Early BDNF, NT-3, and NT-4 signaling events.

Much more is known about nerve growth factor (NGF) signaling than that initiated by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or NT-4. We sought to study early BDNF, NT-3, and NT-4 signaling events. Using TrkB-expressing cells, we found that BDNF and NT-4 individually induced tyrosine phosphorylation of TrkB in a dose-dependent fashion. At maximally effective concentrations, BDNF or NT-4 induced robust TrkB tyrosine phosphorylation at 5 min; this progressively declined at 15, 30, and 60 min. Using immunoprecipitation, PI3-kinase and tyrosine phosphorylated PLC-gamma1 and SHC were shown to be associated with tyrosine phosphorylated TrkB in response to both BDNF and NT-4. BDNF and NT-4 induced similar intensities of phosphorylation of TrkB and signaling intermediates at equivalent doses. NT-3 treatment of TrkC-expressing cells induced very similar patterns for induction of TrkC tyrosine phosphorylation and recruitment of signaling intermediates. BDNF, NT-3, and NT-4 caused rapid tyrosine phosphorylation of ERK and SNT. These data suggest that the earliest signaling events for BDNF, NT-3, and NT-4 are very similar to those for NGF.     

Long-term locomotor training up-regulates TrkB(FL) receptor-like proteins,brain-derived neurotrophic factor,and neurotrophin 4 with different topographies of expression in oligodendroglia and neurons in the spinal cord

Neurotrophins are potent regulators of neuronal survival, maintenance, and synaptic strength. In particular, brain-derived neurotrophic factor (BDNF), acting through full-length TrkB receptor (TrkB(FL)), is implicated in the stimulation of neurotransmission. Physical activity has been reported to increase BDNF expression in the brain and spinal cord. In this study we have evaluated the hypothesis that activation of a spinal neuronal network, due to exercise, affects the entire spinal neurotrophin system acting via TrkB receptors by modulation of BDNF, neurotrophin 4 (NT-4), and their TrkB receptor proteins. We investigated the effect of treadmill walking (4 weeks, 1 km daily) on distribution patterns and response intensity of these proteins in the lumbar spinal cord of adult rats. Training enhanced immunoreactivity (IR) of both neurotrophins. BDNF IR increased in cell processes of spinal gray matter, mainly in dendrites. NT-4 IR was augmented in the white matter fibers, which were, in part, of astrocytic identity. Training strongly increased both staining intensity and number of TrkB(FL)-like IR small cells of the spinal gray matter. The majority of these small cells were oligodendrocytes, representing both their precursor and their mature forms. In contrast, training did not exert an effect on expression of the truncated form of TrkB receptor in the spinal cord. These results show that both neuronal and nonneuronal cells may be actively recruited to BDNF/NT-4/TrkB(FL) neurotrophin signaling which can be up-regulated by training. Oligodendrocytes of the spinal gray matter were particularly responsive to exercise, pointing to their involvement in activity-driven cross talk between neurons and glia.     

Different expressions of BDNF, NT3, and NT4 in muscle and nerve after various types of peripheral nerve injuries

The changes in the expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) in the rat neuromuscular system as a result of three different types of sciatic nerve injuries have been evaluated. The changes in mRNA and protein levels for BDNF, NT-3, and NT-4 in the soleus muscle and sciatic nerve were assessed 4-28 days after sciatic nerve transection (neurotmesis), sciatic nerve crush (axonotmesis), and mild acute compression (neurapraxia). BDNF mRNA levels increased dramatically with nerve transection in the soleus muscle and the sciatic nerve 7-14 days after injury, whereas the changes were low in other types of injury. The changes of protein levels for BDNF were also similar. The mRNA and the protein levels of NT-3 in the soleus muscle did not show any significant difference. The mRNA for NT-4 in the soleus muscle decreased from 4 to 14 days after sciatic nerve transection, and the protein level was also minimum 14 days after sciatic nerve transection. Our results indicate that the neurotrophic factors in the neuromuscular system could play a role in differentiating peripheral nerve injury.     

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.     

Involvement of a spinal brain-derived neurotrophic factor/full-length TrkB pathway in the development of nerve injury-induced thermal hyperalgesia in mice

