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.     

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.     

Biphasic and regionally-restricted chemokine expression in the central nervous system in the Theiler's virus model of multiple sclerosis

Intracerebral infection of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) induces a biphasic disease characterized by acute polioencephalitis followed by chronic demyelination and viral persistence in the spinal cord white matter. There has been limited study of soluble mediators responsible for the initial recruitment of inflammatory cells into the gray matter, and the secondary influx into the white matter during infection with TMEV. We used sensitive and specific RT - PCR/dot blot hybridization assays to quantitate the relative levels of chemokine mRNA in the brains and spinal cords during the acute and chronic phases of TMEV infection in mice susceptible (B10.M, H-2f) and resistant (B10, H-2b) to virus-induced demyelination. TMEV infection resulted in robust expression of mRNA for IP-10, RANTES, and MCP-1, but not GRO-alpha, in brains and spinal cords in both strains of mice within 5 days. By day 21, virus was cleared, inflammation reduced, and expression of all three chemokines subsided to baseline levels in the brains and spinal cords of resistant mice, and the brains of susceptible mice. Chemokine expression was also reduced in the spinal cords of susceptible mice, corresponding to a shift in TMEV replication from the gray to the white matter. During the chronic, demyelinating phase of infection, there was a resurgence in IP-10, RANTES, and MCP-1 mRNA in spinal cords of susceptible B10.M mice. This study demonstrates the coordinated regulation and regionally restricted expression of chemokines in a biphasic disease of the central nervous system and provides greater understanding of the mechanism by which inflammation is established and maintained in the CNS.     

Upregulation of Kv 1.4 protein and gene expression after chronic spinal cord injury.

After spinal cord injury (SCI), white matter tracts are characterized by demyelination and increased sensitivity to the K(+) channel blocker 4-aminopyridine (4-AP). These effects appear to contribute to neurological impairment after SCI, although the molecular changes in K(+) channel subunit expression remain poorly understood. We examined changes in gene expression of the 4-AP-sensitive voltage-gated K(+) channel Kv 1.4 after chronic SCI in the rat. Quantitative immunoblotting showed that Kv 1.4 protein was significantly increased at 6 weeks, but not at 1 week, after SCI in spinal cord white matter. Kv 1.4 was localized to astrocytes, oligodendrocytes, and oligodendrocyte progenitor cells but not to axons in both the normal and the injured spinal cord white matter. Because glial cells proliferate after SCI, we used immunogold electron microscopy to quantify Kv 1.4 protein in individual glial cells and found a sixfold increase of Kv 1.4 in cells of the oligodendrocyte lineage after chronic injury. Finally, quantitative in situ hybridization showed that Kv 1.4 mRNA was significantly upregulated in spinal cord white matter, but not gray matter, after SCI. In summary, we show that Kv 1.4 is expressed in glial cells and not in axons in the rat spinal cord white matter and that its expression is markedly increased in cells of the oligodendrocyte lineage after chronic SCI. Given that K(+) channels play a role in glial cell proliferation, cells exhibiting changes in Kv 1.4 expression may represent proliferating oligodendroglia in the chronically injured spinal cord.     

Down regulation of trophic factors in neonatal rat spinal cord after administration of cerebrospinal fluid from sporadic amyotrophic lateral sclerosis patients

Accumulating evidence supports neuroprotective role of trophic factors in amyotrophic lateral sclerosis (ALS). Previous studies from our laboratory report that the CSF of patients with sporadic ALS (ALS-CSF) induces degenerative changes in the rat spinal motor neurons and reactive astrogliosis in the surrounding gray matter. The present study was aimed to investigate if the ALS-CSF affected the expression of trophic factors namely, brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) in the newborn rat spinal cords. ALS-CSF was intrathecally injected into the neonatal rats and the mRNA levels of the trophic factors were determined by quantitative real-time polymerase chain reaction. Here, we report significant down regulation in the gene expression of trophic factors for BDNF, FGF2 and IGF1. BDNF mRNA levels were found to be reduced by 6.8-fold in the ALS-CSF injected group compared to control groups. The levels of IGF1 and FGF2 mRNA were also decreased by 3.91- and 2.13-fold, respectively, in the ALS group. We further found that exogenous supplementation of BDNF considerably reduced the aberrant phosphorylation of neurofilaments, complementing our earlier findings of restored expression of voltage gated sodium channel. Reduced expression of trophic factors indicates an altered microenvironment of the motor neurons and could possibly be one of the contributing factors in the degeneration process.     

