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Title: Role of Central Nervous System Ceramides and Free Radicals in a Mouse Model of Orofacial Pain
Authors: TANG NING
Keywords: orofacial pain, ceramides, free radicals, sphingomyelinase, serine palmitoyl transferase, patch clamp
Issue Date: 29-Jul-2009
Citation: TANG NING (2009-07-29). Role of Central Nervous System Ceramides and Free Radicals in a Mouse Model of Orofacial Pain. ScholarBank@NUS Repository.
Abstract: Growing evidences have indicated an important role of central nervous system (CNS) lipid mediators and reactive nitrogen species (RNS) in augmenting the sensitivity of sensory neurons and enhancing pain perception. Increased amount of ceramide which is an important sphingolipid signaling molecule and elevated ceramide biosynthetic activity have been shown to contribute to neuronal death in the hippocampus after kainate-induced excitotoxic injury. RNS species such as peroxynitrite (ONOO-) and its derivates can cause lipid oxidation, protein nitration, and DNA damage, leading to changes in the function of signaling molecules. Intracerebroventricular (ICV) injection of inhibitors to ceramide synthetic enzymes into mice was conducted to elucidate possible role of CNS ceramide in orofacial pain induced by facial carrageenan injection. ICV injection of inhibitors to acid sphingomyelinase (ASMase), neutral sphingomyelinase (NSMase), or serine palmitoyltransferase (SPT) significantly reduced allodynic responses in facial carrageenan injected mice. An enzyme activity assay was conducted in the mice brain tissue. Increased ASMase activity was found in the left primary somatosensory cortex at 3 days after facial carrageenan injection. And ICV injection of ASMase inhibitor D609 significantly reduced ASMase activity in all parts of brain examined (i.e., left and right brain stem, thalamus, and primary somatosensory cortex). These results provide a further confirmation that D609 alleviates facial allodynia through the action of ASMase. Since D609 is also found to be a free radical scavenger, phenyl-N-tert-butylnitrone (PBN), a free radical spin trap was ICV injected to elucidate the role of free radicals in nociception. Similar anti-allodynic effect was observed in mice with facial allodynia after PBN treatment. It was also found that C18 ceramide could cause increased hydrogen peroxide production in PC12 cells. This effect could be inhibited by co-treatment with L-type calcium inhibitor (nifedipine), free radical scavengers (D609 or PBN), or mitochondria permeability transition pore blockers (bongkrekic acid or cyclosporine A). Electrophysiological study showed that ceramide has the ability to directly induce exocytosis in cells using membrane capacitance measurement technique (whole-cell patch clamp) and total internal reflection fluorescence microscopy technique. Effects of ceramide were found to be dependent on the integrity of cell membrane lipid raft, as ceramide could not induce exocytosis in cells depleted of membrane cholesterol. Direct application of ceramide can also cause elevated intracellular calcium concentration in PC12 cells. The role of other forms of free radicals such as peroxynitrite in orofacial allodynia was also investigated. Mice behavioral studies showed that ONOO- plays a role in nociception in the CNS in mice with facial allodynia. ICV injection of ONOO- scavenger FeTPPS significantly reduced allodynia in the facial carrageenan injected mice at 3 days after injection. In conclusion, the present study showed a possible role of CNS ceramide and ONOO- in a mouse model of orofacial allodynia.
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