betaII-tubulin and GAP 43 mRNA expression in chronically injured neurons of the red nucleus after a second spinal cord injury

Regeneration by chronically injured supraspinal neurons is enhanced by treatment of a spinal cord lesion site with a variety of neurotrophic and growth factors. The removal of scar tissue, with subsequent reinjury of the spinal cord, is necessary for injured axons to access tissue transplants placed into the lesion to support axon regrowth. The present study examined chronically injured and reinjured rubrospinal tract (RST) neurons to determine if changes in gene expression could explain the failure of these neurons to regenerate without exogenous trophic factor support. Adult female rats were subjected to a right full hemisection lesion via aspiration of the cervical level 3 spinal cord. Using radioactive cDNA probes and in situ hybridization, RST neurons in the contralateral red nucleus were examined for changes in mRNA levels of betaII-tubulin and GAP 43 in an acute injury period (6 h-3 days), a chronic injury period (28 days after spinal cord injury (SCI)) and following a second lesion of the chronic injury site (6 h-7 days). Based upon the analysis of gene expression in single cells, GAP-43 mRNA levels were increased as early as 1 day following the initial SCI, but were no different than uninjured control levels at 28 days postoperative (dpo). The response to relesion was more rapid and higher than that detected after the initial injury with a significant increase in GAP 43 mRNA at 6 h that was maintained for at least 7 days. betaII-tubulin mRNA levels remained unchanged until 3 days after an acute injury followed by a decrease in expression to 30% below uninjured control values at 28 dpo. The expression of betaII-tubulin mRNA was significantly higher within 6 h after a second injury, where it remained stable for 5 days before a second increase occurred at 7 days after reinjury of the spinal cord. Thus, neurons in a chronic injury state retain the ability to respond to a traumatic injury and, in fact, neurons subjected to a second injury exhibit a significantly heightened expression of regeneration-associated genes.     

Exercise-induced gene expression in soleus muscle is dependent on time after spinal cord injury in rats

Cycling exercise attenuates atrophy in hindlimb muscles and causes changes in spinal cord properties after spinal cord injury in rats. We hypothesized that exercising soleus muscle expresses genes that are potentially beneficial to the injured spinal cord. Rats underwent spinal cord injury at T10 and were exercised on a motor-driven bicycle. Soleus muscle and lumbar spinal cord tissue were used for messenger RNA (mRNA) analysis. Gene expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) was elevated 11- and 14-fold, respectively, in soleus muscle after one bout of exercise performed 5 days after spinal cord transection. Also, c-fos and heat shock protein-27 (HSP27) mRNA abundance were increased 11- and 7-fold, respectively. When exercise was started 2 days after the injury, the changes in gene expression were not observed. By contrast, at 2 but not at 5 days after transection, expression of the HSP27 gene was elevated sixfold in the lumbar spinal cord, independent of exercise. Electromyographic activity in soleus muscles was also decreased at 2 days, indicating that the spinal cord was less permissive to exercise at this early time. Long-term exercise for 4 weeks attenuated muscle atrophy equally well in rats started at 2 days or 5 days after injury. We conclude that BDNF and GDNF released from exercising muscle may be involved in exercise-induced plasticity of the spinal cord. Furthermore, the data suggest that the lumbar spinal cord undergoes time-dependent changes that temporarily impede the ability of the muscle to respond to exercise.     

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.     

Expression of CAPON after Spinal Cord Injury in Rats

The adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI) and produces the key signaling molecule nitric oxide (NO). But little is known about the role of CAPON in the pathological process of SCI. The main objective of the present study was to investigate expression of CAPON and nNOS in a spinal cord contusion model in adult rats. Real time-polymerase chain reaction (PCR) and Western blot analysis revealed that mRNA and protein for CAPON increased at 2 h after SCI and reached the peak at 8 h, gradually recovered to the baseline level at 14 days. The expression of nNOS mRNA and protein was similar to that of CAPON. During the peak expression, CAPON mRNA was found in the ventral horn, mediate zone, dorsal horn, and white matter by in situ hybridization. Immunofluorescence showed that CAPON was colocalized with nNOS in neurons, oligodendrocytes, and some astrocytes of spinal cord tissues within 5 mm from the epicenter. Interaction between CAPON and nNOS was also detected by co-immunoprecipitation. Thus, the transient expression of high levels of CAPON may provide new insight into the secondary response after SCI. Chun Cheng and Xin Li contributed equally to this work.     

