Neurotrophin expression by spinal motoneurons in adult and developing rats

Expression of the neurotrophins NT-4, brain-derived neurotrophic factor (BDNF), and NT-3 in adult rat lumbosacral spinal cord motoneurons is reported. A sensitive in situ hybridization procedure demonstrates localization of the mRNA for each of these neurotrophins within spinal motoneurons of the adult and in early postnatal development. A majority of adult rat spinal cord lumbar motoneurons (approximately 63%) express NT-4 mRNA as assessed by counting motoneurons in the L4 and L5 segments of two adult rat spinal cords on adjacent cresyl violet-stained and in situ hybridization sections. Similarly, a majority of lumbar motoneurons (approximately 73%) express BDNF mRNA. Further analyses of adjacent lumbar spinal cord sections revealed that many, although not all motoneurons coexpress both NT-4 and BDNF mRNAs. At birth, the mRNA encoding NT-3 is expressed in motoneurons, but BDNF mRNA is not apparent until postnatal day 5 (P5) and NT-4 mRNA first appears at P9. The potential biological significance of neurotrophin mRNA expression in spinal motoneurons is supported by immunohistochemical localization of each neurotrophin protein in adult motoneurons. We discuss the potential role of spinal cord neurotrophins as autocrine or paracrine factors involved in modulating motoneuron synaptic function.     

Immune activation is required for NT-3-induced axonal plasticity in chronic spinal cord injury

After an unilateral lesion of the corticospinal tract (CST) at the level of the medulla over-expression of Neurotrophin-3 (NT-3) in lumbar spinal cord motoneurons induced axonal sprouting of the intact CST in the acutely injured but not uninjured or chronically injured spinal cord in rats. This suggested that processes associated with immune-mediated wound healing may act with NT-3 to induce neuroplasticity. To test whether immune processes were involved we measured NT-3-induced axonal sprouting in immunosuppressed compared to immunocompetent rats. Rats were immunosuppressed with anti-leukocyte antibodies 1 day before receiving a CST lesion and then 2 weeks later NT-3 was over-expressed in the lumbar spinal motoneurons with an adenoviral vector carrying the NT-3 gene targeted to the motoneurons by retrograde transport. At 35 days post-lesion no axonal sprouting was measured in immunosuppressed rats whereas axonal sprouting was measured in the immunocompetent rats. We then tested whether re-evoking an immune response in chronically lesioned rats would induce neuroplasticity. Rats received CST lesions and then 4 months later were treated with systemic injections of lipopolysaccharide (LPS) 7 days before NT-3 was over-expressed in the lumbar spinal motoneurons. Axonal sprouting was observed in the LPS treated rats but not in control animals that were not treated with LPS. Further studies showed that lesioning the CST activated and LPS reactivated microglia and CD4(+) T-cells in the acutely lesioned and chronically lesioned rats, respectively. However, immunosuppression only decreased the number of activated CD4(+) T-cells suggesting they were responsible for the support of axonal growth. These observations demonstrate that processes associated with immune-mediated wound healing play a role in NT-3-induced neuroplasticity after injury.     

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.     

Temporal changes in the expression of some neurotrophins in spinal cord transected adult rats

Functional recovery of neurons in the spinal cord after physical injury is essentially abortive in clinical cases. As neurotrophins had been reported to be responsible, at least partially, for the lesion-induced recovery of spinal cord, it is not surprising that they have become the focus of numerous studies. Studies on endogenous neurotrophins, especially the three more important ones, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in injured spinal cord might provide some important clues in clinical treatment. Here we investigate the immunohistological expression of the above three factors at lower thoracic levels of the spinal cord as well as changes in the motor functions of the adult rat hindlimbs after cord transection. The injured rats were allowed to survive 3, 7, 14 and 21 days post operation (dpo). Flaccid paralysis was seen at 3 dpo following cord transection, however, hindlimb function showed partial recovery from 7 dpo to 21 dpo. The numbers of NGF, BDNF and NT-3 immunopositive neurons and their optical densities all increased in the lesion-induced cord. The immuno-expression of NGF and BDNF peaked at 7 dpo, while that of NT-3 peaked at 7 dpo and remained so at least up to 14 dpo. These results suggested that neurotrophins might play essential roles in functional recovery of after spinal cord injury, but the time points for the expression of the three factors differed somewhat.     

