMdh'se.v Hospital Medical .S'ehool. Lond~m ( U.K. (Received 4 November 1986: Revised version received 12 December 1986: Accepted 15 I)eccmbcr 1986) Kcr words:
Axonal regeneration: Axotom,,: Enteric glia: Myenteric plexus: Nerve graft: Pcripheral nerve: Mouse
The abilit? of myenteric gila and neurons to support peripheral nerve regeneration was tested b~ grafling pieces of muscularis externa 5 m m long from the dislal colon of inbred CBA mice adjacent to lfic proximal stump of cut c o m m o n peroneal nerves. By two weeks after operation many axons had invaded the plexus and after 3 weeks regeneration c o m m o n peroneal nerve fibres could be identilied in all parts of the plexus throughout the grafts. Some axonal profiles within the plexus appeared to bc m lhc early stages of myelmation by enteric glia. Axons surrounded by compact myelin were found at the pcriphcr? of ganglia, but the cells involved resembled Schwann cells and could not be positively identified as enteric glia. Profiles similar to those of regenerating axons were only very rarely seen in control experiments m which grafts were placed adjacent to intact c o m m o n peroncal nerves. It is suggested that tfie cellular clcnlents of Ifie myemeric plexus can support peripheral nerve regeneration.
Regenerating peripheral nerve fibres are almost always found in contact with Schwann cells [2]. This is even the case when the axons are regenerating through grafts which were initially acellular [2 4, 7], although evidence that some parts of such axons may be naked has been presented [10]. In situations where Schwann cells are not available axonal regeneration is poor [8]. CNS gila do not support regeneration of PNS fibres beyond about 1 mm [1, 4, 13]. Enteric glial cells are derived, like Schwann cells, from neural crest but have some properties in c o m m o n with CNS glia [11]. Little is known about the ability of enteric gila to support the regeneration ot" axons, although it has been shown that adrenergic fibres will regenerate into the myenteric plexus after extrinsic denervation in vivo [9]. However, non-myelinated monoaminergic axons have unusual powers of regeneration even in the CNS [5]. Grafts of myenteric plexus were therefore placed in the vicinity of injured peripheral nerve fibres with a known capacity For regeneration. Adult female inbred CBA mice were used throughout. Segments of distal colon
('orre.vmmh'nee. P.N. Anderson, Department of Anatomy and Biology as Applied to Medicin,.'. Tfic Middlesex tfospital Medical School, London W IP 6DB, U.K.
130
were removed t¥om donor animals killed with an overdose of ether vapour. T'he muscularis externa, containing the myenteric plexus, was dissected free from the mucous membrane and washed in several changes of sterile Hanks' balanced salt solution (Gibco). Segments about 6 mm long were removed from the left common peroneal nerves (LCPN) of host animals under ether anaesthesia. Pieces of muscularis externa about 5 mm long were rolled into a cylindrical shape and placed with their cut ends adjacent to the proximal stump of the LCPN. In control experiments pieces of muscularis externa were placed adjacent to intact LCPN. The animals were perfused through the left ventricle of the heart under deep anaesthesia 1 8 weeks after operation. The fixative contained 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer pH 7.4. The grafts were cut into l-ram segments, osmicated and processed into araldite. Ultrathin sections were examined using a Philips EM300 electron microscope. Grafts placed ad/acent to cut nerves ( 10 gra/ts). One week alter operation the proximal stump of the injured nerve contained swollen axons and new axonal sprouts, but few regenerating axons had entered the grafts. The myenteric plexus contained apparently healthy enteric glia and neurons. Two weeks after operation many axons had entered the grafts, but they were most prominent in the proximal 2 ram. Some regenerating axons were seen in the small branches of the myenteric plexus which innervate the muscle layer. Such axons had the appearence of growth cones or appeared as large electron-lucent profiles in small bundles of