L-carnitine enhances axonal plasticity and improves white-matter lesions after chronic hypoperfusion in rat brain

Chronic cerebral hypoperfusion causes white-matter lesions (WMLs) with oxidative stress and cognitive impairment. However, the biologic mechanisms that regulate axonal plasticity under chronic cerebral hypoperfusion have not been fully investigated. Here, we investigated whether L-carnitine, an antioxidant agent, enhances axonal plasticity and oligodendrocyte expression, and explored the signaling pathways that mediate axonal plasticity in a rat chronic hypoperfusion model. Adult male Wistar rats subjected to ligation of the bilateral common carotid arteries (LBCCA) were treated with or without L-carnitine. L-carnitine-treated rats exhibited significantly reduced escape latency in the Morris water maze task at 28 days after chronic hypoperfusion. Western blot analysis indicated that L-carnitine increased levels of phosphorylated high-molecular weight neurofilament (pNFH), concurrent with a reduction in phosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN), and increased phosphorylated Akt and mammalian target of rapamycin (mTOR) at 28 days after chronic hypoperfusion. L-carnitine reduced lipid peroxidation and oxidative DNA damage, and enhanced oligodendrocyte marker expression and myelin sheath thickness after chronic hypoperfusion. L-carnitine regulates the PTEN/Akt/mTOR signaling pathway, and enhances axonal plasticity while concurrently ameliorating oxidative stress and increasing oligodendrocyte myelination of axons, thereby improving WMLs and cognitive impairment in a rat chronic hypoperfusion model.     

Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord

Apoptosis is an important cause of secondary cell death in spinal cord injury (SCI). SCI induces the expression of the low affinity neurotrophin receptor p75 (p75NTR), that in the absence of the high affinity component, TrkA, can promote cell death by apoptosis. We therefore hypothesized that a reduction of p75NTR expression in SCI may increase tissue sparing and therefore improve recovery of function. As a tool to test our hypothesis we used the synthetic glucocorticoid dexamethasone (DEX) to down-regulate p75NTR expression. A standardized thoracic spinal cord contusion injury was produced in female rats. Laminectomized and SCI rats received various doses of DEX immediately after injury and the treatment was continued daily for 7 days. DEX, given at high doses (20 mg/kg, s.c.) but not at low doses (1 or 8 mg/kg) prevented the increase in p75NTR mRNA and protein in SCI rats, without affecting the expression of TrkA. High doses of DEX also reduced cellular apoptosis both in white and gray matters. This effect correlated with the ability of DEX to accelerate behavioral recovery of function measured by a combined behavioral score. These data suggest that reduction of p75NTR in SCI may be a therapeutic strategy to limit cell and tissue damage and therefore to improve recovery of function in SCI patients.     

Epidural Spinal Cord Stimulation Promotes Motor Functional Recovery by Enhancing Oligodendrocyte Survival and Differentiation and by Protecting Myelin after Spinal Cord Injury in Rats

Epidural spinal cord stimulation (ESCS) markedly improves motor and sensory function after spinal cord injury (SCI), but the underlying mechanisms are unclear.Here, we investigated whether ESCS affects oligodendrocyte differentiation and its cellular and molecular mechanisms in rats with SCI. ESCS improved hindlimb motor function at 7 days, 14 days, 21 days, and 28 days after SCI.ESCS also significantly increased the myelinated area at 28days, and reduced the number of apoptotic cells in the spinal white matter at 7 days. SCI decreased the expression of 20,30-cyclic-nucleotide 30-phosphodiesterase (CNPase,an oligodendrocyte marker) at 7 days and that of myelin basic protein at 28 days. ESCS significantly upregulated these markers and increased the percentage of Sox2/CNPase/DAPI-positive cells (newly differentiated oligodendrocytes) at 7 days. Recombinant human bone morphogenetic protein 4 (rh BMP4) markedly downregulated these factors after ESCS. Furthermore, ESCS significantly decreased BMP4 and p-Smad1/5/9 expression after SCI,and rh BMP4 reduced this effect of ESCS. These findings indicate that ESCS enhances the survival and differentiation of oligodendrocytes, protects myelin, and promotes motor functional recovery by inhibiting the BMP4-Smad1/5/9 signaling pathway after SCI.     

