| Neuropathies can result from any type of neural damage, including that triggered by physical trauma, infection, inflammation, metabolic abnormalities, vascular abnormalities, neurotoxins, chemotherapeutic agents, radiation or autoimmune disease. Neuropathic pain, which is characterized by unexplainable widespread pain, a sensory deficit, a burning sensation, caused by light touch (allodynia), or acute pain in the absence of a noxious stimulus. Furthermore, chronic neuropathic pain can persist for months, without the underlying cause being treatable or identifiable. Traditionally, our understanding about neuronal signaling for pain transmission from the body to the central nervous system (CNS) occurs as a series of relay signals that are eventually processed in the brain。Many changes in neuronal function across the nociceptive system are associated with the genesis of neuropathic pain, including ectopic discharge, exaggerated release of neurotransmitters, and long-term potentiation. Nevertheless, much evidence has suggested that this plasticity does not occur autonomously in neurons, but also depends on glial cells. The role of interactions between glia and neurons in the induction of neuropathic pain has gained much attention in the last decade.In the CNS, there are two categories of cells:neurons and adjacent glial cells. For years, glial cells were thought to be passive cells that had few responses to synaptic activation, and were overlooked as merely supportive cells in the CNS. However, a growing body of evidence has recently suggested that glial cells communicate with one another and with neurons primarily through chemical signals. Glial cells dynamically modulate the function of neurons under both physiological and pathological conditions.There are three general types of glial cells in the CNS:oligodendrocytes, astrocytes and microglia. Microglias are macrophage-like cells in the CNS that priginate from bone marrow-derived monocytes that migrate during perinatal development. Microglia cells are considered the immune cells of the nervous system involved in the repair of the nervous system, immune activity and inflammation. In recent years, people found that microglia activation were ralated with a variety of neurodegenerative diseases and mental diseases, such as PD, AD and depression. Diverse animal models of nerve injury or inflammation, either peripherally or centrally, produce pathological pain states such as thermal hyperalgesia (exaggerated responses to painful heat) and mechanical allodynia. These models have demonstrated the involvement of spinal cord glias which become activated with the development of pain enhancement. Activation of glial cells can release a variety of substances including inflammatory cytokines, prostaglandins (PG), BDNF. These chemical mediators in turn modulate neuronal activity and facilitate pain transmission. Although it has accumulated numerous evidence, the current reseach system still has limitation to explain the mechanism of glia activation and effects, including how did each stimulus active glias(including astrocytes and microglias), through which receptor did the stimulus work. Because pain signal is conveyed only by neurotransmission in the neural circuits, and the mechanisms by which neurotransmitter is only beginning to be understood.The neurotransmitters that carry peripheral injury signals into spinal cord include glutamate, substance P, ATP and CGRP. Tsuda and his colleagues had found ATP could activate microglia and involved in chronic pain. Glutamates are significant excitatory transmitters in the CNS and take part in almost all nervous system function. Substance P is considered nociceptive specific neurotransmitter, and engaged in spinal pain signal transduction and modulation. Whether it could activate microglia and participate in chronic pain is still not clear.Objective:1. Explore the effect of substance P on the activation of spinal microglia, including cell morphology abservation and the release of TNF-a and L-(3from the microglia by ELISA assay.2. Observe the changes of signaling molecules in microglial after substance P incubation.3. Examine whether these activated microglia involved in the themal hyperalgesia.Method:1. Primary culture of spinal microglias were prepared from the spinal cord of0-1d postnatal rats. After cultured for9-14days, microglias were divided into4groups:(1) control group, cells were cultured in cell medium;(2) substance P200μM group, cells were cultured in cell medium+200μM substance P;(3) substance P400μM group, cells were cultured in cell medium+400μM substance P;(5) P substance800μM group, cells were cultured in cell medium+800μM substance P; cells in each group were studied at2h,6h,12h,24h after incubation.2. OX-42specific microglias were analyzed by Immunocytochemistry, to observe the morphologic changes of activated microglias3. The release of TNF-a and IL-(3by activated microglias were determined by ELISA assay, to find out the effective concentration and time points for substance P to activate cultured spinal microglias.4. The change of intracellular cacium were observed by laser scanning confocal microscope. 5. The phosphorylations of p38MAPK in microglias were observed by double immunofluorescence method.6. The spinal microglias, activated by the effective concentration and time points of substance P above, were injected into the lumber enlargement of adult rats by PE-10tube. Paw withdrawal latency (PWL) in response to thermal stimuli was tested after intrathecal insertment of PE-10tube (baseline) and6h,12h and24h after spinal microglias injection.Results:1. The cultured cells were OX-42specific stained micrglias, and the purity of over95%.2. Medium group as control group, for substance P200μM group, there is little changes in morphology in different time point. Substance P400μM incubated cells stated to be rounded and the immunoblotting of OX-42were increase. Substance P800μM incubated cells had higher density of OX-42immunoblotting than400μM group, but the structure of cells lost the integrity.3. TNF-a ELISA assay results showed that there were no significance between substance P200μM group at four time points and the control group, respectively. While groups with400μM and800μM substance P TNF-a release started to increase at6hours (276.63±15.3and447.57±19.36), peaked at12hours(445.45±30.27and759.38±47.64), then decreased after24hours (332.31±16.25and401.48±35.59).3, IL-β ELISA assay results showed that there were no significance between substance P200μM group at four time points and the control group, respectively. While groups with400μM and800μM substance P IL-1β release started to increase at6hours (212.64±15.01and414.69±20.74), peaked at12hours (285.04±20.74å'Œ642.6±11.62), then decreased after24hours (223.97±4.73and367.33±13.42).4, Ca2+imaging results show that incubation of substance P200μM and400μM in microglial cells did not change the concentration of cytoplasmic Ca2+.After substance P800μM incubation, intracellular Ca+decrease rapidly and inrreversiblly, and some of the cells lose the adherent ability.5.15min after substance P400μM incubation, p38MARK in microglia was phosphorylated.6. From the data above, we have found that the concentration of substance P that can induce spinal microglia activated effectively were400μM, and time point is12h. so we activated cultured spinal microglia with substance P (400μM, for12h). we found a reduction in paw withdrawls latency (PWL) after intrathecal injection of substance P-stimulated microglias. The PWL reduction started6h after cell injection and lasted for24h.Conclusion: 1. Substance P can induce spinal microglial activation, the effective concentration should reach400μM, and time should last for6hours.2. Substance P can induce phosphorylation of p38MAPK in microglia, but failed to increase the intracellular calcium concentration in microglia.3. Substance P-induced spinal microglial activation is involved in the formation of thermal hyperalgesia.4. Substance P-induced spinal microglial activation is involved in the formation of thermal hyperalgesia may be associated with p38MAPK pathway and the following release pro-inflammatory cytokines TNF-a and IL-1β. |
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