Survival effects of BDNF and NT-3 on axotomized rubrospinal neurons depend on the temporal pattern of neurotrophin administration

This study shows that both BDNF and NT-3 can prevent cell death in axotomized adult rat rubrospinal neurons (RSNs), but that the efficacy of neuroprotection depends on the temporal pattern of treatment. At 8 weeks after cervical spinal cord injury, 51% of the RSNs had died. Subarachnoidal BDNF infusion into the cisterna magna for 4 weeks resulted in neuronal hypertrophy and 71% survival. Continuous infusion for 8 weeks into the lumbar subarachnoidal space with either BDNF or NT-3 gave similar survival rates, while a combination of BDNF and NT-3 resulted in 96% survival, although the cells were atrophic. When administration of either BDNF or NT-3 was delayed and performed during postoperative weeks 5-8, the number of surviving neurons was increased compared to early treatment. Delayed treatment with a combination of BDNF and NT-3 resulted in complete survival and a reduction in neuronal atrophy. A decreased expression of TrkB receptors and microtubule-associated protein-2 in the RSNs after axotomy was counteracted by BDNF and NT-3. Microglial activity remained increased even when complete cell survival was achieved. Thus, the combination of neurotrophins as well as the temporal pattern of treatment need to be adequately defined to optimize survival of injured spinal tract neurons.     

Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy

Numerous experimental therapies to promote axonal regeneration have shown promise in animal models of acute spinal cord injury, but their effectiveness is often found to diminish with a delay in administration. We evaluated whether brain-derived neurotrophic factor (BDNF) application to the spinal cord injury site 2 months after cervical axotomy could promote a regenerative response in chronically axotomized rubrospinal neurons. BDNF was applied to the spinal cord in three different concentrations 2 months after cervical axotomy of the rubrospinal tract. The red nucleus was examined for reversal of neuronal atrophy, GAP43 and Talpha1 tubulin mRNA expression, and trkB receptor immunoreactivity. A peripheral nerve transplant paradigm was used to measure axonal regeneration into peripheral nerve transplants. Rubrospinal axons were anterogradely traced and trkB receptor immunohistochemistry performed on the injured spinal cord. We found that BDNF treatment did not reverse rubrospinal neuronal atrophy, nor promote GAP-43 and Talpha1 tubulin mRNA expression, nor promote axonal regeneration into peripheral nerve transplants. TrkB receptor immunohistochemistry demonstrated immunoreactivity on the neuronal cell bodies, but not on anterogradely labeled rubrospinal axons at the injury site. These findings suggest that the poor response of rubrospinal neurons to BDNF applied to the spinal cord injury site 2 months after cervical axotomy is not related to the dose of BDNF administered, but rather to the loss of trkB receptors on the injured axons over time. Such obstacles to axonal regeneration will be important to identify in the development of therapeutic strategies for chronically injured individuals.     

Delayed treatment with chondroitinase ABC reverses chronic atrophy of rubrospinal neurons following spinal cord injury

Degradation of extracellular matrix chondroitin sulphate proteoglycans (CSPGs) using Chondroitinase ABC (ChABC) is a promising strategy for the treatment of spinal cord injury, with potent effects on promoting functional recovery and anatomical repair in spinal injured animals. We have previously demonstrated that ChABC treatment prevents atrophy of corticospinal projection neurons following spinal injury in adult YFP-H mice. Here, we investigate whether ChABC-mediated repair of the cell body extends to rubrospinal projection neurons (RSNs), whether neuroprotective effects can be sustained long-term and importantly, whether delayed treatment with ChABC can reverse chronic atrophy. Adult YFP-H mice underwent unilateral rubrospinal tract transection and were treated with ChABC or a control enzyme, delivered either acutely post-injury or after a one month delay. Eight weeks following injury and control treatment, RSNs in the injured red nucleus, identified by YFP label and NeuN immunoreactivity, showed severe atrophy, with ~ 40% loss of mean cell area compared to uninjured neurons in the contralateral red nucleus. Both acute and delayed treatment with ChABC promoted a significant rescue of injured RSNs, restoring cell area to ~ 80% and ~ 70%, respectively, of that in uninjured neurons. Thus, we demonstrate for the first time that CSPG degradation in the injured spinal cord not only promotes sustained rescue of cell atrophy when delivered acutely but can also reverse chronic atrophy in descending projection neurons. Thus, modulation of the extracellular matrix can mediate neuroprotective effects both early and late after spinal cord injury.     

