Differing effects on superior cervical ganglia in neonatal mice produced by antisera to nerve growth factors from mice and snakes.

Rabbit or horse antisera to Nerve Growth Factors from mouse salivary glands and the venoms of five snakes were injected into neonatal mice. The mice were killed 10 days later and the superior cervical ganglia removed, weighed and examined histologically. Treatment with antisera to the snake Nerve Growth Factors had no effect on ganglion weight, maximum neuronal density or mean neurone diameter. This was true even for the one antiserum that in vitro showed a weak cross-reactivity with the mouse antigen. In contrast, treatment with the antiserum to mouse Nerve Growth Factor produced a partial destruction of the superior cervical ganglia (immunosympathectomy). The weights of the ganglia in animals treated for the first 5 days post partum with increasing volumes (a total of 0.5 ml) of antiserum to the mouse factor fell by some 80% the total neurone number by approx 50% the maximum neurone density by 40% and the mean neurone dia. by 17%. The effect was found to be dose dependent.It is considered that caution is required in extrapolating results from in vitro studies on Nerve Growth Factor to the situation obtaining in vivo and that the inability of snake antisera to produce immunosympathectomy in neonatal mice may result from differences in the antigenic determinants of the mouse and snake Nerve Growth Factors. The marked effect of antiserum to the mouse salivary gland factor in neonatal mice reported by earlier workers has been confirmed. No single explanation, however, can be given for the differences in the reduction in neurone numbers found in the present and previous studies. Furthermore, it is concluded that neither the previous nor the present studies afford evidence that the antiserum directly causes neuronal death.     

Effect of sialectomy on the superior cervical ganglion sympathetic neurons in young adult and aged mice.

The dependence of superior cervical ganglion (SCG) adrenergic neurons on their target organ submandibular salivary gland containing high concentrations of nerve growth factor was studied in adult and aged male mice. The submandibular salivary glands were removed (sialectomy) either uni- or bilaterally, and the SCG were studied by fluorescence microscopy and histochemically. Catecholamine fluorescence and tyrosine hydroxylase immunoreactivity decreased after sialectomy, suggesting reduced noradrenaline production. Neuronal density was lower in the aged controls than in the young controls. In both age groups, sialectomy reduced the density of catecholamine-producing neurons. In the mouse SCG, there was remarkable heterogeneity in the size of neuronal somata. In aged control mice there was a greater number of large-size neurons than in young adult control mice. Six weeks postoperatively, no large catecholamine-producing neurons could be observed in the ganglia. Yellow autofluorescent lipopigments accumulated with age in the adrenergic neurons. Sialectomy increased the accumulation of lipopigments in both young and aged neurons. Sialectomy resulted in (a) reduced catecholamine fluorescence, (b) reduced tyrosine hydroxylase immunoreactivity, (c) reduced number of catecholamine neurons, (d) increased autofluorescent lipopigment. Ageing resulted in (a) reduced number of neurons, (b) increased ratio of large to small neurons, (c) increased autofluorescent lipopigment. Alterations after sialectomy were more detrimental in the aged ganglia than in the young adult ganglia. The discontinuation of the retrograde supply of nerve growth factor may contribute to these alterations.     

Prolonged exposure to elevated levels of endogenous nerve growth factor affects the morphological and neurochemical features of sympathetic neurons of postnatal and adult mice

