M. C. Brown

Macrophage dependence of peripheral sensory nerve regeneration: Possible involvement of nerve growth factor

The levels of NGF and NGF receptor mRNA, the degree of macrophage recruitment, and the ability of sensory and motor axons to regenerate were measured in C57BL/Ola mice, in which Wallerian degeneration following a nerve lesion is very slow. Results were compared with those from C57BL/6J and BALB/c mice, in which degeneration is normal. We found that in C57BL/Ola mice, apart from the actual lesion site, recruitment of macrophages was much lower, levels of mRNA for both NGF and its receptor were raised only slightly above normal, and sensory axon regeneration was much impaired. Motor axons regenerated quite well. These results provide in vivo evidence that macrophage recruitment is an important component of NGF synthesis and of sensory (but not motor) axon maintenance and regrowth.

Evidence that Very Slow Wallerian Degeneration in C57BL/Ola Mice is an Intrinsic Property of the Peripheral Nerve.

We have described a mutant mouse, C57BL/Ola, in which Wallerian degeneration following peripheral nerve transection is very slow. Our previous results suggested that recruited monocytes play a role in rapid Wallerian degeneration. The nature of the mutation in C57BL/Ola mice is not known and we have investigated whether the defect is intrinsic to the nerve or due to a defect in the circulating monocytes. We have made chimaeric mice in which bone marrow from histocompatible mice, with rapidly degenerating nerves and normal monocyte recruitment, was used to reconstitute irradiated C57BL/Ola mice and vice‐versa. A substantial degree of donor repopulation of the hosts was confirmed by measures of the levels of glucose‐phosphate isomerase alloenzymes in blood and tissue samples from the two different strains. The rate of degeneration of the transected sciatic nerve was found to be host‐dependent, providing evidence that the mutation affects cell populations intrinsic to the nerve and not the circulating monocytes. We provide additional evidence that the peripheral nerves of C57BL/Ola mice are different from those of other mice as they degenerate at a slower rate in vitro.

Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse.

1. A study of nodal and terminal sprouting in fast and slow muscles of the mouse hind limb has been made using the zinc iodide and osmium tetroxide stain. 2. The terminal sprouting normally elicited by botulinum toxin injection can be prevented by regular and frequent direct electrical stimulation of the muscle fibres. But the number of end‐plates innervated by nodal sprouts in partly denervated spinal preparations was not reduced by direct muscle stimulation. 3. In leg muscles given varying doses of botulinum toxin the amount of terminal sprouting was linearly related to the degree of paralysis. In partly denervated muscles neither the amount of terminal sprouting nor the amount of nodal sprouting was correlated with the degree of denervation. 4. Partial denervation causes relatively more nodal sprouting in the fast muscles peroneus tertius and extensor digitorum longus than in the slower soleus muscle, which itself has considerably more terminal sprouting than the others. The fast muscles can develop as much terminal sprouting as the soleus only in response to full paralysis with botulinum toxin. 5. No evidence could be found for a sprouting signal generated or spreading within the spinal cord. 6. It is concluded in confirmation of earlier work (Duchen & Strich, 1968; Brown & Ironton, 1977 a) that the source of the signal for terminal sprouting is denervated or otherwise inactivated muscle fibres, whose action is boosted by the presence of degenerating nervous tissues. It is suggested that fast muscles probably have less terminal sprouting when partly denervated than slow muscles (a) because of the longer time it takes a fast muscle to undergo the changes associated with inactivity and (b) because of their higher resistance to the effects of nerve degeneration. It does not seem that the signal for nodal sprouting comes from the muscle fibres but further experimentation is needed to establish this firmly.