PTEN knockdown with the Y444F mutant AAV2 vector promotes axonal regeneration in the adult optic nerve

The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. Activating the PI3 K/AKT/m TOR signaling pathway has been shown to enhance the intrinsic growth capacity of neurons and to facilitate axonal regeneration in the central nervous system after injury. The deletion of the m TOR negative regulator phosphatase and tensin homolog(PTEN) enhances regeneration of adult corticospinal neurons and ganglion cells. In the present study, we used a tyrosine-mutated(Y444 F) AAV2 vector to efficiently express a short hairpin RNA(sh RNA) for silencing PTEN expression in retinal ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. The rats received an intravitreal injection of wildtype AAV2 or Y444 F mutant AAV2(both carrying sh RNA to PTEN) 4 weeks before optic nerve axotomy. Compared with the wildtype AAV2 vector, the Y444 F mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy. Moreover, post-axotomy injection of the Y444 F AAV2 vector expressing the sh RNA to PTEN rescued ~19% of retinal ganglion cells and induced axons to regenerate near to the optic chiasm. Taken together, our results demonstrate that PTEN knockdown with the Y444 F AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy. Therefore, the Y444 F AAV2 vector might be a promising gene therapy tool for treating optic nerve injury.     

Single injections of a DNA plasmid that contains the human Bcl-2 gene prevent loss and atrophy of distinct neuronal populations after spinal cord injury in adult rats

Spinal cord injury in adult mammals causes atrophy or loss of axotomized neurons. We have previously found that the product of the antiapoptotic gene Bcl-2, delivered by intraspinal injection of a DNA plasmid, reduces atrophy and loss of axotomized Clarke's nucleus neurons in adult rats. Here we studied whether the same treatment protects axotomized red nucleus (RN) neurons. Two months after the right dorsolateral funiculus was ablated in adult Sprague-Dawley rats by C3/C4 subtotal hemisection, there was approximately 48% loss of RN neurons in the magnocellular portion of the RN contralateral to the lesion and atrophy of many surviving neurons. When a DNA plasmid encoding the human Bcl-2 gene and the bacterial reporter gene LacZ, complexed with cationic lipids, was injected just rostral to the subtotal hemisection site, 87% of RN neurons survived, and there was partial, but robust, protection from atrophy. These and our previous results indicated that intraspinal administration of the Bcl-2 gene can prevent retrograde cell loss and reduce atrophy of axotomized RN and Clarke's nucleus neurons in adult rats and provide an effective means to rescue neurons whose survival depends on different growth factors.     

Promoting axonal regeneration in the central nervous system by enhancing the cell body response to axotomy

Neurons projecting into the peripheral nervous system (PNS) regenerate their axons after injury, in contrast to those confined to the central nervous system (CNS). Both neuronal and nonneuronal factors contribute to the lack of CNS regeneration. In this review we concentrate on the differential gene expression response to axotomy in PNS vs. CNS neurons. In general CNS neurons fail to up-regulate or sustain the expression of regeneration-associated proteins (RAGs), including trophic factors and their receptors. The presumed lack of trophic support of axotomized CNS neurons provided the rationale for the exogenous application of trophic factors, either to the lesion site or to the cell bodies. Here, we review our data on the application of trophic factors to rubrospinal and corticospinal neurons. Cell body treatment of axotomized rubrospinal neurons with brain-derived neurotrophic factor (BDNF) reversed atrophy, increased GAP-43 and Talpha-1 tubulin mRNA expression, and promoted axonal regeneration into peripheral nerve grafts. Importantly, BDNF cell body treatment was still effective in the chronic setting, i.e., when initiated 1 year after injury, but BDNF had no effect when applied to the chronic spinal cord injury site. The ability to promote regeneration in chronically injured neurons will hopefully contribute to the development of treatment strategies for chronic spinal injuries.     

Expression of β-calcitonin gene-related peptide in axotomized rubrospinal neurons and the effect of brain derived neurotrophic factor

