Nitrous oxide plus isoflurane induces apoptosis and increases beta-amyloid protein levels

Abstract

Background: Some anesthetics have been suggested to induce neurotoxicity, including promotion of Alzheimer's disease neuropathogenesis. Nitrous oxide and isoflurane are common anesthetics. The authors set out to assess the effects of nitrous oxide and/or isoflurane on apoptosis and beta-amyloid (Abeta) levels in H4 human neuroglioma cells and primary neurons from naïve mice.

Methods: The cells or neurons were exposed to 70% nitrous oxide and/or 1% isoflurane for 6 h. The cells or neurons and conditioned media were harvested at the end of the treatment. Caspase-3 activation, apoptosis, processing of amyloid precursor protein, and Abeta levels were determined.

Results: Treatment with a combination of 70% nitrous oxide and 1% isoflurane for 6 h induced caspase-3 activation and apoptosis in H4 naïve cells and primary neurons from naïve mice. The 70% nitrous oxide plus 1% isoflurane, but neither alone, for 6 h induced caspase-3 activation and apoptosis, and increased levels of beta-site amyloid precursor protein-cleaving enzyme and Abeta in H4-amyloid precursor protein cells. In addition, the nitrous oxide plus isoflurane-induced Abeta generation was reduced by a broad caspase inhibitor, Z-VAD. Finally, the nitrous oxide plus isoflurane-induced caspase-3 activation was attenuated by gamma-secretase inhibitor L-685,458, but potentiated by exogenously added Abeta.

Conclusion: These results suggest that the common anesthetics nitrous oxide plus isoflurane may promote neurotoxicity by inducing apoptosis and increasing Abeta levels. The generated Abeta may further potentiate apoptosis to form another round of apoptosis and Abeta generation. More studies, especially the in vivo confirmation of these in vitro findings, are needed.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 1. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis, and increases levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. There is no significant difference in amounts of β-actin in the control condition-, staurosporine- or nitrous oxide plus isoflurane-treated H4-APP cells. B. Caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. D. Effects of the nitrous oxide plus isoflurane treatment on apoptosis. E. Effects of the nitrous oxide plus isoflurane treatment on BACE levels. F. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. G. Effects of nitrous oxide plus isoflurane treatment on Aβ40 levels. We have averaged results from ten independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01.

Figure 2. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis in H4 naïve cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. We have averaged results from four independent experiments. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. ** P <0.01

Figure 2. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis in H4 naïve cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. We have averaged results from four independent experiments. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. ** P <0.01

Figure 2. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis in H4 naïve cells

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment on TUNEL positive cells. We have averaged results from four independent experiments. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. ** P <0.01

Figure 3. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis in primary neurons from naïve mice

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment, and staurosporine treatment on TUNEL positive cells. We have averaged results from four independent experiments. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01; ## P < 0.01.

Figure 3. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis in primary neurons from naïve mice

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment, and staurosporine treatment on TUNEL positive cells. We have averaged results from four independent experiments. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01; ## P < 0.01.

Figure 3. Treatment of nitrous oxide plus isoflurane induces caspase-3 activation and apoptosis in primary neurons from naïve mice

A. Effects of 70% nitrous oxide plus 1% isoflurane for six hours, and staurosporine on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide plus isoflurane treatment, and staurosporine treatment on TUNEL positive cells. We have averaged results from four independent experiments. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; FL, full length. * P < 0.05; ** P <0.01; ## P < 0.01.

Figure 4. Nitrous oxide treatment alone induces neither apoptosis nor increases in levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide treatment on apoptosis. We have averaged results from six independent experiments. D. Effects of the nitrous oxide treatment on BACE levels. Each band in the Western blot represents an independent experiment. E. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. F. Effects of the nitrous oxide treatment on Aβ levels. We have averaged results from six independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant; ** P <0.01

Figure 4. Nitrous oxide treatment alone induces neither apoptosis nor increases in levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide treatment on apoptosis. We have averaged results from six independent experiments. D. Effects of the nitrous oxide treatment on BACE levels. Each band in the Western blot represents an independent experiment. E. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. F. Effects of the nitrous oxide treatment on Aβ levels. We have averaged results from six independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant; ** P <0.01

Figure 4. Nitrous oxide treatment alone induces neither apoptosis nor increases in levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide treatment on apoptosis. We have averaged results from six independent experiments. D. Effects of the nitrous oxide treatment on BACE levels. Each band in the Western blot represents an independent experiment. E. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. F. Effects of the nitrous oxide treatment on Aβ levels. We have averaged results from six independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant; ** P <0.01

Figure 4. Nitrous oxide treatment alone induces neither apoptosis nor increases in levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide treatment on apoptosis. We have averaged results from six independent experiments. D. Effects of the nitrous oxide treatment on BACE levels. Each band in the Western blot represents an independent experiment. E. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. F. Effects of the nitrous oxide treatment on Aβ levels. We have averaged results from six independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant; ** P <0.01

Figure 4. Nitrous oxide treatment alone induces neither apoptosis nor increases in levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide treatment on apoptosis. We have averaged results from six independent experiments. D. Effects of the nitrous oxide treatment on BACE levels. Each band in the Western blot represents an independent experiment. E. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. F. Effects of the nitrous oxide treatment on Aβ levels. We have averaged results from six independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant; ** P <0.01

Figure 4. Nitrous oxide treatment alone induces neither apoptosis nor increases in levels of BACE and Aβ in H4-APP cells

A. Effects of 70% nitrous oxide on caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the nitrous oxide treatment on apoptosis. We have averaged results from six independent experiments. D. Effects of the nitrous oxide treatment on BACE levels. Each band in the Western blot represents an independent experiment. E. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. F. Effects of the nitrous oxide treatment on Aβ levels. We have averaged results from six independent experiments. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant; ** P <0.01

Figure 5. Isoflurane treatment alone induces neither caspase-3 activation nor Aβ accumulation in H4-APP cells

A. Effects of 1% isoflurane for six hours on caspase-3 activation. Each band in the estern blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the isoflurane treatment on Aβ levels. We have averaged results from three independent experiments. APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant.

Figure 5. Isoflurane treatment alone induces neither caspase-3 activation nor Aβ accumulation in H4-APP cells

A. Effects of 1% isoflurane for six hours on caspase-3 activation. Each band in the estern blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the isoflurane treatment on Aβ levels. We have averaged results from three independent experiments. APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant.

Figure 5. Isoflurane treatment alone induces neither caspase-3 activation nor Aβ accumulation in H4-APP cells

A. Effects of 1% isoflurane for six hours on caspase-3 activation. Each band in the estern blot represents an independent experiment. B. Quantification of Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of the isoflurane treatment on Aβ levels. We have averaged results from three independent experiments. APP, amyloid precursor protein; Aβ, β-amyloid protein; FL, full length. N.S., not significant.

Figure 6. Caspase inhibitor Z-VAD attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation and Aβ generation in H4-APP cells

A. Effects of Z-VAD on the nitrous oxide plus isoflurane-induced caspase-3 activation in H4-APP cells. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Nitrous oxide plus isoflurane (lanes 1–3) increases levels of APP-N-caspase fragment, which is attenuated by Z-VAD treatment (lanes 4–6) in H4-APP cells. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Z-VAD (black bar) decreases nitrous oxide plus isoflurane-induced increases in the ratio of APP-N-caspase-fragment to FL-APP as compared to nitrous oxide plus isoflurane treatment (white bar), normalized to β-actin levels. We have averaged results from three independent experiments. E. Z-VAD (net bar) reduces the nitrous oxide plus isoflurane-induced increases in Aβ40 levels in H4-APP cells. We have averaged results from six independent experiments. APP, amyloid precursor protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05; ## P < 0.01.

Figure 6. Caspase inhibitor Z-VAD attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation and Aβ generation in H4-APP cells

A. Effects of Z-VAD on the nitrous oxide plus isoflurane-induced caspase-3 activation in H4-APP cells. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Nitrous oxide plus isoflurane (lanes 1–3) increases levels of APP-N-caspase fragment, which is attenuated by Z-VAD treatment (lanes 4–6) in H4-APP cells. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Z-VAD (black bar) decreases nitrous oxide plus isoflurane-induced increases in the ratio of APP-N-caspase-fragment to FL-APP as compared to nitrous oxide plus isoflurane treatment (white bar), normalized to β-actin levels. We have averaged results from three independent experiments. E. Z-VAD (net bar) reduces the nitrous oxide plus isoflurane-induced increases in Aβ40 levels in H4-APP cells. We have averaged results from six independent experiments. APP, amyloid precursor protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05; ## P < 0.01.

Figure 6. Caspase inhibitor Z-VAD attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation and Aβ generation in H4-APP cells

A. Effects of Z-VAD on the nitrous oxide plus isoflurane-induced caspase-3 activation in H4-APP cells. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Nitrous oxide plus isoflurane (lanes 1–3) increases levels of APP-N-caspase fragment, which is attenuated by Z-VAD treatment (lanes 4–6) in H4-APP cells. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Z-VAD (black bar) decreases nitrous oxide plus isoflurane-induced increases in the ratio of APP-N-caspase-fragment to FL-APP as compared to nitrous oxide plus isoflurane treatment (white bar), normalized to β-actin levels. We have averaged results from three independent experiments. E. Z-VAD (net bar) reduces the nitrous oxide plus isoflurane-induced increases in Aβ40 levels in H4-APP cells. We have averaged results from six independent experiments. APP, amyloid precursor protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05; ## P < 0.01.

