Spinal cord injury (SCI) causes a permanent neurological disability, and no satisfactory treatment is currently available. After SCI, pro-nerve growth factor (proNGF) is known to play a pivotal role in apoptosis of oligodendrocytes, but the cell types producing proNGF and the signaling pathways involved in proNGF production are primarily unknown. Here, we show that minocycline improves functional recovery after SCI in part by reducing apoptosis of oligodendrocytes via inhibition of proNGF production in microglia. After SCI, the stress-responsive p38 mitogen-activated protein kinase (p38MAPK) was activated only in microglia, and proNGF was produced by microglia via the p38MAPK-mediated pathway. Minocycline treatment significantly reduced proNGF production in microglia in vitro and in vivo by inhibition of the phosphorylation of p38MAPK. Furthermore, minocycline treatment inhibited p75 neurotrophin receptor expression and RhoA activation after injury. Finally, minocycline treatment inhibited oligodendrocyte death and improved functional recovery after SCI. These results suggest that minocycline may represent a potential therapeutic agent for acute SCI in humans.

After spinal cord injury, the disruption of blood-spinal cord barrier by activation of matrix metalloprotease is a critical event leading to infiltration of blood cells, inflammatory responses and neuronal cell death, contributing to permanent neurological disability. Recent evidence indicates that fluoxetine, an anti-depressant drug, is shown to have neuroprotective effects in ischaemic brain injury, but the precise mechanism underlying its protective effects is largely unknown. Here, we show that fluoxetine prevented blood-spinal cord barrier disruption via inhibition of matrix metalloprotease activation after spinal cord injury. After a moderate contusion injury at the T9 level of spinal cord with an infinite horizon impactor in the mouse, fluoxetine (10 mg/kg) was injected intraperitoneally and further administered once a day for indicated time points. Fluoxetine treatment significantly inhibited messenger RNA expression of matrix metalloprotease 2, 9 and 12 after spinal cord injury. By zymography and fluorimetric enzyme activity assay, fluoxetine also significantly reduced matrix metalloprotease 2 and matrix metalloprotease 9 activities after injury. In addition, fluoxetine inhibited nuclear factor kappa B-dependent matrix metalloprotease 9 expression in bEnd.3, a brain endothelial cell line, after oxygen-glucose deprivation/reoxygenation. Fluoxetine also attenuated the loss of tight junction molecules such as zona occludens 1 and occludin after injury in vivo as well as in bEnd.3 cultures. By immunofluorescence staining, fluoxetine prevented the breakdown of the tight junction integrity in endothelial cells of blood vessel after injury. Furthermore, fluoxetine inhibited the messenger RNA expression of chemokines such as Groα, MIP1α and 1β, and prevented the infiltration of neutrophils and macrophages, and reduced the expression of inflammatory mediators after injury. Finally, fluoxetine attenuated apoptotic cell death and improved locomotor function after injury. Thus, our results indicate that fluoxetine improved functional recovery in part by inhibiting matrix metalloprotease activation and preventing blood-spinal cord barrier disruption after spinal cord injury. Furthermore, our study suggests that fluoxetine may represent a potential therapeutic agent for preserving blood-brain barrier integrity following ischaemic brain injury and spinal cord injury in humans.

The blood-brain barrier (BBB) including blood-spinal cord barrier (BSCB) is a highly specialized brain endothelial structure of the fully differentiated neurovascular system. When damaged by various insults including traumatic spinal cord injury (SCI), the BBB or BSCB disruption elicits blood infiltration and inflammation, thereby generating neurotoxic products that can compromise synaptic and neuronal functions (Hawkins and Davis 2005;Zlokovic 2005;Abbott et al. 2006) and induces the ''programmed death'' of neurons and glia, leading to permanent neurological deficits (Xu et al. 2001;Noble et al. 2002;Gerzanich et al. 2009 Seoul, KoreaAbstract The disruption of blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) elicits an intensive local inflammation by the infiltration of blood cells such as neutrophils and macrophages, leading to cell death and permanent neurological disability. SCI activates matrix metalloprotease-9 (MMP-9), which is known to induce BSCB disruption. Here, we examined whether valproic acid (VPA), a histone deacetylase inhibitor, would attenuate BSCB disruption by inhibiting MMP-9 activity, leading to improvement of functional outcome after SCI. After moderate spinal cord contusion injury at T9, VPA (300 mg/kg) were immediately injected subcutaneously and further injected every 12 h for 5 days. Our data show that VPA inhibited MMP-9 activity after injury, and attenuated BSCB permeability and degradation of tight junction molecules such as occludin and ZO-1. In addition, VPA reduced the expression of inflammatory mediators including tumor necrosis factor-a. Furthermore, VPA increased the levels of acetylated histone 3, pAkt, and heat-shock protein 27 and 70, which have antiapoptotic functions after SCI. Finally, VPA inhibited apoptotic cell death and caspase 3 activation, reduced the lesion volume and improved functional recovery after injury. Thus, our results demonstrated that VPA improves functional recovery by attenuating BSCB disruption via inhibition of MMP-9 activity after SCI.