Compulsive exercise acutely upregulates rat hippocampal brain-derived neurotrophic factor

Summary. This study was to examine the effects of treadmill exercise on the expression of brain-derived neurotrophic factor (BDNF) in rat hippocampus. After 1-wk treadmill familiarization, animals in exercise groups received a 4-wk exercise training or an acute exercise. They were sacrificed 2 h or 2 d after exercise and their hippocampal BDNF mRNA and protein levels were determined. We demonstrated that 1) hippocampal BDNF mRNA and protein levels were both elevated in response to exercise training at 2 h after the last run but not after 2 d; 2) an acute moderate exercise (1 or 3 d) increased BDNF protein levels; 3) acute severe exercise increased BDNF protein and mRNA levels in animals under a familiarization regimen, while suppressed the BDNF mRNA level in rats without treadmill familiarization, paralleling the stress effect of immobilization/water exposure. We conclude that compulsive treadmill exercise with pre-familiarization acutely upregulates rat hippocampal BDNF gene expression.     

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.     

GDNF, RET and GFRalpha-1-3 mRNA expression in the developing human spinal cord and ganglia.

The identification of endogenous neurotrophic factors and their receptors in human spinal cord is important not only to understand development, but also in the consideration of possible future therapies for neurodegenerative disorders and trauma. Using in situ hybridization, the expression of glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), persephin (PSP), GFRalpha-1, GFRalpha-2, GFRalpha-3 and RET mRNA in human fetal spinal cord was studied. Strong GDNF mRNA hybridization signal, presumably restricted to Clarke's nucleus, was detected in the thoracic spinal cord. mRNA encoding GFRalpha-1 was expressed in the entire spinal cord gray matter with particularly high expression in the ventral horn. GFRbeta-1 was also expressed more weakly in dorsal root ganglia. NTN and persephin mRNA were not detected in either the fetal spinal cord or the dorsal root ganglia. mRNA coding for GFRalpha-2, however, was found in most cells of the spinal cord gray matter. A strong expression of GFRalpha-3 mRNA was detected in dorsal root ganglia cells and Schwann cells. The transducing receptor RET was expressed strongly in motorneurons and dorsal root ganglion neurons. We conclude that basic features concerning the role of the GDNF family of ligands and their receptors revealed in rodents applies to humans.     

Amino acids in autopsied human spinal cord

The distribution of glycine, glutamate, aspartate, glutamine, and taurine was measured at autopsy in 10 normal human spinal cords, and in 4 spinal cords from Friedreich’s Ataxia patients, using sensitive double-isotope microassay of their dansyl derivatives. Transverese sections of spinal cord from cervical, thoracic, and lumbar levels were dissected to afford samples of gray matter, posterior columns, dorsal white matter, and ventral white matter. Levels of glycine, glutamate, and glutamine were found to be elevated in lumbar gray matter, being 2–3 times higher than those found in white matter structures. Aspartate and taurine, on the other hand, were found to be distributed more evenly in autopsied human spinal cord. Selective abnormalities of amino acid distribution in Friedreich’s Ataxia included decreased glutamate and glutamine in lumbar gray matter and posterior columns and increased taurine content of lumbar spinal cord. These changes may be of pathophysiological significance in this hereditary neurodegenerative disease.     

