Cerebellar slices were transferred into the pregassed aCSF and the incubation was for 15 min at 37C. results suggest that the BKI-1369 preconditioning-induced neuroprotection by volatile anesthetics is not agent-specific. Mechanisms that are involved in inducing anesthesia may contribute to the induction of preconditioning effect by volatile anesthetics. Modification of glutamate transporter activity may be one of such mechanisms to induce these protective effects. by oxygen-glucose deprivation (OGD) to determine the acute preconditioning effects of volatile reagents. 2. Results The 2 2,3,5-triphenyltetrazolium chloride (TTC) staining method has been a useful technique to quantify infarct volume after focal brain ischemia. Staining the brain tissue with TTC and then measuring the extracted formazan by spectrophotometry has been used to quantify brain injury in rodents after brain ischemia in many publications (Preston and Webster, 2000, Jiang et al., 2005, Weaver et al., 2005). This method could be a relatively simple and objective approach to assess brain injury that is diffused in nature. We applied this method to quantify cell injury in the cerebellar slices after OGD. As shown in physique 1, the longer the OGD was, the greater the cell injury. The formazan production in brain slices exposed to a 10-min OGD was 59 19% of control (P 0.001). We used this duration of OGD for subsequent experiments. Open in a separate window Physique 1 Time-course of oxygen-glucose deprivation (OGD)-induced cell injuryCerebellar slices from adult male rats were subjected to various lengths of OGD. Cell injury BKI-1369 was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) conversion 5 hrs after OGD. Results are means S.D. (n = 6 C 8 per time point). * P 0.001 compared with control. Preconditioning by the four volatile anesthetics induced a concentration-dependent protection against OGD (Fig. 2). Notably, the injury of cerebellar slices exposed to 74 M halothane and then OGD was more severe than that of slices subjected to OGD only (Fig. 2A). The results of isoflurane treated slices had bigger standard BKI-1369 deviations than those of slices treated with other volatile anesthetics (Fig. 2). The reasons for these two phenomena are not known. We performed the experiments in the same way. Low concentrations of halothane have not been shown to be detrimental to the brain after ischemic injury. Future studies are needed to confirm this phenomenon. Open in a separate window Open in a separate window Open in a separate window Open in a separate window Physique 2 Volatile anesthetic preconditioning-induced neuroprotectionCerebellar slices were preconditioned with various concentrations of volatile anesthetics for 15 min before a 10-min oxygen-glucose deprivation (OGD). Cell injury was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) conversion 5 hrs after OGD. Results are means S.D. (n = 5 C 8 per concentration). * P 0.05 compared with OGD without anesthetic pretreatment. The EC50 for the preconditioning-induced neuroprotection by isoflurane, halothane, sevoflurane and desflurane was 221, 173, 184 and 929 M, respectively. Strikingly, this EC50 was linearly related with the aqueous concentration of one minimum alveolar concentrations (MAC) (Fig. 3, r = 0.988 for the positive correlation, P 0.01). However, caution needs to be exercised for this interpretation because 3 out of the 4 data points are close to each other and relationships other than linear correlation between the EC50 and the MAC cannot be reasonably excluded unless more data points are added between the isoflurane and desflurane data points (Fig. 3). However, volatile anesthetics whose aqueous concentrations of MAC are between isoflurane and desflurane are not available yet (Campagna et al., 2003, Sonner et al., 2003). Interestingly, pretreatment of Rabbit polyclonal to AKAP5 the brain slices with the nonimmobilizer F6 did not significantly reduce the injury caused by OGD (Fig. 4). We also failed to find a best-fit curve.