Evidence that the Rate of Wallerian Degeneration is Controlled by a Single Autosomal Dominant Gene

In a substrain of C57BL mice, C57BL/Ola, Wallerian degeneration in the distal segment of the severed sciatic nerve is extremely slow when compared to other mice. Despite this very slow degeneration in the distal segment regeneration of the motor nerves is not impaired. From suitable genetic outcrosses and backcrosses, the authors provide evidence that the rate of Wallerian degeneration in this strain is controlled by a single autosomal gene product. The authors have also shown that the rate of degeneration, in C57BL/Ola mice, is influenced by the environment in which the animals were bred and housed. Wallerian degeneration in the sciatic nerves of mice raised in isolators is slower than in those raised in a conventional animal house. This strain of mouse may prove to be of value in the understanding of nerve degeneration and regeneration.

Further Studies on Motor and Sensory Nerve Regeneration in Mice With Delayed Wallerian Degeneration

The axons of both peripheral and central neurons in C57BL/Wlds (C57BL/Ola) mice are unique among mammals in degenerating extremely slowly after axotomy. Motor and sensory axons attempting to regenerate are thus confronted with an intact distal nerve stump rather than axon‐and myelin‐free Schwann cell‐filled endoneurial tubes. Surprisingly, however, motor axons in the sciatic nerve innervating the soleus muscle regenerate rapidly, and there is evidence that they may use Schwann cells associated with unmyelinated fibres as a pathway. If this is so, motor axon regeneration might be impaired in C57BL/Wlds mice in the phrenic nerve, which has very few unmyelinated fibres. We found that as long as the myelinated axons in the distal stump of the phrenic nerve remained intact (up to 10 days), regeneration of motor axons did not occur, in spite of vigorous production of sprouts at the crush site. In contrast to motor axons, myelinated sensory axons regenerate very poorly in C57BL/Wlds mice, even in the presence of unmyelinated axons. We showed that this was also due to adverse local conditions confronting nerve sprouts, for the dorsal root ganglion cell bodies responded normally to injury with a rapid induction of Jun protein‐like immunoreactivity and when the saphenous nerve was forced to degenerate more rapidly by multiple crush lesions sensory axons regrew much more successfully. The findings show that motor and sensory axons in C57BL/Wlds mice, although very atypical in the way that they degenerate, are able to regenerate normally but only in an appropriate environment. The results also give support to the view that intact peripheral nerves either fail to encourage or actively inhibit axon growth, and that an unsuitable local environment can prevent regeneration even if the cell body is reacting normally to injury.

Macrophages and nerve regeneration.

Macrophages are not only phagocytic cells but also secrete a plethora of growth factors that are potentially important for regeneration. This review will examine the emerging evidence of a likely contribution by macrophages to axonal regeneration.

Radiation‐induced Reductions in Macrophage Recruitment Have Only Slight Effects on Myelin Degeneration in Sectioned Peripheral Nerves of Mice

Macrophage recruitment into the distal nerve stump of the cut or crushed sciatic or saphenous nerves of C57BL/6J mice was reduced by prior whole body irradiation. This procedure was successful in keeping the numbers of cells stained with the mouse macrophage‐specific antibody F4/80 to the levels found in unsectioned nerves. Quantitative image analysis of immunostained sections showed that the rate of loss of myelin basic protein was identical in nerves from irradiated and unirradiated mice up to 5 days but thereafter was slower in macrophage‐deprived nerves. Similar analysis of semithin sections stained with toluidine blue detected more undegenerated myelin in the nerves from irradiated mice 10 days after operation. Quantitative counts made from electron micrographs of the sectioned nerves at 7 days also showed slightly less extensive myelin breakdown in the nerves from irradiated mice. Complete removal of myelin from some Schwann cells can occur without macrophages, but macrophages accelerate the removal of myelin in the later stages of Wallerian degeneration. It is concluded that there are two phases to the breakdown of myelin in peripheral nerves undergoing Wallerian degeneration: an initial stage entirely dependent on the activity of Schwann cells and a later stage dependent on both Schwann cells and the presence of macrophages.

An assessment of the spread of the signal for terminal sprouting within and between muscles.

