| Pain, especially pathologic pain including chronic pain and algesthesia sensity, has becomea serious public issue affecting millions of people worldwide. The well-balanced systemresponsible for the discrimination of noxious and innocuous stimuli may be altered aftertraumatic nerve injury, cancers, fractures, or even after minor fully recoverable injuries and giverise to pathological chronic pain with abnormally increased pain sensation and where innocuousstimuli are perceived as painful. These symptoms are known as hyperalgesia and allodynia,respectively. Chronic pain is not only produced in the injured tissue or territory (innervated bythe injured nerve), but also spread to the adjacent non-injured regions or the extraterritory(extraterritorial pain) and contralateral body (mirror-image pain). This exaggerated pain isthought to result from peripheral sensitization and central sensitization. Furthermore, chornicpain may last for many months when noxious stimuli are absent, which affects people's lifeseriously.It was well known that the accomplishment of pain signal transmission needs three classesneurons from peripheral nervous system(PNS) to central nervous system(CNS). The signal ofpain is recepted by peripheral sensory neuron firstly, then transmited to dorsal horn neurons andlastly transmited to the relative cortex through different pathways. It was reported that functionalalteration of neurons from algethesia conducting system was related to generation of chronicpain closely, whose mechanism includes dystopia discharage of neuron, great releasment ofneurotransmitter and synaptic long time plasticity et al. The chornic pain may be result frominteraction between peripheral sensitization(increase in sensitivity of nociceptive primaryafferent neurons) and central sensitization (hyperexcitability of nociceptive neurons in the CNS).However, abundant reseaches were mainly focus on neurons, such as neuronal plasticity,neurotransmitters and neuronal pathways. And the analgesia results according to neuronalalgethesia theory were not effective. In 1990s, Investigators turned their attention to another cellin nervous system namely glia, and found it paly an important part in pathologic process inchronic pain.Glia was consistently known as subordinate cells in the nervous system in the long timeneuronscience reseach. Astrocyte was considered that it only exert supportive, protective andnutrition effects to neurons. Succedent studies confirmed that in different pain models includingneural injury in PNS or CNS or inflammatory pain, it appears not only the symptom of chronicpain(displaying thermalgsia and allodynia) but also the activation of spinal glia. After damage of nervous tissue and inflammation, astrocytes become activate state from resting state, and releasenumerous nervous active materials, including neurontransmittors(excitatory amino acids, ATPand NO et al.) and cytokines(NO, IL-1β, IL-6和TNF-αet al.) and meanwhile accompanieselevation of neuronal excitatory, accordingly participate in the transimission and regulation ofpain signals. Although it has accumulated numerous evidence, the current reseach system stillhas limitation to explain the mechanism of glia activation and effects, including how did eachstimulus active glias(including astrocytes and microglias), through which receptor did thestimulus work; how did glia interact with neurons to participate in pain, and through whichsubstance(such as cytokine and neurotransmitters), and at which intensity and process could gliainteract with neurons still has no mechanisms.Glutamate, as a significant excitatory transmitters in the CNS, take part in many neuronalphysiologic and pathologic processes through different kinds of receptors(including ionotropicreceptors and metabolic receptors). Whether it could active astrocytes and participate in chronicpain is still not clear. In the process of injury of CNS and inflammatory, glia will release manyinflammatory factors, such as NO,IL-1β,IL-6 and TNF-α, then IL-1βand TNF-αwill activateastrocytes to release IL-6. Thus, astrocytes may be the major resourse of basic IL-6 in brain.Objective:This study explored the direct effect of glutamate activate spinal astrocytes. Furthermore, toexamine whether these activated astrocytes involved in the thermal Hyperalgesia.Methods:1. Primary culture of spinal astrocytes were prepared from the spinal cord of 1-2 postnatalWistar rats. After cultured for 9-14 days, devided astrocytes into 14 groups: (1)control group,cultured in cell medium; (2)ATP group(positive control), medium+50μM ATP for 1 hour(1h);(3)medium+500μM Glutamate(Glu ) for 1 h, 6 h, 12 h and 24 h groups; (4) medium+1000μMGlu for 1 h, 6 h, 12 h and 24 h groups medium+500μM Glu for 6 h group; (5) medium+2000μM Glu for 1 h, 6 h, 12 h and 24 h groups.2. GFAP specific astrocytes were analysed by Immunocytochemistry, to observe themorphologic changes of activated astrocytes.3. The protein expression of IL-6 was determined by ELISA assay, to find out the effectiveconcentration and time points for glutamate to activate cultured spinal astrocytes.4. The mRNA expression of IL-6 was determined by Real time PCR, to observe theeffective concentration and time points for glutamate to activate cultured spinal astrocytes onmRNA level.5. The spinal astrocytes, activated by the effective concentration and time points ofglutamate above, were injected into the lumber enlargement of adult Wistar rats by PE-10 tube. Paw withdrawal latency (PWL) in response to thermal stimuli was tested after intrathecalinsertment of PE-10 tube (baseline) and 2h, 4h and 6h after spinal astrocyte injection.Results:1. The cultured cells were GFAP specific stained astrocytes.2. Medium group as negative control group, ATP group as positive control group, forglutamate group at concentration 500μM for 24 hours, there is little changes in morphology,namely, the soma of spinal astrocytes diversed and synapses contracted. And to concentration of1000μM and 2000μM, the cell body started to be round at 6 hours time point and altereddramatically 24 hours(the last time point we explored) after cells were exposed to glutamate.3. From the protein level, there were no significance between glutamate 500μM at fourtime points and control group(10.81±2.87)(P>0.05). While groups with 1000μM and 2000μMglutamate IL-6 started to increase at 6 hours(153.30±2.08 and 69.41±18.14, respectively), andpresent time-dependnt trend. Compared with group of glutamate 1000μM, the data in group ofglutamate 2000μM reduced significantly. And incubated with 1000μM glutamate for 24 hours,IL-6 reached peak value(426.53±33.89)(P<0.05, One-Way ANOVA).4. From mRNA level, compared with control group(1.05±0.17), IL-6 transcription in groupof glutamate 500μM 24 h increased greatly. groups with 1000μM and 2000μM glutamate IL-6mRNA expression started to increase at 6 hours(4.52±0.96 and 3.47±0.30, respectively). WhileIL-6 mRNA expression in groups incubated with three different concentrations(500μM, 1000μM and 2000μM) of glutamate for 24 hours all shifted sharply(17.77±2.32, 22.43±1.42 and24.12±3.14, respectively), which was the last time point examined, and present dose-dependenttrend(P< 0.05, One-Way ANOVA).5. From the data above, we have found that the concentration of glutamate that can inducespinal astrocytes activated effectively were 1000μM and 2000μM, and time points were 6 h, 12h and 24 h. So we activated cultured spinal astrocytes with ATP(50μM, for 1 h), glutamate(1000μM, for 6 h, 12 h and 24 h) and glutamate(2000μM, for 6 h, 12 h and 24 h). We found adramatic reduction in paw withdrswal latency(PWL) after intrathecal injection ofglutamate-stimulated astrocytes at both 1000μM and 2000μM (P<0.05). The PWL reductionstarted 2 h after astrocyte injection and lasted for 6 h(the last time point we explored), andpresent time-dependent trend.Conclusions:1. Glutamate could activate spinal astrcytes in vitro, and the effective concentration shouldreach 1000μM, and time should last for 6 hours.2. Glutamate activation spinal astrocyte was involved in the thermal hyperalgesia.3. The cytokine such as IL-6 released from spinal astrocytes activated by glutamate played an important roles in the thermal hyperalgesia.
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