Cycling exercise and fetal spinal cord transplantation act synergistically on atrophied muscle following chronic spinal cord injury in rats

The potential of two interventions, alone or in combination, to restore chronic spinal cord transection-induced changes in skeletal muscles of adult Sprague-Dawley rats was studied. Hind limb skeletal muscles were examined in the following groups of animals: rats with a complete spinal cord transection (Tx) for 8 weeks; Tx with a 4-week delay before initiation of a 4-week motor-assisted cycling exercise (Ex) program; Tx with a 4-week delay before transplantation (Tp) of fetal spinal cord tissue into the lesion cavity; Tx with a 4-week delay before Tp and Ex; and uninjured control animals. Muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas were significantly reduced 8 weeks after transection. Whereas transplantation of fetal spinal cord tissue did not reverse this atrophy and exercise alone had only a modest effect in restoring lost muscle mass, the combination of exercise and transplantation significantly increased muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas in both soleus and plantaris muscles. Spinal cord injury (SCI) also caused changes in myosin heavy chain (MyHC) expression toward faster isoforms in both soleus and plantaris and increased soleus myofiber succinate dehydrogenase (SDH) activity. Combined exercise and transplantation led to a change in the expression of the fastest MyHC isoform in soleus but had no effect in the plantaris. Exercise alone and in combination with transplantation reduced SDH activity to control levels in the soleus. These results suggest a synergistic action of exercise and transplantation of fetal spinal cord tissue on skeletal muscle properties following SCI, even after an extended post-injury period before intervention.     

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.     

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.     

Differences in neurotrophic factor gene expression profiles between neonate and adult rat spinal cord after injury

The capacity of the central nervous system for axonal growth decreases as the age of the animal at the time of injury increases. Changes in the expression of neurotrophic factors within embryonic and early postnatal spinal cord suggest that a lack of trophic support contributes to this restrictive growth environment. We examined neurotrophic factor gene profiles by ribonuclease protection assay in normal neonate and normal adult spinal cord and in neonate and adult spinal cord after injury. Our results show that in the normal developing spinal cord between postnatal days 3 (P3) and P10, compared to the normal adult spinal cord, there are higher levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and glial-derived neurotrophic factor (GDNF) mRNA expression and a lower level of ciliary neurotrophic factor (CNTF) mRNA expression. Between P10 and P17, there is a significant decrease in the expression of NGF, BDNF, NT-3, and GDNF mRNA and a contrasting steady and significant increase in the level of CNTF mRNA expression. These findings show that there is a critical shift in neurotrophic factor expression in normal developing spinal cord between P10 and P17. In neonate spinal cord after injury, there is a significantly higher level of BDNF mRNA expression and a significantly lower level of CNTF mRNA expression compared to those observed in the adult spinal cord after injury. These findings suggest that high levels of BDNF mRNA expression and low levels of CNTF mRNA expression play important roles in axonal regrowth in early postnatal spinal cord after injury.     

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.     

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.     

Passive exercise and fetal spinal cord transplant both help to restore motoneuronal properties after spinal cord transection in rats

Spinal cord transection influences the properties of motoneurons and muscles below the lesion, but the effects of interventions that conserve muscle mass of the paralyzed limbs on these motoneuronal changes are unknown. We examined the electrophysiological properties of rat lumbar motoneurons following spinal cord transection, and the effects of two interventions shown previously to significantly attenuate the associated hindlimb muscle atrophy. Adult rats receiving a complete thoracic spinal cord transection (T-10) were divided into three groups receiving: (1) no further treatment; (2) passive cycling exercise for 5 days/week; or (3) acute transplantation of fetal spinal cord tissue. Intracellular recording of motoneurons was carried out 4-5 weeks following transection. Transection led to a significant change in the rhythmic firing patterns of motoneurons in response to injected currents, as well as a decrease in the resting membrane potential and spike trigger level. Transplants of fetal tissue and cycling exercise each attenuated these changes, the latter having a stronger effect on maintenance of motoneuron properties, coinciding with the reported maintenance of structural and biochemical features of hindlimb muscles. The mechanisms by which these distinct treatments affect motoneuron properties remain to be uncovered, but these changes in motoneuron excitability are consistent with influences on ion conductances at or near the initial segment. The results may support a therapeutic role for passive limb manipulation and transplant of stem cells in slowing the deleterious responses of motoneurons to spinal cord injury, such that they remain more viable for subsequent alternative strategies.     

