Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord

The regeneration capacity of spinal cord axons is severely limited. Recently, much attention has focused on promoting regeneration of descending spinal cord pathways, but little is known about the regenerative capacity of ascending axons. Here we have assessed the ability of neurotrophic factors to promote regeneration of sensory neurons whose central axons ascend in the dorsal columns. The dorsal columns of adult rats were crushed and either brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) or a vehicle solution was delivered continuously to the lesion site for 4 weeks. Transganglionic labelling with cholera toxin beta subunit (CTB) was used to selectively label large myelinated Abeta fibres. In lesioned rats treated with vehicle, CTB-labelled fibres were observed ascending in the gracile fasciculus, but these stopped abruptly at the lesion site, with no evidence of sprouting or growth into lesioned tissue. No CTB-labelled terminals were observed in the gracile nucleus, indicating that the lesion successfully severed all ascending dorsal column axons. Treatment with BDNF did not promote axonal regeneration. In GDNF-treated rats fibres grew around cavities in caudal degenerated tissue but did not approach the lesion epicentre. NT-3, in contrast, had a striking effect on promoting growth of lesioned dorsal column axons with an abundance of fibre sprouting apparent at the lesion site, and many fibres extending into and beyond the lesion epicentre. Quantification of fibre growth confirmed that only in NT-3-treated rats did fibres grow into the crush site and beyond. No evidence of terminal staining in the gracile nucleus was apparent following any treatment. Thus, although NT-3 promotes extensive growth of lesioned axons, other factors may be required for complete regeneration of these long ascending projections back to the dorsal column nuclei. The intrathecal delivery of NT-3 or other neurotrophic molecules has obvious advantages in clinical applications, as we show for the first time that dorsal column axonal regeneration can be achieved without the use of graft implantation or nerve lesions.     

Effect of lumbar 5 ventral root transection on pain behaviors: a novel rat model for neuropathic pain without axotomy of primary sensory neurons

A peripheral nerve injury often causes neuropathic pain but the underlying mechanisms remain obscure. Several established animal models of peripheral neuropathic pain have greatly advanced our understanding of the diverse mechanisms of neuropathic pain. A common feature of these models is primary sensory neuron injury and the commingle of intact axons with degenerating axons in the sciatic nerve. Here we investigated whether neuropathic pain could be induced without sensory neuron injury following exposure of their peripheral axons to the milieu of Wallerian degeneration. We developed a unilateral lumbar 5 ventral root transection (L5 VRT) model in adult rats, in which L5 ventral root fibers entering the sciatic nerve were sectioned in the spinal canal. This model differs from previous ones in that DRG neurons and their afferents are kept uninjured and intact afferents expose to products of degenerating efferent ventral root fibers in the sciatic nerve and the denervated muscles. We found that the L5 VRT produced rapid (24 h after transection), robust and prolonged (56 days) bilateral mechanical allodynia, to a similar extent to that in rats with L5 spinal nerve transection (L5 SNT), cold allodynia and short-term thermal hyperalgesia (14 days). Furthermore, L5 VRT led to significant inflammation as demonstrated by infiltration of ED-1-positive monocytes/macrophages in the DRG, sciatic nerve and muscle fibers. These findings demonstrated that L5 VRT produced behavioral signs of neuropathic pain with high mechanical sensitivity and thermal responsiveness, and suggested that neuropathic pain can be induced without damage to sensory neurons. We propose that neuropathic pain in this model may be mediated by primed intact sensory neurons, which run through the milieu of Wallerian degeneration and inflammation after nerve injury. The L5 VRT model manifests the complex regional pain syndrome in some human patients, and it may provide an additional dimension to dissect out the mechanisms underlying neuropathic pain.     

Inflammation near the nerve cell body enhances axonal regeneration

Although crushed axons in a dorsal spinal root normally regenerate more slowly than peripheral axons, their regeneration can be accelerated by a conditioning lesion to the corresponding peripheral nerve. These and other observations indicate that injury to peripheral sensory axons triggers changes in their nerve cell bodies that contribute to axonal regeneration. To investigate mechanisms of activating nerve cell bodies, an inflammatory reaction was provoked in rat dorsal root ganglia (DRG) through injection of Corynebacterium parvum. This inflammation enhanced regeneration in the associated dorsal root, increasing 4-fold the number of regenerating fibers 17 d after crushing; peripheral nerve regeneration was not accelerated. A milder stimulation of dorsal root regeneration was detected after direct injection of isogenous macrophages into the ganglion. It is concluded that changes favorable to axonal regeneration can be induced by products of inflammatory cells acting in the vicinity of the nerve cell body. Satellite glial cells and other unidentified cells in lumbar DRG were shown by thymidine radioautography to proliferate after sciatic nerve transection or injection of C. parvum into the ganglia. Intrathecal infusion of mitomycin C suppressed axotomy-induced mitosis of satellite glial cells but did not impede axonal regeneration in the dorsal root or the peripheral nerve. Nevertheless, the similarity in reactions of satellite glial cells during 2 processes that activate neurons adds indirect support to the idea that non-neuronal cells in the DRG might influence regenerative responses of primary sensory neurons.     

Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A

We have investigated the hypothesis that the chemorepellent Semaphorin3A may be involved in the failure of axonal regeneration after injury to the ascending dorsal columns of adult rats. Following transection of the thoracic dorsal columns, fibroblasts in the dorsolateral parts of the lesion site showed robust expression of Semaphorin3A mRNA. In addition, dorsal root ganglion (DRG) neurons with projections through the dorsal columns to the injury site persistently expressed both Semaphorin3A receptor components, neuropilin-1 and plexin-A1. These ascending DRG collaterals failed to invade scar regions occupied by Semaphorin3A-positive fibroblasts, even in animals which had received conditioning lesions of the sciatic nerve to enhance regeneration. Other axon populations in the dorsal spinal cord were similarly unable to penetrate Semaphorin3A-positive scar tissue. These data suggest that Semaphorin3A may create an exclusion zone for regenerating dorsal column fibres and that enhancing the intrinsic regenerative response of DRG neurons has only limited effects on axonal regrowth. Tenascin-C and chondroitin sulphate proteoglycans were also detected at the injury site, which was largely devoid of central nervous system (CNS) myelin, showing that several classes of inhibitory factors, including semaphorins, with only partially overlapping spatial and temporal patterns of expression are in a position to participate in preventing regenerative axonal growth in the injured dorsal columns. Interestingly, conditioning nerve injuries enabled numerous ascending DRG axons to regrow across areas of strong tenascin-C and chondroitin sulphate proteoglycan expression, while areas containing Semaphorin3A and CNS myelin were selectively avoided by (pre)primed axonal sprouts.     

Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury

Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues.     

BDNF is involved in sympathetic sprouting in the dorsal root ganglia following peripheral nerve injury in rats

Peripheral nerve injury results in sympathetic sprouting around large diameter sensory neurons in the dorsal root ganglia (DRG). The mechanism underlying this pathological phenomenon is not known. Brain-derived neurotrophic factor (BDNF) is up-regulated in large sensory neurons and ensheathing satellite cells following a sciatic nerve injury. In the present study, we investigated the effects of BDNF on the sympathetic sprouting in the DRG, by delivering BDNF antibody or antisense oligodeoxynucleotide to injured DRGs, or by delivering exogenous BDNF to intact DRGs. The sheep antibody to BDNF, characterized by bioassays and dot blots, specifically reacted with BDNF but not other neurotrophins. Noradrenergic fibres were visualized by immunostaining of tyrosine hydroxylase (TH) and quantified by an NIH Imaging program. Two weeks following L5 spinal nerve lesion, a dramatic increase in TH-immunoreac-tive (-ir) fibres was observed in both ipsi- and contralateral DRGs in normal sheep IgG treated rats. BDNF antibody significantly reduced the sprouting of sympathetic nerves in both ipsi- and contra-lateral DRGs by 67% and 42% respectively. BDNF antisense oligodeoxynucleotide, by inhibiting BDNF synthesis in DRGs, also significantly suppressed the sprouting by 67% and 60% respectively in the ipsi- and contralateral DRGs. Delivery of exogenous BDNF into an intact L5 DRGs resulted in an increase in the sprouting by 4.2-fold. Our results clearly indicate that BDNF, synthesized in and secreted from the DRGs, is involved in the sympathetic sprouting in the DRG following the peripheral nerve injury.     

