Molecular and cellular aspects of axon-glia interaction in CNS regeneration

The relationships of neurons and non-neuronal cells are vital for the maintenance and function of neurons. Trauma alters these relationships causing proliferation of non-neuronal cells and, in adult mammalian CNS, presumably disturbs the environmental support needed for regeneration. A supportive environment can be restored by introducing a regenerating nerve to injured mammalian CNS. This response is probably due, at least in part, to diffusible substances secreted by the non-neuronal cells. We have obtained diffusible substances from either regenerating fish optic nerves or neonatal rabbit optic nerves and applied them around crushed adult rabbit optic nerves. This manipulation caused the adult nerve to show regenerative changes: a general increase of protein synthesis in the retinas; selective increase in synthesis of a few polypeptides in the retinas; sprouting from the retinas in vitro; increased viability of nerve fibers as shown by HRP staining; and the appearance of growth cones adjacent to glial limitans in the injured nerves. We termed these diffusible, active substances "Growth Associated Triggering Factors" (GATFs). In addition to the phenomena described above, the active substances (obtained in the form of media conditioned by regenerating fish optic nerve or neonatal rabbit optic nerve) caused various other changes in the injured nerve itself: acceleration of non-neuronal cell proliferation; changes in the protein pattern, e.g. an increase in a 12 kDa polypeptide which might be a second mediator in the cascade of events leading to regeneration; increased laminin immunoreactive sites in the nerve; and the acquisition of growth supportive activity in media conditioned by the implanted injured nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Injury-induced gelatinase and thrombin-like activities in regenerating and nonregenerating nervous systems.

It is now widely accepted that injured nerves, like any other injured tissue, need assistance from their extracellular milieu in order to heal. We compared the postinjury activities of thrombin and gelatinases, two types of proteolytic activities known to be critically involved in tissue healing, in nonregenerative (rat optic nerve) and regenerative (fish optic nerve and rat sciatic nerve) neural tissue. Unlike gelatinases, whose induction pattern was comparable in all three nerves, thrombin-like activity differed clearly between regenerating and nonregenerating nervous systems. Postinjury levels of this latter activity seem to dictate whether it will display beneficial or detrimental effects on the capacity of the tissue for repair. The results of this study further highlight the fact that tissue repair and nerve regeneration are closely linked and that substances that are not unique to the nervous system, but participate in wound healing in general, are also crucial for regeneration or its failure in the nervous system.     

Regulation of Hemopexin Synthesis in Degenerating and Regenerating Rat Sciatic Nerve

Abstract : In injured peripheral nerves, hemopexin mRNA is expressed by fibroblasts, Schwann cells, and invading blood macrophages, and the protein accumulates in the extracellular matrix. This and its absence of regulation in injured central optic nerve suggest that hemopexin could play a positive role in peripheral nerve repair. Here, we studied the regulation of hemopexin expression in degenerating and regenerating nerves. After a sciatic nerve injury, both the synthesis of hemopexin and the level of its mRNA increase sharply during the first 2 days, leading to an accumulation of hemopexin in the nerve. Afterward, hemopexin expression decreases progressively in regenerating nerves. In permanently degenerated nerves, it is again transiently increased and then strongly decreased, whereas hemopexin from blood origin is accumulating. As part of the elucidation of the complex regulation of hemopexin expression in injured nerves, we demonstrate that interleukin-6 increases hemopexin synthesis in intact nerves, whereas adult rat serum, but not purified hemopexin, inhibits it in degenerated nerves. Hemopexin, known as acute-phase protein, is therefore one of the molecules rapidly and specifically up-regulated in injured peripheral nerves. More generally, our findings suggest that the acute phase could be not only a systemic liverspecific response but also a reaction of injured tissues themselves.     