fibres which also contained varicosities typical of autonomic neuromuscular junctions. Regenerating LCPN fibres could also be identified in the ganglia of the plexus in contact with myenteric glia and neurons (Fig. 1). They were larger and more electron lucent that the neurites intrinsic to the plexus. Growth cones were present in the ganglia. Some axons were wrapped by complex or multiple mesaxons which produced an appearence resembling the promyelin stage of myelination [6]. Fenestrated capillaries were occassionally identified in the tissue surrounding the ganglia. Three to 8 weeks after operation increasing numbers of nerve fibres could be identified growing through the grafts. Some axonal profiles were abnormal, being up to 6/~m diameter and largely packed with neurofilaments. Most large electron-lucent profiles within ganglia were typical of normal regenerating PNS axons, however. Myelinated axons were found associated with the myenteric ganglia: most profiles were simply very close to the ganglia, being separated by two layers of basal lamina. Some myelinated axons, however, were seen to extend into the ganglia, the myelin sheath terminating at the periphery of the ganglia with the axons passing into the neuropil as large electron-lucent profiles. It is unlikely that such axons were postganglionic sympathetic. No myelinated internodal axons were seen surrounded by the neuropil of the ganglia. Some regenerating fibres did not grow through the preexisting plexus but formed apparently new routes through the muscle layers. These took the form of minifascicles: small compartments of axons and Schwann cells surrounded by perineurial cells. GraJ?s placed adjacent to intact nerves (5 grafts). Grafts examined 2, 3 and 8 weeks after operation remained viable, but the myenteric plexus was almost entirely devoid
131
Fig. 1. Two ~ecks after grafting, a large rcgeneralmg axon (A), surrounded by enleric glial processes (arrox~s), may bc sccn wi/hin a mycnteric ganglion. The regenerating axon may be compared ~ith the intrinsic mycntcric ncurilcs which form the ad,iacent neuropil. Nolo the basal lamina ~.~fIhc ganglion (arrowheads). Bar 0.5/ml.
of large electron-lucent profiles. Neither growth cones nor myelinated axons entering the plexus were identified in the control grafts. Such structures in the grafts adjacent to cut nerves were therefore likely to have originated from the LCPN. Those rare examples of large electron-lucent axons present in the control experiments presumably originated from nerve fibres accidentally injured during surgery. The prcsent study has demonstrated that peripheral nerve fibres regenerate into all identifiable parts of the myenteric plexus contained within grafts of muscularis externa from the distal colon. However, it is not known how far any individual axon regenerated through the plexus. Nonetheless many more axons were present in these grafts than in CNS tissue under similar conditions [4]. It is reasonable therefore to propose that the elements of the plexus, enteric glia and neurons, may otter a suitable environment to support regenerating peripheral axons. In this regard they are similar to Schwann cells, which in most instances are a prerequisite for peripheral nerve regeneration over macroscopic distances [2, 8]. In contrast, CNS glia in adult mammals provide an environrnent in which axonal regeneration is usually very limited [1, 4, 5]. The environmental factors required for regeneration in vivo remain unknown. Much work has been carried out on trophic substances synthesised by glial cells of all kinds, but cell contact phenomena or even serum-derived trophic factors [12] are
132
just as likely candidates for some part in the process. It may be germane that occasional fenestrated capillaries are found wherever PNS fibres are successfully regenerating [3 5]. The question of whether the enteric gila of mouse distal colon are capable of myelihating peripheral nerve fibres has not been resolved by the present study. The myelin lamellae around axons associated with enteric ganglia had similar spacing to normal PNS myelin and may have been produced by enteric glia or Schwann cells.