Expression of Nemo-like kinase after spinal cord injury in rats

Wnt can induce signal transduction via the canonical pathway, which was involved in many processes in the nervous system. Nemo-like kinase (NLK) acts as a negative regulator of β-catenin/T-cell factor/lymphoid enhancer factor (LEF) and functions downstream of transforming growth factor β-activated kinase-1 in the Wnt signaling pathway. In this study, we performed a spinal cord injury (SCI) test in adult Sprague–Dawley rats and investigated the dynamic changes and role of NLK expression in the spinal cord. Western blot analysis revealed that NLK expression was low in normal spinal cord. It then increased markedly, peaked at 3 days, and declined to basal levels from 5 days after injury. Immunohistochemistry confirmed that NLK immunoactivity was expressed at low levels in gray and white matter under normal conditions and increased prominently in gray matter after the SCI test. Double immunofluorescent staining for NLK, caspase-3, β-catenin, and NeuN (neuronal nuclei) revealed that NLK and β-catenin were markedly increased and colocalized in apoptotic neurons. Coimmunoprecipitation data demonstrated that overexpression of NLK protein reduced β-catenin binding to LEF-1. Our results suggested that NLK was associated with neuronal apoptosis through attenuating the Wnt/β-catenin signaling pathway after SCIs.     

Integrated analysis of microRNA and mRNA expression profiles in the rat spinal cord under inflammatory pain conditions

Recent studies using microarray‐based approaches have demonstrated that microRNAs (miRNAs) are involved in pain processing pathways. However, a significant proportion of computational predictions of miRNA targets are false‐positive interactions. To increase the chance of identifying biologically relevant targets, we performed an integrated analysis of both miRNA and mRNA expression profiles in the rat spinal cord during complete Freund's adjuvant (CFA)‐induced inflammatory pain. We generated miRNA and mRNA arrays from the same corresponding samples on days 5 and 14 after CFA injection. Five miRNAs and 1096 mRNAs in the CFA 5d group and 16 miRNAs and 647 mRNAs in the CFA 14d group were differentially expressed based on a filter of at least a 1.5‐fold change in either direction. An integrated analysis revealed 54 mRNA targets with an inverse correlation to the expression patterns of three miRNAs in the CFA 5d group. Seventy‐five targets were inversely correlated to six miRNAs in the CFA 14d group. The miRNA–mRNA interaction networks revealed significant changes in miR‐124, miR‐149, miR‐3584 and their target genes, IL‐6R, ADAM19, LAMC1 and CERS2, in the CFA 5d group. In the CFA 14d group, significant changes were noted in miR‐124, miR‐29, miR‐34, miR‐30, miR‐338 and their target genes, TIMP2, CREB5 and EFNB1. We also investigated an interaction pair, miR‐124‐3p and IL‐6R, and the results showed that miR‐124‐3p could attenuate inflammatory pain and decrease IL‐6R expression in the spinal cord. These specific miRNAs and their target genes provide possible avenues for the diagnosis and treatment of inflammatory pain.     

Effect of electrical stimulation on neural regeneration via the p38-RhoA and ERK1/2-Bcl-2 pathways in spinal cord-injured rats

Although electrical stimulation is therapeutically applied for neural regeneration in patients, it remains unclear how electrical stimulation exerts its effects at the molecular level on spinal cord injury(SCI). To identify the signaling pathway involved in electrical stimulation improving the function of injured spinal cord, 21 female Sprague-Dawley rats were randomly assigned to three groups: control(no surgical intervention, n = 6), SCI(SCI only, n = 5), and electrical simulation(ES; SCI induction followed by ES treatment, n = 10). A complete spinal cord transection was performed at the 10~(th) thoracic level. Electrical stimulation of the injured spinal cord region was applied for 4 hours per day for 7 days. On days 2 and 7 post SCI, the Touch-Test Sensory Evaluators and the Basso-Beattie-Bresnahan locomotor scale were used to evaluate rat sensory and motor function. Somatosensory-evoked potentials of the tibial nerve of a hind paw of the rat were measured to evaluate the electrophysiological function of injured spinal cord. Western blot analysis was performed to measure p38-RhoA and ERK1/2-Bcl-2 pathways related protein levels in the injured spinal cord. Rat sensory and motor functions were similar between SCI and ES groups. Compared with the SCI group, in the ES group, the latencies of the somatosensory-evoked potential of the tibial nerve of rats were significantly shortened, the amplitudes were significantly increased, RhoA protein level was significantly decreased, protein gene product 9.5 expression, ERK1/2, p38, and Bcl-2 protein levels in the spinal cord were significantly increased. These data suggest that ES can promote the recovery of electrophysiological function of the injured spinal cord through regulating p38-RhoA and ERK1/2-Bcl-2 pathway-related protein levels in the injured spinal cord.     

Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction

Spinal cord injury and cyclophosphamide-induced cystitis dramatically alter lower urinary tract function and produce neurochemical, electrophysiological, and anatomical changes that may contribute to reorganization of the micturition reflex. Mechanisms underlying this neural plasticity may involve alterations in neurotrophic factors in the urinary bladder. These studies have determined neurotrophic factors in the urinary bladder that may contribute to reorganization of the micturition reflex following cystitis or spinal cord injury. A ribonuclease protection assay was used to measure changes in urinary bladder neurotrophic factor mRNA (betaNGF, BDNF, GDNF, CNTF, NT-3, and NT-4) following spinal cord injury (acute/chronic) or cyclophosphamide-induced cystitis (acute/chronic). The correlation between urinary bladder nerve growth factor mRNA and nerve growth factor protein expression was also determined. Each experimental paradigm resulted in significant (P 相似文献   

Upregulation of the HLH Id gene family in neural progenitors and glial cells of the rat spinal cord following contusion injury.

Spinal cord injury (SCI) leads to a complex sequence of cellular responses, including astrocyte activation, oligodendrocyte death, and ependymal cell proliferation. Inhibitors of DNA binding (Id1, Id2, Id3) belong to a helix-loop-helix (HLH) gene family. Id genes have been implicated in playing a vital role in the proliferation of many cell types, including astrocytes and myoblasts. In the present study, the expression of Id family members in spinal cord after contusion injury was investigated by in situ hybridization. Id1, Id2, and Id3 mRNA expression was upregulated 5 mm rostral and caudal to the lesion center, and reached maximal levels 3 days after SCI. In addition, cell populations expressing Id1, Id2, and Id3 mRNA were maximally increased 3 days after SCI. The increase in Id2 and Id3 mRNA expression and Id2 and Id3 mRNA+ cells was still observed at 8 days. The Id mRNA expressing cells were phenotyped by combining immunostaining of cell-specific markers with in situ hybridization. Glial fibrillary acidic protein (GFAP)+ astrocytes were found to express all three Id mRNA, whereas S-100alpha+ astrocytes only expressed high levels of Id2 and Id3 mRNA. Cells having a neural progenitor morphology and the marker nestin appeared after SCI and they expressed Id1, Id2, and Id3 mRNA. Interestingly, some Rip+ oligodendrocytes located in the areas close to the central canal expressed Id3 mRNA after injury. In conclusion, Id genes are upregulated in a time-dependent manner in astrocytes, oligodendrocytes, and neural progenitor subpopulations after SCI, suggesting that they play major roles in cellular responses following SCI.     

Spinal cord injury in neonates alters respiratory motor output via supraspinal mechanisms

Upper cervical spinal cord injury (SCI) alters respiratory output and results in a blunted respiratory response to pH/CO2. Many SCI studies have concentrated on respiratory changes in neural function caudal to injury; however few have examined whether neural plasticity occurs rostral to SCI. Golder et al. (2001a) showed that supraspinal changes occur to alter respiratory output after SCI. Furthermore, Brown et al. (2004) showed that neural receptors change rostral to a thoracic SCI. We hypothesized that SCI in neonates will alter supraspinal output, show a blunted response to pH and alter receptor protein levels in the medulla. On postnatal day 0/1, a C2 SCI surgery was performed. Two days later, neonates were anesthetized and brainstem-spinal cords removed. Respiratory-related activity was recorded using the in vitro brainstem-spinal cord preparation and the superfusate pH was changed (pH 7.2, 7.4 and 7.8). The respiratory-like frequency was significantly reduced in SCI rats indicating supraspinal plasticity. Increasing the pH decreased respiratory-like frequency and peak amplitude in injured and sham controls. Increasing the pH increased burst duration and area in sham controls, whereas in injured rats, the burst duration and area decreased. Western blot analysis demonstrated significant changes in glutamate receptor subunits (NR1, NR2B and GluR2), adenosine receptors (A1, A2A), glutamic acid decarboxylase (65) and neurokinin-1 receptors in medullary tissue ipsilateral and contralateral to injury. These data show that supraspinal plasticity in the respiratory system occurs after SCI in neonate rats. The mechanisms remain unknown, but may involve alterations in receptor proteins involved in neurotransmission.     