The sudanophilic lipid of normal and regenerating limb tissues of the adult newt,Diemictylus viridescens

The most intense and widely distributed sudanophilic responses of cryostat-sectioned newt limb tissues were obtained with a simultaneous fix and stain procedure of 1:1 10% formal-calcium and sudan black B. Droplets and globules of lipid mixtures and rodlets (mitochondria) were typical responses distributed within the epidermis, subcutaneous glands, dermis and other connective tissues, striated muscle (also with positive fibrils), tunics of blood vessels, and blood cells. A prominent droplet response was located subjacent to the adepidermal basement membrane. The myelin of brachial nerve stained intensely. In regenerating limbs, the wound epithelium response was comparable to that of epidermis. Post-amputational lipophanerosis of injured muscle and brachial nerves was observed. The retrograde degeneration of nerve myelin was extensive, and continued into the early differentiative phase of regeneration. Lipid-engorged macrophages were prominent among the injured tissues, distal to these, and within the wound epithelium. The regeneration blastema revealed a large quantity of sudanophilic lipid. Prominent droplet and rodlet responses were typical of the myelinating regenerating nerves. The response of regenerating muscle equaled that of the mature stump fibers. The cells of the regenerating chondroskeleton contained sudanophilic lipid. Organic solvents such as acetone, ether, chloroform and chloroform:methanol reduced or prevented the sudanophilic responses. Sudan red 7B revealed less lipid than did sudan black B. A fixation effect was demonstrated with post-chromated formalcalcium, and chromic-formalin fixed sections. In the latter preparations, swollen-bodies, identified as mitochondria, stained intensely.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

Axonal Transport of Polyamines in Intact and Regenerating Axons of the Rat Sciatic Nerve

The axonal transport of putrescine or its polyamine derivatives spermidine or spermine is a subject of some debate. We investigated this question by injecting [3H]putrescine into the lumbar spinal cord of the rat and measuring the accumulation of radioactivity central to ligatures placed on intact and regenerating sciatic nerves. In normal nerves, approximately twice as much radioactivity built up proximal to these ligatures 2 or 3 days after injection than at more distal ligatures used to control for accumulation of radioactivity which might be due to tissue damage alone. In regenerating nerves the amount of radioactivity accumulating at the ligature was approximately five times that at the distal ligature and two to three times greater than in intact nerves. The identity of the radioactivity in regenerating nerves, determined on an amino acid analyzer, was found to be primarily spermidine and an unknown compound that migrated as a frontal elution peak. Autoradiographic analysis showed that the radioactivity was largely confined to axons, but a significant amount of the silver grains was associated with Schwann cells and myelin sheaths surrounding labeled axons in both intact and regenerating nerves. The data indicate that polyamine derivatives of putrescine are transported axonally in rat sciatic nerves, and some of this transported material accumulates in Schwann cells surrounding the labeled axons. These processes are apparently augmented during regeneration of the injured axons.     

Competitive reinnervation of salamander skin by regenerating and intact mechanosensory nerves

We have investigated in the salamander the possibility that regenerating mechanosensory nerves might prefer the epidermal Merkel cells (their specific targets) that are located within their segmental domain to those within a "foreign" domain. Since regerating nerves cross domain boundaries with no evidence of the marked delay exhibited by intact sprouting nerves, we examined situations in which the regenerating axons of one segmental nerve were effectively in equal competition for denervated skin with those of another segmental nerve. Additionally, we investigated whether there were differences between regenerating axons and intact sprouting axons of the same segmental nerve, in their ability to innervate available skin both inside and outside the parent domain. No preference was detected of any type of nerve, regenerating or intact, for particular skin regions, or for Merkel cells as indicated by the numbers of mechanosensory thresholds of the touch spots that developed in reinnervated skin. Neither was there any indicating of displacement of "foreign" nerves from a particular region by appropriate axons. When regenerating and intact (sprouting) axons invaded denervated skin more or less simultaneously, the former appeared to have a slight advantage since a significantly greater proportion of skin was innervated by regenerated fibres. With this one exception, all the results were explained most simply by assuming that the axon that first arrives at a denervated Merkel cell establishes a permanent association with that cell and at the same time causes it to lose its "target character" for other axons.     