The authors gratefully acknowledge support from the Weilcome Trust. I Aguayo, A.J,, David, S., Richardson, P. and Bray, G.M., Axonal elongation in peripheral and central nervous system transplants. In S. Fedoroff and L. Hertz (Ed.), Advances in Cellular Neurobiotogy, Vol. 3, Academic, New York, 1982, pp 215 234. 2 Anderson, P.N., Mitchell, J., Mayor, D. and Stauber, V.V., An ultrastructural study of the early stages of axonal regeneration through rat nerve grafts, Neuropathol. Appl. Neurobiol., 9 (1983) 455 466. 3 Anderson, P.N. and Turmaine, M., Axonal regeneration through arterial grafts, J. Anat., t47 (1986) 73 82. 4 Anderson, P.N. and Turmaine, M.~ Peripheral nerve regeneration through grafts of living and freezedried CNS tissue, Neuropathol. Appt. Neurobiol., 12 (1986) 389 399. 5 Berry, M., Regeneration of axons in the central nervous system, Prog. Anat., 3 (1983) 213 233. 6 Freide, R.L. and Samorajski, T., Myelin formation in the sciatic nerve of the rat a quantitative electron microscopic, histochemical and radioautographic study, J. Neuropathol. Exp. Neurol., 27 (1968) 546 570. 7 ttall, S.M., Regeneration in cellular and acellular autografts in the peripheral nervous system, Neuropathol. Appl. Neurobiol., 12 (1986) 27 46. 8 Hall, S.M., The effect of inhibiting Sehwann cell mitosis on the reinnervation of aceltular autografts in the peripheral nervous system of the mouse, Neuropathol. Appl. Neurobiol., 12 (1986) 401 414. 9 Hill, ('.[~., Hirst, G.D.S., Ngu, M.('. and van Helden, D.["., Sympathetic postganglionic reinnervation of mesenteric arteries and enteric ncurones of the ileum of the rat, J. Auton. Nerv. Sysl,, 14 (1985) 317 334. 10 ldc, C., Tohyama, K., Yakota, R., Nitatori, T. and Odonera, S., Schwann cell basal lamina and nerve regeneration, Brain Res., 288 (1983) 61 75. 1 l Jessen, K.R. and Mirsky, R., Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia, J. Neurosci., 3 (1983) 2206 2218. t2 Kierqan, J.A., ifypotheses concerned with axonal regeneration in the mammalian nervous system, Biol, Rev., 54 (1979) 155 197. 13 Weinberg, E.L. and Spencer, P.S., Studies on the control of myelinogenesis. 3. Signalling of oligodend rocyte myelination by regenerating peripheral axons, Brain Res., 162 (1979) 273 279.
129
NSL 04534
Peripheral nerve fibres regenerate through myenteric plexus P . N . A n d e r s o n a n d M. T u r m a i n e I)~7~arttn('nl ol'.411atonly and Biolog, y as Applied to Medieim'. The MMdh'se.v Hospital Medical .S'ehool. Lond~m ( U.K. (Received 4 November 1986: Revised version received 12 December 1986: Accepted 15 I)eccmbcr 1986) Kcr words:
Axonal regeneration: Axotom,,: Enteric glia: Myenteric plexus: Nerve graft: Pcripheral nerve: Mouse
The abilit? of myenteric gila and neurons to support peripheral nerve regeneration was tested b~ grafling pieces of muscularis externa 5 m m long from the dislal colon of inbred CBA mice adjacent to lfic proximal stump of cut c o m m o n peroneal nerves. By two weeks after operation many axons had invaded the plexus and after 3 weeks regeneration c o m m o n peroneal nerve fibres could be identilied in all parts of the plexus throughout the grafts. Some axonal profiles within the plexus appeared to bc m lhc early stages of myelmation by enteric glia. Axons surrounded by compact myelin were found at the pcriphcr? of ganglia, but the cells involved resembled Schwann cells and could not be positively identified as enteric glia. Profiles similar to those of regenerating axons were only very rarely seen in control experiments m which grafts were placed adjacent to intact c o m m o n peroncal nerves. It is suggested that tfie cellular clcnlents of Ifie myemeric plexus can support peripheral nerve regeneration.
Regenerating peripheral nerve fibres are almost always found in contact with Schwann cells [2]. This is even the case when the axons are regenerating through grafts which were initially acellular [2 4, 7], although evidence that some parts of such axons may be naked has been presented [10]. In situations where Schwann cells are not available axonal regeneration is poor [8]. CNS gila do not support regeneration of PNS fibres beyond about 1 mm [1, 4, 13]. Enteric glial cells are derived, like Schwann cells, from neural crest but have some properties in c o m m o n with CNS glia [11]. Little is known about the ability of enteric gila to support the regeneration ot" axons, although it has been shown that adrenergic fibres will regenerate into the myenteric plexus after extrinsic denervation in vivo [9]. However, non-myelinated monoaminergic axons have unusual powers of regeneration even in the CNS [5]. Grafts of myenteric plexus were therefore placed in the vicinity of injured peripheral nerve fibres with a known capacity For regeneration. Adult female inbred CBA mice were used throughout. Segments of distal colon
('orre.vmmh'nee. P.N. Anderson, Department of Anatomy and Biology as Applied to Medicin,.'. Tfic Middlesex tfospital Medical School, London W IP 6DB, U.K.