BDNF is involved in sympathetic sprouting in the dorsal root ganglia following peripheral nerve injury in rats

Peripheral nerve injury results in sympathetic sprouting around large diameter sensory neurons in the dorsal root ganglia (DRG). The mechanism underlying this pathological phenomenon is not known. Brain-derived neurotrophic factor (BDNF) is up-regulated in large sensory neurons and ensheathing satellite cells following a sciatic nerve injury. In the present study, we investigated the effects of BDNF on the sympathetic sprouting in the DRG, by delivering BDNF antibody or antisense oligodeoxynucleotide to injured DRGs, or by delivering exogenous BDNF to intact DRGs. The sheep antibody to BDNF, characterized by bioassays and dot blots, specifically reacted with BDNF but not other neurotrophins. Noradrenergic fibers were visualized by immunostaining of tyrosine hydroxylase (TH) and quantified by an NIH Imaging program. Two weeks following L5 spinal nerve lesion, a dramatic increase in TH-immunoreactive (-ir) fibres was observed in both ipsi- and contralateral DRGs in normal sheep IgG treated rats. BDNF antibody significantly reduced the sprouting of sympathetic nerves in both ipsi- and contra-lateral DRGs by 67% and 42% respectively. BDNF antisense oligodeoxynucleotide, by inhibiting BDNF synthesis in DRGs, also significantly suppressed the sprouting by 67% and 60% respectively in the ipsi- and contra-lateral DRGs. Delivery of exogenous BDNF into an intact L5 DRGs resulted in an increase in the sprouting by 4.2-fold. Our results clearly indicate that BDNF, synthesized in and secreted from the DRGs, is involved in the sympathetic sprouting in the DRG following the peripheral nerve injury.     

Pericellular Griffonia simplicifolia I isolectin B4-binding ring structures in the dorsal root ganglia following peripheral nerve injury in rats

Patients with a peripheral nerve injury often suffer from persistent chronic pain, but the underlying mechanism remains largely unknown. The persistent nature of the pain suggests injury-induced profound structural changes along the sensory pathways. In the present study, using the plant Griffonia simplicifolia I isolectin B4 (IB4) as a marker for nonpeptidergic small sensory neurons, we sought to examine whether these neurons sprout in the dorsal root ganglia (DRG) in response to peripheral nerve injury. The lumbar 5 (L5) spinal nerve was transected, and rats were allowed to survive for varying lengths of time before IB4 histology was performed. We found that a subpopulation of IB4-positive sensory neurons sprouted robustly after spinal nerve injury. Twelve weeks after spinal nerve injury, the IB4-positive ring structures became dramatic and encircled both large and small neurons in the DRG. The aberrant sprouting of small sensory neurons was also demonstrated by retrograde labeling. The processes of satellite cells surrounding large sensory neurons also became IB4 positive, and 87.8% of perineuronal IB4-positive ring structures intermingled and/or coexpressed with glial fibrillary acidic protein-positive satellite cells. Thus, the sprouting axons of IB4-positive neurons were intermingled with IB4-positive satellite cells, forming perineuronal ring structures surrounding large-diameter neurons. Ultrastructural examinations further confirmed that IB4-positive nerve terminals were entangled with satellite cells and IB4-negative unmyelinated sprouting fibers around sensory neurons. These studies have provided the first evidence that a subpopulation of IB4-binding small sensory neurons sprouts and forms perineuronal ring structures together with IB4-positive satellite cells in response to nerve injury. The significance of the sprouting of IB4-positive neurons remains to be determined.     

Differences in Sympathetic Innervation of Mouse DRG Following Proximal or Distal Nerve Lesions

Nerve injury leads to novel sympathetic innervation of the dorsal root ganglion (DRG). We have hypothesized previously that the degenerating nerve increases the sympathetic sprouting in the DRG and pain after chronic sciatic constriction injury (CCI) by virtue of its influence on sensory and sympathetic axons spared by the injury. However, L5 spinal nerve ligation and transection (SNL) results in the complete isolation of the L5 DRG from the degenerating stump, yet sympathetic axons invade the ganglion, and sympathetically dependent pain develops. We investigated the role of Wallerian degeneration in both sympathetic sprouting and neuropathic pain in these two models of painful peripheral neuropathy by comparing responses of normal C57B1/6J and C57B1/Wld^smice in which degeneration is impaired. After CCI, Wld^smice, unlike 6J mice, did not develop thermal or mechanoallodynia or sympathetic innervation of the L5 DRG. After SNL, both strains developed mechanoallodynia and sympathetic sprouts in L5, but only 6J mice developed thermal allodynia. Observation of the origins of the invading sympathetic axons revealed that after CCI, sympathetics innervating blood vessels and dura (probably intact) sprouted into the ganglion, but after SNL sympathetics (probably axotomized) invaded from the injured spinal nerve. Based on these findings, we hypothesize that there are two mechanisms for sympathetic sprouting into DRG, differentially dependent on Wallerian degeneration. Analysis of pain behavior in these animals reveals that (i) mechanoallodynia and sympathetic innervation of the DRG tend to coincide and (ii) thermal allodynia and Wallerian degeneration, but not sympathetic innervation of the DRG tend to coincide.     