It is well documented that acute increases of target-derived nerve growth factor affect the morphological and neurochemical features of post-ganglionic sympathetic neurons. It has yet to be determined, however, whether similar changes are still evident after prolonged exposure to increased levels of endogenous nerve growth factor. Using a transgenic line of mice which overexpresses nerve growth factor in the brain commencing after the first week of postnatal life and continuing into adulthood, we have shown previously that sympathetic axons sprout into the nerve growth factor-rich cerebellum of these animals; no such axons are seen in the cerebellum of age-matched wild type animals. The aim of this study was to examine and characterize the effects of chronically elevated levels of endogenous nerve growth factor on sympathetic neurons of the superior cervical ganglion. In comparison to adult wild type mice, adult transgenic animals possessed hypertrophied ganglia which displayed both an increase in sympathetic somal size and a decrease in their density. At the electron microscope level, sympathetic somata of the adult transgenic animals had numerous electron-dense lysosome-like structures in the cytoplasm, as compared to that seen in the sympathetic somata of adult wild type animals. Immunodetection of nerve growth factor in the sympathetic somata revealed that the staining intensity in postnatal (day 28) transgenic mice was greater than that in age-matched wild type mice. By adulthood, however, such differences in the intensities of nerve growth factor immunostaining were no longer evident. In situ hybridization analyses of trkA receptor messenger RNA revealed that levels of expression among somata of similar sizes were comparable between the transgenic and wild type neuronal populations of both postnatal day 28 and adult animals. A small subpopulation of sympathetic somata in postnatal transgenic mice displayed a marked increase in p75NTR messenger RNA expression in comparison to somata of a similar size in age-matched wild type animals. By adulthood, the proportion of sympathetic somata in the transgenic animals possessing elevated levels of p75NTR messenger RNA expression had increased. These results reveal that chronically elevated levels of endogenous nerve growth factor in the postnatal and adult mouse brain can induce both structural and neurochemical remodelling of sympathetic neurons. The preferential increase in p75NTR messenger RNA expression among sympathetic somata of transgenic mice may be required for their growth of collateral axons into the nerve growth factor-rich cerebellum during postnatal development and may facilitate the increased immunodetection of nerve growth factor on these aberrant sympathetic axons in adult transgenic animals.     

Effects of nerve growth factors from mouse salivary glands and snake venom on the sympathetic ganglia of neonatal and developing mice.

1. Quantitative histological analysis has been made of the effects of nerve growth factor (NGF) from mouse submaxillary gland and from the venom of Vipera russelli on superior cervical ganglia of neonatal mice. 2. The hypertrophic and hyperplastic effects reported by other workers have been confirmed. 3. The hypertrophic effect arises from an increase in the rate at which the sympathetic neurones attain their mature size. The size at maturity is never exceeded. 4. The hyperplastic effect arises from an increase in the rate of production of neurones from less differentiated cells. In the developmental period, the number of neurones can exceed that found at maturity. If injection of NGF is discontinued the excess neurones disappear. 5. If injection of NGF is continued to maturity, the excess number of neurones is maintained.     

The effects of postganglionic axotomy and nerve growth factor on the superior cervical ganglia of developing mice

Summary Sectioning of the two major outflows from the superior cervical ganglia in two week mice results in a marked drop in the number of neurons within one week of operation and a smaller drop over the following two weeks. In animals receiving daily injections of nerve growth factor (NGF), the effect of axotomy is modified. One week after axotomy, the number of neurons in the axotomized ganglia is approxima the same in NGF treated animals as in the control, sham operated ganglia. Over the next two weeks, however, the cell death that results from axotomy is no longer prevented by treatment with NGF. The normal, hyperplastic response to NGF appears to occur independently of the cell reaction caused by axotomy.     

Cell division in the developing sympathetic nervous system

Summary Cell division in the rat superior cervical and stellate ganglia has been followed from 6 days before birth until 14 days after birth by [³H] thymidine autoradiography. Neuronal division ceased at the postnatal day 4 in superior cervical ganglia and postnatal day 3 in stellate ganglia, whereas division of non-neuronal cells continued throughout the entire period studied. Dividing cells in both ganglia were predominantly neuronal before birth and non-neuronal after the postnatal day 3.Nerve growth factor treatment resulted in an increase in the number of neurons in the ganglia when given either over the first four postnatal days while neuronal division was still occurring or from postnatal days 6–8 after neuronal division had ceased. The increase in neuron numbers was not due to increased neuronal division and was apparently due to an increased survival rate of differentiated neurons. An increase in the rate of division of non-neuronal cells accompanied the administration of nerve growth factor.     

Some studies on the mechanism of action of antibodies to nerve growth factor.

The effects of antibodies to the nerve growth factor from mouse salivary gland were examined in vitro and in vivo. Treatment of explants of receptive ganglia with antibody and complement did not produce cell damage as judged by the ability of the tissue to respond to nerve growth factor. New-born mice experimentally depleted of or genetically deficient in key complement components were susceptible to the action of the antiserum.These results show that the effect of the antibody is independent of complement and are consistent with the view that it acts by neutralization of endogenous nerve growth factor.     

Neonatal synapse elimination in the rat submandibular ganglion: effect of retarded target growth

1. We have studied synapse elimination in the submandibular ganglion of neonatal rats to determine the effects of retarded target growth on synaptic development. Neurons of this ganglion provide parasympathetic innervation to the submandibular and sublingual salivary glands. 2. Ligating the main salivary ducts 2-4 days after birth at a point where nerve fibers were not damaged reduces gland weight by 55% during the 2nd wk after birth and 80% by adulthood. 3. In control animals, the average number of preganglionic inputs/neuron normally declines steadily during the first few weeks after birth, before stabilizing during the 5th wk at the control adult level. Between birth and adulthood, the number of ganglionic neurons increases by 150%. 4. Ganglia from duct-ligated animals showed an acceleration in the process of synapse elimination. Input number in experimental ganglia reached the control adult level during the 3rd wk after birth. This acceleration is confined solely to ganglia that innervate the underdeveloped glands. 5. The loss of inputs was not further enhanced by prolonged target atrophy. Thus average input numbers to neurons of 5th wk or adult experimental ganglia were not different from age-matched control values. 6. No differences from control values were seen in most cases for resting potentials, input resistances, or cell size. However, the increase in neuron number was retarded in experimental animals, and the number of synapses/neuronal profile was reduced in the adult animals. 7. Thus subnormal target growth leads to an acceleration in the process of synaptic elimination in neonatal rats. This acceleration may be mediated by alterations in the level of trophic factors emanating from the target.     

Nerve growth factor-like activity in the rat pituitary intermediate lobe

Nerve growth factor (NGF)-like immunoreactivity was demonstrated in the rat pituitary intermediate lobe by indirect immunofluorescence method using antisera specific to beta-NGF isolated from adult male mouse submaxillary salivary gland. Co-culture of frozen or fresh intermediate lobes with newborn rat superior cervical ganglion resulted in marked fiber growth from the ganglion, which was totally inhibited by NGF antiserum, suggesting the presence in situ and secretion in vitro of biologically active pituitary NGF. Pituitary stalk transection caused decrease in both the NGF immunoreactivity and biological activity. These findings suggest that pituitary NGF level is under neural regulation.     

Effects of conditioned media on extension of substance P-immunoreactive neurites from cultured mouse sensory ganglia

Media conditioned by heart or stomach smooth muscle cells stimulated elongation of neurites with substance P-immunoreactivity from explants of mouse dorsal root ganglia in culture, but nerve growth factor (NGF) had no effect. However, NGF is known to be needed for sensory neurone survival; at least two types of factor may therefore be required for the normal development of substance P-containing sensory neurones.     

Generation and phenotypic characterization of a galanin overexpressing mouse

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.     

The role of post-synaptic neurones in the biochemical maturation of presynaptic cholinergic nerve terminals in a mouse sympathetic ganglion

1. The role of post-synaptic adrenergic neurones in the biochemical maturation of presynaptic cholinergic nerve terminals has been investigated in mouse superior cervical ganglion in vivo.2. Selective destruction of ganglion adrenergic neurones chemically, with 6-hydroxydopamine (6-OH-DA), or immunologically, with nerve growth factor antiserum (NGF-antiserum) prevented the normal maturation of choline acetyl transferase (ChAc) activity in presynaptic endings during development. Enzyme activity remained depressed for at least 2 months.3. 6-OH-DA treatment failed to alter ChAc activity in the developing duodenum or diaphragm, organs in which cholinergic fibres do not synapse with adrenergic neurones, suggesting that destruction of post-synaptic neurones per se inhibited presynaptic maturation.4. Similarly, NGF-antiserum, which does not destroy adrenergic neurones in the adult did not alter ChAc activity in adult mouse ganglia.5. These observations suggest that post-synaptic adrenergic neurones regulate the biochemical development of presynaptic cholinergic nerve terminals.     

