title: Isoflurane neurotoxicity is mediated by p75NTR-RhoA activation and actin depolymerization.
abstract: BACKGROUND
  The mechanisms by which isoflurane injured the developing brain are not clear. Recent work has demonstrated that it is mediated in part by activation of p75 neurotrophin receptor. This receptor activates RhoA, a small guanosine triphosphatase that can depolymerize actin. It is therefore conceivable that inhibition of RhoA or prevention of cytoskeletal depolymerization might attenuate isoflurane neurotoxicity. This study was conducted to test these hypotheses using primary cultured neurons and hippocampal slice cultures from neonatal mouse pups.
  
  
  METHODS
  Primary neuron cultures (days in vitro, 4-7) and hippocampal slice cultures from postnatal day 4-7 mice were exposed to 1.4% isoflurane (4 h). Neurons were pretreated with TAT-Pep5, an intracellular inhibitor of p75 neurotrophin receptor, the cytoskeletal stabilizer jasplakinolide, or their corresponding vehicles. Hippocampal slice cultures were pretreated with TAT-Pep5 before isoflurane exposure. RhoA activation was evaluated by immunoblot. Cytoskeletal depolymerization and apoptosis were evaluated with immunofluorescence microscopy using drebrin and cleaved caspase-3 staining, respectively.
  
  
  RESULTS
  RhoA activation was increased after 30 and 120 min of isoflurane exposure in neurons; TAT-Pep5 (10 μm) decreased isoflurane-mediated RhoA activation at both time intervals. Isoflurane decreased drebrin immunofluorescence and enhanced cleaved caspase-3 in neurons, effects that were attenuated by pretreatment with either jasplakinolide (1 μm) or TAT-Pep5. TAT-Pep5 attenuated the isoflurane-mediated decrease in phalloidin immunofluorescence. TAT-Pep5 significantly attenuated isoflurane-mediated loss of drebrin immunofluorescence in hippocampal slices.
  
  
  CONCLUSIONS
  Isoflurane results in RhoA activation, cytoskeletal depolymerization, and apoptosis. Inhibition of RhoA activation or prevention of downstream actin depolymerization significantly attenuated isoflurane-mediated neurotoxicity in developing neurons.
authors.affiliation: Department of Anesthesiology and Veterans Affairs San Diego Healthcare System, University of California, San Diego, La Jolla, California 92161-5085, USA. ppatel@ucsd.edu
authors.forename: Brian P
authors.initials: BP
authors.lastname: Lemkuil
authors.forename: Brian P
authors.initials: BP
authors.lastname: Head
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Pearn
authors.forename: Hemal H
authors.initials: HH
authors.lastname: Patel
authors.forename: John C
authors.initials: JC
authors.lastname: Drummond
authors.forename: Piyush M
authors.initials: PM
authors.lastname: Patel
mesh_terms: D000199:Actins / Q000187:drug effects / Q000378:metabolism*; D018685:Anesthetics, Inhalation / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D007530:Isoflurane / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D020258:Neurotoxicity Syndromes / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
chemical_list: D000199:Actins; D018685:Anesthetics, Inhalation; D017475:Receptors, Nerve Growth Factor; C517634:Ngfr protein, mouse; D007530:Isoflurane; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein