Numbers of regenerating axons in parent and tributary peripheral nerves in the rat

This study is concerned with numerical parameters of axonal regeneration in peripheral nerves. Our first finding is that the number of axons that regenerate into the distal stump of a somatic nerve at a particular time after transection is partially dependent on the type of lesion used to interrupt the axons. The second question concerns the proportion of axons that regenerate into the distal stump of a parent nerve compared to the proportions that regenerate into tributary nerves that arise from the parent. The proportions of regenerated myelinated axons in the nerve to the medial gastrocnemius muscle and myelinated and unmyelinated axons in the sural nerve are the same as the proportions of myelinated and unmyelinated axons that regenerate into the distal stump of the sciatic nerve for the crush, 0 and 4 mm gap transections. Proportionally fewer axons regenerate into the tributary nerves following the 8 mm gap transection, however. This implies that the length of the gap has an influence on whether or not axons in tributary nerves regenerate in concert with axons in the distal stump of the parent nerve. The unmyelinated fibers in the nerve to the medial gastrocnemius muscle are different because they do not regenerate in proportion to those in the distal stump of the sciatic nerve. We also provide evidence to indicate that myelinated axons branch whereas unmyelinated fibers end blindly when they enter the distal stump after crossing a sciatic nerve transection. Finally the normal arrangement of perineurial cells seems to be disrupted after the sciatic nerve regenerates across a gap.     

Regeneration of axons after nerve transection repair is enhanced by degradation of chondroitin sulfate proteoglycan

Our past work indicates that growth-inhibiting chondroitin sulfate proteoglycan (CSPG) is abundant in the peripheral nerve sheaths and interstitium. In this study we tested if degradation of CSPG by chondroitinase enhances axonal regeneration through the site of injury after (a) nerve crush and (b) nerve transection and coaptation. Adult rats received the same injury bilaterally to the sciatic nerves and then chondroitinase ABC was injected near the injury site on one side, and the contralateral nerve was injected with vehicle alone. Nerves were examined 2 days after injury in the nerve crush model and 4 days after injury in the nerve transection model. Chondroitinase-dependent neoepitope immunolabeling showed that CSPG was thoroughly degraded around the injury site in the chondroitinase-treated nerves. Axonal regeneration through the injury site and into the distal nerve was assessed by GAP-43 immunolabeling. Axonal regeneration after crush injury was similar in chondroitinase-treated and control nerves. In contrast, axonal regrowth through the coaptation of transected nerves was markedly accelerated and the ingress of axons into the distal segment was increased severalfold in nerves injected with chondroitinase. On the basis of these results we concluded that growth inhibition by CSPG contributes critically to the poor regenerative growth of axons in nerve transection repair. In addition, degradation of CSPG by injection of chondroitinase ABC at the site of nerve repair increased the ingress of axonal sprouts into basal laminae of the distal nerve segment, presumably by enabling more latitude in growth at the interface of coapted nerve. This suggests that chondroitinase application may be used clinically to improve the outcome of primary peripheral nerve repair.     

Effects of sciatic nerve regeneration on axonal populations in tributary nerves

The present study tests 2 hypotheses: (1) that the numbers of axons that regenerate into a tributary nerve are in part dependent on the type of lesion used to transect the axons in the parent nerve; and (2) that the numbers of axons that regenerate will be different in different tributary nerves. Axons were counted in the sural nerve and the nerve to the medial gastrocnemius muscle 8 weeks following crush, simple transection, transection with removal of 4 mm and transection with removal of 8 mm of the sciatic nerve in the rat. The counts of myelinated and unmyelinated axons are presented in the text. If axon numbers in the 2 nerves are normalized, the proportion of regenerated to normal myelinated axon numbers are approximately the same in the 2 nerves, with more regenerated axons than normal following crush, simple transection, or 4 mm gap transection and fewer following 8 mm gap transection. The unmyelinated axons behave differently. In the nerve to the medial gastrocnemius muscle, the numbers of unmyelinated axons are greater than or equal to the normal numbers following our various surgical paradigms whereas in the sural nerve there are always fewer unmyelinated axons than normal. These findings indicate that the above hypotheses are correct for the nerves tested in the rat.     