The mRNA levels for α- and β-calcitonin gene-related peptide (CGRP) in rat rubrospinal neurons were studied by in situ hybridization 3, 7, 14, 28 and 56 days following cervical spinal hemisection. CGRP-like immunoreactivity (LI) in the rubrospinal neurons and the rubrospinal tract in cervical spinal cords were examined using immunohistochemistry. There was almost no signal for α- and β-CGRP mRNAs and undetectable level of CGRP-LI in the rubrospinal neurons ipsilateral to cervical spinal hemisection (control side). Fourteen days after spinal hemisection, the rubrospinal neurons contralateral to cervical hemisection (axotomized side) showed CGRP-LI in their cell bodies, and CGRP containing fibers were observed in the lateral funiculi just proximal, but not distal, to the injury sites. In situ hybridization showed upregulation of β-CGRP mRNA in a subpopulation of the rubrospinal neurons on the axotomized side. The proportion of β-CGRP mRNA-expressing neurons reached its maximum (approximately 19%) 4 days following axotomy and slowly decreased to about 5% 56 days after axotomy. The percentage of α-CGRP mRNA-expressing neurons was much lower than that of β-CGRP mRNA (maximum about 2.6% 4 days after axotomy) and not significantly different from the control side throughout the time period studied. These data indicate that axotomy induces de novo synthesis of the CGRP β-subtype in rubrospinal neurons and that the β-CGRP is transported to the injury site through the rubrospinal tract. In addition, we studied the effect of the intracerebral injections of brain derived neurotrophic factor (BDNF). BDNF treatment fully reversed the severe cell atrophy that followed axotomy and increased the number of neurons labeled for β-CGRP mRNA, but did not increase the percentage of rubrospinal neurons expressing β-CGRP mRNA. Thus, topical application of BDNF does not have direct modulatory effect on CGRP induction in axotomized neurons in the red nucleus.     

BDNF and NT-3, but not NGF, Prevent Axotomy-induced Death of Rat Corticospinal Neurons In Vivo

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been identified as survival factors for adult axotomized rat corticospinal neurons (CSN) in vivo. Axotomy of corticospinal neurons at the level of the internal capsule induced death of 46% of the CSN within the first week after axotomy. The surviving population of CSN displayed severe atrophy with mean cross-sectional area 49% of their unlesioned contralateral counterparts 7 days after axotomy. Using in situ hybridization to assess the expression of the receptors for the family of neurotrophins, we found trkB and trkC but not trkA mRNA expression in CSN. Intraparenchymal application of BDNF or NT-3 at doses of 12 μg/day for 7 days via an osmotic minipump fully prevented the axotomy-induced death of CSN. Interestingly, no neuronal atrophy was seen after BDNF application while NT-3 had only a partial effect on the size of the axotomized CSN. Nerve growth factor did not prevent death or cell atrophy, consistent with the lack of trkA mRNA expression in these neurons. These findings show that BDNF and NT-3 are survival factors for adult rat CSN in vivo , and may contribute to the development of therapeutic strategies aiming at the prevention of CSN degeneration in human motor neuron diseases.     

Up- and down-regulation of BDNF mRNA in distinct subgroups of rat sensory neurons after axotomy

Anterograde transport of BDNF is enhanced by axotomy in the rat sciatic nerve. However, the changes in BDNF gene expression in dorsal root ganglion (DRG) neurons after axotomy are not known. We examined this issue using in situ hybridization histochemistry. BDNF mRNA was detected in 35-40% of DRG neurons (L5) of control rats. Most of these neurons are small. BDNF gene expression in these neurons was down-regulated after application of capsaicin to the sciatic nerve. Transection of the sciatic nerve induced the up-regulation of BDNF mRNA. The intensely labeled neurons were mainly large and immunoreactive for neuropeptide Y. These results suggest that up- and down-regulation of BDNF gene expression in distinct subgroups of rat DRG neurons are caused by damage to the peripheral nerve.     

Undesired effects of a combinatorial treatment for spinal cord injury--transplantation of olfactory ensheathing cells and BDNF infusion to the red nucleus

Transplantations of olfactory ensheathing cells (OECs) have been reported to promote axonal regeneration and functional recovery after spinal cord injury, but have demonstrated limited growth promotion of rat rubrospinal axons after a cervical dorsolateral funiculus crush. Rubrospinal neurons undergo massive atrophy after cervical axotomy and show only transient expression of regeneration-associated genes. Cell body treatment with brain-derived neurotrophic factor (BDNF) prevents this atrophy, stimulates regeneration-associated gene expression and promotes regeneration of rubrospinal axons into peripheral nerve transplants. Here, we hypothesized that the failure of rubrospinal axons to regenerate through a bridge of OEC transplants was due to this weak intrinsic cell body response. Hence, we combined BDNF treatment of rubrospinal neurons with transplantation of highly enriched OECs derived from the nasal mucosa and assessed axonal regeneration as well as behavioral changes after a cervical dorsolateral funiculus crush. Each treatment alone as well as their combination prevented the dieback of the rubrospinal axons, but none of them promoted rubrospinal regeneration beyond the lesion/transplantation site. Motor performance in a food-pellet reaching test and forelimb usage during vertical exploration (cylinder test) were more impaired after combining transplantation of OECs with BDNF treatment. This impaired motor performance correlated with lowered sensory thresholds in animals receiving the combinatorial therapy - which were not seen with each treatment alone. Only this combinatorial treatment group showed enhanced sprouting of calcitonin gene-related peptide-positive axons rostral to the lesion site. Hence, some combinatorial treatments, such as OECs with BDNF, may have undesired effects in the injured spinal cord.     

Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43.

Neurons confined within the mammalian CNS usually do not regenerate after axonal injury, while axonal regeneration is the rule in the PNS. It has been hypothesized that this may be related to differences in the microenvironment of the PNS versus CNS and to differences in the neuronal response to injury. In order to test the latter hypothesis, we compared changes in gene expression after axotomy in two populations of neurons: rat facial motoneurons and rat rubrospinal neurons. In situ hybridization with cDNA probes for the medium and light neurofilament protein revealed a reduced mRNA content in both facial and rubrospinal neurons at all times investigated (i.e., 1, 2, and 3 weeks after axotomy). On the other hand, mRNAs for actin and tubulin were increased in both neuronal populations during the first week after axotomy. While this increase was sustained in facial motoneurons for several weeks, total tubulin mRNA and actin mRNA were decreased in rubrospinal neurons at 2 and 3 weeks after axotomy, coincident with their atrophy. The developmentally regulated T alpha 1 tubulin mRNA, which was previously shown to be reexpressed in facial motoneurons after axotomy, was elevated severalfold in axotomized rubrospinal neurons, and increased levels persisted in some rubrospinal neurons as late as 7 weeks after axotomy. Similarly, the developmentally regulated GAP-43 mRNA increased in both axotomized facial and rubrospinal neurons, and increased levels were sustained in some axotomized rubrospinal neurons for at least 7 weeks. The response of rubrospinal neurons to axotomy in the cervical spinal cord is, in the first week, qualitatively similar to the response of facial motoneurons. However, by 2 weeks after axotomy there is a generalized reduction in mRNA levels for all three cytoskeletal proteins that is associated with neuronal atrophy. During this period, mRNA levels for the two specific markers of the growth state, T alpha 1 tubulin and GAP-43, remain elevated. Thus, axotomy of rubrospinal neurons appears to set in motion two independent events. First, an axotomy signal initiates a cell-body reaction similar to that of PNS neurons, including increased mRNA levels for T alpha 1 tubulin and GAP-43. Later, a generalized cellular atrophy and decrease in mRNA levels occur without reversing the specific responses of T alpha 1 and GAP-43 to axotomy. We conclude that the failure of rubrospinal neurons to regenerate is not due to a failure to initiate gene-expression changes characteristic of regenerating peripheral neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function

Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNF in vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that approximately 7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons.     

Brain-derived neurotrophic factor modulates GAP-43 but not tα1 expression in injured retinal ganglion cells of adult rats

The administration of neurotrophins affects neuronal survival and growth, but less is known about their ability to modify the expression of growth associated genes following injury to CNS neurons. Here we characterize the effect of brain-derived neurotrophic factor (BDNF) on mRNA levels for Tα1 α-tubulin, and for GAP-43, two genes whose expression levels in retinal ganglion cells (RGC) tend to correlate with growth. We first determined that most adult rat RGCs can retrogradely transport BDNF by injecting ¹²⁵I-BDNF into RGC target sites in vivo. We then used quantitative in situ hybridization to characterize the effect of axotomy, or axotomy and BDNF administration on mRNA levels for GAP-43 and Tα1. Axotomy alone resulted in a general decrease in Tα1 α-tubulin mRNA levels by 2 weeks, and elicited an increase in GAP-43 mRNA levels in an average of 30% of surviving RGCs. The intravitreal administration of a single dose of BDNF (5 μg) to axotomized RGCs on the day of injury did not affect Tα1 α-tubulin mRNA levels, but was followed by a moderate (approximately 80%), and short-lasting enhancement of GAP-43 mRNA levels in most RGCs during the first week after axotomy. No significant increase in GAP-43 mRNA levels was observed when BDNF was injected into the uninjured eye. We conclude that BDNF specifically enhances GAP-43 but not Tα1 mRNA levels in injured RGCs. Because BDNF is known to stimulate branch length of injured RGCs, we suggest that changes in the expression of GAP-43, but not Tα1 tubulin, correlate with branching of injured neurons as opposed to long distance regrowth. J. Neurosci. Res. 47:561–572, 1997. © 1997 Wiley-Liss, Inc.     