Certain muscles of the mouse and rat have been studied in order to assess how far a signal from denervated muscle can spread to elicit terminal sprouting from intact endplates. Denervation of the muscles surrounding the rat foot 4th lumbrical muscle caused no terminal sprouting in the 4th lumbrical itself. In the hemidenervated mouse gluteus maximus terminal sprouting was restricted to the central region of the muscle where innervated and denervated fibres intermingle. There was no enhancement of such sprouting if the underlying and closely apposed gluteus medius was simultaneously denervated. Hemidenervation of the mouse diaphragm and interscutularis, where intact endplates lie near to denervated muscle fibres, produced no terminal sprouting. Hemidenervation of the mouse platysma, where intact endplates often lie adjacent to denervated muscle fibres, similarly produced no significant response. However, all muscles were capable of producing extensive terminal sprouting in response to paralysis induced by botulinum toxin. The stimulus for terminal sprouting produced by an inactive muscle fibre must therefore be effective only on the fibres own terminal or immediately adjacent terminals.

Comparison of matrix metalloproteinase expression during wallerian degeneration in the central and peripheral nervous systems

The matrix metalloproteinases (MMPs) are a large family of zinc-dependent enzymes which are able to degrade the protein components of the extracellular matrix. They can be placed into subgroups based on structural similarities and substrate specificity. Aberrant expression of these destructive enzymes has been implicated in the pathogenesis of immune-mediated neuroinflammatory disorders. In this study we investigate the involvement of MMPs, from each subgroup, in Wallerian degeneration in both the central and peripheral nervous systems. Wallerian degeneration describes the process initiated by transection of a nerve fibre and entails the degradation and removal of the axon and myelin from the distal stump. A similar degenerative process occurs as the final shared pathway contributing to most common neuropathies. MMP expression and localisation in the peripheral nervous system are compared with events in the CNS during Wallerian degeneration. Within 3 days after axotomy in the peripheral nervous system, MMP-9, MMP-7 and MMP-12 are elevated. These MMPs are produced by Schwann cells, endothelial cells and macrophages. The temporospatial expression of activated MMP-9 correlates with breakdown of the blood-nerve barrier. In the CNS, 1 week after optic nerve crush, four MMPs are induced and primarily localised to astrocytes, not microglia or oligodendrocytes. In the degenerating optic nerve, examined at later time points (4, 8, 12 and 18 weeks), MMP expression was down-regulated. The absence of MMPs in oligodendrocytes and mononuclear phagocytes during Wallerian degeneration may contribute to the slower removal of myelin debris observed in the CNS. The low level of the inactive pro-form of MMP-9 in the degenerating optic nerve may explain why the blood-brain barrier remains intact, while the blood-nerve barrier is rapidly broken down. We conclude that the difference in the level of expression, activation state and cellular distribution of MMPs may contribute to the different sequence of events observed during Wallerian degeneration in the peripheral compared to the CNS.

The pattern of axonal degeneration in the peripheral nervous system varies with different types of lesion

Degeneration of axons in the mouse sciatic nerve was examined using conventional silver staining and by noting the presence or absence of a compound action potential on stimulating the nerve distal to the point of crush or cut. The presence of myeloperoxidase-positive cells was also examined in frozen sections of the nerve. In all experiments the distal, disconnected segment was studied. Degeneration after crushing with fine watchmakers forceps always began in the most distal part of the nerve and proceeded in a distoproximal direction, from the nerve entry point into a muscle back to the crush site. Myeloperoxidase-positive cells were also recruited into the nerve starting at the distal rather than proximal (the originally injured) end. This result favours the view that degeneration is triggered by lack of trophic support from the cell body rather than entry of deleterious substances at the site of the injury, for the terminals furthest from both the source of supply and the injury are affected first. Degeneration following nerve section was always more rapid than after crushing. The rate of axonal sealing at the injury was, however, no slower than after crushing, so it does not seem likely that greater entry of possible degeneration-triggering material at the injury site is the explanation for this. Crush lesions in which the perineurium was also cut open and the blood supply at the site was damaged, degenerated at the slower rate characteristic of simple crushes.(ABSTRACT TRUNCATED AT 250 WORDS)