Long-term effects of spinal cord transection on fast and slow rat skeletal muscle: II. Morphometric properties

Morphometric properties of rat soleus and extensor digitorum longus muscles were studied 1 year following complete thoracic spinal cord transection (spinal cord level T9). Both muscles demonstrated almost complete type 1 to type 2 muscle fiber type conversion after 1 year. Muscle fiber atrophy was observed in both muscles. Type 2 fiber atrophy occurred to about the same extent in both muscles. Atrophy was most severe for the soleus type 1 fibers (50% decrease in size). Calculations based on the fiber type and size changes observed indicate that the percentage of the muscle cross-sectional area occupied by each fiber type was almost the same for both muscles 1 year after transection. Discriminant analysis of the data indicated that the percentage of type 2 fibers present in the muscle was the best discriminator between the various groups. These morphometric data provided a basis for understanding the contractile results presented in the previous study as well as insights into the mechanism of transformation in skeletal muscle. Furthermore, inherent differences between type 1 and type 2 fibers were demonstrated between predominantly slow and predominantly fast muscles. Thus, after almost one-half a lifetime of transection, rat muscles are almost completely transformed to fast muscle, and, regardless of initial conditions, have nearly identical properties.     

Effects of fetal spinal cord tissue transplants and cycling exercise on the soleus muscle in spinalized rats.

Studies were carried out to determine if an intraspinal transplant (Trpl) of fetal spinal cord tissue or hind limb exercise (Ex) affected the changes in myosin heavy chain (MyHC) composition or myofiber size that occur following a complete transection (Tx) of the lower thoracic spinal cord of the adult rat. In one group of animals, transplants were made acutely, whereas in a second group, daily cycling exercise was initiated 5 days after injury, with animals in both groups being sacrificed 90 days after injury. The soleus muscle is normally composed of myofibers expressing either type I (90%) or type IIa (10%) MyHC. Following a spinal transection, expression of type I MyHC isoform decreased (18% of myofibers), type IIa MyHC expression increased (65% of myofibers), and the majority of myofibers (80%) expressed type IIx MyHC. Most myofibers coexpressed multiple MyHC isoforms. Compared with Tx only, with Ex or with Trpl, there was a decrease in the number of myofibers expressing type I or IIa isoforms but little change in expression of IIx MyHC. Myofibers expressing the IIb isoform appeared in several transplant recipients but not after exercise. Transection resulted in atrophy of type I myofibers to approximately 50% of normal size, whereas myofibers were significantly larger after exercise (74% of control) and in Trpl recipients (77% of control). Type IIa myofibers also were significantly larger in Trpl recipients compared with the Tx only group. Overall, the mean myofiber size was significantly greater after exercise and in Trpl recipients compared with myofibers in Tx only animals. Thus, although neither strategy shifted the MyHC profile towards the control, both interventions influenced the extent of atrophy observed after spinalization. These data suggest that palliative strategies can be developed to modulate some of the changes in hind limb muscles that occur following a spinal cord injury.     

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

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

Brain-Derived Neurotrophic Factor mRNA Levels Are Up-Regulated in Hindlimb Skeletal Muscle of Diabetic Rats: Effect of Denervation

In a previous study the levels of brain-derived neurotrophic factor (BDNF) mRNA were shown to be elevated in skeletal muscle of the diabetic rat compared with age-matched control animals. It was proposed that diabetes-induced changes in nerve function may initiate changes in nerve/muscle contact akin to those following denervation of target skeletal muscle. In this study hindlimb skeletal muscles were denervated by sciatic nerve crush or transection and the effect on BDNF mRNA levels in control and diabetic rats was observed using Northern blotting. Contralateral to the side of nerve injury, the levels of BDNF mRNA in soleus muscle of diabetic rats were higher than in controls (three- to sevenfold), as has been seen previously in diabetic rats without any axotomy. Sciatic nerve crush or transection, of 1 week or of 3 weeks duration, lowered the levels of BDNF mRNA by 50% in ipsilateral soleus muscle of diabetic rats. BDNF mRNA levels in contralateral gastrocnemius muscle were not similarly raised in diabetic rats compared with controls and nerve injury had no effect. In control animals, ipsilaterally, the BDNF mRNA levels of soleus muscle were raised approximately twofold at 1 week and were lowered by approximately 50% at 3 weeks following nerve injury. Neurotrophin-3 mRNA levels were reduced approximately 50% in soleus muscle of diabetic rats compared with control rats, and nerve injury had no significant effect. The specific up-regulation of BDNF mRNA in soleus muscle of diabetic rats is discussed in terms of a proposed diabetes-induced ischemia within hindlimb skeletal muscle, with a protective role for BDNF in muscle and/or nerve being introduced.     

Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vector-mediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor

Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.     

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 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.     