Age-related structural and neurochemical changes of the human superior cervical ganglion.

The aim of this study was to investigate age-related morphological and neurochemical changes in the human superior cervical ganglion (SCG). Thirty-seven superior sympathetic human cervical ganglia of young, adult, and aged subjects were examined using morphometric analysis, biotin-streptavidin immunohistochemistry for detecting neurofilament, myelin protein, protein gene product 9.5, nerve growth factor receptor p75 in sympathetic neurons and nerve fibers. Morphometric parameters of neurons (area, long and short axis, shape factor of the neuron body, nucleus, cytoplasm, and lipofuscin) were investigated in every sixth serial section of the ganglion. Seven hundred neurons with clearly visible nuclei were measured in each studied group. The present study showed that human SCG of older subjects had larger areas of neuron body, cytoplasm and nucleus, a lower shape factor, an increased amount of lipofuscin, and a greater number of large-size neurons, as compared to SCG obtained from young subjects. Neuronal cytoskeletal alterations manifested themselves through a decreased number of neurofilament-positive neurons were detected in old human SCG. The amount of myelinated fibers decreased with age, although the amount of myelinated fibers in the young and the adult subjects varied from few to a moderate number. PGP 9.5 immunoreactivity varied in different age groups. A marked reduction of nerve growth factor receptor p75 in old human sympathetic neurons was detected. In conclusion, the findings of this study confirm age-related morphological changes in the human SCG. Structural neuronal changes may influence the deterioration of neuronal functional capacity, neuronal plasticity, and regenerative characteristics.     

Pathologic alterations in pre- and postsynaptic elements in aged mouse sympathetic ganglia

Summary Dysfunction of the sympathetic autonomic nervous system is an increasingly recognized, although poorly understood, complication of increasing age in experimental animals and man. In this study of young adult (4–6 months old) and aged (12– 24 months old) mice we have examined the ultrastructural appearance of perikarya, dendritic processes, preterminal axons, and synapses in selected sympathetic ganglia as well as the three-dimensional structure of the dendritic arborizations of principal sympathetic neurons using intracellular injections of Lucifer Yellow. Ultrastructural examination demonstrated numerous markedly enlarged presynaptic terminal axons and synapses which distorted the contours of perikarya and dendrites of neurons within the prevertebral celiac/superior mesenteric and paravertebral superior cervical and stellate sympathetic ganglia of aged mice. Dilated preterminal axons had the distinctive ultrastructural appearance of neuroaxonal dystrophy, a pathologic process described in a wide variety of clinical and experimental entities. Dystrophic axons were identical in ultrastructural appearance in young and old animals, differing only in frequency. A distinctive type of ultrastructural alteration, characterized by markedly distended neurites containing numerous vacuoles, was confined to the superior cervical ganglia and also increased in frequency with aging. Although many intraganglionic vacuolated processes disappeared with surgical interruption of the cervical sympathetic trunk, which contains the preganglionic axons innervating the superior cervical ganglia, others persisted. In addition, the presence in some processes of admixed ribosomes, lipofuscin, or continuity with the cell body indicated that numerous neuritic alterations within aged sympathetic ganglia were likely of dendritic origin. Intracellular injections of Lucifer Yellow into principal sympathetic neurons demonstrated that the dendritic arborizations of the celiac/superior mesenteric ganglia neurons of young adult mice were significantly more complex and extensive than those of the superior cervical ganglia. Sympathetic neurons of aged superior cervical ganglia, but not superior mesenteric ganglia, appeared significantly smaller with regard to total dendritic length, extent, and branching when compared to those of young animals. In the aged superior cervical ganglia, short, stunted dendritic processes also exhibited large, focal, often multiple, swellings, a phenomenon infrequently observed in the superior cervical ganglia of young animals. The celiac/ superior mesenteric ganglia of aged or young adult mouse failed to exhibit comparable dendritic swellings. These observations may provide a neuropathological basis for understanding age-related changes in autonomic function observed in animals and man, and provide a model system in which age-related pathogenetic mechanisms resulting in neuroaxonal dystrophy and dendritic alterations can be studied.     