Regeneration of primary sensory axons into the adult rat spinal cord via a peripheral nerve graft bridging the lumbar dorsal roots to the dorsal column

This study investigated the feasibility of using a peripheral nerve autograft (NAG) to promote and guide regeneration of sensory axons from the caudal lumbar dorsal roots to the rostral dorsal column following a lower thoracic cordotomy in adult rats. After a left hemicordotomy at the T13 vertebra level and ipsilateral L3 and L4 rhizotomies, a peripheral NAG (peroneal nerve) was connected to the distal roots stumps, then implanted into the left dorsal column 10 mm rostral to hemicordotomy site (n = 12). After surgery, all animals of the experimental group experienced complete anesthesia in their left hindlimb. Three months later, a slight response to nociceptive stimulation reappeared in L3 and/or L4 dermatomes in 6 of the 12 experimental animals. None of these animals exhibited self-mutilation. Nine months after surgery, we performed retrograde tracing studies by injecting horseradish peroxidase (HRP) into the left dorsal column 30 mm rostral to the NAG implantation site. In eight animals, we found HRP-stained neurons in the left L3 and/or L4 dorsal root ganglia (DRG). The mean number of HRP-stained neurons per DRG was 71 +/- 92 (range 2-259). In control groups, no HRP-stained neurons were found in L3 or L4 DRG. Histological analysis of the NAG showed evidence of axonal regeneration in all 8 animals with positive retrograde labeling of DRG neurons. However, we did not find a statistical correlation between the number of HRP-stained neurons and the degree of sensory recovery. This study demonstrates that an NAG joining dorsal roots to the dorsal column, thus shunting the original CNS-PNS junction, can support regeneration of central axons from DRG primary sensory neurons into the dorsal column over distances of at least 30 mm despite the inhibitory influence of the CNS white matter.     

Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord

We have investigated the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on the intraspinal regeneration of anterogradely labeled axotomized ascending primary sensory fibers in the adult rat. These fibers were allowed to grow across a predegenerated peripheral nerve graft and back into the thoracic spinal cord. In control animals that had been infused with vehicle for two weeks into the dorsal column, 3 mm rostral to the nerve graft, essentially no fibers had extended from the nerve graft back into the spinal cord. The number of sensory fibers in the rostral end of the nerve graft was not significantly different between control and neurotrophin-infused animals. With infusion of NGF, 37+/-2% of the fibers at the rostral end of the graft had grown up to 0.5 mm into the dorsal column white matter, 30+/-2% up to 1 mm, 19+/-3% up to 2 mm and 8+/-2% up to 3 mm, i.e., the infusion site. With infusion of NT-3, sensory fiber outgrowth was similar to that seen with NGF, but with BDNF fewer fibers reached farther distances into the cord. Infusion of a mixture of all three neurotrophins did not increase the number of regenerating sensory fibers above that seen after infusion of the individual neurotrophins. These findings suggest that injured ascending sensory axons are responsive to all three neurotrophins and confirm our previous findings that neurotrophic factors can promote regeneration in the adult central nervous system.     

Lumbar 5 ventral root transection-induced upregulation of nerve growth factor in sensory neurons and their target tissues: a mechanism in neuropathic pain

We have previously demonstrated that profound and persistent neuropathic pain as displayed by mechanical and cold allodynia and thermal hyperalgesia can be produced by a lumbar 5 ventral root transection (L5 VRT) model in adult rats in which only the motor nerve fibers were injured without axotomy of sensory neurons. However, the underlying mechanisms remain to be determined. In this study, by examining its changes in expression and by inhibiting its functions using a neutralizing antibody, we have investigated whether nerve growth factor (NGF), a neurotrophic factor known to have a function in regulating nerve injury-induced pain, is involved in the development of neuropathic pain induced by L5 VRT. Motor nerve injury by L5 VRT resulted in a de novo expression of NGF mRNA in a subpopulation of small sensory neurons and pericellular satellite cells in ipsilateral L5 dorsal root ganglion. NGF protein expression was also increased by sensory neurons with various sizes and by keratinocytes in the target tissue ipsilateral skin. Systemic administration of NGF antiserum twice within 17 days markedly attenuated L5 VRT-induced mechanical allodynia but not the cold allodynia and thermal hyperalgesia. These findings suggest that NGF is an important pain mediator in the generation of mechanical sensitivity induced by L5 VRT.     

Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo

In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.     

p38 activation in uninjured primary afferent neurons and in spinal microglia contributes to the development of neuropathic pain induced by selective motor fiber injury

Compelling evidence shows that the adjacent uninjured primary afferents play an important role in the development of neuropathic pain after nerve injury. The underlying mechanisms, however, are largely unknown. In the present study, the selective motor fiber injury was performed by L5 ventral root transection (L5 VRT), and p38 activation in dorsal root ganglia (DRG) and L5 spinal dorsal horn was examined. The results showed that phospho-p38 immunoreactivity (p-p38-IR) was increased in both L4 and L5 DRGs, starting on day 1 and persisting for nearly 3 weeks (P<0.05) following L5 VRT and that the activated p38 was confined in neurons, especially in IB4 positive C-type neurons. L5 VRT also induced p38 activation in L5 spinal dorsal horn, occurred at the first day after the lesion and lasted for 2 weeks (P<0.05). The activated p38 is restricted entirely in spinal microglia. In contrast, selective injury of sensory neurons by L5 dorsal root transection (L5 DRT) failed to induce behavioral signs of neuropathic pain and activated p38 only in L5 DRG but not in L4 DRG and L5 spinal dorsal horn. Intraperitoneal injection of thalidomide, an inhibitor of TNF-alpha synthesis, prevented p38 activation in DRG and spinal cord. Intrathecal injection of p38 inhibitor SB203580, starting before L5 VRT, inhibited the abnormal pain behaviors. Post-treatment with SB203580 performed at the 1st day or at the 8th day after surgery also reduced established neuropathic pain. These data suggest that p38 activation in uninjured DRGs neurons and in spinal microglia is necessary for the initiation and maintenance of neuropathic pain induced by L5 VRT.     

Guiding adult Mammalian sensory axons during regeneration

Misdirection of axons after nerve injury impairs successful regeneration of adult neurons. Investigations of axon guidance in development have provided an understanding of pathfinding, but their relevance to regenerating adult axons is unclear. We investigated adult mammalian axon guidance during regeneration after peripheral nerve injury and focused on the effects of the prototypic guidance molecule nerve growth factor (NGF). Adult rat sensory neurons from dorsal root ganglia that expressed the NGF receptor tropomyosin-related kinase A (trkA) were presented with a point source of NGF in vitro. Naive trkA neurons had no net turning response to NGF, but if they had been preconditioned by a peripheral nerve transection in vivo before culturing, their growth cones were attracted toward the NGF gradient. A laminin substrate was required for this behavior and an anti-trkA antibody interrupted turning. These data demonstrate that injured adult mammalian axons can be guided as they regenerate. Moreover, despite the downregulation of trkA mRNA and protein levels within the dorsal root ganglion after injury, sensory neurons retain and increase trkA protein at the injury site where the regenerating axons are found. This may enhance the axonal response to NGF and allow guidance along an NGF gradient created in vivo in the distal nerve stump.     

Sciatic nerve transection triggers release and intercellular transfer of a genetically expressed macromolecular tracer in dorsal root ganglia

We recently developed a genetic transneuronal tracing approach that allows for the study of circuits that are altered by nerve injury. We generated transgenic (ZW-X) mice in which expression of a transneuronal tracer, wheat germ agglutinin (WGA), is induced in primary sensory neurons, but only after transection of their peripheral axon. By following the transneuronal transport of the tracer into the central nervous system (CNS) we can label the circuits that are engaged by the WGA-expressing damaged neurons. Here we used the ZW-X mouse line to analyze dorsal root ganglia (DRG) for intraganglionic connections between injured sensory neurons and their neighboring "intact" neurons. Because neuropeptide Y (NPY) expression is strongly induced in DRG neurons after peripheral axotomy, we crossed the ZW-X mouse line with a mouse that expresses Cre recombinase under the influence of the NPY promoter. As expected, sciatic nerve transection triggered WGA expression in NPY-positive DRG neurons, most of which are of large diameter. As expected, double labeling for ATF-3, a marker of cell bodies with damaged axons, showed that the tracer predominated in injured (i.e., axotomized) neurons. However, we also found the WGA tracer in DRG cell bodies of uninjured sensory neurons. Importantly, in the absence of nerve injury there was no intraganglionic transfer of WGA. Our results demonstrate that intraganglionic, cell-to-cell communication, via transfer of large molecules, occurs between the cell bodies of injured and neighboring noninjured primary afferent neurons.     

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).