title: Characterization and quantification of isoflurane-induced developmental apoptotic cell death in mouse cerebral cortex.
abstract: BACKGROUND
  Accumulating evidence indicates that isoflurane and other, similarly acting anesthetics exert neurotoxic effects in neonatal animals. However, neither the identity of dying cortical cells nor the extent of cortical cell loss has been sufficiently characterized. We conducted the present study to immunohistochemically identify the dying cells and to quantify the fraction of cells undergoing apoptotic death in neonatal mouse cortex, a substantially affected brain region.
  
  
  METHODS
  Seven-day-old littermates (n = 36) were randomly assigned to a 6-hour exposure to either 1.5% isoflurane or fasting in room air. Animals were euthanized immediately after exposure and brain sections were double-stained for activated caspase 3 and one of the following cellular markers: Neuronal Nuclei (NeuN) for neurons, glutamic acid decarboxylase (GAD)65 and GAD67 for GABAergic cells, as well as GFAP (glial fibrillary acidic protein) and S100β for astrocytes.
  
  
  RESULTS
  In 7-day-old mice, isoflurane exposure led to widespread increases in apoptotic cell death relative to controls, as measured by activated caspase 3 immunolabeling. Confocal analyses of caspase 3-labeled cells in cortical layers II and III revealed that the overwhelming majority of cells were postmitotic neurons, but some were astrocytes. We then quantified isoflurane-induced neuronal apoptosis in visual cortex, an area of substantial injury. In unanesthetized control animals, 0.08% ± 0.001% of NeuN-positive layer II/III cortical neurons were immunoreactive for caspase 3. By contrast, the rate of apoptotic NeuN-positive neurons increased at least 11-fold (lower end of the 95% confidence interval [CI]) to 2.0% ± 0.004% of neurons immediately after isoflurane exposure (P = 0.0017 isoflurane versus control). In isoflurane-treated animals, 2.9% ± 0.02% of all caspase 3-positive neurons in superficial cortex also coexpressed GAD67, indicating that inhibitory neurons may also be affected. Analysis of GABAergic neurons, however, proved unexpectedly complex. In addition to inducing apoptosis among some GAD67-immunoreactive neurons, anesthesia also coincided with a dramatic decrease in both GAD67 (0.98 vs 1.84 ng/mg protein, P < 0.00001, anesthesia versus control) and GAD65 (2.25 ± 0.74 vs 23.03 ± 8.47 ng/mg protein, P = 0.0008, anesthesia versus control) protein levels.
  
  
  CONCLUSIONS
  Prolonged exposure to isoflurane increased neuronal apoptotic cell death in 7-day-old mice, eliminating approximately 2% of cortical neurons, of which some were identified as GABAergic interneurons. Moreover, isoflurane exposure interfered with the inhibitory nervous system by downregulating the central enzymes GAD65 and GAD67. Conversely, at this age, only a minority of degenerating cells were identified as astrocytes. The clinical relevance of these findings in animals remains to be determined.
authors: Department of Anesthesia, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
authors: George K
authors: GK
authors: Istaphanous
authors: Christopher G
authors: CG
authors: Ward
authors: Xinyu
authors: X
authors: Nan
authors: Elizabeth A
authors: EA
authors: Hughes
authors: John C
authors: JC
authors: McCann
authors: John J
authors: JJ
authors: McAuliffe
authors: Steve C
authors: SC
authors: Danzer
authors: Andreas W
authors: AW
authors: Loepke
mesh_terms: D018685:Anesthetics, Inhalation / Q000633:toxicity*; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000378:metabolism; D007150:Immunohistochemistry; D007395:Interneurons / Q000187:drug effects; D007530:Isoflurane / Q000633:toxicity*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010641:Phenotype; D009418:S100 Proteins / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000502:physiology
chemical_list: D018685:Anesthetics, Inhalation; D005904:Glial Fibrillary Acidic Protein; D009418:S100 Proteins; D005680:gamma-Aminobutyric Acid; D007530:Isoflurane; D053148:Caspase 3; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2