Differential expression of growth-associated protein (GAP-43) mRNA in rat primary sensory neurons after peripheral nerve lesion: a non-radioactive in situ hybridisation study

An alkaline phosphatase-labelled anti-sense oligodeoxynucleotide probe specific for growth-associated protein messenger RNA (GAP-43 mRNA) was used for non-radioactive in situ hybridisation histochemistry to follow relative changes in GAP-43 mRNA content in lumbar primary sensory neurons (L4-6) after unilateral ligation of the sciatic nerve. In normal dorsal root ganglia (DRG) 16% of neurons expressed GAP-43 mRNA, and these cells belonged to a sub-group of intermediate-sized (32-50 microns diameter) and large (> 50 microns) neurons. The hybridisation signal detected in these cells was weak to moderate. One day after nerve ligature a significant increase in the number of GAP-43 mRNA expressing neurons in the ipsilateral DRG was detected involving particularly the very small (12-20 microns) cells, and small cell population (20-32 microns), though the hybridisation signal was less pronounced in this latter cell group. A significant increase in the cellular content of GAP-43 mRNA was detected in both cell groups when compared to the normal DRG by 2 days after the lesion. At later times (4, 7, and 10 days postinjury) the intermediate-sized and large cell subpopulations also showed an increase in the number of GAP-43 mRNA positive neurons, followed by a significant rise in their content of GAP-43 mRNA. However, they did not reach the same intensity of hybridisation signal as seen in the small and very small neurons. All DRG neurons showed a maximum of GAP-43 mRNA expression by 10 days postsurgery. At longer times there was a slight decrease in the content of GAP-43 mRNA towards 14 days postinjury, but mRNA levels remained elevated up to 28 days after nerve ligature, the longest time point examined in this study. The different onset and levels of GAP-43 gene expression in the rat primary sensory neurons after lesion of their peripheral branch axons further characterize the different subclasses of these cells and may reflect their different involvement in the plastic changes following peripheral nerve injury.     

Intrinsic versus extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurons: The influence of a peripheral nerve graft

The relative contribution of intrinsic growth capacity versus extrinsic growth-promoting factors in determining the capacity of transected dorsal root axons to regenerate long distances was studied. L4 dorsal root axons regenerating into 4-cm peripheral nerve grafts on transected dorsal roots were counted. Few dorsal root myelinated axons regenerated to the distal end of the grafts by 10 weeks unless the sciatic nerve was also crushed. Regeneration of unmyelinated axons was also increased by peripheral lesions. Crush or transection of the dorsal roots without grafting did not alter GAP-43 mRNA expression in L4 dorsal root ganglion (DRG) cells. Grafting a peripheral nerve onto the cut end of an L4 dorsal root doubled the number of DRG cells expressing high levels of GAP-43 mRNA after a delay of several weeks. Peripheral nerve crush at the time of nerve grafting resulted in a very rapid rise in GAP-43 mRNA expression, which then declined to a steady level, twice that of controls, by 7 weeks. Thus, the rapid increase in the number of DRG neurons expressing high levels of GAP-43 mRNA after peripheral but not central axotomy correlates with the regeneration of central axons through nerve grafts. Because GAP-43 mRNA is slowly upregulated in a subpopulation of sensory neurons in response to exposure of their central axons to a peripheral nerve environment, environments favourable for axonal growth may act by increasing the intrinsic growth response of neurons. Lack of intrinsic growth capacity may contribute to the failure of dorsal root axons to regenerate into the spinal cord. © 1996 Wiley-Liss, Inc.     

Constitutive expression of GAP-43 correlates with rapid, but not slow regrowth of injured dorsal root axons in the adult rat

It has been postulated that the neuronal growth-associated protein GAP-43 plays an essential role in axon elongation. Although termination of developmental axon growth is generally accompanied by a decline in expression of GAP-43, a subpopulation of dorsal root ganglion (DRG) neurons retains constitutive expression of GAP-43 throughout adulthood. Peripheral nerve regeneration occurring subsequent to injury of the peripheral axon branches of adult DRG neurons is accompanied by renewed elevation of GAP-43 expression. Lesions of DRG central axon branches in the dorsal roots are also followed by some regenerative growth, but little or no increase in GAP-43 expression above the constitutive level is observed. To determine whether dorsal root axon regeneration occurs only from neurons which constitutively express GAP-43, we have used retrograde fluorescent labeling to identify those DRG neurons which extend axons beyond a crush lesion of the dorsal root. Only GAP-43 immunoreactive neurons supported axon regrowth of 7 mm or greater within the first week. At later times, axon regrowth is seen to occur from neurons both with and without GAP-43 immunoreactivity. We conclude that regeneration of injured axons within the dorsal root is not absolutely dependent on the presence of GAP-43, but that expression of GAP-43 is correlated with a capacity for rapid growth.     

Fate of GAP-43 in ascending spinal axons of DRG neurons after peripheral nerve injury: delayed accumulation and correlation with regenerative potential.

Proteins characteristic of growing axons often fail to be induced or transported along axons that have been interrupted far from their cell bodies in the adult mammalian CNS. Here, we inquire whether long axons in the mammalian CNS can support efficient axonal transport and deposition of one such protein, GAP-43, when the protein is induced in neuron cell bodies. We have used immunocytochemistry to follow the fate of GAP-43 in dorsal column axons ascending the rat spinal cord from dorsal column axons ascending the rat spinal cord from dorsal root ganglion (DRG) neurons, after synthesis of the protein is induced in these cells by peripheral nerve injury. Sciatic nerve lesions do lead to an accumulation of GAP-43 in dorsal column axons derived from the lumbar DRG. However, in distal segments of these CNS axons, accumulation of GAP-43 is apparent only after a delay of 1-2 weeks, in contrast to its rapid accumulation in axon segments within the PNS environment, suggesting that deposition and stabilization of GAP-43 can be limited by local, posttranslational regulation. GAP-43 immunoreactivity subsides to control levels within 8 weeks after crush lesions that permit peripheral axon regeneration, but remains robust 8 weeks after resection lesions that prevent peripheral regeneration. Accumulation of GAP-43 in cervical dorsal column axons after peripheral nerve injury is closely correlated with the ability of these axons to respond to local cues capable of eliciting axon growth (Richardson and Verge, 1986).     

Role of the peripheral benzodiazepine receptor in sensory neuron regeneration

Peripheral benzodiazepine receptor (PBR) expression increases in small dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury. To determine the functional significance of this induction, we evaluated the effects of PBR ligands on rodent sensory axon outgrowth. In vitro, Ro5-4864, a PBR agonist, enhanced outgrowth only of small peripherin-positive DRG neurons. When DRG cells were preconditioned into an active growth state by a prior peripheral nerve injury Ro5-4864 augmented and PK 11195, a PBR antagonist, blocked the injury-induced increased outgrowth. In vivo, Ro5-4864 increased the initiation of regeneration after a sciatic nerve crush injury and the number of GAP-43-positive axons in the distal nerve while PK 11195 inhibited the enhanced growth produced by a preconditioning lesion. These results show that PBR has a role in the early regenerative response of small caliber sensory axons, the preconditioning effect, and that PBR agonists enhance sensory axon regeneration.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging

We have here sought to cross-correlate the expression of immunoreactivities for several neuropeptides, nitric oxide synthase (NOS) and the growth associated protein GAP-43 in subpopulations of dorsal root ganglion (DRG) neurons tagged by the selective markers isolectin B4 and the neurofilament antibody RT97, selective for, respectively, subpopulations of small and large DRG neurons. By use of double- and triple-labeling immunohistochemistry, non-manipulated and sciatic nerve transected young adult rats as well as aged (30-months-old) rats were examined using a confocal microscope equipped with enhanced spectral separation. In young adult rats, the DRG neuron profiles could be divided into three subpopulations (B4 binding (B4+) approximately 50%; RT97-immunoreactive (RT97+) approximately 35%; B4-/RT97- approximately 15%). Calcitonin gene-related peptide (CGRP) is expressed in all three subpopulations. Galanin message-associated peptide (GMAP) colocalize with CGRP (100%) but is not expressed in RT97+ profiles. NOS is present in the RT97- subpopulations and frequently colocalize with CGRP (92%). GAP-43 is expressed in all three DRG subpopulations and colocalize with CGRP (88%), GMAP (38%) and/or NOS (22%). Only very small differences were seen among the young adult rats, implicating that the size of respective subpopulation as well as the expression pattern for neuropeptides, NOS and GAP-43 are fairly stable. Sciatic nerve transection reduced B4-binding but not RT97-like immunoreactivity. Distinct changes in the expression of neuropeptides, NOS and GAP-43 were evident in the DRG subpopulations and, furthermore, the regulatory changes were very similar among the lesioned animals. The relative size of the DRG subpopulations was unaffected by aging, while the expression of neuropeptides was altered showing similarities with the changes induced by axotomy in young adult rats.     

Increased expression and localization of the RNA-binding protein HuD and GAP-43 mRNA to cytoplasmic granules in DRG neurons during nerve regeneration

The neuronal-specific RNA-binding protein, HuD, binds to a U-rich regulatory element of the 3' untranslated region (3' UTR) of the GAP-43 mRNA and delays the onset of its degradation. We have recently shown that overexpression of HuD in embryonic rat cortical cells accelerated the time course of normal neurite outgrowth and resulted in a twofold increase in GAP-43 mRNA levels. Given this evidence, we sought to investigate the involvement of HuD during nerve regeneration. It is known that HuD protein and GAP-43 mRNA are expressed in the dorsal root ganglia (DRG) of adult rat and that GAP-43 is upregulated in DRG neurons during regeneration. In this study, we examined the expression patterns and levels of HuD and GAP-43 mRNA in DRG neurons following sciatic nerve injury using a combination of in situ hybridization, immunocytochemistry, and quantitative RT-PCR. GAP-43 and HuD expression increased in the ipsilateral DRG during the first 3 weeks of regeneration, with peak values seen at 7 days postcrush. At this time point, the levels of HuD and GAP-43 mRNAs in the ipsilateral DRG increased by twofold and sixfold, respectively, relative to the contralateral DRG. Not only were the temporal patterns of expression of HuD protein and GAP-43 mRNA similar, but also they were found to colocalize in the cytoplasm of DRG neurons. Moreover, both molecules were distributed in cytoplasmic granules containing ribosomal RNA. In conclusion, our results suggest that HuD is involved in the upregulation of GAP-43 expression observed at early stages of peripheral nerve regeneration.     

Peripheral axotomy affects nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthases in the spinal cord of the rabbit

Using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry combined with radioassay of calcium-dependent NOS activity, we examined the occurrence of NADPHd staining and NOS immunoreactivity (NOS-IR) in the dorsal root ganglia (DRG) neurons, dorsal root afferents, and axons projecting via gracile fascicle to gracile nucleus 14 days after unilateral sciatic nerve transection in the rabbit. Mild to moderate NADPHd staining and NOS-IR appeared in a large number of small and medium-sized to large neurons in the ipsilateral L4-L6 DRG, accompanied by enhanced NOS-IR of thick myelinated fibers in the ipsilateral L4-L6 dorsal roots. A noticeable increase in the density of punctate NADPHd staining occurred throughout laminae I-IV in the ipsilateral medial part of the dorsal horn in L4-L6 segments. Concurrently, a statistically significant decrease in the number of small NADPHd-exhibiting neurons in laminae I-II and, in contrast to this, a statistically significant increase of medium-sized to large NADPHd-stained somata in the ipsilateral laminae III-VI of L4-L6 segments were found. A detailed compartmentalization of L4-L6 segments into gray and white matter regions disclosed substantially increased catalytic NOS activity and inducible NOS mRNA levels in the dorsal horn and dorsal column ipsilaterally to the peripheral injury. A noticeable increase in the number of thick myelinated NADPHd-exhibiting and NOS-IR axons was noted in the ipsilateral gracile fascicle, terminating in dense, punctate NADPHd staining in the neuropil of the gracile nucleus. These observations indicate that the de novo-synthesized NOS in the lesioned primary afferent neurons resulting after sciatic nerve transection may be involved in an increase in NADPHd staining and NOS-IR in the ipsilateral dorsal roots and dorsal horn of L4-L6 segments, whence NOS could be supplied to ascending axons of the gracile fascicle.     

GAP-43 mRNA and Calcitonin Gene-Related Peptide mRNA Expression in Sensory Neurons Are Increased Following Sympathectomy

Sympathectomy has been shown to result in an increased density of fibers immunoreactive for sensory peptides in peripheral targets innervated by both sensory and sympathetic neurons, providing evidence for functional interactions between sympathetic and sensory systems. These findings provided the background for examining the hypothesis that axonal outgrowth is induced from sensory neurons following sympathectomy. We examined the expression of GAP-43 mRNA, a specific marker for axonal outgrowth, in cervical (C3, C7, C8) and thoracic (T1, T2) dorsal root ganglia (DRG) of the rat following bilateral removal of the superior cervical ganglion, to assess whether the described increases in peptidergic afferent fibers reflected axonal outgrowth. In situ hybridization was used with ³⁵S labeled riboprobes complementary to GAP-43 mRNA, and to calcitonin gene-related peptide (CGRP) mRNA, a marker for a major subset of thin-fiber sensory neurons. The density of GAP-43 mRNA nearly doubled by 18 h following sympathectomy and reached a threefold increase by 3 days. By 45 days following surgery, the GAP-43 mRNA level was still nearly twice that of normal animals. CGRP immunoreactivity was also examined: the density of fibers in the iris and cornea of sympathectomized animals was considerably greater from two weeks to 45 days following surgery, than in sham-operated controls. Concomitantly, there was a slight but significant increase in CGRP mRNA expression in T1 and C3 DRG 14 days postsympathectomy. Quantitative computerized image analysis demonstrated that GAP 43 mRNA expression in sympathectomized animals was 1.5 times greater in medium-sized DRG neurons and almost fourfold greater in small DRG neurons than in control rats. These results indicate that sympathetic denervation elicits axonal outgrowth in the population of sensory neurons that give rise to the small unmyelinated and thinly myelinated axons of peripheral nerves. Copyright © 1997 Elsevier Science Inc.     

Sympathetic sprouting in the dorsal root ganglion after spinal nerve ligation: evidence of regenerative collateral sprouting

It is well documented that there is an increase in the number of sympathetic fibers within the dorsal root ganglion (DRG) after a peripheral nerve injury. The present study examined the numbers and distribution of sympathetic fibers in the DRG and their sprouting routes by utilizing various surgical manipulations and retrograde tracing and immunohistochemical staining methods in spinal nerve-ligated neuropathic rats. The appearance of many double immunostained fibers with antibodies to tyrosine hydroxylase (TH) and growth associated protein-43 (GAP-43) in the L5 DRG 1 week after L5 spinal nerve ligation, indicated sprouting of sympathetic fibers. The confined location of early sprouting sympathetic fibers in the distal half of the L5 DRG confirmed that sprouting fibers come primarily from the injured spinal nerve. A second cut proximal to the previously ligated L5 spinal nerve -- a process which would transect the regenerating sympathetic fibers extending from the injury site -- did not change the density of sympathetic fibers in the L5 DRG. When retrograde tracers (fast blue and diamidino yellow) were injected into the L5 spinal nerve and DRG, respectively, the number of double-labeled sympathetic postganglionic neurons was greatly increased after spinal nerve ligation, suggesting the increased number of sympathetic neurons projecting to both the spinal nerve and DRG. All these results indicate that many sympathetic fibers in the DRG are regenerating branches that are sprouting from the proximal part of the injured spinal nerve (regenerative collateral sprouting).     

Either nitric oxide or nerve growth factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development

During early embryonic (E12) development almost all dorsal root ganglion (DRG) neurons express the neuronal isoform of nitric oxide synthase (nNOS). At this stage, the axons of these neurons are rudimentary and have not made contact with peripheral tissue targets. As their axons establish contact with peripheral targets such as the skin, the number of neurons expressing nNOS decrease that correspond to increased immunoreactivity for nerve growth factor (NGF) in the skin, and its high affinity receptor, tyrosine kinase A (trkA) in both skin and DRG neurons. During late postnatal development, very few DRG neurons express nNOS; however, axotomy or NGF deprivation of cultured DRG neurons induce nNOS and NOS blockade causes neuronal death. In contrast, NGF-deprived embryonic and neonatal DRG neurons die by apoptosis, while NOS blockade has no effect. Overall, these observations suggest that NGF and nitric oxide (NO) interact during embryonic and postnatal development to facilitate neuronal selection and survival. The roles of NO, NGF and its receptor trkA in DRG neurons during different stages of development are discussed.     

Cavernous nerve injury elicits GAP-43 mRNA expression but not regeneration of injured pelvic ganglion neurons

Recovery of erectile dysfunction after cavernous nerve injury takes a long period. To elucidate this mechanism, unilateral cavernous nerve of male rat was cut, and the expression level of a nerve regeneration marker, the growth associated protein-43 (GAP-43) mRNA was evaluated by in situ hybridization and RT-PCR. While GAP-43 mRNA expression was transiently increased in the injured neurons of the major pelvic ganglion (MPG) at 7 days after nerve injury, continuous increase of GAP-43 mRNA was observed in the contralateral MPG from 7 days to 6 months after the nerve injury. Histochemical double-labeling studies for either neuronal NOS (nNOS) or tyrosine hydroxylase (TH) and the GAP-43 mRNA expression demonstrated that in injured MPG the transient up-regulation of GAP-43 mRNA was mainly seen in nNOS negative and/or TH positive neurons, suggesting non-parasympathetic post-ganglionic neurons, and also demonstrated that in contralateral MPG GAP-43 mRNA positive neurons were gradually increased in nNOS positive but TH negative neurons, suggesting parasympathetic post-ganglionic neurons. When a retrograde tracer Fluorogold (FG) was injected into the penile crus 7 days before histological experiments, FG-positive neurons were, if any, hardly seen in nNOS-positive neurons of the injured MPG for at least 6 months, whereas numerous FG-positive cells were seen in nNOS-positive neurons of the contralateral MPG. These results suggest that post-ganglionic projecting neurons of the intact side, which express increased GAP-43 mRNA, would be most likely to contribute to the recovery of the erectile function after unilateral cavernous nerve injury possibly by a plastic change such as nerve sprouting.     

Nogo-A expression in the intact and injured nervous system

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Treatment of chronically injured spinal cord with neurotrophic factors stimulates betaII-tubulin and GAP-43 expression in rubrospinal tract neurons

Exogenous neurotrophic factors provided at a spinal cord injury site promote regeneration of chronically injured rubrospinal tract (RST) neurons into a peripheral nerve graft. The present study tested whether the response to neurotrophins is associated with changes in the expression of two regeneration-associated genes, betaII-tubulin and growth-associated protein (GAP)-43. Adult female rats were subjected to a right full hemisection lesion via aspiration of the C3 spinal cord. A second aspiration lesion was made 4 weeks later and gel foam saturated in brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), or phosphate-buffered saline (PBS) was applied to the lesion site for 60 min. Using in situ hybridization, RST neurons were examined for changes in mRNA levels of betaII-tubulin and GAP-43 at 1, 3, and 7 days after treatment. Based on analysis of gene expression in single cells, there was no effect of BDNF treatment on either betaII-tubulin or GAP-43 mRNA expression at any time point. betaII-Tubulin mRNA levels were enhanced significantly at 1 and 3 days in animals treated with GDNF relative to levels in animals treated with PBS. Treatment with GDNF did not affect GAP-43 mRNA levels at 1 and 3 days, but at 7 days there was a significant increase in mRNA expression. Interestingly, 7 days after GDNF treatment, the mean cell size of chronically injured RST neurons was increased significantly. Although GDNF and BDNF both promote axonal regeneration by chronically injured neurons, only GDNF treatment is associated with upregulation of betaII-tubulin or GAP-43 mRNA. It is not clear from the present study how exogenous BDNF stimulates regrowth of injured axons.     

Expression of three forms of nitric oxide synthase in peripheral nerve regeneration

Nitric oxide (NO) is a short‐lived molecule with messenger and cytotoxic functions in nervous, cardiovascular, and immune systems. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis, exists in three different forms: the neuronal (nNOS), present in discrete neuronal populations; the endothelial (eNOS), present in vascular endotheliun, and the inducible isoform (iNOS), expressed in various cell types when activated, including macrophages and glial cells. In this study, we have investigated the possible involvement of NO in Wallerian degeneration and the subsequent regeneration occurring after sciatic nerve ligature, using histochemistry and immunocytochemistry for the three NOS isoforms, at different postinjury periods. Two days after lesion, the three NOS isoforms are overexpressed, reaching their greatest expression during the second week. nNOS is upregulated in dorsal root ganglion neurons, centrifugally transported and accumulated in growing axons. eNOS is overexpressed in vasa nervorum of the distal stump and around ligature, and iNOS is induced in recruited macrophages. These findings indicate that different cellular sources contribute to maintain high levels of NO at the lesion site. The parallelism between NOS inductions and well‐known repair phenomena suggests that NO, acting in different ways, may exert a beneficial effect on nerve regeneration. J. Neurosci. Res. 55:198–207, 1999. © 1999 Wiley‐Liss, Inc.     

Cell body response to injury in motoneurons and primary sensory neurons of a mutant mouse,ola (Wld), in which wallerian degeneration is delayed

We examined the response to axon injury in the facial motoneurons and dorsal root ganglion (DRG) neurons of C57BL/Ola (Wld) mice, compared with the responses of C57BL/6J mice. The peripheral nerves of Ola mutants undergo remarkably slowed and muted Wallerian degeneration after injury. The increase in GAP-43 mRNA levels in facial motoneurons and DRG neurons was similar in both strains of mice, as was the initial decrease in medium-weight neurofilament (NFM) mRNA in facial motoneurons, and the increase in JUN immunoreactivity in both types of neurons. However, the subsequent recovery to normal low levels of JUN and GAP-43 mRNA expression and high levels of NFM mRNA was delayed in Ola motoneurons. We ascribe this delay to the slow regeneration and target reinnervation of facial axons in the Ola mice. These results show that absence of rapid Wallerian degeneration does not affect the initial cell body response to axonal injury. They also provide further evidence that restoration of normal levels of expression of GAP-43 and NFM mRNAs is dependent on target reinnervation and/or trophic factors provided by the distal nerve, Impaired regeneration in the Ola mouse does not seem to be a consequence of a defective cell body response to injury, and our results illustrate the general principle that, even if there is a vigorous cell body response to injury, normal axonal regeneration requires the additional provision of a favorable environment for growth. © 1995 Wiley-Liss, Inc.     

Growth-associated protein-43 expression in cocultures of dorsal root ganglion neurons and skeletal muscle cells with different neurotrophins

Introduction: Both target skeletal muscle (SKM) cells and neurotrophins (NTs) are essential for the maintenance of neuronal function and nerve–muscle communication. The effects of different NTs and SKM cells on growth-associated protein-43 (GAP-43) expression in dorsal root ganglion (DRG) neurons have not been clarified. Methods: The morphological relationship between DRG neurons and SKM cells in neuromuscular cocultures was observed by scanning electron microscopy. The levels of GAP-43 and its mRNA were determined after administration of different NTs. Results: DRG neurons demonstrated dense neurite outgrowth in the presence of NTs. Distinct NTs promoted GAP-43 and its mRNA expression in neuromuscular cocultures of DRG neurons and SKM cells. Conclusions: These results offer new clues for a better understanding of the effects of distinct NTs on GAP-43 expression in DRG sensory neurons in the presence of target SKM cells and implicate NTs and target SKM cells in DRG neuronal regeneration. Muscle Nerve 47: 909–915, 2013