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1 045 myelinated axons and 4 160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2 476 in the proximal stump to one of 2 219.

The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones.

These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.     

Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography.

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.     

Functional aspects of the regeneration of unmyelinated axons in the rat saphenous nerve

Electrophysiological experiments have been carried out to investigate aspects of unmyelinated axon regeneration in a transected cutaneous nerve. Some comparisons with regeneration of myelinated axons in the same nerve have also been made.

By 3 months after injury approximately 80% of the unmyelinated axons that had survived in the proximal stump had regenerated into the distal stump. About the same proportion of myelinated axons had regrown into the distal stump by this time. With both groups of axons there was no marked increase in the amount of regeneration across the injury site with longer recovery times. Conduction velocities in the regenerated unmyelinated axons tended to be slower across the injury site than proximally; the proximal conduction velocities did not differ from those in control nerves. The unmyelinated axons seemed to take longer to resupply the skin than did the myelinated ones, but in both cases the extent of skin innervation had reached about 60% of control values by 6 months after the injury.     

An ultrastructural study of schwann cell response to axonal degeneration

Adult Sprague-Dawley rats were used to prepare isolated and distal segments of ovarian, vagus and saphenous nerves. Isolated segments were prepared by cutting between double ligatures followed by retracting and anchoring the proximal and distal segments 0.5 cm or more away from the isolated piece. After various intervals between 3 and 123 days the rats were perfused with buffered glutaraldehyde and nerves were processed for electron microscopic examination. Cross sections of isolated and distal segments of all nerves displayed an abundance of mostly circular profiles containing microtubules and filaments that were indistinguishable from those in unmyelinated nerve fibers. Many such profiles were nested against perinuclear Schwann cell cytoplasm, others were present in isolated clusters enclosed only by basement membrane. Examples of continuity between the nerve-like profiles and perinuclear cytoplasm of Schwann cells were found in longitudinal sections. In time, the number of such processes gradually diminished, but 123 days after transection they still could be found. It was concluded that Schwann cells in isolated and distal segments of transected nerves develop great numbers of small cylindrical processes that are arranged in bundles parallel to the long axis of the degenerated nerve. These processes had an arrangement of cytoplasmic organelles similar to axons of the proximal segment and the contralateral nerve. However the processes differed from most mature axons in that they were not ensheathed by Schwann cell cytoplasm with a mesaxon.     

Growth of injured rabbit optic axons within their degenerating optic nerve

Spontaneous growth of axons after injury is extremely limited in the mammalian central nervous system (CNS). It is now clear, however, that injured CNS axons can be induced to elongate when provided with a suitable environment. Thus injured CNS axons can elongate, but they do not do so unless their environment is altered. We now show apparent regenerative growth of injured optic axons. This growth is achieved in the adult rabbit optic nerve by the use of a combined treatment consisting of: (1) supplying soluble substances originating from growing axons to be injured rabbit optic nerves (Schwartz et al., Science, 228:600-603, 1985), and (2) application of low energy He-Ne laser irradiation, which appears to delay degenerative changes in the injured axons (Schwartz et al., Lasers Surg. Med., 7:51-55, 1985; Assia et al., Brain Res., 476:205-212, 1988). Two to 8 weeks after this treatment, unmyelinated and thinly myelinated axons are found at the lesion site and distal to it. Morphological and immunocytochemical evidence indicate that these thinly myelinated and unmyelinated axons are growing in close association with glial cells. Only these axons are identified as being growing axons. These newly growing axons transverse the site of injury and extend into the distal stump of the nerve, which contains degenerating axons. Axons of this type could be detected distal to the lesion only in nerves subjected to the combined treatment. No unmyelinated or thinly myelinated axons in association with glial cells were seen at 6 or 8 weeks postoperatively in nerves that were not treated, or in nerves in which the two stumps were completely disconnected. Two millimeters distal to the site of injury, the growing axons are confined to a compartment comprising 5%-30% of the cross section of the nerve. A temporal analysis indicates that axons have grown as far as 6 mm distal to the site of injury, by 8 weeks postoperatively. Anterograde labeling with horseradish peroxidase, injected intraocularly, indicates that some of these newly growing axons arise from retinal ganglion cells.     