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

Following cervical spinal cord injury at C(2) (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability.     

Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats

We compared the effect of viral administration of brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT-3) on locomotor recovery in adult rats with complete thoracic (T10) spinal cord transection injuries, in order to determine the effect of chronic neurotrophin expression on spinal plasticity. At the time of injury, BDNF, NT-3 or green fluorescent protein (GFP) (control) was delivered to the lesion via adeno-associated virus (AAV) constructs. AAV-BDNF was significantly more effective than AAV-NT-3 in eliciting locomotion. In fact, AAV-BDNF-treated rats displayed plantar, weight-supported hindlimb stepping on a stationary platform, that is, without the assistance of a moving treadmill and without step training. Rats receiving AAV-NT-3 or AAV-GFP were incapable of hindlimb stepping during this task, despite provision of balance support. AAV-NT-3 treatment did promote the recovery of treadmill-assisted stepping, but this required continuous perineal stimulation. In addition, AAV-BDNF-treated rats were sensitized to noxious heat, whereas AAV-NT-3-treated and AAV-GFP-treated rats were not. Notably, AAV-BDNF-treated rats also developed hindlimb spasticity, detracting from its potential clinical applicability via the current viral delivery method. Intracellular recording from triceps surae motoneurons revealed that AAV-BDNF significantly reduced motoneuron rheobase, suggesting that AAV-BDNF promoted the recovery of over-ground stepping by enhancing neuronal excitability. Elevated nuclear c-Fos expression in interneurons located in the L2 intermediate zone after AAV-BDNF treatment indicated increased activation of interneurons in the vicinity of the locomotor central pattern generator. AAV-NT-3 treatment reduced motoneuron excitability, with little change in c-Fos expression. These results support the potential for BDNF delivery at the lesion site to reorganize locomotor circuits.     

Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury

We have conducted studies to determine the potential of exercise to benefit the injured spinal cord using neurotrophins. Adult rats were randomly assigned to one of three groups: (1) intact control (Con); (2) sedentary, hemisected at a mid-thoracic level (Sed-Hx), or (3) exercised, hemisected (Ex-Hx). One week after surgery, the Ex-Hx rats were exposed to voluntary running wheels for 3, 7, or 28 days. BDNF mRNA levels on the lesioned side of the spinal cord lumbar region of Sed-Hx rats were approximately 80% of Con values at all time points and BDNF protein levels were approximately 40% of Con at 28 days. Exercise compensated for the reductions in BDNF after hemisection, such that BDNF mRNA levels in the Ex-Hx rats were similar to Con after 3 days and higher than Con after 7 (17%) and 28 (27%) days of exercise. After 28 days of exercise, BDNF protein levels were 33% higher in Ex-Hx than Con rats and were highly correlated (r=0.86) to running distance. The levels of the downstream effectors for the action of BDNF on synaptic plasticity synapsin I and CREB were lower in Sed-Hx than Con rats at all time points. Synapsin I mRNA and protein levels were higher in Ex-Hx rats than Sed-Hx rats and similar to Con rats at 28 days. CREB mRNA values were higher in Ex-Hx than Sed-Hx rats at all time points. Hemisection had no significant effects on the levels of NT-3 mRNA or protein; however, voluntary exercise resulted in an increase in NT-3 mRNA levels after 28 days (145%). These results are consistent with the concept that synaptic pathways under the regulatory role of BDNF induced by exercise can play a role in facilitating recovery of locomotion following spinal cord injury.     