Protein Synthesis and Axonal Transport During Nerve Regeneration

Abstract— Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [³⁵S]-methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two-dimensional polyacryl-amide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Pulsed magnetic fields enhance the rate of recovery of damaged nerve excitability

Pulsed magnetic fields (PMFs) have well‐known beneficial effects on nerve regeneration. However, little research has examined the nerve conduction characteristics of regenerating peripheral nerves under PMF. The main goal of this study was to examine the conduction characteristics of regenerating peripheral nerves under PMFs. The sucrose‐gap recording technique was used to examine the conduction properties of injured sciatic nerves of rats exposed to PMF. Following the injury, peripheral nerves were very sensitive to repetitive stimulation. When the stimulation frequency was increased, the amplitude of the compound action potential (CAP) decreased more at 15 days post‐crush injury (dpc) than at 38 dpc. PMF treatment for 38 days after injury caused significant differences in the conduction of CAPs. Moreover, application of PMF ameliorated the abnormal electrophysiological activities of nerves such as hyperpolarizing afterpotentials and delayed depolarizations that were revealed by 4‐aminopyridine (4‐AP). Consequently, characteristic findings in impulse conduction of recovered nerves under PMF indicate that the observed abnormalities in signaling or aberrant ion channel functions following injury may be restored by PMF application. Bioelectromagnetics 32:200–208, 2011. © 2010 Wiley‐Liss, Inc.     

The interaction of nerves of opposite regenerating polarity in fused newt forelimbs

Previous studies involving nerve interactions and limb regenerative processes were carried out on adult newts after their forelimbs were amputated through the distal radius and ulna and fused end-to-end. On the basis of limb regeneration results at the junction of the fused limbs, it was postulated that regenerating nerves from each limb (i.e., nerves of opposite polarity) would not invade the foreign territory of the contralateral limb if it were already normally innervated. A direct study of this nerve interaction, however, was not made in this earlier study. The present investigation was designed to obtain direct histological and electrophysiological evidence for the interaction of nerves of opposite regenerating polarity in fused newt forelimbs. The primary objective was to determine how the regenerating nerves would interact in the establishment of innervation territories-first, at the fusion zone, which represents the junction of the normal innervation territories of the nerves of each limb; and secondly, half way up one of the limbs, where interaction would occur in a territory normally innervated by only one of the regenerating nerves. The results showed that when nerves of opposite regenerating polarity approached one another at the junction of the fused limbs a discontinuation of axonal growth occurred; no indication of overlap of nerves into foreign territory was seen. When the nerves were allowed to interact within one of the fused limbs, however, an overlap of nerve fibers and a functional "double innervation" of that limb was demonstrated. These results are discussed in terms of possible mechanisms for the establishment of innervation territories in salamander limbs. The question of nerve-muscle reinnervation specificity is also raised.     

Optic Nerve Regeneration in Adult Fish and Apolipoprotein A-I

Fish optic nerves, unlike mammalian optic nerves, are endowed with a high capacity to regenerate. Injury to fish optic nerves causes pronounced changes in the composition of pulse-labeled substances derived from the surrounding non-neuronal cells. The most prominent of these injury-induced changes is in a 28-kilodalton (kDa) polypeptide whose level increases after injury, as revealed by one-dimensional gel electrophoresis and autoradiography. The present study identified as apolipoprotein A-I (apo-A-I) a polypeptide of 28 kDa in media conditioned by regenerating fish optic nerves. The level of this polypeptide increased after injury by approximately 35%. Apo-A-I was isolated by gel-permeation chromatography from delipidated high-density lipoproteins (HDL) that had been obtained from carp plasma by sequential ultracentrifugation. Further identification of the purified protein as apo-A-I was based on its molecular mass (28 kDa) as determined by gel electrophoresis, amino acid composition, and microheterogeneity studies. The isolated protein was further analyzed by immunoblots of two-dimensional gels and was found to contain six isoforms. Western blot analysis using antibodies directed against the isolated plasma protein showed that the 28-kDa polypeptide in the preparation of soluble substances derived from the fish optic nerves (conditioned media, CM) cross-reacted immunologically with the isolated fish plasma apo-A-I. Immunoblots of two-dimensional gels revealed the presence of three apo-A-I isoforms in the CM of regenerating fish optic nerves (pIs: 6.49, 6.64, and 6.73). At least some of the apo-A-I found in the CM is derived from the nerve, as was shown by pulse labeling with [35S]methionine, followed by immunoprecipitation. The apo-A-I immunoactive polypeptides in the CM of the fish optic nerve were found in high molecular-weight, putative HDL-like particles. Immunocytochemical staining revealed that apo-A-I immunoreactive sites were present in the fish optic nerves. Higher labeling was found in injured nerves (between the site of injury and the brain) than in non-injured nerves. The accumulation of apo-A-I in nerves that are capable of regenerating may be similar to that of apo-E in sciatic nerves of mammals (a regenerative system); in contrast, although its synthesis is increased, apo-A-I does not accumulate in avian optic nerves nor does apo-E in rat optic nerves (two nonregenerative systems).     

