Isoflurane neuroprotection in hypoxic hippocampal slice cultures involves increases in intracellular Ca2+ and mitogen-activated protein kinases.
BACKGROUND
The volatile anesthetic isoflurane reduces acute and delayed neuron death in vitro models of brain ischemia, an action that the authors hypothesize is related to moderate increases in intracellular calcium concentration ([Ca2+]i). Specifically, the authors propose that during hypoxia, moderate increases in [Ca2+]i in the presence of isoflurane stimulates the Ca2+-dependent phosphorylation of members of the mitogen-activated protein kinase (MAP) kinase Ras-Raf-MEK-ERK pathway that are critical for neuroprotective signaling and suppression of apoptosis.


METHODS
Death of CA1, CA3, and dentate neurons in rat hippocampal slice cultures was assessed by propidium iodide fluorescence 48-72 h after 60-75 min of hypoxia. [Ca2+]i in CA1 neurons was measured with fura-2 and fura-2 FF. Concentrations of the survival-signaling proteins Ras, MEK, MAP kinase p42/44, and protein kinase B (Akt) were assessed by immunostaining, and specific inhibitors were used to ascertain the role of Ca2+ and MAP kinases in mediating survival.


RESULTS
Isoflurane, 1%, decreased neuron death in CA1, CA3, and dentate gyrus neurons after 60 but not 75 min of hypoxia. Survival of CA1 neurons required an inositol triphosphate receptor-dependent increase in [Ca2+]i of 30-100 nm that activated the Ras-Raf-MEK-ERK (p44/42) signaling pathway. Isoflurane also increased the phosphorylation of Akt during hypoxia.


CONCLUSIONS
Isoflurane stimulates the phosphorylation of survival signaling proteins in hypoxic neurons. The mechanism involves a moderate increase in [Ca2+]i from release of Ca from inositol triphosphate receptor-dependent intracellular stores. The increase in [Ca2+]i sets in motion signaling via Ras and the MAP kinase p42/44 pathway and the antiapoptotic factor Akt. Isoflurane neuroprotection thus involves intracellular signaling well known to suppress both excitotoxic and apoptotic/delayed cell death.
Gray
Jonathan J
JJ
Department of Anesthesia and Perioperative Care, University of California at San Francisco, 94143-0542, USA.
Bickler
Philip E
PE
Fahlman
Christian S
CS
Zhan
Xinhua
X
Schuyler
Jennifer A
JA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D006624:Hippocampus / Q000187:drug effects* / Q000473:pathology; D007530:Isoflurane / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
D018696:Neuroprotective Agents; D011518:Proto-Oncogene Proteins; D007530:Isoflurane; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium