Traumatic spinal cord injury (SCI) leads to neurological deficits and motor and sensory dysfunctions. In the United States alone, there were approximately 270,000 people living with SCI in 2012, and an additional 12,000 new SCI cases occur every year, most of them younger than 30 years (https://www.nscisc.uab.edu). To date, there is no effective pharmacological treatment for SCI. SCI is caused by mechanical damage that triggers cellular events culminating in the secondary injury phase, which provides an important therapeutic window for neuroprotective strategies to improve recovery of function after SCI. Previous studies indicate that multiple injury mechanisms, including inflammation, oxidative stress, and glutamate excitotoxicity,[3-6] are involved in the secondary injury process after initial trauma, but exact mechanisms remain to be fully elucidated.
Several lines of evidence suggest that phospholipase A2 (PLA2) may play a key role in mediating multiple injury insults, as mentioned above, after SCI.[7-11] PLA2 is a diverse family of enzymes that hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids.[8, 12, 13] These products are precursors of bioactive eicosanoids and platelet activating factor, which are well-known mediators of inflammation and tissue damage implicated in pathological states of several acute and chronic neurological disorders.[8, 12-14] Our previous study showed that PLA2 activity and expression increased after SCI in rats. Injections of exogenous PLA2 or melittin, a potent activator of endogenous PLA2, into the normal spinal cord resulted in inflammation and tissue damage. Administration of annexin A1, a nonselective inhibitor of PLA2, inhibited SCI-induced inflammation and reduced tissue damage after SCI. These findings suggest that PLA2 may be a potential therapeutic target for SCI.
PLA2 can be broadly classified into 3 major categories: secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and Ca2+-independent PLA2 (iPLA2). Among them, cPLA2 is considered to be the most important PLA2 isoform, because it has been implicated as an effector in receptor-mediated release of arachidonic acid (AA) and exhibits strong preference for deacylation of AA over other fatty acids.[13, 17] However, the role of cPLA2 in the pathogenesis of SCI has not yet been fully understood, and is even controversial.[15, 18] Here, we report that SCI significantly induced cPLA2 activation and expression. Blocking cPLA2 pharmacologically and genetically ameliorated motor deficits, and reduced cell loss and tissue damage after SCI in mice. Thus, cPLA2 may represent a therapeutic target for treatment after traumatic SCI.
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The goal of this study was to determine whether targeting cPLA2 could be an effective strategy for functional repair after SCI. Our study showed that SCI induced an elevation of cPLA2 expression and activation. The elevated cPLA2 was localized mainly in neurons and oligodendrocytes. We also showed that the SCI-induced cPLA2 activation is mediated, at least in part, by ERK signal, revealing a molecular mechanism of cPLA2 activation. In vitro studies demonstrated that cPLA2 activation induced cultured spinal cord neuronal death. Most importantly, both pharmacological blockade and genetic deletion of cPLA2 significantly reduced inflammation, cell death, and tissue damage, as well as improved behavioral recovery after SCI. These findings collectively suggest that modulation of cPLA2 could represent a new therapeutic strategy for treatment of SCI.
SCI significantly induced cPLA2 activation, which was observed as early as 8 hours postinjury and peaked at 7 days postinjury. The activated cPLA2 was mainly localized in neurons and oligodendrocytes. The expression of cPLA2 mRNA was increased in the injured cord, which correlated well with increased expression of cPLA2 protein. Several earlier investigators found that AA and eicosanoids, metabolites of cPLA2, increased within 30 minutes after SCI.[45, 46] Others reported that increased eicosanoids were persistent at least for 3 days (the longest time point studied) after SCI. Furthermore, Demediuk et al reported that induced concentrations of free fatty acid quickly increased after SCI, peaked at 3 days, and remained significantly high at 7 days after SCI. The induction profiles of these PLA2 metabolites are similar to that of cPLA2 after SCI in the present study. These results indicate that a prolonged effect of cPLA2 exists after SCI, which suggests that there may be a unique therapeutic window for intervention. The finding that cPLA2 was mainly localized in neurons and oligodendrocytes is particularly interesting, because these 2 cell types not only play important roles in normal central nervous system (CNS) function but also are the most vulnerable cell types to injuries as compared to other CNS cell types such as astrocytes and microglia.
Although cPLA2 activation and expression were significantly increased after SCI, the mechanism(s) by which they were increased remains to be determined. Our in vivo experiments revealed that ERK1/2 signaling pathway mediated SCI-induced cPLA2 activation. Our previous in vitro experiments also showed that ERK1/2 signaling pathway mediated cPLA2 phosphorylation, induced by glutamate and H2O2, two important injury mediators of secondary SCI5. We and others have reported that cPLA2 is induced by several toxic factors that are generated in the injured cord, including inflammatory cytokines,[8, 17] free radicals,[7-9, 11] and excitatory amino acids.[7, 8, 10] Therefore, cPLA2 may serve as a central or convergence molecule that mediates multiple key mechanisms of secondary injury, making it an attractive therapeutic target to improve tissue protection and function recovery.
Our results clearly demonstrated that cPLA2 activation induced spinal cord neuronal death. This was in agreement with our previous finding that cPLA2 activation mediated cultured spinal cord neuronal death induced by glutamate and H2O2.7 Apoptosis has been considered a key mechanism of cell death following SCI.[49, 50] Caspase-3 plays a central role in the execution phase of apoptosis and is responsible for the cleavage of proteins such as the nuclear enzyme PARP. Our results showed that cPLA2 activation induced the expression of active caspase-3 and PARP-1. TUNEL staining further confirmed that cPLA2 activation induced neuronal apoptosis, which was supported by cPLA2-mediated neural apoptosis induced by Aβ. These results suggest that cPLA2 activation induced neuronal death through apoptosis, at least in part.
A significant finding of this study is that pharmacological blockade of cPLA2 with AACOCF3 inhibited inflammation and membrane injury, reduced tissue damage, and improved behavioral recovery in C57BL/6 mice after SCI. Notably, the cPLA2 inhibitor was administered after trauma. Our results showed a long beneficial effect of targeting cPLA2 on anatomical and functional recoveries. In agreement with our observation, several studies have reported a detrimental effect of cPLA2 in other CNS diseases such as ischemia,[20, 52] experimental autoimmune encephalomyelitis,[29, 53] and Alzheimer disease. In contrast, there is a recent report showing that activation of cPLA2 is beneficial. In that study, both BALB/c mice treated with AX059, a cPLA2 inhibitor, and cPLA2-null BALB/c mice displayed greater neuronal and myelin loss after SCI. The contrary results between this mouse strain and others remain unclear, and they may be related to different mouse backgrounds (C57BL/6 vs BALB/c) and inhibitors (AACOCF3 vs AX059) that were used. It has been reported that different strains of mice display distinctly different responses to trauma injury, including inflammation, histology, and behavioral recovery.[30, 55-57] For example, post-traumatic inflammation was markedly reduced in BALB/c mice compared with C57BL/6 mice. After SCI, a densely packed cellular matrix fills necrotic cavities. The magnitude of this response was greatest for C57BL/6 mice and least for BALB/c mice. Kipnis and colleagues also showed that BALB/c mice exhibited a T-cell–dependent neuroprotective response, whereas trauma- or glutamate-mediated neuronal cell loss was enhanced in C57BL/6 mice. These results suggest that genetic differences may confer different responses to traumatic injury, which may modify the secondary injury processes after SCI. Thus, the contrary results from C57BL/6 and BALB/c mice may be related to genetic differences including sPLA2.
We previously demonstrated increased sPLA2 expression following SCI. Injection of sPLA2 into the normal spinal cord resulted in tissue damage, demyelination, and behavioral impairment in vivo. Importantly, administration of a sPLA2 inhibitor GK511 in BALB/c mice reduced tissue damage and improved behavioral recovery after SCI. In the current study, sPLA2 action was excluded in both sham and SCI groups, because C57BL/6 mice have a naturally occurring null mutation of sPLA2. iPLA2 is generally considered to be a housekeeping enzyme for the maintenance of membrane phospholipids. Lopez-Vales et al reported that iPLA2 was upregulated after SCI and was expressed mainly in oligodendrocytes. Using FKGK11, a potent and highly selective iPLA2 inhibitor in BALB/c mice with SCI, they further showed that iPLA2 appeared to play a minor detrimental role in SCI. Although AACOCF3 has been reported to be a weak inhibitor of iPLA2,[41-43] our results showed that there was no significant difference of iPLA2 activity between the SCI and AACOCF3-treated groups. These results suggest that AACOCF3 exerts neuroprotection mainly via inhibition of cPLA2.
A definitive finding of the present study is that genetic deletion of cPLA2 resulted in neuroprotection and behavioral recovery following SCI. Genetic deletion of cPLA2 also inhibited the expression of active caspase-3 after SCI, suggesting that cPLA2 activation mediates neural apoptosis. Our observation is supported by several other reports that cPLA2−/− mice show protection in ischemic brain damage, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, and neurodegeneration.[20, 54, 58] cPLA2−/− mice also show significant reductions in AA release and eicosanoid production in response to a variety of stimuli.[19, 20] cPLA2 may contribute to injury by a direct effect on cell membranes and/or indirectly through generation of its metabolites, which are inflammatory and vasoconstrictive mediators.[8, 59] Our results suggest that cPLA2 contributes to the pathogenesis of SCI and that targeting cPLA2 could be an effective therapeutic strategy for intervention after SCI.
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This work was supported by the NIH NINDS (1R01 NS059622, 1R01 NS050243, 1R01 NS073636; X.-M.X.), the Indiana Clinical and Translational Sciences Institute Collaboration in Biomedical/Translational Research Pilot Grant Program (RR025761; X.-M.X.), the Indiana Spinal Cord and Brain Injury Research Foundation, Mari Hulman George Endowment Funds (X.-M.X.), the Indiana State Department of Health (A70-2-079609, A70-9-079138; N.-K.L.), and the NIH National Institute of Diabetes and Digestive and Kidney Diseases (DK39773, DK 054741; J.V.B.).
We thank Dr S. Gao, a biostatistician, for statistical assistance; P. Raley, a medical editor, for critical reading of the manuscript; and Dr C. L. Hammer, a medical fellow, for some in vitro experiments.