[Show abstract][Hide abstract] ABSTRACT: Functional neuroimaging studies have implicated the prefrontal cortex (PFCTX) in descending modulation of pain and the placebo effect. This study was performed to elucidate comprehensive PFCTX gene expression in an animal model of persistent trigeminal pain. Adult male C57BL/6J mice received facial carrageenan injection and showed sustained increase in nociceptive responses. Microarray analyses of differentially expressed genes in the PFCTX at 3 d after injection showed "immune system process" as the dominant ontology term and increased mRNA expression of S100a8, S100a9, Lcn2, Il2rg, Fcgr1, Fcgr2b, C1qb, Ptprc, Ccl12, and Cd52 were verified by RT-PCR. Upregulation of S100A8, S100A9, and lipocalin 2 (LCN2) were confirmed by Western blots, and cells in the PFCTX were double immunolabeled with MPO, indicating they were neutrophils. Analyses of blood of facial carrageenan-injected mice also showed increased mRNA expression of these markers, suggesting transmigration of activated neutrophils into the brain. Other immune-related genes, Il2rg, Fcgr2b, C1qb, Ptprc, and Ccl12 were upregulated in the PFCTX but not blood. Approximately 70% of S100A9-positive cells in the PFCTX of carrageenan-injected mice were located in capillaries adherent to endothelial cells, whereas 30% were within the brain parenchyma. Carrageenan-injected mice showed significantly reduced nociceptive responses after injection of C terminus of murine S100A9 protein in the lateral ventricles and PFCTX but not somatosensory barrel cortex. Together, these findings demonstrate activation of immune-related genes in the PFCTX during inflammatory pain and highlight an exciting role of neutrophils in linking peripheral inflammation with immune activation of the PFCTX and antinociception.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2012; 32(1):35-45. DOI:10.1523/JNEUROSCI.2389-11.2012 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of the prefrontal cortex occurs during acute and chronic pain and models of experimental hyperalgesia. The present study was carried out to determine possible miRNA changes in the prefrontal cortex, after inflammatory pain induced by facial carrageenan injection in mice. miRNA microarray analyses showed significantly increased levels of miR-155 and miR-223 in the prefrontal cortex of carrageenan-injected mice. The changes were verified by real-time RT-PCR, and shown to occur bilaterally. The potential targets of the two miRNAs were predicted, and changes in two of the miRNA targets, c/ebp Beta and granulocyte colony-stimulating factor (GCSF) verified by real-time RT-PCR. Significantly downregulated c/ebp Beta but upregulated GCSF, accompanied by increased immunolabeling with an antibody to myeloperoxidase were found in the prefrontal cortex of facial carrageenan treated mice. It is postulated that this could lead to increased inflammation and activation of the prefrontal cortex. Further studies are necessary to determine if specific miRNAs could be useful as therapeutic molecules for pain.
European journal of pain (London, England) 03/2011; 15(8):801.e1-12. DOI:10.1016/j.ejpain.2011.02.002 · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was carried out to elucidate the effects of calcium independent phospholipase A(2) (iPLA(2)) on mitochondrial function and exocytosis in neuroendocrine cells. iPLA(2) mRNA and protein were detected in cell lysates and mitochondria from PC12 cells. Treatment of cells with the iPLA(2) inhibitor, bromoenol lactone (BEL), resulted in reduction in the mitochondrial membrane potential. Increase in membrane capacitance and number of spikes at amperometry, indicating exocytosis, were detected from PC12 cells after treatment with BEL. The induced exocytosis was abolished by pre-incubation of cells with the antioxidant, glutathione monoethyl ester, spin-trap/free radical scavenger, PBN, or inhibitors of the mitochondrial permeability transition pore, cyclosporine A and bongkrekic acid. These findings indicate that inhibition of iPLA(2) results in excessive exocytosis through increased oxidative damage (or failure to repair such damage) and defects in mitochondrial function. A similar process may occur in neurons with mutations in iPLA(2), leading to neuronal injury.
