Fig. 2. Tissue plasminogen activator (tPA) decreases isoflurane-induced cell death in primary neurons 5 days in vitro (DIV5). Primary neurons were exposed to 1.4% isoflurane for 4 h, and the media were frozen 2 h after exposure at −80°C before tPA ELISA. ( A ) The media from neurons exposed to isoflurane (1.4%, 4 h; 2 h post) contained significantly (n = 6, * P =0.001) less tPA compared with media from nonexposed cells (approximately 45% decrease; 1.40 ng/ml BASAL vs . 0.77 ng/ml isoflurane). ( B ) Neurons were pretreated with increasing doses of tPA (0.03 to 3 μg/ml) before isoflurane (1.4%, 4 h; 2 h post), and neuronal cell death was assessed 2 h after exposure with the apoptotic marker, cleaved caspase 3 (Cl-Csp3). ( Ba and Bb ) Increasing doses of tPA decreased isoflurane-induced Cl-Csp3 with a maximum decrease at 3 μg/ml. ( C ) tPA (3 μg/ml) (n = 3, # P = 0.02), but not matrix metalloproteinases (MMP)-7 (2 μg/ml) or MMP-9 (1 μg/ml), significantly decreased isoflurane-induced Cl-Csp3. MMP-9 significantly elevated Cl-Csp3 under basal conditions ( P = 0.01). * Isoflurane alone versus control alone (n = 3, P = 0.01). λ Isoflurane versus control (n = 3, P = 0.01). μ Isoflurane versus control (n = 3, P = 0.01). Δ MMP-9 basal versus basal alone (n = 3, P = 0.02). ( D ) tPA significantly (n = 3, ϕ P = 0.002) enhanced the prosurvival kinase phosphorylated Akt (P-Akt) even in the presence of isoflurane. ( E ) Application of the serine protease inhibitor, aprotinin (Apo) (3 μg/ml), blocked the protective effect of 3 μg/ml plasmin (P) ( upper blots ) and tPA ( lower blots ) (n = 3, # P = 0.004). Error bars = SEM. T-Csp3 = total caspase 3.