Partial sciatic nerve ligation in mice caused a marked and persistent decrease in the latency of paw withdrawal from a thermal stimulus only on the ipsilateral side. This thermal hyperalgesia was abolished by repeated intrathecal pretreatment with a specific antibody to brain-derived neurotrophic factor (BDNF), but not neurotrophin-4, just before and after the nerve ligation. These results provide direct evidence that BDNF within the spinal cord may contribute to the development of thermal hyperalgesia caused by nerve injury in mice. We previously reported that protein level of full-length TrkB, which contains the cytoplasmic protein tyrosine kinase domain, were clearly increased on the ipsilateral side of spinal cord membranes obtained from sciatic nerve-ligated mice. In the present study, we further demonstrated that the increased in the protein level of full-length TrkB is completely reversed by concomitant intrathecal injection of BDNF antibody. Furthermore, thermal hyperalgesia induced by nerve ligation was completely suppressed by repeated intrathecal injection of a specific antibody to full-length TrkB and an inhibitor of the protein tyrosine kinase activity for the neurotrophin receptor, K-252a. However, repeated intrathecal injection of a specific antibody to truncated TrkB, which lacks the cytoplasmic protein tyrosine kinase domain, failed to reverse thermal hyperalgesia observed in nerve-ligated mice. These findings suggest the possibility that the binding of BDNF to full-length TrkB and subsequent its activation may play a critical role in the development of neuropathic pain-like thermal hyperalgesia induced by nerve injury in mice.     

Differential regulation by exercise of BDNF and NT-3 in rat spinal cord and skeletal muscle

We have investigated the impact of neuromuscular activity on the expression of neurotrophins in the lumbar spinal cord region and innervating skeletal muscle of adult rats. Rats were exercised on a treadmill for 1 day or 5 consecutive days and euthanized at 0, 2 or 6 h after the last bout of exercise. By Day 1, there was no clear evidence of an increase in brain-derived neurotrophic factor (BDNF) mRNA in the spinal cord or the soleus muscle. By Day 5, there was a significant increase in BDNF mRNA in the spinal cord at 2 h post-training, and the soleus muscle showed a robust increase between 0 and 6 h post-training. Immunoassays showed significant increases in BDNF protein in the soleus muscle by training Day 5. Immunohistochemical analyses showed elevated BDNF levels in motoneuron cell bodies and axons in the ventral horn. Neurotrophin-3 (NT-3) mRNA was measured to determine whether selected neurotrophins respond with a selective pattern of induction to neuromuscular activity. In the spinal cord, there was a progressive post-training decrease in NT-3 mRNA following a single bout of training, while there was a significant increase in NT-3 mRNA at 2 h post-training by Day 5. The soleus muscle showed a progressive increase in NT-3 mRNA by Days 1 and 5 following training. These results show that neuromuscular activity has specific effects on the BDNF and NT-3 systems, and that repetitive exercise affects the magnitude and stability of these responses.     

Sprouting in intact sartorius muscles of the frog following contralateral axotomy

The intact hind limb sartorius muscle of the frog was examined anatomically for sprouting following axotomy of the sciatic nerve that contains motor axons innervating the contralateral sartorius muscle. The incidence of sprouting in intact muscles of experimental animals increased more than 3-fold over normal. Thus, contralateral axotomy produces sprouting in intact sartorius muscles much the same as it does in cutaneous-pectoris and piriformis muscles of the frog.     

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.     

The role of neurotrophins in the maintenance of the spinal cord motor neurons and the dorsal root ganglia proprioceptive sensory neurons

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non-muscle- and a few muscle-innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double-mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT-4/5, TrkB and TrkC, and not NT-3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT-4/5 and TrkC, and not NT-3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT-3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).     

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.     

Effect of NT-4 and BDNF delivery to damaged sciatic nerves on phenotypic recovery of fast and slow muscles fibres

We investigated whether neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF) affected the reinnervation of slow and fast motor units. Neurotrophin-impregnated or plain fibronectin (FN) conduits were inserted into a sciatic nerve gap. Fast extensor digitorum longus (EDL) and slow soleus muscles were collected 4 months postsurgery. Muscles were weighed and fibre type proportion and mean fibre diameters were derived from muscle cross-sections. All fibre types in muscles from FN animals were severely atrophied and this correlated well with type 1 fibre loss and atrophy in soleus and type 2b loss and atrophy in EDL. Treatment with NT-4 reversed soleus but not EDL mass loss above the FN group by significantly restoring type 1 muscle fibre proportion and diameters towards those of normal unoperated animals. BDNF did not increase muscle mass but did have minor effects on fibre type and diameter. Thus, NT-4 significantly improved slow motor unit recovery, and provides a basis for therapies intended to aid the functional recovery of muscles after denervating injury.     

BDN F Down-regulates Neurotrophin Responsiveness, TrkB Protein and TrkB mRNA Levels in Cultured Rat Hippocampal Neurons

Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal TrkB protein does not appear to be just a developmental phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro , this leads to long-term functional desensitization to BDNF. NT-3 and NT-4/5. as well as down-regulation of trkB and trkC mRNA.     