Neurochemical Research 02/2011; 36(2):347-54. DOI:10.1007/s11064-010-0340-y · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secretory phospholipase A2 (sPLA2) activity is present in the CNS and the sPLA2-IIA isoform has been shown to induce exocytosis in cultured hippocampal neurons. However, little is known about possible contributions of various lysophospholipid species to exocytosis in neuroendocrine cells. This study was therefore carried out to examine the effects of several lysophospholipid species on exocytosis on rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion, indicating exocytosis, was observed in PC12 cells after external infusion of lysophosphatidylinositol (LPI), but not lysophosphatidylcholine or lysophosphatidylserine by total internal reflection microscopy. Similarly, external infusion of LPI induced significant increases in capacitance, or number of spikes detected at amperometry, indicating exocytosis. Depletion of cholesterol by pre-incubation of cells with methyl beta cyclodextrin and depletion of Ca2+ by thapsigargin and incubation in zero external Ca2+ resulted in attenuation of LPI induced exocytosis, indicating that exocytosis was dependent on the integrity of lipid rafts and intracellular Ca2+. Moreover, LPI induced a rise in intracellular Ca2+ suggesting that this could be the trigger for exocytosis. It is postulated that LPI may be an active participant in sPLA2-mediated exocytosis in the CNS.
[Show abstract][Hide abstract] ABSTRACT: Phospholipases A(2) (PLA(2)) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A(2) (sPLA(2)) isoforms in the normal rat CNS with focus on sPLA(2)-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA(2)-IB expression was low throughout the CNS, sPLA(2)-IIA expression was high in the brainstem and spinal cord, sPLA(2)-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA(2)-V expression was high in the olfactory bulb and cerebellum, and sPLA(2)-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA(2)-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA(2)-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA(2)-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA(2)-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA(2) in nociceptive transmission.
Journal of chemical neuroanatomy 02/2010; 39(4):242-7. DOI:10.1016/j.jchemneu.2010.02.002 · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate possible effects of the intracerebroventricular (icv) injection of either O-Tricyclo [18.104.22.168(2,6)] dec-9-yl dithiocarbonate potassium salt (D609), a potent antioxidant and inhibitor of phosphatidylcholine specific phospholipase C (PtdCho-PLC) and acid sphingomyelinase (ASMase), or the spin trap/free radical scavenger N-tert-Butyl-alpha-phenylnitrone (PBN), on mechanical allodynia induced by facial carrageenan injection in mice.
Balb/c mice received icy injection of D609/PBN plus facial carrageenan injection, and the number of face wash strokes to von Frey hair mechanical stimulation of the maxillary skin was quantified. PtdCho-PLC and ASMase activities were also assayed in the brainstem, thalamus, and somatosensory cortex.
Mice that received the icy injection of 10 nmol D609 plus facial carrageenan injection showed significantly fewer face wash strokes evoked by von Frey hair stimulation (indicating reduced mechanical allodynia) at 1 and 3 days post-injection, compared to mice that received icy injection of isotonic saline plus facial carrageenan injection. Mice that received icy injection of 1.13 micromol PBN plus facial carrageenan injection likewise showed significantly fewer face wash strokes after facial carrageenan injection, compared to isotonic saline-injected plus carrageenan-injected controls. D609 injection also resulted in significantly reduced ASMase activity in the brainstem, thalamus, and somatosensory cortex 3 days after injection, compared to controls.
The icv injections of D609 and PBN were effective in reducing mechanical allodynia after facial carrageenan injection-induced pain. Together, the results point to a possible role of central nervous system sphingolipids and/or free radicals in orofacial pain.
Journal of orofacial pain 02/2009; 23(2):167-73. · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to examine global gene expression in the brainstem, in a mouse facial carrageenan injection model of orofacial pain. Mice that received facial carrageenan injection showed increased mechanical allodynia, demonstrated by increased responses to von Frey hair stimulation of the face. The brainstem was harvested at 3 days post-injection, corresponding to the time of peak responses, and analyzed by Affymetrix Mouse Genome 430 2.0 microarrays. We sought to identify common genes that are changed in the respective sides of the brainstem after either right- or left-sided facial carrageenan injection. The result is a relatively small list of genes (22 genes), which were then classified using DAVID software. Many of them fell into the categories of "response to stress", "defence response", "response to biotic stimulus", "cell adhesion" and "leukocyte adhesion". Of these, increased expression of P-selectin, ICAM-1 and CCL12 after carrageenan injection could be verified by real-time RT-PCR on both the right and left sides, and increased in P-selectin and ICAM-1 further verified by Western blot analysis. P-selectin and ICAM-1 were immunolocalized to endothelial cells, and were double labelled with von Willebrand factor. Intraperitoneal injection of the P-selectin inhibitor KF38789 significantly reduced mechanical allodynia in the facial carrageenan-injected mice. P-selectin mediates the capturing of leukocytes from the bloodstream and rolling of leukocytes along the endothelial surface. We hypothesize that increased nociceptive input to the brainstem could attract circulating macrophages into the brain, resulting in neuroinflammation and pain.
[Show abstract][Hide abstract] ABSTRACT: Peroxynitrite (ONOO(-)) and species derived from it can oxidize and nitrate lipids, proteins and DNA leading to changes in signaling molecules. The present study was carried out to elucidate possible effects of CNS peroxynitrite in a mouse model of orofacial pain. Mice that received facial carrageenan injection + intracerebroventricular (i.c.v.) injection of the peroxynitrite scavenger [5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato iron (III), chloride] (FeTPPS) showed significantly fewer face wash strokes upon probing the inflamed area of the face with a von Frey hair at 6 h after injection, compared to mice that received facial carrageenan alone, or facial carrageenan injection + i.c.v. injection of saline. Mice that received i.c.v. injection of FeTPPS without facial carrageenan injection showed no significant difference in response to von Frey hair stimulation, compared to mice that received i.c.v. injection of saline without facial carrageenan injection. These results indicate an anti-nociceptive effect of the peroxynitrite scavenger FeTPPS in carrageenan induced facial pain but no effect on normal tactile sensation and point to an important role of CNS peroxynitrite in nociception.
Experimental Brain Research 02/2008; 184(3):435-8. DOI:10.1007/s00221-007-1211-x · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increases in several ceramide species have been shown by non-targeted lipid profiling (lipidomics) of the rat hippocampus after kainate lesions (Guan et al. FASEB J 20:1152-1161, 2006). This study was carried out to examine possible effects of ceramide species on exocytosis. Significant increase in membrane capacitance in voltage-clamped rat pheochromocytoma (PC12) cells, an indication of exocytosis, was detected immediately after external application of C2, C6, and C18 ceramide. In contrast, no increase in capacitance was found after addition of C16 and C20 ceramide, or DMSO vehicle. The effect of ceramide on exocytosis was dependent on the integrity of lipid rafts. Treatment of cells with the cholesterol binding agent/disruptor of lipid rafts, methyl beta cyclodextrin, prior to addition of C18 ceramide suppressed the increase in capacitance induced by this lipid species. The ability of C2, C6 and C18 ceramide to trigger exocytosis was confirmed using total internal reflection fluorescence microscopy (TIRFM) experiments. External application of these species caused an exponential decrease in the number of subplasmalemmal neuropeptide Y (NPY)-enhanced green fluorescence protein (EGFP) labeled vesicles, indicating exocytosis. Interestingly, C18 is also the ceramide species that showed the greatest increase in the rat hippocampus after kainate excitotoxicity. It is postulated that C18 ceramide might facilitate exocytosis of glutamate from damaged neurons, thus propagating neuronal injury.
Experimental Brain Research 12/2007; 183(2):241-7. DOI:10.1007/s00221-007-1036-7 · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to elucidate the distribution of calcium-independent phospholipase A(2) (iPLA(2)) in the normal monkey brain. iPLA(2) immunoreactivity was observed in structures derived from the telencephalon, including the cerebral neocortex, amygdala, hippocampus, caudate nucleus, putamen, and nucleus accumbens, whereas structures derived from the diencephalon, including the thalamus, hypothalamus and globus pallidus were lightly labeled. The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled. Immunoreactivity was observed on the nuclear envelope of neurons, and dendrites and axon terminals at electron microscopy. Western blot analysis showed higher levels of iPLA(2) protein in the cytosolic, than the nuclear fraction, but little or no protein in the membrane fraction. Similarly, subcellular fractionation studies of iPLA(2) activity in rat brain cortical cell cultures showed greater enzymatic activity in the cytosolic, than the nuclear fraction, and the least activity in non-nuclear membranes. The association of iPLA(2) with the nuclear envelope suggests a role of the enzyme in nuclear signaling, such as during neuronal proliferation and differentiation or death. In addition, the localization of iPLA(2) in dendrites and axon terminals suggests a role of the enzyme in neuronal signaling.