Combination strategies for repair, plasticity, and regeneration using regulation of gene expression during the chronic phase after spinal cord injury

Although recovery after spinal cord injury (SCI) is rare in humans, recent literature indicates that some patients do recover sensorimotor function years after the trauma. This study seeks to elucidate the genetic underpinnings of SCI repair through the investigation of neurodegenerative and regenerative associated genes involved in the response to SCI during the chronic phase in adult rats. Intervention on the level of gene regulation focused on enhancing naturally attempting SCI regenerative genes has the potential to promote SCI repair. Our aim was to analyze gene expression characteristics of candidate genes involved in the neuro-degenerative and -regenerative processes following various animal models of SCI. We compiled data showing gene expression changes after SCI in adult rats and created a chronological time-line of candidate genes differentially expressed during the chronic phase of SCI. Compiled data showed that SCI induced a transient upregulation of endogenous neuro-regenerative genes not only within a few hours but also within a few days, weeks, and months after SCI. For example, gene controlling growth-associated protein-43 (GAP-43), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and others, showed significant changes in mRNA accumulation in SCI animals, from 48 hours to 12 weeks after SCI. Similarly, inhibitory genes, such as RhoA, LINGO-1, and others, were upregulated as late as 4 to 14 days after injury. This indicates that gene specific regulation changes, corresponding to repair and regenerative attempts, are naturally orchestrated over time after injury. These delayed changes after SCI give ample time for therapeutic gene modulation through upregulation or silencing of specific genes responsible for the synthesis of the corresponding biogenic proteins. By following the examination of differential gene regulation during the chronic phase, we have determined times, successions, co-activations, interferences, and dosages for potential therapeutic synchronized interventions. Finally, local cellular specificities and their neuropathophysiologies have been taken into account in the elaboration of the combination treatment strategy we propose. The interventions we propose suggest the delivery of exogenous therapeutic agents to upregulate or downregulate chosen genes or the expression of the downstream proteins to revert the post-traumatic stage of SCI during the chronic phase. The proposed combination and schedule of local cell-specific treatment should enhance intrinsic regenerative machinery and provide a promising strategy for treating patients sustaining chronic SCI.     

NGF message and protein distribution in the injured rat spinal cord

Nerve growth factor (NGF) content of the spinal cord is increased after cord injury. NGF can cause central sprouting of sensory fibers after spinal cord injury (SCI), leading to autonomic dysfunction and pain. NGF also can promote the death of oligodendroglia after SCI. Knowing the source of intraspinal NGF would benefit strategies for minimizing abnormal plasticity and cell death after SCI. We identified these sources, using RNA in situ hybridization to detect NGF mRNA and double-labeling immunocytochemistry for NGF and cell-marking antigens. In uninjured and sham-injured rats, we identified NGF mRNA in leptomeningeal cells and in neurons in the intermediate grey matter, whereas NGF protein was observed only in leptomeningeal cells. At 3-7 days after transection or clip-compression SCI, NGF mRNA and protein were expressed in the lesion and throughout the intermediate grey matter and white matter rostral and caudal to the injury site. Transection-SCI was used to permit comparisons to previous studies; clip-compression injury was used as a more clinically relevant model. mRNA and protein in adjacent sections were expressed in ramified microglia, astrocytes, intermediate grey neurons, pial cells, and leptomeningeal and Schwann cells in the lateral white matter and the lesion site. Rounded macrophages in the lesion were immunoreactive (Ir) for NGF, but the cells expressing NGF mRNA were not in the same areas of the lesion and were not stained by a macrophage marker. Our data demonstrate that glia, neurons, meningeal cells and Schwann cells but not macrophages contribute to the increased intraspinal NGF after SCI.