Effects of Exercise Following Lateral Fluid Percussion Brain Injury in Rats

Previous studies have suggested that brain-derived neurotrophic factor (BDNF) is involved in memory and learning, and may be neuroprotective following various brain insults. Exercise has been found to increase BDNF mRNA levels in various brain regions, including specific subpopulations of hippocampal neurons. In the present study, we were interested in whether following traumatic brain injury, exercise could increase BDNF mRNA expression, attenuate neuropathology, and improve cognitive and neuromoter performance. We subjected adult male Sprague-Dawley rats to a fluid percussion brain injury, followed by either 18 days of treadmill exercise or handling. Spatial memory was evaluated in a Morris Water Maze (MWM) and motor function was evaluated with a battery of neuromotor tests. Neuropathology was evaluated by measuring the cortical lesion volume and the extent of neuronal loss in the hipocampus. Expression of BDNF mRNA in the hippocampus was assessed with in situ hybridization and densitometry. Hybridization signal for BDNF mRNA was significantly increased bilaterally in the exercise group in hippocampal regions CA1 and CA3 (p<0.05), but not in the granule cell layer of the dentate gyrus. No significant differences were observed between the groups in neuropathology, spatial memory, or motor performance. This study suggests that after traumatic brain injury, exercise elevates BDNF mRNA in specific regions of the hippocampus.     

Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter

Myelencephalon-specific protease (MSP) is a novel serine protease that is expressed predominantly in the nervous system. In the adult rat spinal cord, MSP mRNA expression was dramatically upregulated, in both the white and gray matter, after systemic exposure to the glutamate receptor agonist, kainic acid (KA) (Scarisbrick et al. J Neurosci 17: 8156-8168, 1997b). To determine the cell-specific expression patterns of MSP, we generated MSP-specific monoclonal antibodies. These have been used in immunohistochemical and in situ hybridization colocalization studies, to demonstrate that MSP mRNA and protein are produced predominantly by CNP-immunoreactive oligodendroglia, but not by GFAP-immunoreactive astrocytes, in the white matter of the normal adult cord. In vitro, the soma of oligodendrocytes were also densely MSP immunoreactive, as were their growth tips, while astrocytes were associated with lower levels. These findings suggest that the enzymatic activity of MSP is likely to be important in the biology of oligodendrocytes and/or in the maintenance of the nerve fiber tracts of the adult spinal cord.     

Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in binding to some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipids of the PNS.     

nov基因在不同种属动物脊髓中的表达

用地高辛标记的cRNA探针原位杂交方法研究了鲢、青蛙、蛇、鸡、牛、犬和猫脊髓中肾母细胞瘤过度表达基因（nov）mRNA阳性神经元的种系发育。结果显示，低等脊椎动物鲢、青蛙和蛇脊髓中仅有少量novmRNA阳性神经元，分布于灰质腹角。鸡脊髓中阳性神经元除主要分布于脊髓腹角外，中央灰质也有少量分布。哺乳动物牛、大和猫脊髓灰质中novmRNA阳性神经元分布广泛，背腹角、中央灰质及中央核区都检测到很强的杂交信号。以上结果表明，nov基因在从低等脊椎动物到高等脊椎动物的进化过程中非常保守，这种保守性提示nov基因在脊髓神经元发育、分化及正常生理功能中可能具有重要作用。     

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.     

NGF, NT-3 and Trk C mRNAs, but not TrkA mRNA, are upregulated in the paraventricular structures in experimental hydrocephalus

OBJECTS: This study was designed to detect possible alterations in the expression of neurotrophins and trks in kaolin-induced hydrocephalus by in situ hybridization. METHODS AND RESULTS: Sixteen rats were treated by injection of 25 mg kaolin suspended in 0.1 ml of physiological saline into the cisterna magna. Four rats were injected with saline and served as controls. The kaolin-treated rats were divided into two groups studied 1 and 4 weeks after treatment. Rats were anesthetized and killed, and their brains were rapidly dissected and frozen. DNA oligonucleotide probes for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and trkA, trkB, and C were labeled with [(35)S]dATP using terminal deoxyribonucleotidyl transferase for in situ hybridization. Hydrocephalic brains were also classified according to the degree of ventricular enlargement. The results observed were as follows. (1) The medial septal and striatal NGF mRNA levels increased with severity in animals. (2) Hippocampal trkB and BDNF mRNA levels increased with time in animals with moderate ventricular enlargement. (3) Expression of hippocampal trkB, trkC, and NT-3 mRNA increased in animals with moderate ventricular enlargement, while it apparently decreased in the large ventricular enlargement group reaching normal ranges. (4) In the corpus callosum there was an apparent increase in NGF, NT-3 and trkC mRNA, but not in trkA, in hydrocephalic animals. NT-3 EIA confirmed the presence of NT-3 protein increases in corpus callosum. It is therefore possible that simultaneous NGF, NT-3, and trkC receptor upregulation occurred in glial elements of the white matter. CONCLUSIONS: These results demonstrate that neurotrophins and their receptors are overexpressed in many damaged structures of the severely hydrocephalic brain. There were discrepancies in the distribution of NGF and trkA mRNA, and we hypothesize that NGF mRNA in the damaged white matter structure might be due to the reduced availability of other receptors, such as the low-affinity NGF receptors.     