130
were removed t¥om donor animals killed with an overdose of ether vapour. T'he muscularis externa, containing the myenteric plexus, was dissected free from the mucous membrane and washed in several changes of sterile Hanks' balanced salt solution (Gibco). Segments about 6 mm long were removed from the left common peroneal nerves (LCPN) of host animals under ether anaesthesia. Pieces of muscularis externa about 5 mm long were rolled into a cylindrical shape and placed with their cut ends adjacent to the proximal stump of the LCPN. In control experiments pieces of muscularis externa were placed adjacent to intact LCPN. The animals were perfused through the left ventricle of the heart under deep anaesthesia 1 8 weeks after operation. The fixative contained 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer pH 7.4. The grafts were cut into l-ram segments, osmicated and processed into araldite. Ultrathin sections were examined using a Philips EM300 electron microscope. Grafts placed ad/acent to cut nerves ( 10 gra/ts). One week alter operation the proximal stump of the injured nerve contained swollen axons and new axonal sprouts, but few regenerating axons had entered the grafts. The myenteric plexus contained apparently healthy enteric glia and neurons. Two weeks after operation many axons had entered the grafts, but they were most prominent in the proximal 2 ram. Some regenerating axons were seen in the small branches of the myenteric plexus which innervate the muscle layer. Such axons had the appearence of growth cones or appeared as large electron-lucent profiles in small bundles of fibres which also contained varicosities typical of autonomic neuromuscular junctions. Regenerating LCPN fibres could also be identified in the ganglia of the plexus in contact with myenteric glia and neurons (Fig. 1). They were larger and more electron lucent that the neurites intrinsic to the plexus. Growth cones were present in the ganglia. Some axons were wrapped by complex or multiple mesaxons which produced an appearence resembling the promyelin stage of myelination [6]. Fenestrated capillaries were occassionally identified in the tissue surrounding the ganglia. Three to 8 weeks after operation increasing numbers of nerve fibres could be identified growing through the grafts. Some axonal profiles were abnormal, being up to 6/~m diameter and largely packed with neurofilaments. Most large electron-lucent profiles within ganglia were typical of normal regenerating PNS axons, however. Myelinated axons were found associated with the myenteric ganglia: most profiles were simply very close to the ganglia, being separated by two layers of basal lamina. Some myelinated axons, however, were seen to extend into the ganglia, the myelin sheath terminating at the periphery of the ganglia with the axons passing into the neuropil as large electron-lucent profiles. It is unlikely that such axons were postganglionic sympathetic. No myelinated internodal axons were seen surrounded by the neuropil of the ganglia. Some regenerating fibres did not grow through the preexisting plexus but formed apparently new routes through the muscle layers. These took the form of minifascicles: small compartments of axons and Schwann cells surrounded by perineurial cells. GraJ?s placed adjacent to intact nerves (5 grafts). Grafts examined 2, 3 and 8 weeks after operation remained viable, but the myenteric plexus was almost entirely devoid
131
Fig. 1. Two ~ecks after grafting, a large rcgeneralmg axon (A), surrounded by enleric glial processes (arrox~s), may bc sccn wi/hin a mycnteric ganglion. The regenerating axon may be compared ~ith the intrinsic mycntcric ncurilcs which form the ad,iacent neuropil. Nolo the basal lamina ~.~fIhc ganglion (arrowheads). Bar 0.5/ml.
