Desflurane/hypoxia induces caspase-3 activation, affects APP processing, and increases Aβ levels in H4-APP cells. A, treatment with 12% desflurane/hypoxia (18%) for 6 h (lanes 4–6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in the amounts of β-actin in control conditions or desflurane/hypoxia-treated H4-APP cells. B, quantification of the Western blot shows that desflurane/hypoxia treatment (black bar) (**, p = 0.002) increases caspase-3 activation compared with control conditions (white bar), normalized to β-actin levels. C, desflurane/hypoxia (lanes 4 and 5) reduces levels of APP-CTFs, but not FL-APP, as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or desflurane/hypoxia-treated H4-APP cells. D, quantification of the Western blot shows that desflurane/hypoxia (black bar) does not alter levels of FL-APP as compared with control conditions (white bar)(p = 0.789, NS), normalized to β-actin levels. E, quantification of the Western blot shows that desflurane/hypoxia (black bar) decreases levels of APP-CTFs as compared with control conditions (white bar) (*, p = 0.019), normalized to β-actin levels. F, desflurane/hypoxia (black bar) increases generation of both Aβ40 (**, p = 0.002) and Aβ42 (*, p = 0.018) as compared with control conditions (white bar).

Desflurane/hypoxia treatment induces caspase-3 activation independent of APP processing in H4 naïve cells. A, treatment with desflurane (12%) plus hypoxia (18%) for 6 h (lanes 3 and 4) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) in H4 naïve cells. The same treatment (lanes 7 and 8) also induces caspase-3 activation in H4-APP cells as compared with control conditions (lanes 5 and 6). B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (*, p = 0.025), normalized to β-actin levels, in H4 naïve cells. C, quantification of the Western blot shows that desflurane/hypoxia (black bar) (**, p = 0.007) increases caspase-3 activation as compared with control conditions (white bar), normalized to β-actin levels, in H4-APP cells. D, desflurane/hypoxia (lane 2) does not alter levels of APP-CTFs as compared with control conditions (lane 1) in H4 naïve cells. Desflurane/hypoxia (lane 4) reduces levels of APP-CTFs as compared with control conditions (lane 3) in H4-APP cells. E, quantification of the Western blot shows that desflurane/hypoxia (black bar) does not alter ratio of APP-CTFs to FL-APP as compared with control conditions (white bar) in H4 naïve cells (p = 0.35, NS). F, quantification of the Western blot shows that desflurane/hypoxia (black bar) decreases ratio of APP-CTFs to FL-APP in H4-APP cells (*, p = 0.03).

γ-Secretase inhibitor L-685,458 and Aβ aggregation inhibitor CQ reduce the desflurane/hypoxia-induced caspase-3 activation, but Aβ potentiates the desflurane/hypoxia-induced caspase-3 activation in H4-APP cells. A, treatment with desflurane (12%) plus hypoxia (18%) for 6 h (lanes 5 and 6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) or L-685,458 (0.5 μm) treatment (lanes 3 and 4). L-685,458 (0.5 μm) treatment (lane 7 and 8) attenuates caspase-3 cleavage induced by desflurane/hypoxia. There is no significant difference in amounts of β-actin in H4-APP cells with above treatments. B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (**, p = 0.0001), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is reduced by L-685,458 (0.5 μm) (net bar) (#, p = 0.018). C, L-685,458 (0.5 μm) reduces the desflurane/hypoxia-induced increases in secreted Aβ40 (**, p = 0.00003) and Aβ42 (**, p = 0.0009) levels in H4-APP cells. D, desflurane/hypoxia (lanes 5 and 6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) or CQ (1 μm) treatment (lanes 3 and 4). CQ (1 μm)(lanes 7 and 8) treatment attenuates caspase-3 cleavage induced by desflurane/hypoxia. There is no significant difference in amounts of β-actin in H4-APP cells with above treatments. E, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (*, p = 0.035), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is attenuated by treatment of CQ (1 μm) (net bar) (#, p = 0.046). F, desflurane/hypoxia (lane 3) induces caspase-3 cleavage (activation) as compared with control conditions (lane 1). Aβ40 (7.5 μm) plus Aβ42 (7.5 μm) treatment potentiates caspase-3 activation induced by desflurane/hypoxia (lane 4). There is no significant difference in amounts of β-actin in H4-APP cells with the above treatments. G, quantification of the Western blot shows that desflurane/hypoxia (black bar; *, p = 0.024) increases caspase-3 activation as compared with control conditions (white bar), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is potentiated by treatment of Aβ (net bar) (##, p = 0.0012). DMSO, dimethyl sulfoxide.

