///2009 Abstract Details
2009 Abstract Details2019-08-03T15:55:31-06:00

Isoflurane Inhibits Allopregnanolone-Induced Proliferation of Embryonic Neural Stem/ Progenitor Cells In Vitro

Abstract Number: 2
Abstract Type: Original Research

Arvind Palanisamy MBBS, MD, FRCA1 ; Deborah J Culley MD2; Lawrence C Tsen MD3; Gregory Crosby MD4

Background: Accumulating evidence suggests general anesthetics may be neurotoxic to the developing brain by inducing apoptosis. In addition, preliminary work from our lab indicates isoflurane (ISO) inhibits proliferation of neural stem/progenitor cells (NPCs) in vitro. Whether there is risk to the fetal brain associated with maternal anesthesia for non-obstetric or fetal surgery is unclear, however, partly because pregnancy is characterized by marked elevation of progesterone and its neuroactive metabolite, allopregnanolone (AP). These hormones are protective in several models of neural injury and enhance proliferation of NPCs, important since neurogenesis is a prominent feature of fetal neurodevelopment. Accordingly, we designed an in vitro study to test whether physiologically relevant concentrations of AP prevent the anti-proliferative effect of ISO on NPCs.

Methods: Rat NPCs obtained on embryonic day 14 were maintained in an undifferentiated state. Cells were subsequently plated (1,000,000 cells/ 96 well plate, n = 2 plates) and incubated for 24 h in a range of AP doses (0, 10, 50, 100, or 500 nM in 0.1% DMSO) before placing them in identical anesthesia chambers containing 21% oxygen and 5% CO2 with/without 1.4% ISO (ISO; control) for 6 h. Cell division was assessed using EdU (5-ethynl-2-deoxyuridine; 10 μM), a thymidine analogue that incorporates into the DNA of dividing cells, by adding it to the media prior to anesthesia. After treatment, cells were fixed and processed for EdU immunofluorescence and counterstained with nestin, a marker of NPCs, and Hoechst, a nuclear dye. Six images per well per fluorophore were acquired in an unbiased fashion from ten wells per group using an IN Cell Analyzer 1000 and Workstation 3.5 analysis software (GE Healthcare). The percentages of EdU+ cells per treatment were analyzed by Students t-test with Bonferronis correction for multiple comparisons.

Results: As expected, ISO decreased proliferation of NPCs compared to control (p < 0.0001) and AP, at 10 and 100 nM (p< 0.05, p< 0.01), stimulated it (Fig 1). Contrary to our hypothesis, AP did not prevent the anti-neurogenic effect of ISO.

Conclusions: These data indicate that isoflurane inhibits proliferation of NPCs in vitro and prevents the pro-neurogenic action of AP. Since neurogenesis is prominent during fetal development, our results suggest the fetal brain may be at risk for isoflurane-induced neurotoxicity despite elevated levels of maternal hormones.

SOAP 2009