Acute up-regulation of brain-derived neurotrophic factor expression resulting from experimentally induced injury in the rat spinal cord

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family of trophic factors, has multiple functions including a role in the promotion of neuronal survival and nerve fiber elongation in both the central and the peripheral nervous systems. We assessed the expression of endogenous BDNF following an experimentally induced compression injury to the spinal cord. Expression of BDNF mRNA was increased following the spinal cord injury; reaching maximum levels 24 h after the injury. Expression of BDNF mRNA returned to the levels observed in sham-operated control animals within 3 days of the injury. Using the in situ hybridization technique, we observed a wide distribution of BDNF expression among the different cell types in the spinal cord, including motor and sensory neurons, and in glia cells, including astrocytes. We also observed expression of BDNF in putative macrophages and/or microglia; however, this effect was not observed until day 7 following spinal cord injury. These results suggest that BDNF is synthesized in both neurons and astrocytes during the acute response to injury to the spinal cord, functioning in a mainly neuroprotective role. This is followed by a later phase of expression in which BDNF is produced by macrophages and/or microglia, apparently functioning in a restorative capacity.     

Compression analysis of the gray and white matter of the spinal cord

The spinal cord is composed of gray matter and white matter.It is well known that the properties of these two tissues differ considerably.Spinal diseases often present with symptoms that are caused by spinal cord compression.Understanding the mechanical properties of gray and white matter would allow us to gain a deep understanding of the injuries caused to the spinal cord and provide information on the pathological changes to these distinct tissues in several disorders.Previous studies have reported on the physical properties of gray and white matter,however,these were focused on longitudinal tension tests.Little is known about the differences between gray and white matter in terms of their response to compression.We therefore performed mechanical compression test of the gray and white matter of spinal cords harvested from cows and analyzed the differences between them in response to compression.We conducted compression testing of gray matter and white matter to detect possible differences in the collapse rate.We found that increased compression(especially more than 50%compression)resulted in more severe injuries to both the gray and white matter.The present results on the mechanical differences between gray and white matter in response to compression will be useful when interpreting findings from medical imaging in patients with spinal conditions.     

A comparison of the regeneration potential of dorsal root fibers into gray or white matter of the adult rat spinal cord.

To assess the role of white matter inhibition as a barrier to neurite outgrowth in vivo, we unilaterally transected three consecutive lumbar dorsal roots (L4-L6), incised the spinal cord, and transplanted the peripheral stump of L4 either medially onto the white matter of the dorsal columns or laterally, just superficial to the gray matter of the dorsal horn at the level of L5. Three weeks to seven months later, the translocated root was retransected, and its central stump was anterogradely labeled with HRP. The staining pattern demonstrated that regenerating sensory axons had entered the spinal cord from both medially and laterally placed roots. Axonal staining from medially placed dorsal roots (onto the white matter of the dorsal columns) was sparse and limited to the white matter. Staining of laterally placed roots revealed a small subpopulation of regenerating axons which had entered the gray matter and formed terminal arbors. Successful axonal regeneration into the gray matter, albeit minimal, was associated with a localized and limited inflammatory response near the sites of axonal ingrowth.