| Summary: | 博士 === 長庚大學 === 臨床醫學研究所 === 93 === Ischemic stroke is a serious neurological disease. It has been the second leading cause of death in Taiwan after 1983 and the leading killer for those aged over 65. The patients with stroke often have variable functional disability, resulting in many socioeconomic problems. Thus, how to minimize the injury induced by cerebral ischemia and promote functional recovery after stroke has been a great challenge for clinical practice. In the past years, the therapy for stroke is restricted to secondary prevention. Recent advances in searching neuroprotective agents provide a new target to ameliorate the sequelae caused by ischemic injury. Therefore, to find a more effective drug, it is important to investigate and understand the cellular and molecular mechanisms of cerebral ischemia.
The cerebral ischemia caused by occluding the blood vessel results in neuronal death. Substantial studies indicated that excitotoxicity induced by glutamate is important for neuronal injury caused by cerebral ischemia. L-glutamate is the major excitatory neurotransmitter in the mammalian brain and plays essential roles in neural plasticity, neural development and neurodegeneration. Glutamate activates ionotropic glutamate receptors (iGluRs) to open cationic channels. While larger amount of glutamate released, postsynaptic metabotropic glutamate receptors (mGluRs) including mGluR1 and mGluR5, which are localized at the perisynaptic junction in brain, are activated and subsequently induce G-protein linked secondary messenger cascades. The released glutamate is reuptaked by astroglial glutamate transporters GLT-1 or GLAST. Overactivation of ionotropic and metabotropic glutamate receptors causes enhancement of cell excitability as well as substantial increase of intracellular Ca2+ and finally results in delayed neuronal death. Accordingly, excessive extracellular glutamate caused by dysfunction of astroglial glutamate transporters could deteriorate the ischemic neuronal injury. Thus, the aims of this project are to investigate functional change of glutamate transporters in astrocytes and of group I metabotropic glutamate receptor in CA1 pyramidal neurons after global ischemia.
Astroglial glutamate transporters, GLT-1 and GLAST, play a crucial role in removing released glutamate from the extracellular space and are essential for maintaining a low concentration of extracellular glutamate in the brain. It was hypothesized that impaired function of glial glutamate transporters induced by cerebral ischemia may lead to an elevated level of extracellular glutamate and subsequent excitotoxic neuronal death. Since glutamate transporters mediate transport of glutamate accompanied by the cotransport of 3 Na+ and 1 H+, and the countertransport of 1 K+, the function of astroglial glutamate transporter can be investigated by recording the transporter current. In the present study we performed whole-cell patch-clamp recording of hippocampal CA1 astrocytes in control or postischemic slices, and measured glutamate transporter activity by recording glutamate-evoked transporter currents. Six to twenty-four hours after global ischemia, maximal amplitude of glutamate transporter currents recorded from postischemic CA1 astrocytes was significantly reduced. Western blotting analysis indicated that transient global ischemia decreased the protein level of GLT-1 in the hippocampal CA1 area without affecting GLAST protein level. Further TaqMan real-time quantitative RT-PCR assays showed that global ischemia resulted in a decrease in GLT-1 mRNA level of hippocampal CA1 region. Global ischemia-induced reduction in GLT-1 expression and glutamate transporter function of CA1 astrocytes precedes the initiation of delayed neuronal death in CA1 pyramidal layer of the rat hippocampus. The present study provides the evidence that transient global ischemia downregulates glutamate transporter function of hippocampal CA1 astrocytes by decreasing mRNA and protein levels of GLT-1, which could lead to delayed neuronal death.
The activation of postsynaptic mGluR1 or mGluR5 increases the neuronal excitability, elevates intracellular Ca2+ concentration, and potentiates NMDA receptor-mediated response. It was hypothesized that overactivation of postsynaptic mGluRs results in excitotoxic neuronal death following the transient global ischemia. Within the hippocampus, electrophysiological and immunohistochemical studies showed that mGluR5 is the major postsynaptic mGluR expressed in CA1 pyramidal neurons. To better understand the role of mGluR5 in ischemia-induced neuronal death, we investigated the functional change of mGluR5 in CA1 pyramidal neurons of control and postischemic hippocampal slices using whole-cell patch-clamp recordings. Our results indicated that 6 to 24 hours after transient global ischemia, mGluR5-induced cationic currents and mGluR5-mediated enhancement of NMDA-evoked currents in CA1 pyramidal neurons were significantly reduced. Further TaqMan real-time quantitative RT-PCR assay showed that mGluR5 mRNA expression in hippocampal CA1 region or single CA1 pyramidal neurons was markedly downregulated following ischemic insults. Global ischemia-induced reduction in mGluR5 mRNA levels and function precedes the initiation of delayed neuronal death in CA1 pyramidal layer. The present study suggests that transient global ischemia downregulates mGluR5 function of CA1 pyramidal neurons by decreasing mGluR5 mRNA and that the resulting reduced mGluR5-mediated excitotoxicity could contribute to the survival of CA1 pyramidal neurons after ischemic insult.
In conclusion, the present results demonstrate that global ischemia downregulates functions of GLT-1 glutamate transporter in CA1 astrocytes and of postsynaptic mGluR5 in CA1 pyramidal neurons. These findings indicate that astroglial GLT-1 glutamate transporter and mGluR5 in CA1 pyramidal neurons play important roles in delayed neuronal death following ischemic insults. Our results will provide a novel target for developing new neuroprotective agents for stroke therapy in the future.
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