Changes in myelinated and unmyelinated axon numbers in the proximal parts of rat sural nerves after two types of injury

Counts of myelinated and unmyelinated axon profiles have been made from normal, uninjured rat sural nerves and from nerves injured 6 months earlier in one of two ways. In one group of rats the nerve was simply cut and left to regenerate, leading to the development of a neuroma in continuity, while in the second group the nerve was cut but then ligated as well to prevent regeneration; this led to stump neuroma formation. After nerve transection and regeneration, with subsequent formation of a neuroma in continuity, there was no change in the number of myelinated axon profiles found 25 mm proximal to the old injury site when compared with control, but there was an 18% reduction (P < 0.05) in the number of unmyelinated axon profiles. Immediately proximal to the injury site the picture was similar, with there still being the same number of myelinated axon profiles as in control material but here the reduction in unmyelinated axon numbers was slightly greater at 24% (P < 0.05). In the proximal part of nerves that had been cut and stump neuroma formation induced there was a large increase (33%) in myelinated axon profiles over and above control values (P < 0.001) but the number of unmyelinated profiles was the same as in controls. Closer to the stump neuroma the number of myelinated axon profiles had increased yet further to be 88% (P < 0.001) above control while the number of unmyelinated ones remained no different from control. Our interpretation of these results is that after nerve transection and regeneration there is no loss of peripheral neurons supporting myelinated axons but some loss of those supporting unmyelinated ones. If a cut nerve is prevented from regenerating and a stump neuroma forms, however, a vigorous sprouting response is triggered in neurons with myelinated axons while those supporting unmyelinated axons are possibly prevented from dying. The reaction of peripheral neurons to injury is such that the number of axons they support varies along the nerve as one goes disto-proximally away from the injury site. Thus discrepancies in results from different laboratories have come about because material for axon counting has been taken from different points along the nerve relative to the injury site and also because the material has been taken from nerves injured in different ways.     

Axonal branching following crush lesions of peripheral nerves of rat

Branching of myelinated and unmyelinated nerve fibers in normal and regenerating personal and soleus nerves was studied by light and electron microscopy. There were at most 2% more myelinated and 13% more unmyelinated axons in the distal as compared with the proximal nerve segments. Two to four weeks after a crush lesion the distal axons became 2-3 times more numerous; thereafter their number decreased. The number of axons in the proximal nerve segment did not change. The number of myelinated sprouts in most regenerated nerves equalled the number of myelinated fibers in the proximal nerve, while the number of unmyelinated axons after 12-19 weeks was 18-60% higher than normal. Branching was not restricted to the crush region. The results indicate that following a crush lesion all axons branch but only branches of unmyelinated fibers persist for a prolonged period of time. It is tentatively suggested that regenerating axons branch when searching for a target and that when contact is made with the target this prevents additional branching and eliminates redundant branches. Myelinated axons are guided by existing Schwann cells, whereas unmyelinated axons do not follow predetermined pathways; this may explain their greater tendency to form permanent branches.     

Myelinated fiber regeneration after crush injury is retarded in sciatic nerves of aging mice

To compare nerve regeneration in young adult and aging mice, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two weeks later, both groups of mice were perfused with an aldehyde solution, and, after additional fixation, the sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. The same level was sectioned in unoperated contralateral nerves; these nerves served as controls. Electron micrographs and the Bioquant Image Analysis System IV were used to measure areas of posterior tibial fascicles and count the number of myelinated axons, the number of unmyelinated axons, and their frequency in Schwann cell units. In aging mice, the total number of regenerating myelinated axons was significantly reduced, but totals of regenerating unmyelinated axons in aging and young adults did not differ significantly. In aging mice, the frequency of Schwann cells that contained a single unmyelinated axon was greater, suggesting that before myelination began, Schwann cell ensheathment of axons also was slowed. After axotomy by a crush injury, the area of the posterior tibial fascicle was less than that in young adults and the distal disintegration of myelin sheath remnants also appeared to be retarded. The results indicate that responses of neurons, axons, and Schwann cells could be important in slowing the regeneration of myelinated fibers found in sciatic nerves from aging mice.     

