The volatile anaesthetic agents isoflurane and sevoflurane have been shown to induce a preconditioned state in cardiac tissue protecting against ischaemia (Novalija et al., 1999). Previous workers have demonstrated that prior exposure to isoflurane for 3h preconditions rat cortical neurones in vitro, assayed by release of lactate dehydrogenase, a marker of cell damage (Kapinya et al. 2002). In this present study, we investigated whether the newer volatile anaesthetic agent sevoflurane also preconditioned neural cells in vitro, and whether this preconditioning could be illustrated with a marker of cell viability rather than cell damage.

Rat primary cortical neurones were prepared by trituration of embryonic male Sprague Dawley rat brain cortices in Hanks balanced salt solution, and seeded at a density of 1000 cells/mm² in Neurobasal medium/ B27 supplement, containing 2mM of L-glutamine on poly–D–lysine-coated 24-well tissue culture plates. L-glutamate (25 μM) was included in the media for P1-3 in culture, and cells were fed on P3 and P7. On P8, plates were placed in a chamber containing volatile anaesthetic agent (isoflurane 1.4% or sevoflurane 2.1%), CO₂ 5%/O₂ 20%/N₂ 80%, and incubated at 37 °C for 3h. Plates were then returned to a 5% CO₂ in an air incubator at 37°C. 24h post-preconditioning, cells were washed and rinsed in a physiological saline solution devoid of glucose, then placed in 200 μl of this solution and maintained in an environment of 95% N₂ /5% CO₂ for 2h at 37°C. Control cells were maintained in identical physiological saline solution to which glucose (20 mM) had been supplemented in standard incubator conditions. After OGD or control conditions, 250 μl of the original media and 250 μl of fresh media was added to each well. 24h after oxygen-glucose deprivation, cell viability was assessed using a standard MTT assay (Denizot & Lang, 1986). Data were analysed using an unpaired Student’s test with a level of significance set at p<0.001 (n = 22 separate wells).

Both isoflurane (mean absorbance 0.200, 95% CI 0.1873-0.2131) and sevoflurane (mean absorbance 0.171, 95% CI 0.1618-0.1812) elicited highly significant preconditioning compared to control (mean absorbance 0.156, 95% CI 0.1457-0.1655) cells when subjected to oxygen-glucose deprivation. Therefore, we show, for the first time, that prior exposure to the volatile anaesthetic agents, isoflurane and sevoflurane increases the viability of rat primary brain cortical neurones grown in culture subjected to an oxygen-glucose deprivation insult.

Novalija E et al. (1999) Anesthesiology 91, 701-12.
Kapinya K et al. (2002) Stroke 33, 1889-98.
Denizot F. & Lang R. (1986) J. Immunol Methods 89, 271-7.

This work was supported by The Royal College of Anaesthetists, BUPA, an NRF scholarship (South Africa), Durham School of Health, the ORS, and BBSRC.