Changes in brain-derived neurotrophic factor and trkB receptor in the adult Rana pipiens retina and optic tectum after optic nerve injury

In this study we used immunocytochemistry to investigate the distribution of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase (trkB) in retina and optic tectum of the frog Rana pipiens during regeneration after axotomy. We also measured changes in BDNF mRNA in retina and tectum. Retrograde labeling was used to identify retinal ganglion cells (RGCs) prior to quantification of the BDNF immunoreactivity. In control animals, BDNF was found in the majority of RGCs and displaced amacrine cells and in some cells in the inner nuclear layer (INL). After axotomy, BDNF immunoreactivity was reduced in RGCs but increased in the INL. BDNF mRNA levels in the retina remained high before and after axotomy. Three months after axotomy, after reconnection to the target, the staining intensity of many of the surviving RGCs had partially recovered. In the control tectum, BDNF staining was present in ependymoglial cells and in neurons throughout layers 4, 6, 8, and 9. After axotomy, BDNF staining in tectal neurons became more intense, even though mRNA synthesis was transiently down-regulated. In control retinas, trkB receptor immunostaining was present in most RGCs; no significant changes were observed after axotomy. In control tectum, trkB was detected only in ependymoglial cells. After axotomy, many neuronal cell bodies were transiently labeled. Our data are consistent with the hypothesis that a considerable fraction of the BDNF normally present in RGCs is acquired from their targets in the tectum. However, there are also intraretinal sources of BDNF that could contribute to the survival of RGCs.     

Brain-derived neurotrophic factor spares choline acetyltransferase mRNA following axotomy of motor neurons in vivo

Choline acetyltransferase (ChAT) is a functional and specific marker gene for neurons such as primary motor neurons that synthesize and release acetylcholine as a neurotransmitter. In adult mammals, transection of the peripheral nerve results in a loss of immunoreactivity for ChAT in the injured motor neurons without affecting their cell number. Using a quantitative RNase protection assay, we have investigated dynamic changes in ChAT mRNA levels following axotomy of motor neurons in the brainstem of adult rats. One week after transection of the left hypoglossal nerve, levels of ChAT mRNA in the ipsilateral side of the hypoglossal motor nucleus decreased dramatically to around 10% when compared to the uninjured contralateral side. When cut axons were chronically exposed to brain-derived neurotrophic factor (BDNF) for 1 week, ChAT mRNA levels were maintained at 63% of control levels. Thus, BDNF can abrogate the injury-induced loss of ChAT mRNA in mature motor neurons in vivo. In contrast, neither neurotrophin 4/5 nor nerve growth factor could prevent the decrease in message. This effect of BDNF on ChAT mRNA levels following peripheral injury to motor neurons demonstrates the existence of regulatory pathways responsive to neurotrophic factors that can “rescue” or “protect” cholinergic gene expression. J. Neurosci. Res. 47:134–143, 1997. © 1997 Wiley-Liss, Inc.     

NT‐3, but not BDNF, prevents atrophy and death of axotomized spinal cord projection neurons

Following spinal cord injury, projection neurons are frequently axotomized and many of the cells subsequently die. One goal in spinal injury research is to preserve damaged neurons so that ultimately they are accessible to regeneration‐promoting strategies. Here we ask if neurotrophin treatment can prevent atrophy and death of axotomized sensory projection neurons. In adult rats, a hemisection was made in the thoracic spinal cord and axotomized neurons were retrogradely labelled with Fluoro‐Gold. Four distinct populations of cells were identified in the lumbar spinal cord, and both numbers and sizes of labelled cells were assessed at different time points postlesion. A progressive and significant degeneration was observed over time with severe atrophy apparent in all cell populations and significant cell loss evident by 4 weeks postlesion. This time point was used to assess neurotrophin effects. Hemisected rats were treated with either neurotrophin 3 (NT‐3) or brain‐derived neurotrophic factor (BDNF, 12 μg/day for each), or a vehicle solution, delivered continuously to the lesion site via an osmotic minipump. Treatment with NT‐3, but not BDNF, completely reversed cell atrophy in three of the four cell populations and also induced a significant increase in the number of surviving cells. In situ hybridization experiments showed trkB and trkC mRNA to be expressed in the majority of ascending spinal projection neurons, suggesting that these cells should be responsive to both BDNF and NT‐3. However, only NT‐3 treatment was neuroprotective, indicating that BDNF may not have reached the cell bodies of injured neurons. These results demonstrate that NT‐3 may be of benefit in preventing the secondary cell loss that occurs following spinal injury.     

Axotomy-induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus.

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high-affinity PACAP receptors, PAC(1) and VPAC(2), in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time-dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20-fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10-fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC(1) and VPAC(2) receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC(2) receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC(1) receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses.     

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.