Hypothetical pathway by which desflurane/hypoxia induces a vicious cycle of apoptosis and Aβ generation/aggregation. Desflurane/hypoxia induces caspase-3 activation/apoptosis. Caspase activation, in turn, increases BACE levels, which serves to increase Aβ generation. Desflurane/hypoxia also enhances Aβ aggregation, which further induces caspase-3 activation and apoptosis. Elevated Aβ generation and Aβ aggregation then further induces apoptosis leading to a vicious cycle of desflurane/hypoxia-induced apoptosis and Aβ generation/aggregation.

Desflurane does not affect caspase-3 activation, APP processing, nor Aβ levels in H4-APP cells. A, treatment with 12% desflurane for 6 h (lanes 4–6) does not induce caspase-3 cleavage (activation) as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in the amounts of β-actin in control conditions or desflurane-treated H4-APP cells. B, caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots. Quantification of the Western blot shows that desflurane treatment (black bar) does not increase caspase-3 activation compared with control conditions (white bar)(p = 0.14, NS), normalized to β-actin levels. C, desflurane (lanes 3 and 4) does not affect APP processing as compared with control conditions (lanes 1 and 2) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or desflurane-treated H4-APP cells. D, APP processing assessed by quantifying levels of FL-APP in the Western blots. Quantification of the Western blot shows that desflurane treatment (black bar) does not alter levels of FL-APP as compared with control conditions (white bar) (p = 0.11, NS), normalized to β-actin levels. E, APP processing assessed by quantifying levels of APP-CTFs in the Western blots. Quantification of the Western blot shows that desflurane treatment (black bar) does not alter levels of APP-CTFs as compared with control conditions (white bar)(p = 0.09, NS), normalized to β-actin levels. F, desflurane (black bar) does not increase generation of Aβ40 (p = 0.10, NS) and Aβ42 (p = 0.56, NS) as compared with control conditions (white bar).

Caspase inhibitor Z-VAD attenuates caspase-3 activation induced by desflurane/hypoxia in H4-APP cells. A, treatment with desflurane (12%) plus hypoxia (18%) for 6 h (lanes 5 and 6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) or Z-VAD (50 μm) treatment (lanes 3 and 4). Z-VAD treatment (lane 7 and 8) attenuates caspase-3 cleavage induced by desflurane/hypoxia. There is no significant difference in amounts of β-actin in H4-APP cells with above treatments. B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (*, p = 0.016), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is reduced by Z-VAD (50 μm) treatment (net bar; ##, p = 0.009). C, desflurane/hypoxia (black bar) increases secreted Aβ levels in H4-APP cells as compared with control conditions (white bar) (**, p = 0.0032). Z-VAD (net bar) reduces the desflurane/hypoxia-induced increases in secreted Aβ levels in H4-APP cells (##, p = 0.00038). DMSO, dimethyl sulfoxide.

Desflurane/hypoxia increases levels of BACE in H4-APP cells. A, desflurane/hypoxia (lanes 4 and 5) increases levels of BACE (65 kDa) as compared with control conditions (lanes 1–3). There is no significant difference in amounts of β-actin in control conditions or desflurane/hypoxia-treated H4-APP cells. B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases BACE levels as compared with control conditions (white bar) (**, p = 0.006), normalized to β-actin levels.

Hypoxia (18%) does not affect caspase-3 activation, APP processing, nor Aβ generation in H4-APP cells. A, hypoxia treatment alone (lanes 4–6) does not induce caspase-3 cleavage (activation) as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or hypoxia-treated H4-APP cells. B, quantification of the Western blot shows that hypoxia treatment (black bar) does not increase caspase-3 activation compared with control conditions (white bar)(p = 0.16, NS), normalized to β-actin levels. C, hypoxia (lanes 4–6) does not affect APP processing as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or hypoxia-treated H4-APP cells. D, quantification of the Western blot shows that hypoxia treatment (black bar) does not alter levels of FL-APP as compared with control conditions (white bar) (p = 0.85, NS), normalized to β-actin levels. E, quantification of the Western blot shows that hypoxia treatment (black bar) does not alter levels of APP-CTFs as compared with control conditions (white bar)(p = 0.24, NS), normalized to β-actin levels. F, hypoxia (black bar) does not increase generation of Aβ40 as compared with control conditions (white bar)(p = 0.45, NS).