of large electron-lucent profiles. Neither growth cones nor myelinated axons entering the plexus were identified in the control grafts. Such structures in the grafts adjacent to cut nerves were therefore likely to have originated from the LCPN. Those rare examples of large electron-lucent axons present in the control experiments presumably originated from nerve fibres accidentally injured during surgery. The prcsent study has demonstrated that peripheral nerve fibres regenerate into all identifiable parts of the myenteric plexus contained within grafts of muscularis externa from the distal colon. However, it is not known how far any individual axon regenerated through the plexus. Nonetheless many more axons were present in these grafts than in CNS tissue under similar conditions [4]. It is reasonable therefore to propose that the elements of the plexus, enteric glia and neurons, may otter a suitable environment to support regenerating peripheral axons. In this regard they are similar to Schwann cells, which in most instances are a prerequisite for peripheral nerve regeneration over macroscopic distances [2, 8]. In contrast, CNS glia in adult mammals provide an environrnent in which axonal regeneration is usually very limited [1, 4, 5]. The environmental factors required for regeneration in vivo remain unknown. Much work has been carried out on trophic substances synthesised by glial cells of all kinds, but cell contact phenomena or even serum-derived trophic factors [12] are
132
just as likely candidates for some part in the process. It may be germane that occasional fenestrated capillaries are found wherever PNS fibres are successfully regenerating [3 5]. The question of whether the enteric gila of mouse distal colon are capable of myelihating peripheral nerve fibres has not been resolved by the present study. The myelin lamellae around axons associated with enteric ganglia had similar spacing to normal PNS myelin and may have been produced by enteric glia or Schwann cells.
The authors gratefully acknowledge support from the Weilcome Trust. I Aguayo, A.J,, David, S., Richardson, P. and Bray, G.M., Axonal elongation in peripheral and central nervous system transplants. In S. Fedoroff and L. Hertz (Ed.), Advances in Cellular Neurobiotogy, Vol. 3, Academic, New York, 1982, pp 215 234. 2 Anderson, P.N., Mitchell, J., Mayor, D. and Stauber, V.V., An ultrastructural study of the early stages of axonal regeneration through rat nerve grafts, Neuropathol. Appl. Neurobiol., 9 (1983) 455 466. 3 Anderson, P.N. and Turmaine, M., Axonal regeneration through arterial grafts, J. Anat., t47 (1986) 73 82. 4 Anderson, P.N. and Turmaine, M.~ Peripheral nerve regeneration through grafts of living and freezedried CNS tissue, Neuropathol. Appt. Neurobiol., 12 (1986) 389 399. 5 Berry, M., Regeneration of axons in the central nervous system, Prog. Anat., 3 (1983) 213 233. 6 Freide, R.L. and Samorajski, T., Myelin formation in the sciatic nerve of the rat a quantitative electron microscopic, histochemical and radioautographic study, J. Neuropathol. Exp. Neurol., 27 (1968) 546 570. 7 ttall, S.M., Regeneration in cellular and acellular autografts in the peripheral nervous system, Neuropathol. Appl. Neurobiol., 12 (1986) 27 46. 8 Hall, S.M., The effect of inhibiting Sehwann cell mitosis on the reinnervation of aceltular autografts in the peripheral nervous system of the mouse, Neuropathol. Appl. Neurobiol., 12 (1986) 401 414. 9 Hill, ('.[~., Hirst, G.D.S., Ngu, M.('. and van Helden, D.["., Sympathetic postganglionic reinnervation of mesenteric arteries and enteric ncurones of the ileum of the rat, J. Auton. Nerv. Sysl,, 14 (1985) 317 334. 10 ldc, C., Tohyama, K., Yakota, R., Nitatori, T. and Odonera, S., Schwann cell basal lamina and nerve regeneration, Brain Res., 288 (1983) 61 75. 1 l Jessen, K.R. and Mirsky, R., Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia, J. Neurosci., 3 (1983) 2206 2218. t2 Kierqan, J.A., ifypotheses concerned with axonal regeneration in the mammalian nervous system, Biol, Rev., 54 (1979) 155 197. 13 Weinberg, E.L. and Spencer, P.S., Studies on the control of myelinogenesis. 3. Signalling of oligodend rocyte myelination by regenerating peripheral axons, Brain Res., 162 (1979) 273 279.