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.     

Biological demonstration of nerve growth factor in the rat pituitary gland

Explants of anterior, intermediate and posterior pituitary lobes of rats at various ages evoked an intense nerve fibre outgrowth when co-cultured with neonatal sympathetic superior cervical ganglia in a collagen gel medium. Freezing and thawing of pituitary lobes, prior to culture, did not abolish their growth-promoting effect. Since the addition of antiserum to nerve growth factor in the culture medium inhibited totally the stimulative action of both fresh and frozen pituitary explants it is suggested that the stimulation is mediated by nerve growth factor or an immunologically related molecule. Based on the present results it is concluded that nerve growth factor is synthesized by both the anterior, intermediate and posterior lobes throughout the postnatal period, as well as in adult rats. The function of nerve growth factor in the pituitary gland is discussed.     

Distribution of mouse cytomegalovirus in organs of white mice experimentally infected by natural route

One, 10, 21-day-old and adult mice were inoculated by peroral and/or intranasal routes with mouse cytomegalovirus (MCMV). In animals surviving generalized infection, the virus could be demonstrated in salivary glands up to 123 days postinfection (p.i.). In mouse females which had eaten their infected and diseased offspring, the virus was detectable in salivary glands up to day 121, p.i. On day 16 p.i., the virus was present in salivary glands, lungs and kidneys of mice of different age groups, but no virus was recovered from their Gasserian ganglia. These results were compared with those obtained after infection with murine alpha herpesvirus.     

Retrograde transport of horseradish peroxidase in sensory and adrenergic neurons following injection into the anterior eye chamber

Summary Horseradish peroxidase (HRP) was injected into the anterior eye chamber of rats and mice and frozen sections from both superior cervical and trigeminal ganglia were incubated to demonstrate neurons accumulating the tracer by retrograde axonal transport. Labelled cells were observed only in ganglia ipsilateral to the HRP injection. Within the trigeminal ganglion, peroxidase-containing neurons were restricted to the medial ophthalmic area, whereas labelled cells in the superior cervical ganglion were more widely distributed.With the use of a new and more sensitive technique for the demonstration of HRP in neurons, it was possible to show retrograde transport also of small amounts of peroxidase injected into the anterior eye chamber. In addition, this technique enabled identification of the central and peripheral processes of neurons in the trigeminal ganglion and the dendrites and axons of sympathetic ganglion cells.The rate of retrograde HRP transport in rats was calculated to approximately 4–5 mm/h for both sensory and adrenergic nerves, which is consistent with previous estimates for this protein. It differs from the transport rate reported for nerve growth factor (NGF) and macromolecular toxins in sensory and adrenergic nerves of the same species. These rates were, however, obtained with a different method and in a different population of sensory neurons and are, therefore, not directly comparable.After treatment with 6-hydroxydopamine the number of HRP-labelled cells in the superior cervical ganglion was significantly reduced compared to controls. Cell counts from trigeminal ganglia showed no significant difference between controls and treated animals.     

NGF enhances neurite regeneration from nerve-transected terminals of young adult and aged mouse dorsal root ganglia in vitro

Dorsal root ganglia with nerve fibers, dissected from 3-month-old young adult mice and 26-month-old aged mice, were embedded in collagen gel. In the young adult mice regenerating neurites appeared from nerve-transected terminals after 12-24 h in culture, regardless of the length of fibers, but in the aged mice, regenerating neurites were first seen after 12-48 h. Administration of nerve growth factor to the explants markedly enhanced the growth of regenerating neurites from both the young adult and aged mice.     

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.