脑源性神经生长因子与脊髓全横断大鼠的神经可塑性

The present study employed a rat model of T10 spinal cord transection.Western blot analyses revealed increased brain-derived neurotrophic factor(BDNF) expression in spinal cord segments caudal to the transection site following injection of replication incompetent herpes simplex virus vector(HSV-BDNF) into the subarachnoid space.In addition,hindlimb locomotor functions were improved.In contrast,BDNF levels decreased following treatment with replication defective herpes simplex virus vector construct small interference BDNF(HSV-siBDNF).Moreover,hindlimb locomotor functions gradually worsened.Compared with the replication incompetent herpes simplex virus vector control group,extracellular signal regulated kinase1/2 expression increased in the HSV-BDNF group on days 14 and 28 after spinal cord transection,but expression was reduced in the HSV-siBDNF group.These results suggested that BDNF plays an important role in neural plasticity via extracellular signal regulated kinase1/2 signaling pathway in a rat model of adult spinal cord transection.     

Temporal changes in the expression of TGF-beta 1 and EGF in the ventral horn of the spinal cord and associated precentral gyrus in adult Rhesus monkeys subjected to cord hemisection

It is well known that some growth factors can not only rescue neurons from death, but also improve motor functions following spinal cord injury. However, their cellular distribution in situ and temporal expressions following spinal cord injury have not been determined, especially in primates. This study investigated the temporal changes in the expression of two growth factors--epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta1) in the injured motoneurons of the spinal cord and the associated precentral gyrus in adult Rhesus monkeys subjected to spinal cord hemisection. Animals were allowed to survive 7, 14, 30 and 90 days post operation (dpo). Functional recovery of the hindlimbs was assessed using Tarlov scale. The immunohistological expressions of EGF and TGF-beta1 in the ventral horn motoneurons decreased sharply at 7 dpo in the cord segments caudal to the lesion site, which was followed by an increase and a peak between 14 and 30 dpo for EGF and at 90 dpo for TGF-beta1. Changes in the expression of EGF in the precentral gyrus were similar to that in the spinal cord. No TGF-beta1 immunoreactive neurons were detected in the precentral gyrus. In the spinal segments rostral to the lesion, the expressions of EGF and TGF-beta1 peaked at 30 dpo. The mRNA of EGF was detected in both spinal motoneurons and the precentral gyrus, while that of TGF-beta1, only in the spinal motoneuons, suggesting that the spinal motoneurons themselves could synthesize both the growth factors. Partial locomotor recovery in hindlimbs was seen, especially after 14 dpo. It was concluded that a possible association existed between the modulation of EGF and TGF-beta1 and the recovery of locomotor function, and their roles differed somewhat in the neuroplasticity observed after spinal cord injury in primates.     

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.     

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.     

驽药针刺对大鼠脊髓损伤后运动功能及BDNF表达的影响

目的研究驽药针刺在大鼠脊髓损伤后运动功能变化以及BDNF表达的变化。方法采用脊髓半横断损伤模型。100只SD大鼠随机分为对照组、假手术组、脊髓损伤组、单纯针刺组、驽药针刺组,每组分为3天、7天、14天、21天共4个亚组,每组5只。BBB法评定大鼠后肢运动功能变化,免疫组化法检测大鼠脊髓中BDNF的表达变化。结果 BBB评分显示驽药针刺组的各时间点评分均高于脊髓损伤组(P0.05),驽药针刺组7、14、21d的BDNF表达均高于脊髓损伤组(P0.05),且与BBB评分呈正相关(r=0.717,P0.05)。结论驽药针刺可明显改善脊髓损伤大鼠的运动功能,并可明显促进大鼠脊髓损伤后BDNF的表达。     

Changes in truncated trkB and p75 receptor expression in the rat spinal cord following spinal cord hemisection and spinal cord hemisection plus neurotrophin treatment

Although numerous studies have examined the effects of neurotrophin treatment following spinal cord injury, few have examined the changes that occur in the neurotrophin receptors following either such damage or neurotrophin treatment. To determine what changes occur in neurotrophin receptor expression following spinal cord damage, adult rats received a midthoracic spinal cord hemisection alone or in combination with intrathecal application of brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Using immunohistochemical and in situ hybridization techniques, p75, trkA, trkB, and trkC receptor expression was examined throughout the spinal cord. Results showed that trkA, full-length trkB, and trkC receptors were not present in the lesion site but had a normal expression pattern in uninjured parts of the spinal cord. In contrast, p75 receptor expression occurred on Schwann cells throughout the lesion site. BDNF and NT-3 (but not saline) applied to the lesion site increased this expression. In addition, the truncated trkB receptor was expressed in the border between the lesion and intact spinal cord. Truncated trkB receptor expression was also increased throughout the white matter ipsilateral to the lesion and BDNF (but not NT-3 or saline) prevented this increase. The study is the first to show changes in truncated trkB receptor expression that extend beyond the site of a spinal cord lesion and is one of the first to show that BDNF and NT-3 affect Schwann cells and/or p75 expression following spinal cord damage. These results indicate that changes in neurotrophin receptor expression following spinal cord injury could influence the availability of neurotrophins at the lesion site. In addition, neurotrophins may affect their own availability to damaged neurons by altering the expression of the p75 and truncated trkB receptor.     