The reciprocal change of neurotrophin-4 and glial cell line-derived neurotrophic factor protein in the muscles,spinal cord and cerebellum of the dy mouse

Laminin alpha2 (merosin)-deficient congenital muscular dystrophy (CMD) patients show progressive muscle fiber necrosis and ineffective muscle regeneration, probably due to a lower formation of multinucleated myotubes due to an adhesion defect of myoblasts to each other. Some recent studies found that CMD patients have a white matter disorder and cerebellum atrophy. In the spinal cord of dy mice, a model of CMD, inducible nitric oxide synthase (iNOS) was markedly expressed. Using Western blotting and immunohistochemical analyses, we investigated the levels of neurotrophin-4 (NT-4), brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the central nervous system and skeletal muscles of dy mice. In the dy mice, the microtubule-associated protein-2 (MAP-2) protein level was markedly decreased in the Purkinje and granule cells of the cerebellum, and in lumbar motoneurons of the spinal cord. The motoneurons and axons of dy mice possessed lower expressions of phosphorylated tau. The amount of NT-4 was markedly lower in the cerebellum, spinal cord and hindlimb muscles of dy mice. In dy mice, GDNF was markedly enhanced in the Purkinje and granule cells of the cerebellum, in many lumbar motoneurons, and in the regenerating atrophied fibers. The CNTF protein level did not differ in the hindlimb muscles between the normal and dy mice. Therefore, GDNF could act to inhibit the death of Purkinje and granular neurons, and motoneurons, and to promote the remodeling of the neuromuscular junction of atrophied muscle fibers of dy mice. Furthermore, dy mice include neurogenic abnormalities in the cerebellum and spinal cord along with myogenic disorder of muscle fibers.     

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.     

The adaptive response of transforming growth factor-β2 and -βRII in the overloaded, regenerating and denervated muscles of rats

Using a muscle cell line and satellite cell cultures, it has been shown that transforming growth factor-β (TGF-β) has a powerful inhibitory effect on myoblast replication and differentiation. However, little work has been done on the possible role of TGF-β in adult muscle in vivo. Using Western blot and immunohistochemical analyses, we investigated normal distribution of TGF-β2 and TGF-βRII proteins between slow and fast-type muscles, and the adaptive response of these proteins in the mechanically overloaded muscles, in the regenerating muscles following bupivacaine injection and in the denervated muscle after section of sciatic nerve. Slight TGF-β2 immunoreactivity was detected both in slow- and fast-type muscles of mature rat. The amount of TGF-βRII protein was markedly greater in fast-type muscles. In the overloaded muscle, immunohistochemical analysis showed a marked increase in TGF-β2 immunoreactivity in the mononuclear cells (probably endothelial and perithelial or smooth muscle cells of endomysial capillaries) of the extracellular space at 3 and 6 days post surgery. Rapid increase of TGF-β2 protein and concomitant decrease of the receptor (TGF-βRII) were observed in the mechanically overloaded and regenerating muscles. On the other hand, denervation of slow- and fast-type muscles showed a rapid increase in TGF-β2 protein, but did not elicit a concomitant decrease of TGF-βRII. These results indicate that TGF-βRII is preferentially distributed in fast-type muscles. Furthermore, TGF-β2 may play an important role in muscle hypertrophy and regeneration by the usage of TGF-βRII. Received: 13 January 1999 / Revised: 22 April/15 June 1999 / Accepted: 17 June 1999     

Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei

Brain derived neurotrophic factor (BDNF) is normally expressed by a small number of predominantly trkA-expressing dorsal root ganglion cells. Using immunocytochemistry and in situ hybridization, we have examined the effect of sciatic nerve section on the expression of BDNF in the adult rat. Following axotomy there was a long lasting (4-week) increase in BDNF mRNA and protein in large-diameter, trkB- and trkC-expressing dorsal root ganglion cells. By 2 days postaxotomy, expression of BDNF mRNA had increased from 2% of trkB cells to 50%, and from 18% of trkC cells to 56%. In contrast, BDNF expression in most trkA cells was unchanged, although was increased in the small population of medium- and large-sized trkA cells. Following axotomy, BDNF-immunoreactive terminals appeared in the central axonal projections of large-diameter cells, including the deep dorsal horn and gracile nucleus. Neuropeptide Y was also upregulated following axotomy and was coexpressed with BDNF in the cell bodies and central terminals of the large cells. Ultrastructural analysis in lamina IV of the spinal cord revealed that BDNF terminals in these central projections establish synaptic contacts. Immunoreactivity at 4 weeks was also observed in pericellular baskets that contained calcitonin gene-related peptide (CGRP) and surrounded trkA- and trkB-expressing cells in L4 and L5 lumbar ganglia. These baskets are likely to arise from local, highly immunoreactive, BDNF/CGRP/trkA-expressing cells. Our results identify several novel targets for BDNF and imply that it acts locally in both autocrine and paracrine modes, as well as centrally in a synaptic mode, to modulate the response of somatosensory pathways in nerve injury.     

