| Background: AMPA receptor mediates the overwhelming majority of fast excitatory neurotransmission in the mammalian central nervous system, and influences transmission efficacy of individual synapse, integrative functions of neuron, and synapse plasticity. Applied modern methodology, the structure of AMPA receptor was clarified. AMPA receptor is assembled from subunits encoded by four genes, GluR1-4. The presence of edited GluR2 can block the Ca~2+ influx .Hence, more and more researchers focus attention on its effect in ischemia insult. In hippocampus, most of neurons express abundant GluR2 and exhibit low AMPA receptor Ca~2+ permeability. Reduction of AMPA receptor GluR2 subunit on postsynaptic membrane causes increase of GluR2-lacking AMPA receptor. The GluR2-lacking AMPA receptor containing high Ca~2+ permeability can produce toxic Ca~2+ input, leading to neuronal injury. Base on the characteristics above-mentioned, especially the ratio change of presence or absence of the GluR2 subunit, AMPA receptor plays a critical role in modulating the injury caused by ischemia insult. But, the detailed molecular mechanisms were unclear, such as the remodeling and phosphorylation of AMPA receptor in cerebral ischemia. The divaricationon whether GluR2 mRNA edited or not in cerebral ischemia was still in existence. Moreover, electrophysiological characteristic changes of AMPA receptor channel, including of miniature excitatory postsynaptic currents (mEPSCs) and calcium permeability mediated by AMPA receptor, also need to be further confirmed.In this study, the structural and functional changes of AMPA receptor were explored together. The experimental design was described in brief as follow. Based on the ischemic model of cultured hippocampal neuron in vitro, which was quite simlar with the clinical global cerebral ischemia process, the multiple immunofluorescence labeling, laser confocal microscope (LCM), RT-PCR and digestion with special restriction endonuclease were applied to quantify the AMPA receptor subunits on postsynaptic membrane and explore the expressional change of subunits and edition of GluR2 mRNA in ischemia. The electrophysiological characteristics of AMPA receptor channel were detected with whole-cell patch-clamp recording. Moreover, the phosphorylation of AMPA receptor was also assessed by phosphoryl transfer pieces and Western Blot analysis. We hope to provide more data to understand the effects of AMPA receptor in cerebral ischemia.Parti:Culture of hippocampal neurons and damage from oxygen and glucose deprivation (OGD) in vitroObjective: To establish the ischemic culture system in vitro of rat hippocampal neurons and observe the morphologic changes, apoptosis and intracellular calcium overload of cultured rat hippocampal neurons subjected to ischemia and reperfusion. Methods: (DPrimary hippocampal cultures were prepared from day-18 SD rat embryos. Hippocampal neurons were dissociated by incubation in typsin and purified with arabinosylcytosin (Ara-c), which could inhibit the proliferation of neuroglia. Then, neuron was identified by immunofluorescence staining with special neuronal nuclei (NeuN) antibody.? The ischemia and reperfusion of cultured rat hippocampal neurons were simulated by oxygen-glucose deprivation (OGD) and recovery .The neurons were dividedinto control group and experimental group, the latter was subdivided into 5 subgroups according to the ischemic time (0.5h, l.Oh, 1.5, 2.0h, 2.5h and 3.0h) The same recovery (24h) of oxygen and glucose were designed. Necrosis and apoptosis of neurons prelabeled with Annexin V/PI were detected with and Flow Cytometry (FCM) ?The intracellular calcium of ischemia and reperfusion injured neurons prelabeled with Fulo-4 AM were observed with Laser Confocal Microscope (LCM). The intracellular calcium concentrations were indicated via fluorescence intensity.Results: ?After purification, the hippocampal neurons were predominant in cultured system. Neurons presented shuttle and triangle in shape with microstructures including the dendrites, dendritic spine and axon. ?At different time points(0~3h) after OGD, neurons apoptosis rate were 3.24±0.58^ 6.56±2.38> 6.73±2.42^ 6.95±1.89, 7.16±2.82^ 6.93±2.56 and 7.23±2.89, necrosis rate were 0.87±0.16^ 3.71±0.34> 16.47±1.27> 18.73±2.02, 19.77±2.32> 22.13+2.12 and 28.78±2.69, there were statistically significant differences in neurons necrosis rate (p<0.05), but not in apoptosis rate (p>0.05). When the recovery was take placed, neurons apoptosis rate were 0.98±0.2Ck 8.46±1.37^ 17.68±2.97, 20.79±3.16> 21.67±3.05, 35.53±3.33 and 50.22±4.19, necrosis rate were 3.41±0.9K 14.92±2.64> 28.67±3.05> 44.67±3.78> 61.67±4.89, 27.76±3.15 and 12.67±2.27, survival rate of hippocampal neurons further decreased with necrosis rate and apoptosis rate increased. Furthermore, necrosis rates of different time differed greatly (p<0.05), and apoptosis rate gradually increase with significant difference among those of different time points (p<0.05). At ischemic time behind 2h, the apoptosis rates after recovery decreased markedly. ?The calcium fluorescence intensity of neurons at different postischemic time(0~3h)were20.64±4.31> 36.23±4.62, 63.87±12.59> 123.28±18.35^ 216.64±21.34, 217.31±20.97 and 223.21±23.79, When the recovery was take placed, they were 20.95±3.87, 42.67±4.92, 75.25±13.43^ 176.78±20.13. 243.72±24.57, 247.95±22.34 and 258.59±23.43. At 30min postischemia, the calcium fluorescence intensity of ischemic neurons increased significantly and slowed behind 2h. At same postischemia, the calcium intensity in ischemia and reperfusion injured neurons was higher than those in singleischemic neurons with similar tendency (p<0.05).Conclusion: ?Damage from oxygen and glucose deprivation and recovery in cultured rat hippocampal neurons can stimulate veritably the brain injury caused by ischemia and reperfusion. ?Both of necrosis and apoptosis were noted in acute ischemic neuronal injury and necrosis overshadowed apoptosis. And, apoptosis was predominant pathway in selective delayed neuronal injury caused by the ischemia and reperfusion. The difference of apoptosis rate ahead and behind 2h in ischemia and reperfusion injured rat hippocampal neurons suggest that the therapeutic time window should be within 2h after cerebral ischemia. ?The intracellular calcium overloaded in ischemic neurons and enhanced following the prolongation of the ischemic time. Furthermore, the calcium overloading in ischemia and reperfusion injured neurons was more severe than those in ischemic neurons alone.Part II:Quantitive change of postsynaptic membrane AMPA receptor subunits and modulation of protein kinaseObjective: To explore the quantitive changes of ischemic postsynaptic membrane AMPA receptor subunits and the modulation of protein kinase during it.Methods: The postischemia time was simplified to 30min and 60min, the parameters were detected as follow: ?The indexes including the total surface subunits, the number of synapse with different subunits and the quanity of different subunits in synapse in different postischemia time of GluRl, GluR2 and GluR3 on post-synaptic membrane were quantified with double immuno-fluorescence labeling and LCM. ?Change of PKC activity in plasma and membrane were assessed by phosphoryl transfer pieces and expression of PKCa protein was measured by Western Blot analysis. Results: ?GluR2 numbers of control groupon. Ischemia 30min group and Ischemia 60min group on neuron surface(S/T, %) were 59.95±13.0K 36.98±7.49 and 17.89±3.33, synapse containing GluR2 / total synapse(G/Sy, %) were 64.26±12.1K 42.01±7.13 and22.87±4.65, GluR2 of synapse / GluR2 of neuron membrane (G/S, %) were 80.76±11.39^ 39.36±7.26 and 27.77±5.90. S/T of GluR3 were 34.06±8.3K 58.44±11.95 and 65.57±12.83, G/Sy were 38.34±9.68 , 50.29±10.42 and 60.47±12.37, G/S were 59.13±11.08 ^ 73.46±17.25 and 89.61±20.59. S/T of GluRl were 23.12±3.93, 41.73±8.52 and 43.57±9.14, G/Sy were 21.12±3.56> 39.26±5.31 and 40.51±9.87, G/S were 35.61 ±7.58> 62.15+11.42 and 65.36±12.73. Ischemia and reperfusion injury decreased the quantity of three indexes in GluR2 and increased those in GluR3. These changes presented severe tendency following the elongation of postischmia time. Significant differences could be found between different postischmia time groups (p<0.05) As to GluRl, quantitive increase was also observed with alleviative tendency. ?The activity of cytosolic PKC of control groupon> Ischemia 30min group and Ischemia 60min group were 6.24±0.27^ 3.26±0.21 and 3.05±0.17, the activity of membrane PKC were 2.63±0.^ 8.85±0.32 and 10.63±0.35. When the recovery was take placed for Ischemia groups, the activity of cytosolic PKC were 0.97±0.19 and 0.82±0.16, the activity of membrane PKC were 12.38±0.39 and 12.66±0.99. Ischemia and reperfusion injury significantly increased the activity of membrane PKC and decreased that of cytosolic PKC with severe tendency following the prolongation of ischmia duration. The membrane PKCa proteins were 23.18±3.23, 17.97±2.18 and 9.35±0.94, the plasma PKCa proteins were 20.25±3.46> 26.31±4.14 and 36.57±4.39, the similar results were also shown in the expression of PKCa protein. Conclusion: ?Ischemia and reperfusion injury induced the reduction of GluR2 and increase of GluR3 at postsynaptic membrane resulted in the formation of GluR2-lacking AMPA receptor, which was replaced with GluR3 principally. ?Ischemia reperfusion injury of rat hippocampal neurons results in translocational activation of PKC, especially the PKCa and the activation might damage the neurons through promoting calcium overload.Part III:Expression of AMPA receptor subunits mRNA in ischemic hippocampalneurons in vitro and molecular mechanismsObjective: To explore the expressional change of AMPA receptor subunits including the GluRl, GluR2 and GluR3 and possible molecular mechanism in brain ischemic injury. Methods: ?The expressional changes of AMPA subunits were assessed by RT-PCR. The densities of electrophoresis bands that indicate the expression of AMPA subunits in different postischemia time were scanned with image analysis system. Compared with that in control group, the changes of ratios presented the expressional change. (2)The edition of GluR2 m RNA were analyzed with digestion of Tsel.Results: (D Compared with the density in control group, the ratios of GluR2 were decrease to 0.83±0.19 (postischemic 30min) and 0.76±0.14 (postischemic 60min), the ratios of GluR3 were decreased to 0.89±0.14 (postischemic 30min) and 0.83±0.16 (postischemic 60min). Significant differences were found between those in control group and in experimental groups. Furthermore, the statistical differences also were shown between the experimental groups. The ratios of GluRl were 1.01 ±0.18 (postischemic 30min) and 0.98±0.17 (postischemic 60min) with no significant difference. ?After digestion of Tsel to GluR2 PCR products, two (440bp and 190bp) not three fragmentations were found in control group, as well as in experimental groups.Conclusion: ?Reduction of ratios suggests that ischemia down-regulate both GluR2 and GluR3 expression. GluRl expression and (2)The two fragmentations obtained from the digestion of Tsel indicates that ischemia do not effect the GluR2 m RNA edition.Part IV:Changes of the permeability of AMPA receptor channel after ischemic insult in cultured hippocampal neuron in vitro.Objective: To explore the changes of miniature excitatory postsynaptic currents (mEPSCs)and relative calcium permeability mediated by AMPA receptor in ischemic hippocampalneurons.Methods: Based on the application of varied antagonists to block unnecessary