.¥euro.wiem'e l.elter.s'. 76 (1987) 129 132 129

Elsevier Scientific Publishers Ireland Ltd.

NSL 04534

Peripheral nerve fibres regenerate through myenteric plexus

P.N. A n d e r s o n and 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~ grafl-

ing pieces of muscularis externa 5 mm long from the dislal colon of inbred CBA mice adjacent to lfic

proximal s tump of cut common peroneal nerves. By two weeks after operation many axons had invaded

the plexus and after 3 weeks regeneration common 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 common peroncal nerves. It is suggested that tfie cellular clc-

nlents 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 regene- ration 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.

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were removed t¥om donor animals killed with an overdose of ether vapour. T'he mus- cularis 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 mus- cularis 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 ope- ration. 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 prox- imal 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 neuromuscu- lar 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]. Fenes- trated capillaries were occassionally identified in the tissue surrounding the ganglia.

Three to 8 weeks after operation increasing numbers of nerve fibres could be iden- tified 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 post- ganglionic sympathetic. No myelinated internodal axons were seen surrounded by the neuropil of the ganglia. Some regenerating fibres did not grow through the pre- existing plexus but formed apparently new routes through the muscle layers. These took the form of minifascicles: small compartments of axons and Schwann cells sur- rounded 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

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Fig. 1. Two ~ecks after grafting, a large rcgeneralmg axon (A), surrounded by enleric glial processes (ar-

rox~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 ~.~f Ihc 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 presuma- bly 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 rege- neration 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

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just as likely candidates for some part in the process. It may be germane that occasio- nal fenestrated capillaries are found wherever PNS fibres are successfully regenerat- ing [3 5].

The question of whether the enteric gila of mouse distal colon are capable of myeli- hating 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.

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