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Spinal cord injury (SCI) induces massive cell death, leading to permanent neurological disability. No satisfactory treatment is currently available. Ghrelin, a gastric hormone, is known to stimulate GH release from the hypothalamus and pituitary gland. Here, we report that ghrelin administration improves functional recovery after SCI in part by inhibiting apoptosis of neurons and oligodendrocytes. Ghrelin was not detected in normal, uninjured spinal cords, but spinal cord neurons and oligodendrocytes expressed the ghrelin receptor. Ghrelin significantly inhibited apoptotic cell death of neurons and oligodendrocytes, release of mitochondrial cytochrome c, and activation of caspase-3 after moderate contusion SCI. Ghrelin also significantly increased the level of phosphorylated ERK but decreased the level of phosphorylated p38MAPK. In addition, ghrelin increased the level of ERK-dependent brain-derived neurotrophic factor expression and decreased the level of pronerve growth factor expression. Furthermore, the neuroprotective effects of ghrelin were mediated through the ghrelin receptor. Finally, ghrelin significantly improved functional recovery and reduced the size of the lesion volume and the loss of axons and myelin after injury. These results suggest that ghrelin may represent a potential therapeutic agent after acute SCI in humans.

Our previous study showed that, after spinal cord injury (SCI) in rats, estrogen provides neuroprotection through expression of Bcl-2. However, molecular targets that mediate estrogen-induced expression of Bcl-2 are not fully understood. Here, we investigated whether, after SCI, the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways are involved in estrogen-induced expression of Bcl-2. Both Akt and ERK were activated and peaked at 8 h after SCI. Treatment with estrogen significantly increased the level of phosphorylated Akt (pAkt) and ERK (pERK) after injury. Cyclic-AMP response element binding protein (CREB) transcription factor was also activated and peaked at 8 h after SCI. Treatment with estrogen significantly increased the level of phosphorylated CREB (pCREB) after injury. Administration of LY294002, an inhibitor of PI3K/Akt, decreased the level of pCREB after SCI, whereas PD98059, an inhibitor of ERK, showed no significant effect. Also, treatment with LY294002 significantly inhibited expression of Bcl-2, but PD98059 showed no significant effect. Furthermore, treatment with estrogen inhibited apoptotic cell death, whereas treatment with LY294002 or PD98059 increased apoptotic cell death after SCI. Together, these data indicate that estrogen's neuroprotection is mediated in part by induction of Bcl-2 through PI3K/Akt-dependent CREB activation.

Several lines of evidence suggest that neurotrophins (NTs) potentiate or cause neuronal injury under various pathological conditions. Since NTs enhance survival and differentiation of cultured neurons in serum or defined media containing antioxidants, we set out experiments to delineate the patterns and underlying mechanisms of brain-derived neurotrophic factor (BDNF)–induced neuronal injury in mixed cortical cell cultures containing glia and neurons in serum-free media without antioxidants, where the three major routes of neuronal cell death, oxidative stress, excitotoxicity, and apoptosis, have been extensively studied. Rat cortical cell cultures, after prolonged exposure to NTs, underwent widespread neuronal necrosis. BDNF-induced neuronal necrosis was accompanied by reactive oxygen species (ROS) production and was dependent on the macromolecular synthesis. cDNA microarray analysis revealed that BDNF increased the expression of cytochrome b558, the plasma membrane-spanning subunit of NADPH oxidase. The expression and activation of NADPH oxidase were increased after exposure to BDNF. The selective inhibitors of NADPH oxidase prevented BDNF-induced ROS production and neuronal death without blocking antiapoptosis action of BDNF. The present study suggests that BDNF-induced expression and activation of NADPH oxidase cause oxidative neuronal necrosis and that the neurotrophic effects of NTs can be maximized under blockade of the pronecrotic action.