ionchannels, the whole-cell patch-clamp was utilized to measure mEPSCs. To the latter, thesolution was natrium free.Results: Application of varied antagonists separated inward current mediated by calcium.Compared with uninjured neurons, the amplitude of mEPSCs in ischemia neurons wereincreased from 18.3±2.1pA to 29.6±2.3p A ( postischemic 30min) and 34.9±2.0pA(postischemic 60min) respectively with significant difference. The enhancement tendencyfollowing the ischemia duration was observed. Statistical difference was found betweenthose in control and in experimental groups (p<0.05).Conclusion: A rapid calcium influx mediate by GluR2-lacking AMPA with high calciumpermeability occurs in ischemia neurons, resulting in the formation of a novel current.Furthermore, the amplitudes of mEPSCs were correlated nearly with the ischemic degrees.Part V:Experimental study on effects of AMPA antagonist in ischemic rat hippocampal neuronsObjective: To explore the effects of AMPA antagonists in ischemic rat hippocampal neurons in vitro.Methods: The cultured rat hippocampal neurons were randomly divided into ischemia groups and therapy groups. Both of these were subdivided into subgroups according to postischemia time. The lOumol/L CNQX, an AMPA receptor antagonist was added aforehand into the medium of therapy groups. The annexin V/PI labeling and FCM were used to detect the neurons apoptosis, Fulo-4 AM labeling and LCM were applied to measure the intracellular calcium, the whole-cell patch-clamp was utilized the exam the mEPSCs. The postischemia time in the latter were simplified to 30min and 60min. Results: ? After AMPA antagonist CNQX were added, the neurons apoptosis rates at different postischemic time(0~3h) were 2.97±0.57> 5.83±0.68, 5.62±0.55, 5.73±0.72> 6.07±1.31> 6.23±1.21 and 6.36±1.24, when the recovery was take placed, neurons apoptosis rate were 2.98±0.73, 10.52±1.83, 21.32±3.(^ 28.83±4.59, 32.29±4.27,21.04±3.52 and 7.34±1.25. CNQX decrease the apoptosis rates not only in the neurons caused by ischemia only but also in the neurons caused by ischemia and reperfusion injury. Compared with the ischemia subgroups at same postischemia time, there is no statistical difference in apoptosis rates were found in therapy subgroups neurons caused by ischemia only (p>0.05). On the contrary, the decrease of CNQX on apoptosis rates was more strongly in the neurons caused by the ischemia and reperfusion injury. Compared with the ischemia subgroups at same postischemia time, there is significant difference in apoptosis rates were shown in therapy subgroups neurons caused by ischemia and reperfusion injury (p<0.05). (2) In CNQX treatment subgroups, intracellular calcium intensity were 19.89±3.34> 21.36±3.95, 45.16±8.72, 53.24±10.14, 71.27+11.63. 71.53+11.58 and 71.93±11.37, they were decreased. At same postischemia time, there are significant difference was found between the therapy subgroups and ischemia subgroups (p<0.05).But, the calcium intensity in treatment subgroups were higher than that in uninjured neurons.? Compared with the ischemia group, the amplitude of mEPSCs in therapy groups were decrease from 29.6±2.3 and 34.9±2.0pAto 21.8±1.9 and 22.4±2.2pA respectively at postichemic 30min and 60min. . Furthermore, there are statistical difference in current amplitude between the each therapy subgroups and ischemia group (p<0.05), whereas, no significant difference in current amplitude was found between the therapy subgroups (p>0.05). The current frequency of mEPSCs in therapy groups were not changed compared with that in ischemia groups.Conclusion: Decrease of apoptosis rates suggest that AMPA receptor antagonist CNQX possess neuroprotective effect in cerebral ischemia due to blockage of rapid calcium influx through Glu2-lacking AMPA receptor illuminate by reduction of intracellular calcium.
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