Regeneration of unmyelinated and myelinated sensory nerve fibres studied by a retrograde tracer method

Regeneration of myelinated and unmyelinated sensory nerve fibres after a crush lesion of the rat sciatic nerve was investigated by means of retrograde labelling. The advantage of this method is that the degree of regeneration is estimated on the basis of sensory somata rather than the number of axons. Axonal counts do not reflect the number of regenerated neurons because of axonal branching and because myelinated axons form unmyelinated sprouts. Two days to 10 weeks after crushing, the distal sural or peroneal nerves were cut and exposed to fluoro-dextran. Large and small dorsal root ganglion cells that had been labelled, i.e., that had regenerated axons towards or beyond the injection site, were counted in serial sections. Large and small neurons with presumably myelinated and unmyelinated axons, respectively, were classified by immunostaining for neurofilaments. The axonal growth rate was 3.7 mm/day with no obvious differences between myelinated and unmyelinated axons. This contrasted with previous claims of two to three times faster regeneration rates of unmyelinated as compared to myelinated fibres. The initial delay was 0.55 days. Fewer small neurons were labelled relative to large neurons after crush and regeneration than in controls, indicating that regeneration of small neurons was less complete than that of large ones. This contrasted with the fact that unmyelinated axons in the regenerated sural nerve after 74 days were only slightly reduced.     

Local administration of vasoactive intestinal peptide after nerve transection accelerates early myelination and growth of regenerating axons

Our goal was to determine whether local injections of vasoactive intestinal peptide (VIP) promote early stages of regeneration after nerve transection. Sciatic nerves were transected bilaterally in 2 groups of 10 adult mice. In the first group, 15 microg (20 microL) of VIP were injected twice daily into the gap between transected ends of the right sciatic nerve for 7 days (4 mice) or 14 days (6 mice). The same number of mice in the second group received placebo injections (20 microL of 0.9% sterile saline) in the same site, twice daily, for the same periods. After 7 days, axon sizes, relationships with Schwann cells and degree of myelination were compared in electron micrographs of transversely sectioned distal ends of proximal stumps. Fourteen days after transection, light and electron microscopy were used to compare and measure axons and myelin sheaths in the transection gap, 2-mm distal to the ends of proximal stumps. Distal ends of VIP-treated proximal stumps contained larger axons 7 days after transection. More axons were in 1:1 relationships with Schwann cells and some of them were surrounded by thin myelin sheaths. In placebo-treated proximal stumps, axons were smaller, few were in 1:1 relationships with Schwann cells and no myelin sheaths were observed. In VIP-treated transection gaps, measurements 14 days after transection showed that larger axons were more numerous and their myelin sheaths were thicker. Our results suggest that in this nerve transection model, local administration of VIP promotes and accelerates early myelination and growth of regenerating axons.     

Specificity in regenerative outgrowth and target reinnervation by mammalian peripheral axons

There are indications that specific factors are present in the distal stump of transected nerves which preferentially attract axons of the corresponding proximal stump into the distal nerve stumps. However, the impact of these factors is unclear, since there is abundant evidence that numerous regenerating motor and sensory axons are topographically misdirected after nerve transection and repair. Topographic reinnervation is improved after fascicular repair of fasciculated nerves, and quite precise after nerve crush. The latter may not be true, however, for non-myelinated axons, which show a high degree of aberrant growth even after crush. In contrast, regenerative outgrowth appears to be topographically specific after neonatal nerve transection. Reinnervation of muscle fibers appears to be unspecific in adult mammals, but specific after neonatal injury under certain circumstances. Some preference for reinnervation of the appropriate sensory receptors seems to exist although this preference does not preclude reinnervation of receptors by 'foreign' sensory fibers. In conclusion, incorrect topographic and target reinnervation commonly occurs after peripheral regeneration in adult mammals, and most certainly explains some of the functional disturbances after peripheral nerve lesions. Topographic regeneration appears to be better after nerve injury in developing mammals indicating that mechanisms from the developmental period may persist and aid in accurate regenerative outgrowth.     