Lentiviral vectors enveloped with rabies virus glycoprotein can be used as a novel retrograde tracer to assess nerve recovery in rat sciatic nerve injury models

Retrograde labeling has become the new “gold standard” technique to evaluate the recovery of injured peripheral nerves. In this study, lentiviral vectors with rabies virus glycoprotein envelop (RABV-G-LV) and RFP genes are injected into gastrocnemius muscle to determine the location of RFP in sciatic nerves. We then examine RFP expression in the L4-S1 spinal cord and sensory dorsal root ganglia and in the rat sciatic nerve, isolated Schwann cells, viral dose to expression relationship and the use of RABV-G-LV as a retrograde tracer for regeneration in the injured rat sciatic nerve. VSV-G-LV was used as control for viral envelope specificity. Results showed that RFP were positive in the myelin sheath and lumbar spinal motorneurons of the RABV-G-LV group. RFP gene could be detected both in myelinated Schwann cells and lumbar spinal motor neurons in the RABV-G-LV group. Schwann cells isolated from the RABV-G-LV injected postnatal Sprague Dawley rats were also RFP-gene positive. All the results obtained in the VSV-G-LV group were negative. Distribution of RFP was unaltered and the level of RFP expression increasing with time progressing. RABV-G-LV could assess the amount of functional regenerating nerve fibers two months post-operation in the four models. This method offers an easy-operated and consistent standardized approach for retrograde labeling regenerating peripheral nerves, which may be a significant supplement for the previous RABV-G-LV-related retrograde labeling study.     

Posttranslational Protein Modification by Amino Acid Addition in Regenerating Optic Nerves of Goldfish

Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000-g supernatant of optic nerves was analyzed for incorporation of 3H-amino acids into proteins. No incorporation of 3H-amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S-200 column (removing molecules less than 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4-5 times for Lys and 2-13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5-15 times more [3H]Lys and [3H]Leu into proteins than did the shafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

The long-term influence of decentralisation or preganglionic hypogastric nerve stimulation in vivo on the reinnervation of minced vas deferens in the guinea-pig

Previous studies have shown that minced regenerating smooth muscle of the guinea-pig vas deferens becomes reinnervated by nerves growing in from the surrounding intact vas deferens. Using electron microscopy, we have examined the effect of altering activity in the preganglionic nerves, either by decentralisation, or by chronic stimulation of the hypogastric nerve, in vivo, on the reinnervation of regenerating smooth muscle cells. Chronic stimulation induced earlier reinnervation than that seen in unstimulated (sham-operated) or decentralised preparations; the number of nerve profiles present in four preparations stimulated for up to 7 days was approximately 10-20 times that seen in unstimulated or decentralised preparations. However, electron micrographs revealed that "empty" nerve terminals were a feature following stimulation for longer periods. Decentralised preparations showed little change of reinnervation, at least up to 7 weeks. Compensatory changes in the density of innervation were found in the unstimulated contralateral vas deferens.     

Fast axonal transport in motor nerve regeneration.

This report describes the fast transport of [3H]-leucine-labeled proteins in regenerating rat sciatic motor nerves. A normal rate of fast transport (383 +/- 33 mm/day) was present in the regenerating sprouts, as well as in the central stumps. The rapidly transported proteins passed the level of axotomy without impediment, and accumulated in the endings of the regenerating sprouts, as shown by electron microscope autoradiography. In addition, transported proteins accumulated in terminal neuromas. The relative amount of protein-incorporated radioactivity in the crest of transport in the regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating sprouts as in normal axons; during regeneration fast transport appears to add newly synthesized materials to the growing tip.     

Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme

Monoclonal antibody 9G1 (mAb 9G1) is reactive to the wound epithelium of axolotl larvae and therefore provided the opportunity to examine the interaction between the wound epithelium, nerves, and blastemal mesenchyme during axolotl limb regeneration. In unamputated limbs, mAb 9G1 is reactive to most or all cells of the dermis, skeletal elements, blood vessels, and nerves, to a few unidentified cells in muscle, and to none in epidermis. During regeneration of axolotl limbs, mAb 9G1 reacts strongly to an intracellular antigen of the blastemal mesenchyme and of the distal-most portion of the wound epithelium, the so-called apical epithelial cap (AEC). Because this thickened wound epithelium of regenerating amphibian limbs has been suggested as functioning in a manner similar to the apical ectodermal ridge (AER) of embryonic limb buds, it was of interest to further examine the reactivity of mAb 9G1 during various stages of regeneration. Whether mAb 9G1 reactivity in the AEC depended on mesenchyme and/or nerves was also tested. Monoclonal antibody 9G1 reactivity appears in the AEC of regenerating limbs prior to outgrowth of the blastema and persists throughout blastemal stages. Apical epithelial cap reactivity to mAb 9G1 is nerve dependent during early stages of blastema development and becomes nerve-independent at later stages. When epithelium-free blastemal mesenchyme is grafted onto injured flank musculature, ectopic limb regeneration occurs and the AEC derived from flank epidermis exhibits mAb 9G1 reactivity. These results show that a mAb 9G1 reactive AEC is characteristic of regenerating limbs and that expression of the 9G1 antigen by the AEC is dependent upon underlying blastemal mesenchyme and nerves.     

Cerebral Vulnerability Is Associated with Selective Increase in Extracellular Glutamate Concentration in Experimental Thiamine Deficiency

Abstract: The concentration of apolipoprotein E (apoE), a high-affinity ligand for the low-density lipoprotein receptor, increases dramatically in peripheral nerve following injury. This endoneurial apoE is thought to play an important role in the redistribution of lipids from the degenerating axonal and myelin membranes to the regenerating axons and myelin sheaths. The importance of apoE in nerve repair was examined using mutant mice that lack apoE. We show that at 2 and 4 weeks following sciatic nerve crush, regenerating nerves in apoE-deficient mice were morphologically similar to regenerating nerves in control animals, indicating that apoE is not essential for peripheral nerve repair. Moreover, cholesterol synthesis was reduced in regenerating nerves of apoE-deficient mice as much as in regenerating nerves of control animals. These results suggest that the intraneural conservation and reutilization of cholesterol following nerve injury do not require apoE.     

SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves

Two tetrodotoxin-resistant voltage-gated sodium channels, SNS/PN3 and SNS2/NaN, have been described recently in small-diameter sensory neurones of the rat, and play a key role in neuropathic pain. Using region-specific antibodies raised against different peptide sequences of their alpha subunits, we show by Western blot evidence for the presence of these channels in human nerves and sensory ganglia. The expected fully mature 260 kDa component of SNS/PN3 was noted in all injured nerve tissues obtained from adults; however, for SNS2/NaN, smaller bands were found, most likely arising from protein degradation. There was increased intensity of the SNS/PN3 260 kDa band in nerves proximal to the site of injury, whereas it was decreased distally, suggesting accumulation at sites of injury; all adult patients had a positive Tinel's sign at the site of nerve injury, indicating mechanical hypersensitivity. Injured nerves from human neonates showed similar results for both channels, but neonate neuromas lacked the SNS2/NaN 180 kDa molecular form, which was strongly present in adult neuromas. The distribution of SNS/PN3 and SNS2/NaN sodium channels in injured human nerves indicates that they represent targets for novel analgesics, and could account for some differences in the development of neuropathic pain in infants.