BDNF abolishes the survival effect of NT-3 in axotomized Clarke neurons of adult rats

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) have previously been shown to support survival and axonal regeneration in various types of neurons. Also, synergistic neuroprotective effects of these neurotrophins have been reported in descending rubrospinal neurons after cervical spinal cord injury (Novikova et al., [2000] Eur. J. Neurosci. 12:776-780). The present study investigates the effects of intrathecally delivered NT-3 and BDNF on the survival and atrophy of ascending spinocerebellar neurons of Clarke nucleus (CN) after cervical spinal cord injury in adult rats. At 8 weeks after cervical spinal cord hemisection, 40% of the axotomized CN neurons had been lost, and the remaining cells exhibited marked atrophy. Microglial activity was significantly increased in CN of the operated side. Intrathecal infusion of NT-3 for 8 weeks postoperatively resulted in 91% cell survival and a reduction in cell atrophy, but did not reduce microglial activity. In spite of the fact that the CN neurons expressed both TrkC and TrkB receptors, only NT-3 had a neuroprotective effect, whereas BDNF was ineffective. Furthermore, when a combination of BDNF and NT-3 was administered, the neuroprotective effect of NT-3 was lost. The present results indicate a therapeutic potential for NT-3 in the treatment of spinal cord injury, but also demonstrate that in certain neuronal populations the neuroprotection obtained by a combination of neurotrophic factors may be less than that of a single neurotrophin.     

Expression of neurotrophins in skeletal muscle: Quantitative comparison and significance for motoneuron survival and maintenance of function

Neurotrophins play a crucial role in the regulation of survival and maintenance of specific functions of various populations of neurons. Brain-derived neurotrophic factor (BDNF), newrotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4) have been shown to support motoneuron survival during embryonic development and, after birth, to protect motoneurons from degeneration after nerve lesion. We have compared the levels of these neurotrophins in skeletal muscle by quantitative Northern blot analysis, both during embryonic development and postnatally. We localized the sites of expression of these neurotrophins by in situ hybridisation and analysed the expression of trkB in the spinal cord by in situ hybridisation and immunohistochemistry. NT-3 is most abundantly expressed both during embryonic development and in the postnatal phase, followed by NT-4. The levels of BDNF are very low, in particular after birth. After nerve lesion, NT-3 mRNA essentially remained unchanged, whereas NT-4 mRNA rapidly decreased. The slow increase in BDNF expression seems to be essentially due to the expression in Schwann cells rather than skeletal muscle, demonstrated by in situ hybridisation. Our data indicate that motoneurons can receive trophic support from several members of the neurotrophin gene family during the period of naturally occurring cell death. Postnatally, the predominant ligand acting via trkB on motoneurons is NT-4, whereas BDNF expression seems to play a role mainly after nerve lesion. © 1995 Wiley-Liss, Inc.     

Neurotrophic factors expressed in both cortex and spinal cord induce axonal plasticity after spinal cord injury

We reported recently that overexpression of neurotrophin-3 (NT-3) by motoneurons in the spinal cord of rats will induce sprouting of corticospinal tract (CST) axons (Zhou et al. [2003] J. Neurosci. 23:1424-1431). We now report that overexpression of brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF) in the rat sensorimotor cortex near the CST neuronal cell bodies together with overexpression of NT-3 in the lumbar spinal cord significantly increases axonal sprouting compared to that induced by NT-3 alone. Two weeks after unilaterally lesioning the CST at the level of the pyramids, we injected rats with saline or adenoviral vectors (Adv) carrying genes coding for BDNF (Adv.BDNF), GDNF (Adv.GDNF) or enhanced green fluorescent protein (Adv.EGFP) at six sites in the sensorimotor cortex, while delivering Adv.NT3 to motoneurons in each of these four groups on the lesioned side of the spinal cord by retrograde transport from the sciatic nerve. Four days later, biotinylated dextran amine (BDA) was injected into the sensorimotor cortex on the unlesioned side to mark CST axons in the spinal cord. Morphometric analysis of axonal sprouting 3 weeks after BDA injection showed that the number of CST axons crossing the midline in rats treated with Adv.BDNF or Adv.GDNF were 46% and 52% greater, respectively, than in rats treated with Adv.EGFP or PBS (P < 0.05). These data demonstrate that sustained local expression of neurotrophic factors in the sensorimotor cortex and spinal cord will promote increased axonal sprouting after spinal cord injury, providing a basis for continued development of neurotrophic factor therapy for central nervous system damage.     

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤*☆

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。 关键词：坐骨神经损伤;重组腺病毒;脑源性神经营养因子;基因转染;免疫组织化学;原位分子杂交;神经再生