A comparative study of the effects of chronic axotomy, crush lesion and re-anastomosis of the rat sural nerve on horseradish peroxidase labelling of primary sensory neurons

Chronic axotomy is detrimental to the incorporation of horseradish peroxidase (HRP) by neurons of the central and peripheral nervous system. Using the rat sural nerve as a model, this study aimed to determine the effects of other types of nerve injury on the peroxidase labelling of dorsal root ganglion (DRG) cells. Compared to the decreased labelling occurring shortly after permanent transection of the sural axons at the ankle, crush injury of the nerve had no effect on the number and size distribution of peroxidase-stained cells. Re-anastomosing the sural nerve to its own distal segment or to the tibial nerve delayed the changes in HRP neuronal labelling, which subsequently were less severe in neurons allowed to reinnervate their own nerve. It also sustained the incorporation of HRP by many large DRG neurons, a function which is lost shortly after these cells are chronically axotomized. Nerve re-anastomosis also prevented the retrograde atrophy of myelinated and unmyelinated nerve fibers which is triggered by permanent transection. Based on the preservation of fiber counts in the sural nerves proximal to the site of surgery, with no evidence of degeneration, our observations possibly reflect alterations in the peroxidase metabolism of DRG neurons depending on the type of axonal injury they sustained and the possibility they had upon regeneration to contact endoneurial tubes and ultimately their original end-organs.     

Success of regeneration of peripheral nerve axons in rats after injury at different postnatal ages

Electrophysiological experiment have been carried out on rats to see if the age at which a peripheral nerve injury occurs influences the success of regeneration. The assessment was made on the basis of two measures of peripheral nerve regeneration; the extent to which axons manage to grow across the injury site and into the distal stump, and their ability to resupply cutaneous structures with functional endings. Regeneration after nerve transection of both myelinated and unmyelinated axons was studied. The results showed that, apart from rats injured when 2 weeks old, the age at which injury occurred, over the range 4–40 weeks, had little bearing on the overall success of skin reinnervation. The 2-week-old rats showed significantly poorer recovery.     

Degeneration and regeneration of peripheral nerve in the rat trigeminal system. II. Response to nerve lesions

The course of vibrissa sensory receptor denervation and subsequent reinnervation was studied following transection or crush of the rat infraorbital nerve. Eighteen hours after nerve lesion, the large-diameter myelinated nerves supplying the lanceolate receptors of the intermediary zone and the Merkel cells of the stratum basale contained areas of focal axoplasmic abnormalities, and some of the nerve terminals exhibited vacuolization, mitochondrial swelling, and disruption of the neurofilament pattern. The Merkel cells and lanceolate receptors of the intermediary zone were completely deafferented by 24 hours after the nerve injuries. The Ruffini complex, free nerve endings and lanceolate receptors of the inner conical body, as well as the free nerve endings and lanceolate receptors of the connective tissue below the Ringwulst, were completely normal 24 hours after crush or transection of the nerve. These receptors underwent progressive degeneration from days 2 through 6 and the vibrissa was totally denervated by day 7. Regenerating axons were first seen entering the vibrissae 2 weeks after the crush lesion and 1 month following nerve transection. Except for a slight decrease in the percentage of Merkel cells innervated, vibrissae from post-crush animals were virtually indistinguishable from normal by 3 months. In contrast, vibrissae from rats subjected to the transection lesion exhibited evidence of misdirected axons and abnormally reinnervated receptors throughout the course of regeneration. Axons entering the hairs with the main vibrissal nerve were observed contributing to the innervation of the inner conical body, an area normally supplied exclusively by the conus nerve. Many of the lanceolate receptors contained multiple unmyelinated axons, and the usually highly ordered circular innervation of the inner conical body was markedly abnormal. It is suggested that these results may help explain the faulty sensory localization and abnormal sensations reported by patients suffering a peripheral nerve injury.     