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.     

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.     

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.     

A possible role for BDNF, NT-4 and TrkB in the spinal cord and muscle of rat subjected to mechanical overload, bupivacaine injection and axotomy.

Neurotrophins play a crucial role in the regulation of survival and the maintenance of specific functions for various populations of neurons. Neurotrophin-4 (NT-4) is most abundant in skeletal muscle, and is thought to promote sciatic nerve sprouting, inhibit agrin-induced acetylcholine receptor (AChR) clustering, evoke postsynaptic potentiation and induce mitochondrial proliferation. Using Western blot analysis, immunoprecipitation and immunohistochemistry, we investigated the distribution of NT-4 in slow- and fast-type muscles. We also tested the adaptive response of this protein in the mechanically overloaded muscle, in the regenerating muscle following bupivacaine injection and in the denervated muscle. Additionally, we investigated whether TrkB phosphorylation in the spinal cord and in the sciatic nerve occurs through the interaction with BDNF or NT-4 when the innervating muscle is damaged. Markedly more NT-4 was expressed in fast-type muscles compared with the slow types. TrkB protein was more frequently observed around the edge of myofibers (neuromuscular junction) of the soleus muscle compared with the gastrocnemius muscle. TrkB tyrosine phosphorylation occurred in the spinal cord but not in the sciatic nerve 24 h after bupivacaine injection of the innervating muscle. At the same time, the amount of TrkB co-precipitating with BDNF was markedly increased in the spinal cord. A rapid activation of TrkB (1-8 h) was also observed in the spinal cord after axotomy,while the amount of TrkB co-precipitating with NT-4 was markedly lower after axotomy. These results indicate that NT-4 is preferentially distributed in fast-type muscles. Furthermore, by interacting with BDNF and NT-4, the TrkB in the spinal cord may be important for the survival of motoneurons and outgrowth of injured peripheral axons following muscle damage.     

Fibroblast growth factor-2 mRNA expression in the brainstem and spinal cord of normal and chronic spinally transected urodeles

Descending pathways in the spinal cord of adult urodele amphibians show a high regenerative ability after body spinal cord transection; regenerated axons regrow into the transected spinal cord, and hindlimb locomotor recovery occurs spontaneously. Little is currently known about the molecular basis of spinal cord regeneration in urodeles, but it is believed that fibroblast growth factor-2 (FGF2) may play an important role by inducing proliferation of neural progenitor cells. The aim of our study, using in situ hybridization in adult Pleurodeles waltlii, was twofold: 1) to document FGF2 mRNA expression pattern along the brainstem-spinal cord of intact salamanders and 2) to investigate the changes in this pattern in animals unable to display hindlimb locomotor movements and in animals having fully recovered hindlimb locomotor activity after body spinal cord transection. This design establishes a firm basis for further studies on the role of FGF2 in functional recovery of hindlimb locomotion. Our results revealed a decreasing rostrocaudal gradient in FGF2 mRNA expression along the brainstem-spinal cord in intact animals. They further demonstrated a long-lasting up-regulation of FGF2 mRNA expression in response to spinal transection at the midtrunk level, both in brainstem and in the spinal cord below the injury. Finally, double immunolabeling showed that FGF2 was up-regulated in neuroglial, presumably undifferentiated, cells. Therefore, we propose that FGF2 may be involved in cell proliferation and/or neuronal differentiation after body spinal cord transection in salamander and could thus play an important role in functional recovery of locomotion after spinal lesion.     

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.