ERK1/2 and p38 mitogen-activated protein kinase mediate iNOS-induced spinal neuron degeneration after acute traumatic spinal cord injury
Introduction
It is known that acute spinal cord injury (SCI) initiates a series of cellular and molecular cascade events (Tator, 1995, Xu et al., 2001, Hausmann, 2003, Beattie, 2004, Gris et al., 2004) and progressive neural injury results from a combination of secondary injury factors including ischemia, biochemical alterations, apoptosis, excitotoxicity, neurotransmitter accumulation, lipid peroxidation/free radical injury, and inflammatory responses (Carlson and Gorden, 2002). It is believed that inflammatory and immune responses are a major component of secondary injury and play a central role in regulating the pathogenesis of acute and chronic SCI (Anderson, 2002, Bethea and Dietrich, 2002, Yang et al., 2004), and effective intervention of inflammatory and immune responses may help to attenuate degeneration of neurons and be of benefit to tissue repair (Brewer et al., 1999, Hauben and Schwartz, 2003, Stirling et al., 2004).
Resident microglia and macrophages originating from blood are two pivotal cell types related to the occurrence of neuronal degeneration in CNS after traumatic injury. When SCI occurs, microglia in parenchyma are activated and macrophages in circulation get across blood–brain barrier (BBB) to act as intrinsic spinal phagocytes. These cells can secrete some neurotrophic peptides such as BDNF, GDNF (Batchelor et al., 2002) and laminin (Rabchevsky and Streit, 1997). All of these factors serve as excellent substrates for growing neurites. Concomitantly, multiple cytotoxic substances such as proinflammatory cytokines (Yang et al., 2004) and reactive nitrogen intermediates, nitric oxide (NO) (Kwak et al., 2005), are also produced. NO is known to be closely involved in the development of pathological processes in vivo such as post-traumatic spinal cord cavitation (Matsuyama et al., 1998). It is confirmed in vitro that NO-induced cell injury is mediated via either the necrotic or apoptotic pathway, depending upon the severity of the cellular damage (Dawson et al., 1996). A recent study clearly demonstrates that NO produced by inducible nitric oxide synthase (iNOS) in macrophages causes apoptosis following traumatic SCI (Satake et al., 2000).
Studies mostly done in vitro have disclosed a three-step cascade event related to the occurrence of neuronal damage following SCI: activation of MAPKs, initiation of inflammatory responses, and degeneration of neuron. It is reported that phosphorylation of ERK1/2 and p38 MAPK results in expression of genes mediating the inflammatory responses, such as tumor necrosis factor-alpha (TNF-α) and NO (Bhat et al., 1998, Wang et al., 2004a). In the other hand, administration of p38 and JNK inhibitors partially rescued neurons from death in the LPS-treated microglia-neuron co-culture (Xie et al., 2004), and inhibition of ERK1/2 activation may also reduce IL-1-induced cortical neuron damage (Lu et al., 2005). These findings suggest that initiation of inflammatory responses in CNS is related to activation of MAPKs, especially ERK1/2 and p38 MAPK, and whether or not these kinases are activated would be a determinant for neuronal death or survival on certain occasions. However, in vivo evidence linking MAPK activation, NO production and neuronal survival in post-traumatic pathophysiological process of SCI is as yet absent.
The purpose of present research is to investigate the relationship of NO production and MAPKs phosphorylation following traumatic SCI and their effect on the survival of neurons in the injured area. A rat model of complete transection of spinal cord was used. In view of the technical difficulty of measurement of NO, which requires accuracy in the time of sampling and prompt measurement immediately after sampling due to the instability of NO and nitrite, we observed the level of iNOS to represent NO change, as previously reported (Satake et al., 2000, Wang et al., 2004a). The temporal and spatial patterns of iNOS gene expression were investigated with immunohistochemistry, Western blot as well as RT-PCR techniques and the role of NO in induction of neuronal apoptosis was evaluated by systemic application of a NOS inhibitor, l-NAME. Furthermore, the spatial and temporal patterns of phosphorylated ERK1/2 (phospho-ERK1/2) and phosphorylated p38 (phospho-p38) after SCI were examined and their role in NO induction and neuronal destiny was also addressed.
Section snippets
Animal model of spinal cord injury
All experiments were done in accordance with the guidelines established by the Committee of Fourth Military Medical University on Animal Use in Research and Education. Male Sprague Dawley rats (230–250 g) were used and divided into two groups: SCI group and sham-operated group. All the rats were anesthetized with pentobarbital (50 mg/kg, i.p), and laminectomy was executed at the level of the 10th thoracic vertebra (T10). The spinal cord of SCI group was completely transected with a specific
Activation of microglia/macrophages after SCI
Microglia/macrophages were detected in the sections of sham-operated and transection-injured spinal cord at various time intervals after surgery in a 10-mm long segment containing 5 mm rostral and 5 mm caudal from the lesion center with immunohistochemical staining for OX-42, a marker for both microglia and macrophages. In the spinal cord of sham operated animals, the majority of OX-42 positive microglia/macrophages exhibited resting morphological feature; their cell bodies were small with
Discussion
The present study demonstrates in vivo that acute traumatic SCI-induced apoptosis and loss of neurons in the area adjacent to the lesion epicenter are mediated by over-expression of iNOS, a synthase of NO, in the microglia/macrophages in the lesion area, and preliminary administration of NOS inhibitor (l-NAME) can partially rescue neurons from apoptosis and death. This finding underscores the pathological significance of iNOS expression in microglia/macrophages in occurrence of secondary damage
Acknowledgements
The work is supported by the National Natural Science Foundation of China (No 30471787, 30471663 and 30500451).
References (37)
- et al.
Macrophages and microglia produce local trophic gradients that stimulate axonal sprouting toward but not beyond the wound edge
Molecular and Cellular Neuroscience
(2002) Inflammation and apoptosis: linked therapeutic targets in spinal cord injury
Trends in Molecular Medicine
(2004)- et al.
Neuroprotective effects of interleukin-10 following excitotoxic spinal cord injury
Experimental Neurology
(1999) - et al.
Current developments in spinal cord injury research
The Spine Journal
(2002) - et al.
Roles of nitric oxide in compression injury of rat spinal cord
Free Radical Biology & Medicine
(1996) - et al.
Therapeutic vaccination for spinal cord injury: helping the body to cure itself
Trends in Pharmacological Sciences
(2003) - et al.
Extracellular signal-regulated kinase-mediated IL-1-induced cortical neuron damage during traumatic brain injury
Neuroscience Letters
(2005) - et al.
Nitric oxide via macrophage iNOS induces apoptosis following traumatic spinal cord injury
Brain Research. Molecular Brain Research
(2000) - et al.
c-Jun N-terminal kinase and, to a lesser extent, p38 mitogen-activated protein kinase regulate inducible nitric oxide synthase expression in hyaluronan fragments-stimulated BV-2 microglia
Journal of Neuroimmunology
(2004) - et al.
Inhibition of MEK/ERK 1/2 pathway reduces pro-inflammatory cytokine interleukin-1 expression in focal cerebral ischemia
Brain Research
(2004)
Mechanisms and pathways of inflammatory responses in CNS trauma: spinal cord injury
The Journal of Spinal Cord Medicine
Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity
Journal of Neuroscience
Targeting the host inflammatory response in traumatic spinal cord injury
Current Opinion in Neurology
Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures
Journal of Neuroscience
Activated microglia mediate neuronal cell injury via a nitric oxide mechanism
Journal of Immunology
Thrombin-induced microglial activation produces degeneration of nigral dopaminergic neurons in vivo
Journal of Neuroscience
Beta-Amyloid stimulation of microglia and monocytes results in TNFalpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis
Journal of Neuroscience
Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice
Journal of Neuroscience
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