Categories of axons in the inferior cardiac nerve of the cat

Myelinated and unmyelinated axons in the inferior cardiac nerve of the cat were examined to determine how many axons were (1) sensory, (2) preganglionic sympathetic, and (3) postganglionic sympathetic. In one group of cats, a segment was removed from the middle of the inferior cardiac nerve as a control, and the proximal and distal stumps of the nerve were examined one week later. In another group of cats, the control segment of nerve was removed and the first thoracic white ramus communicans and sympathetic trunk were cut proximal to the stellate ganglion, followed in one week by examination of the proximal and distal stumps of the inferior cardiac nerve. In still another group of cats, the first five thoracic spinal nerves were cut just distal to the dorsal root ganglion. The counts of myelinated and unmyelinated axons after these surgical procedures indicated that, in the cat inferior cardiac nerve, all or almost all of the approximately 30,000 unmyelinated axons and 10 percent of the myelinated axons are postganglionic sympathetic fibers, and that approximately 90 percent of the myelinated axons are sensory.     

An experimental painful peripheral neuropathy due to nerve constriction. I. Axonal pathology in the sciatic nerve.

A constriction injury to the sciatic nerve of the rat produces a painful peripheral neuropathy that is similar to the conditions seen in man. The pathology of the sciatic nerve in these animals was examined at 10 days postinjury, when the abnormal pain sensations are near maximal severity. The nerves were examined with (1) complete series of silver-stained longitudinal sections of pieces of the nerve (3 cm or more) that contained the constriction injury in the center, (2) toluidine blue-stained semithin sections taken at least 1 cm proximal and 1 cm distal to the constriction, and (3) EM sections taken adjacent to those stained with toluidine blue. One centimeter or more proximal to the constriction, both myelinated and unmyelinated axons were all normal. Nearer to the constriction, extensive degeneration of myelinated axons became increasingly common, as did signs of endoneurial edema. Distal to the constriction, the nerve was uniformly edematous and full of myelinic degeneration. There was a profound loss of large myelinated axons and a distinctly less severe loss of small myelinated and unmyelinated axons. These observations show that at 10 days postinjury the constriction produces a partial and differential deafferentation of the sciatic nerve's territory. The absence of degeneration in the nerve 1 cm proximal to the constriction indicates the survival of the primary afferent neurons whose axons are interrupted.     

Long-term patterns of axon regeneration in the sciatic nerve and its tributaries

The present study determines the numbers of axons that regenerate after sciatic nerve transection in the rat. The transections are done by removing either 4 mm or 8 mm of the nerve. The axons are counted in the gap and distal stump of the sciatic nerve and in 5 of its tributaries. Survival time is 9 months which we define as long-term to allow comparison with short-term data obtained after a much shorter survival. The first findings is that the numbers of axons in the gap and distal stump are different in the 2 transection paradigms. For the 4 mm paradigm, more axons than normal appear in the gap and only a fraction of these pass into the distal stump. For the 8 mm paradigm, the numbers of axons in the gap are normal and the numbers in the distal stump do not deviate far from these. Thus by changing only the length of the segment of removed nerve, one causes major differences in the numbers of axons that regenerate. Second the numbers of axons that regenerate in tributary nerves that innervate muscle have a different pattern than the numbers that regenerate into cutaneous nerves. Thus the factors control axonal numbers must be different in the 2 types of nerves. Finally, axons that regenerate into tributary nerves do not, by and large, regenerate in concert with those in the distal stump of the parent nerve. Thus the factors that control axonal numbers in the tributary nerves must be different from those that control the numbers in the distal stump of the parent nerve.