The Role Of Glial Adenosine Signaling In Epileptogenesis After SC

PARTⅠ THE DYNAMIC CHANGES OF ASTROCYTES ANDNEURONS IN HIPPOCAMPUS AFTER STATUSCONVULSIONObjective: To observe the dynamic changes of astrocytes and neuronsin hippocampus after Status convulsion（SC）.Methods: SC were induced by lithium-pilocarpine intraperitoneally inadult SD rats for60min. Rats were sacrificed at4time points (1d,5d,4w,8w) after SC. The dynamic changes of astrocytes and neurons inhippocampus were observed by immunofluorescence double-labeling forGFAP and NeuN. The expression of GFAP was also detected by westernblot and RT-qPCR.Results:(1) Neuronal cell death were found in hippocampus CA1andCA3in1d after SC, and neuronal cell loss can be seen in8w after SC;(2)The number of GFAP-positive cells first increased in5d after SC (P<0.05),and persisting increased until8w after SC (P<0.01); Reactive astrocyteswere found by cellular swelling, cells hypertrophy, the development of large and thickening extensions, and glial scar were found in neuronal lossregion;(3) The expression of GFAP-mRNA elevated in1d after SC, andpersisting elevated in5d,4w and8w after SC (P<0.01);(4) The expressionof GFAP protein began to increase in5d after SC, and persisting increasedin4w and8w after SC (P<0.01).Conclusion: Neuronal cell death and reactive astrocytes were found inhippocampus after SC, the changes continued to chronic epileptic period,and glial scar can be seen in neuronal loss region; The process of astrocyteactivation started prior to epileptogenesis period. PARTⅡ THE DYNAMIC CHANGES OF GLIALADENOSINE SIGNALING PATHWAY AFTER STATUSCONVULSIONObjective: To explore the dynamic Changes of glial adenosinesignaling pathway after SC.Methods: SC were induced by lithium-pilocarpine intraperitoneally inadult SD rats for60min. Rats were sacrificed at4time points (1d,5d,4w,8w) after SC. The concentration of adenosine in hippocampus wasmeasured by high performance liquid chromatography. The expression of ADK in astrocytes and neurons was observed by immunofluorescencedouble-labeling for ADK/GFAP and ADK/NeuN. The expression of ADKwas also detected by western blot and RT-qPCR. The expression ofadenosine receptors (A1R, A2aR, A2bR, A3R) were detected by westernblot.Results:(1) The concentration of adenosine in hippocampus increasedand reached the peak in1d after SC (P<0.01), and soon declined from5dafter SC; and even lower than the baseline in8w after SC (P<0.01);(2) Thenumber of ADK/GFAP-positive cells increased from5d to8w after SC, andreached the peak in4w after SC (P <0.01), ADK localized in the cytoplasmof astrocytes respectively; the number of ADK/NeuN-positive cells alsoincreased from5d to8w after SC (P <0.01), AdK localized in the nucleusof neurons, especially in CA1and CA3of hippocampus;(3) Theexpression of ADK-mRNA and protein began to increase in5d after SC,and persisting increased in4w and8w after SC (P<0.01);(4) Theexpression of adenosine receptors were different, the expression of A1Rincreased in1d after SC, but decreased from5d to8w after SC (P<0.01);while the expression of A2aR increased in1d after SC, reached the peak in4w after SC, and persisting increased to8w after SC (P<0.01); theexpression of both A2bR and A3R decreased from1d after SC, andcontinued to8w after SC (P<0.01).Conclusion: The concentration of adenosine in hippocampus increased sharply during the acute phase after SC, but went back to thebaseline during epileptogenesis period, this phenomenon may be caused bythe increased expression of ADK; the disturbance of adenosine receptorA1R/A2aR balance may contribute to the epileptogenesis. PART Ⅲ THE EFFECT OF ADENOSIN SIGNALINGMEDIATED BY ASTROCYTES ON SEIZURESUSCEPTIBILITYObjective: To explore the effect of adenosine signaling mediated byastrocytes on seizure susceptibility and the possible mechanism.Methods: Using patch clamp recording spontaneous dischargeinduced by Mg2+-free artificial cerebrospinal fluid(ACSF) in hippocampusslices, the latency and persisting period of spontaneous discharge weredetected to explore the effect of ADK inhibitor, A1R antagonist, A2aRantagonist on hippocampal seizure susceptibility; A neuron-astrocyteco-culture system was used, the expression of ADK in neurons andastrocytes were detected by immunofluorescence double-labeling forADK/GFAP and ADK/NeuN. Using patch clamp recording repeatedspontaneous spike discharge induced by Mg2+-free media, the frequency of spontaneous spike discharge were detected to explore the effect of ADKinhibitor, A1R antagonist, A2aR antagonist on the excitability of neurons.Results:(1) Spontaneous discharge of brain slices can be seen byinduced with Mg2+-free ACSF, the latency of spontaneous discharges wereprolonged and the persisting periods of spontaneous discharges wereshortened by ADK inhibitor and A2aR antagonist (P<0.01), and A1Rantagonists failed to change the latency and the persisting periods ofspontaneous discharges (P>0.05);(2) The expression of ADK can be seenin neurons and astrocytes of primary culture, localized in the nucleus ofneurons, and localized in the nucleus and cytoplasm of astrocytesrespectively;(3) Spontaneous spike discharge of neurons can be seen byinduced with Mg2+-free media, the frequency ofspontaneous spike discharge were reduced by ADK inhibitor in co-cultureof neurons and astrocytes（P<0.01）, while ADK inhibitor could not affectthe frequency of spontaneous spike discharge in culture of neurons only(P>0.05);(4) The frequency of spontaneous spike discharge were reducedby A2aR antagonist（P<0.01）, while A1R antagonists failed to change thefrequency of spontaneous spike discharge (P>0.05).Conclusion: Astrocytic ADK regulates the metabolism of adenosine,ADK inhibitors can decrease seizure susceptibility; the effect ofanticonvulsant of adenosine may be mainly mediated by A1R, and regulation the level of A2aR may be helpful to decrease excitability ofneurons, and may be helpful to prevent epileptogenesis.