Figure 6. Caspase inhibitor Z-VAD attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation and Aβ generation in H4-APP cells

A. Effects of Z-VAD on the nitrous oxide plus isoflurane-induced caspase-3 activation in H4-APP cells. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Nitrous oxide plus isoflurane (lanes 1–3) increases levels of APP-N-caspase fragment, which is attenuated by Z-VAD treatment (lanes 4–6) in H4-APP cells. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Z-VAD (black bar) decreases nitrous oxide plus isoflurane-induced increases in the ratio of APP-N-caspase-fragment to FL-APP as compared to nitrous oxide plus isoflurane treatment (white bar), normalized to β-actin levels. We have averaged results from three independent experiments. E. Z-VAD (net bar) reduces the nitrous oxide plus isoflurane-induced increases in Aβ40 levels in H4-APP cells. We have averaged results from six independent experiments. APP, amyloid precursor protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05; ## P < 0.01.

Figure 6. Caspase inhibitor Z-VAD attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation and Aβ generation in H4-APP cells

A. Effects of Z-VAD on the nitrous oxide plus isoflurane-induced caspase-3 activation in H4-APP cells. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot, normalized to β-actin levels. We have averaged results from three independent experiments. C. Nitrous oxide plus isoflurane (lanes 1–3) increases levels of APP-N-caspase fragment, which is attenuated by Z-VAD treatment (lanes 4–6) in H4-APP cells. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Z-VAD (black bar) decreases nitrous oxide plus isoflurane-induced increases in the ratio of APP-N-caspase-fragment to FL-APP as compared to nitrous oxide plus isoflurane treatment (white bar), normalized to β-actin levels. We have averaged results from three independent experiments. E. Z-VAD (net bar) reduces the nitrous oxide plus isoflurane-induced increases in Aβ40 levels in H4-APP cells. We have averaged results from six independent experiments. APP, amyloid precursor protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05; ## P < 0.01.

Figure 7. γ-Secretase inhibitor L-685,458 attenuates, but Aβ potentiates, the nitrous oxide plus isoflurane-induced caspase-3 activation

A. Effects of L-685,458 on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot shows that L-685,458 treatment (net bar) attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of Aβ on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Aβ treatment potentiates the nitrous oxide plus isoflurane-induced caspase-3 activation. We have averaged results from three independent experiments. Aβ, β-amyloid protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05.

Figure 7. γ-Secretase inhibitor L-685,458 attenuates, but Aβ potentiates, the nitrous oxide plus isoflurane-induced caspase-3 activation

A. Effects of L-685,458 on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot shows that L-685,458 treatment (net bar) attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of Aβ on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Aβ treatment potentiates the nitrous oxide plus isoflurane-induced caspase-3 activation. We have averaged results from three independent experiments. Aβ, β-amyloid protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05.

Figure 7. γ-Secretase inhibitor L-685,458 attenuates, but Aβ potentiates, the nitrous oxide plus isoflurane-induced caspase-3 activation

A. Effects of L-685,458 on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot shows that L-685,458 treatment (net bar) attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of Aβ on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Aβ treatment potentiates the nitrous oxide plus isoflurane-induced caspase-3 activation. We have averaged results from three independent experiments. Aβ, β-amyloid protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05.

Figure 7. γ-Secretase inhibitor L-685,458 attenuates, but Aβ potentiates, the nitrous oxide plus isoflurane-induced caspase-3 activation

A. Effects of L-685,458 on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. B. Quantification of the Western blot shows that L-685,458 treatment (net bar) attenuates the nitrous oxide plus isoflurane-induced caspase-3 activation, normalized to β-actin levels. We have averaged results from three independent experiments. C. Effects of Aβ on the nitrous oxide plus isoflurane-induced caspase-3 activation. Each band in the Western blot represents an independent experiment. D. Quantification of the Western blot shows that Aβ treatment potentiates the nitrous oxide plus isoflurane-induced caspase-3 activation. We have averaged results from three independent experiments. Aβ, β-amyloid protein. DMSO, dimethyl sulfoxide; FL, full length. * P < 0.05; ** P <0.01; # P < 0.05.

Figure 8. Hypothetical pathway by which nitrous oxide plus isoflurane induces apoptosis and Aβ generation

Nitrous oxide plus isoflurane induces apoptosis. Apoptosis, in turn, increases BACE levels, which serves to facilitate APP processing and to increase Aβ generation. Elevated Aβ generation then further induces apoptosis leading to another round of nitrous oxide plus isoflurane-induced apoptosis and Aβ generation. BACE, β-site amyloid precursor protein-cleaving enzyme; APP, amyloid precursor protein; Aβ, β-amyloid protein.