Fibrillation potentials following spinal cord injury: improvement with neurotrophins and exercise

Fibrillation potentials and positive sharp waves (spontaneous potentials) are the electrophysiological hallmark of denervated skeletal muscle, and their detection by intramuscular electromyography (EMG) is the clinical gold standard for diagnosing denervated skeletal muscle. Surprisingly, spontaneous potentials have been described following human and experimental spinal cord injury (SCI) in muscles innervated by spinal cord segments distal to the level of direct spinal injury. To determine whether electrophysiological abnormalities are improved by two therapeutic interventions for experimental SCI, neurotrophic factors and exercise training, we studied four representative hindlimb muscles in adult domestic short-hair cats following complete transection of the spinal cord at T11-T12. In untreated cats, electrophysiological abnormalities persisted unchanged for 12 weeks postinjury, the longest duration studied. In contrast, fibrillations and positive sharp waves largely resolved in animals that underwent weight-supported treadmill training or received grafts containing fibroblasts genetically modified to express brain-derived neurotrophic factor and neurotrophin-3. These findings suggest that neurotrophins and activity play an important role in the poorly understood phenomenon of fibrillations distal to SCI.     

Repair of spinal cord injury by neural stem cells modified with BDNF gene in rats

Objective To explore repair of spinal cord injury by neural stem cells （NSCs） modified with brain derived neurotrophic factor （BDNF） gene （BDNF-NSCs） in rats. Methods Neural stem cells modified with BDNF gene were transplanted into the complete transection site of spinal cord at the lumbar 4 （L4） level in rats. Motor function of rats＇ hind limbs was observed and HE and X-gal immunoeytochemical staining, in situ hybridization, and retrograde HRP tracing were also performed. Results BDNF-NSCs survived and integrated well with host spinal cord. In the transplant group, some X-gal positive, NF-200 positive, GFAP positive, BDNF positive, and BDNF mRNA positive cells, and many NF-200 positive nerve fibers were observed in the injury site. Retrograde HRP tracing through sciatic nerve showed some HRP positive cells and nerve fibers near the rostral side of the injury one month after transplant and with time, they increased in number. Examinations on rats＇ motor function and behavior demonstrated that motor function of rats＇ hind limbs improved better in the transplant group than the injury group. Conclusion BDNF-NSCs can survive, differentiate, and partially integrate with host spinal cord, and they significantly ameliorate rats＇ motor function of hind limbs, indicating their promising role in repairing spinal cord injury.     

Repair of spinal cord injury by neural stem cells modified with BDNF gene in rats

Objective To explore repair of spinal cord injury by neural stem cells (NSCs) modified with brain derived neurotrophic factor (BDNF) gene (BDNF-NSCs) in rats. Methods Neural stem cells modified with BDNF gene were transplanted into the complete transection site of spinal cord at the lumbar 4 (L4) level in rats. Motor function of rats' hind limbs was observed and HE and X-gal immunocytochemical staining, in situ hybridization, and retrograde HRP tracing were also performed, Results BDNF-NSCs survived and integrated well with host spinal cord. In the transplant group, some X-gal positive, NF-200 positive, GFAP positive, BDNF positive, and BDNF mRNA positive cells, and many NF-200 positive nerve fibers were observed in the injury site. Retrograde HRP tracing through sciatic nerve showed some HRP positive cells and nerve fibers near the rostral side of the injury one month after transplant and with time, they increased in number. Examinations on rats' motor function and behavior demonstrated that motor function of rats' hind limbs improved better in the transplant group than the injury group. Conclusion BDNF-NSCs can survive, differentiate, and partially integrate with host spinal cord, and they significantly ameliorate rats' motor function of hind limbs, indicating their promising role in repairing spinal cord injury.     

Bone marrow stromal cells promote neurite outgrowth of spinal motor neurons by means of neurotrophic factors in vitro

Transplantation of bone marrow stromal cells (BMSCs) into spinal cord injury models has shown significant neural function recovery; however, the underlying mechanisms have not been fully understood. In the present study we examined the effect of BMSCs on neurite outgrowth of spinal motor neuron using an in vitro co-culture system. The ventral horn of the spinal grey matter was harvested from neonatal Sprague–Dawley rats, cultured with BMSCs, and immunostained for neurofilament-200 (NF-200). Neurite outgrowth of spinal motor neurons was measured using Image J software. ELISA was used to quantify neurotrophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in culture media, and antibodies or exogenous neurotrophic factors were used to block or mimic the effect of BMSCs on neurite outgrowth, respectively. The results showed that neurite outgrowth significantly increased in spinal motor neurons after co-cultured with BMSCs, while the secretion level of BDNF, GDNF and NGF was dramatically elevated in co-culture. However, the neurite outgrowth-promoting effect of BMSCs was found to significantly reduced using antibodies to BDNF, GDNF and NGF. In addition, a fraction of BMSCs was found to exhibit NF-200 immunoreactivity. These results indicated that BMSCs could promote neurite outgrowth of motor neurons by means of neurotrophic factors. The findings of the present study provided new cues for the treatment of spinal cord injury.     

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.     

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.