mixed cortical cell cultures containing both glia and neurons , were prepared from icr mouse brains at 15 - 16 days of gestation . briefly , dissociated neocortical cells ( 3.510 cells / well ) were plated onto primaria - coated 24-well plates ( falcon ) containing a glial bed in plating medium consisting of eagle 's minimal essential medium ( mem ; earle 's salts , supplied glutamine - free ) supplemented with 20 mm glucose , 2 mm l - glutamine , 5% fetal bovine serum , and 5% horse serum . cytosine arabinoside ( 10 m ) was added 5 days after plating to halt the growth of non - neuronal cells . cultures were maintained at 37 in a humidified co2 incubator and used for experiments between days 12 and 14 in vitro . glial cultures were prepared from postnatal mice ( 1 - 3 days ) and plated at 110 cells / well in plating medium supplemented with 10% horse serum , 10% fetal bovine serum , and 10 ng / ml epidermal growth factor . after 2 weeks in vitro , cytosine arabinoside was added to the cultures , which were fed weekly with the same medium with 10% horse serum used for mixed cultures . we pre - incubated cultures with esculetin for 2 h before inducing neurotoxicity . for measuring ca uptake , cultured cells were washed with hepes - buffered control salt solution ( hcss ) , and then incubated with nmda ( 150 m ) in the presence of mk-801 ( an nmda receptor inhibitor , 10 m ) or esculetin ( 10 , 100 m ) in hcss containing cacl2 ( final activity : 1.0 ci / ml ) . after 5 min , exposure solution was washed away using four boluses of hcss and cells were lysed by addition of 0.2% sodium dodecyl sulfate solution . aliquots of lysed cells were added to scintillation counting solution for the counting of beta emissions . for protein preparation , cells from three culture plates were pooled in 0.5 ml of 0.1 m phosphate buffer ( ph 7.4 ) and homogenized . the homogenate was centrifuged for 30 min at 3,000 g at 4 and the supernatant , consisting of the cytosolic+mitochondrial fractions , was used in enzyme assays . glutathione peroxidase ( gpx ) activity was measured using an nadph reduction assay following the technique of lawrence and burke . soluble cell proteins were added to a reaction mixture containing reduced glutathione , glutathione reductase , and nadph in phosphate buffer . the reaction was initiated by adding h2o2 , the absorbance decrease at 340 nm was recorded , and the activity in the absence of cellular protein was subtracted . one unit of gpx is defined as moles nadph oxidized / min , and results are expressed in moles / min / mg protein . glutathione reductase ( gr ) activity was measured using an nadph reduction assay following the technique of eklw et al . gr activity was monitored by decreases in nadph absorbance at 340 nm at 37. results were based on a molar extinction coefficient for nadph of 6.2210 m cm . one unit of gr is defined as moles nadph oxidized / min , and results are expressed in moles / min / mg protein . protein content was measured by the method of lowry et al   with bovine serum albumin as a standard . neuronal damage was assessed by measuring the amount of ldh released into the medium by damaged cells 24 h after nmda or bso treatment . data are expressed as the meanstandard error of the mean ( sem ) and analyzed for statistical significance by one - way anova using a post - hoc student - neuman - keuls test for multiple comparisons . cultured cortical cells exposed to 150 m nmda or 50 m kainic acid ( ka ) generated moderate ( 50 - 70% ) neuronal damage as indicated by increased ldh release into the bathing medium after 24 h. pre - exposure of esculetin ( 10 , 100 m ) for 2 h attenuated nmda neurotoxicity by 16% and 29% , respectively , but did not show any inhibitory effect against ka neurotoxicity ( table 1 ) . at the highest concentration , esculetin esculetin dose - dependently and significantly inhibited the neurotoxicity induced by h2o2 or superoxide anions ( o2 ) produced by xanthine / xanthine oxidase co - treatment ( table 2 ) . morphologically , nmda - induced neurotoxicity manifested as loss of cell bodies ( figure 1a ) ; esculetin rescued many neurons from nmda neurotoxicity ( figure 1b ) . to determine whether esculetin modulates nmda receptors , we tested the effects of nmda alone or nmda+esculetin . mk-801 , a non - competitive nmda receptor antagonist , blocked ca uptake as well as ldh release . at 100 m , esculetin significantly ameliorated the nmda - induced rise in ca uptake ( figure 2 ) . to investigate the interrelationship between lipoxygenase ( lox ) inhibition and glutathione metabolism , we measured esculetin modifiable gsh depletion - induced neurotoxicity . after 2-h pretreatment with esculetin or vehicle , we exposed the cultured cells to 1 mm bso for 24 h. esculetin , a lox inhibitor ( 1 , 10 or 30 m ) significantly attenuated bso - induced neuronal injury by 46 , 62 , and 73% , respectively ( figure 3a ) . we morphologically confirmed the protective effect of 30 m esculetin ( figures 3b-3d ) . at 150 m arachidonic acid , cycloheximide , an inhibitor of de novo protein synthesis , and vitamin e , an antioxidant , reduced neuronal damage by 25% ( figure 4 ) . in contrast , esculetin ( 100 m ) significantly inhibited the neuronal injury by 90% ( figure 4 ) . gpx activity was decreased by bso treatment , but bso did not change gr activity at 24 h compared to the normal group ( figures 5a and 5b ) . here , esculetin at 3 - 30 m concentration - dependently elevated gr activity compared to bso alone ( figure 5b ) but caused little change in gpx activity ( figure 5a ) . these findings suggest that esculetin inhibits oxidized glutathione ( gssg ) accumulation by increasing gr activity , thus sparing the pool of reduced glutathione ( gsh ) . ischemic injury , especially nmda neurotoxicity , is known to activate calcium - dependent lipoxygenase pathways . so far , almost all nmda antagonists have failed to protect neuronal cells from cerebral ischemia clinically due to their psychiatric side effects . in this study , , we confirmed that the protective effect of esculetin against nmda toxicity can be partly attributed to its ability to modulate nmda receptors directly or indirectly , as demonstrated by the results for calcium uptake . when nmda receptor overactivation is initiated by excitotoxic insults , release of arachidonic acid and increases in lipoxygenase activity can occur . as in other reports , the antioxidant vitamin e and the de novo protein synthesis inhibitor cycloheximide exhibited only weak protective effects . this means that esculetin may contribute to the diminution of neuronal injury that occurs in ischemic insults by attenuating the nmda receptor - mediated arachidonic acid cascade . interestingly , esculetin also had a sparing effect on the pool of gsh by halting gsh turnover to gssg . furthermore , esculetin may contribute to the rescue of neuronal cells from nmda neurotoxicity by scavenging h2o2 or o2 we conclude that esculetin is a potential neuroprotectant in cerebral ischemia that appears to works by modulating nmda receptors and the metabolism of arachidonic acid or glutathione .