Journal of Neurocytology 12/2006; 34(6):447-58. DOI:10.1007/s11068-006-8730-4 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to determine the effects of central nervous free fatty acids, lysophospholipids, or platelet activating factor (PAF), in a mouse facial carrageenan injection model of orofacial pain. Mice that received intracerebroventricular (I.C.V.) injection of arachidonic acid or oleic acid showed significantly reduced allodynia and behavioral responses to von Frey hair stimulation of a carrageenan-injected area of the face, at 8 h post-injection, compared to controls that received I.C.V. injection of vehicle. In contrast to free fatty acids, increased responses were observed in mice at 72 h after I.C.V. lysophosphatidic acid or lysophosphatidylcholine injection, and at 8 and 24 h after PAF injection, compared vehicle injected controls. Information regarding pro-nociceptive effect of specific brain lipids may be a useful basis for further studies to explore mechanism.
Experimental Brain Research 11/2006; 174(4):781-5. DOI:10.1007/s00221-006-0672-7 · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fusion of synaptic vesicles with the plasma membrane during exocytosis can be recorded by membrane capacitance measurements under voltage-clamp conditions. These measurements enable high time-resolution quantitation of exocytosis. The present study was carried out using the above technique to elucidate the effects of various polyunsaturated fatty acids on exocytosis in a neuroendocrine cell, the rat pheochromocytoma-12 (PC12) cell. External application of eicosapentaenoic acid and arachidonic acid resulted in an increase in capacitance of PC12 cells, indicating fusion of secretory vesicles with cell membranes and exocytosis. In contrast, docosahexaenoic acid, linoleic acid, oleic acid, and vehicle control had no significant effect on capacitance. The above findings show differential effects of polyunsaturated fatty acids on exocytosis in PC12 cells. It is postulated that besides arachidonic acid, eicosapentaenoic acid could also play an important role in exocytosis and neurotransmitter release, in neurons and hormone-secreting cells.
Neurochemical Research 02/2006; 31(1):41-8. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to elucidate the effect of a single episode of oxidative stress on pyramidal neurons of the rat hippocampus. A significant increase in the number of neurons that were immunolabeled for the toxic lipid peroxidation product, 4-hydroxynonenal (HNE) was observed in field CA3 of the hippocampus, at 1 day, 7 days and 14 days after intracerebroventricular injection of 1 microL of 5 mM ferrous ammonium citrate, compared to ammonium citrate injected controls at these time points. The number of HNE positive cells was fewer at 14 days, compared to 1 day, after ferrous ammonium citrate injection. The changes in HNE immunoreactivity were paralleled by changes in cytoplasmic phospholipase A2 (cPLA2) labeling in the pyramidal neurons in adjacent sections, suggesting that some of the HNE could have arisen as a result of peroxidation of arachidonic acid that was released by cPLA2. Interestingly, despite the HNE and cPLA2 labeling, no loss of neurons was observed in adjacent Nissl and Fluoro-Jade stained sections. Electron microscopy also showed that the HNE or cPLA2 labeled cells had features of injured neurons, rather than necrotic neurons. The reduction of HNE immunoreactivity in neurons at 14 days after oxidative injury, and the absence of cell loss at any of the time intervals, shows that hippocampal pyramidal neurons have remarkable ability to recover from a single episode of oxidative stress, if repeated injury such as seizures / excitotoxicity could be avoided.
Reproduction Nutrition Development 09/2005; 45(5):647-62. DOI:10.1051/rnd:2005051 · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out, using inhibitors to secretory phospholipase A2 (sPLA2, 12-epi-scalaradial), cytosolic phospholipase A2 (cPLA2, AACOCF3), or calcium-independent phospholipase A2 (iPLA2, bromoenol lactone), to compare possible contributions of central nervous PLA2 isoforms to the development of allodynia after facial carrageenan injection in mice. C57BL/6J (B6) mice showed increased responses to facial stimulation using a von Frey hair (1 g force), at 8 h, 1 day, and 3 days after facial carrageenan injection. On the other hand, BALB/c mice did not show increased responses at any of the time points. In both B6 and BALB/c mice, intracerebroventricular injection of inhibitors to each of the three PLA2 isoforms significantly reduced responses to von Frey hair stimulation at 8 h and 1 day after facial carrageenan injection, but at 3 days after injection, only the sPLA2 inhibitor had an effect. Since BALB/c mice did not show increased responses after facial carrageenan injection, the reduction in responses actually indicates that there is loss of normal sensitivity to von Frey hair stimulation after intracerebroventricular injection of each of these inhibitors, in this strain of mice. The effects of PLA2 inhibitors are unlikely to be due simply to inhibition of arachidonic acid generation, since intracerebroventricular injection of arachidonic acid also had an anti-nociceptive effect. The above results support an important role of central nervous PLA2s in neurotransmission and pain transmission.