title: GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons' exposure to isoflurane.
abstract: BACKGROUND
  Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. γ-Aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, depolarizing the membrane potential and inducing a cytosolic Ca2+ increase ([Ca2+]i), because of a reversed transmembrane chloride gradient. Recent experimental data from several rodent studies have demonstrated that exposure to isoflurane during an initial phase causes neuronal excitotoxicity and apoptosis. GABAA receptor-mediated synaptic voltage-dependent calcium channels' (VDCCs) overactivation and Ca2+ influx are involved in these neural changes.
  
  
  METHODS
  We monitored [Ca2+]i using Fluo-4 AM fluorescence imaging. Using whole-cell patch clamp techniques, IVDCC (voltage-dependent calcium channel currents) were recorded from primary cultures of rat hippocampal neurons (5-day culture) exposed to isoflurane. To further investigate the neurotoxicity of high cytosolic-free calcium after isoflurane in a dose- and time-dependent manner, the possibility of increased caspase-3 levels was evaluated by Western blot and quantitative real-time polymerase chain reaction. Statistical significance was assessed using the Student t test or 1-way analysis of variance followed by the Tukey post hoc test.
  
  
  RESULTS
  Under control conditions, isoflurane enhanced the GABA-induced [Ca2+]i increase in a dose-dependent manner. Dantrolene and nicardipine markedly inhibited this enhancement mediated by isoflurane. Moreover, in Ca2+-free media, pretreatment with isoflurane did not show any influence on the caffeine-induced increase of [Ca2+]i. Similarly, using whole-cell recording, isoflurane increased the peak amplitude of IVDCC in the cultured neurons from rat hippocampus by depolarization pulses. Isoflurane (0.25, 0.5, 0.75, and 1 minimum alveolar concentration [MAC]) potentiated IVDCC peak current amplitude by 109.11%±9.03%, 120.56%±11.46%, 141.33%±13.87%, and 146.78%±15.87%, respectively. To analyze variation in protein levels, the effect of treatments with isoflurane on caspase-3 activity was dose- and time-dependent, reaching a maximal caspase-3 activity after exposure to 1 MAC for 6 hours (P<0.001). However, in the mRNA levels, hippocampal caspase-3 mRNA levels began to be significantly increased in isoflurane-treated developing rat hippocampal neurons after 6 hours of exposure to 0.25 MAC isoflurane (P<0.001).
  
  
  CONCLUSIONS
  Isoflurane-mediated enhancement of GABA-triggered [Ca2+]i release results from membrane depolarization with subsequent activation of VDCCs and further Ca2+-induced Ca2+ release from the ryanodine-sensitizing Ca2+ store. An increase in [Ca2+]i, caused by activation of the GABAA receptor and opening of VDCCs, is necessary for isoflurane-induced calcium overload of immature rat hippocampal neurons, which may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.
authors.affiliation: Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 JieFang Rd., Wuhan, Hubei, 430030, China.
authors.forename: Y L
authors.initials: YL
authors.lastname: Zhao
authors.forename: Q
authors.initials: Q
authors.lastname: Xiang
authors.forename: Q Y
authors.initials: QY
authors.lastname: Shi
authors.forename: S Y
authors.initials: SY
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Tan
authors.forename: J T
authors.initials: JT
authors.lastname: Wang
authors.forename: X G
authors.initials: XG
authors.lastname: Jin
authors.forename: A L
authors.initials: AL
authors.lastname: Luo
mesh_terms: D018685:Anesthetics, Inhalation / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D053148:Caspase 3 / Q000096:biosynthesis; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects*; D007530:Isoflurane / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D017966:Pyramidal Cells / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000633:toxicity*
chemical_list: D018685:Anesthetics, Inhalation; D015220:Calcium Channels; D018690:Excitatory Amino Acid Agonists; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; D005680:gamma-Aminobutyric Acid; D007530:Isoflurane; D053148:Caspase 3; D002118:Calcium