We have previously shown that the commonly used inhalational anesthetic isoflurane can induce caspase activation and apoptosis and increase Aβ generation in H4-APP cells.19,24,33,37
However, it is unknown whether other inhalational anesthetics can also promote AD neuro-pathogenesis. Here we assessed the effects of sevoflurane, currently the most commonly used inhalational anesthetic, on caspase activation, apoptosis, APP processing, and Aβ levels in H4 cells and naive mice.
First, we found that sevoflurane can induce caspase-3 activation and apoptosis in H4-APP cells. Given that isoflurane, desflurane plus hypoxia, and ischemia have all been shown to enhance BACE and Aβ levels subsequent to caspase activation,15,18,19
we next asked whether sevoflurane has similar effects. We were able to show that a clinically relevant regimen of sevoflurane anesthesia enhanced BACE levels, altered APP processing, and increased Aβ levels in H4-APP cells. These results, along with previous findings, indicate that AD neuropatho-genesis can be promoted by multiple inhalational anesthetics, suggesting that it may be prudent to carry out systematic and comprehensive assessment of the effects of all currently used inhalational anesthetics on AD-related neuropathogenic events.
Because the above findings were in vitro–based, we next sought in vivo confirmation of sevoflurane effects in naive mice. We found that a clinically relevant concentration of sevoflurane induces caspase activation and PARP cleavage, and elevates levels of caspase-cleaved APP–N-fragment, BACE, and Aβ for up to 24 hours after the anesthesia. However, sevoflurane anesthesia did not significantly alter blood pressure and blood gas in naive mice (). These findings suggest that a clinically relevant regimen of sevoflurane anesthesia induces a time-dependent cascade of caspase activation and elevated BACE and Aβ levels in vivo.
We next showed that the broad caspase activation inhibitor Z-VAD could attenuate sevoflurane-induced caspase-3 activation, indicating that sevoflurane-induced alterations in APP processing and Aβ levels are at least partially dependent on caspase activation. We also showed that the γ-secretase inhibitor L-685,458 reduced Aβ levels (data not shown) and attenuated sevoflurane-induced caspase activation and apoptosis in H4-APP cells. In contrast, exogenously added Aβ potentiated sevoflurane-induced caspase-3 activation in naive H4 cells. These data suggest that enhanced Aβ generation, subsequent to sevoflurane-induced caspase-3 activation, can lead to further caspase-3 activation, resulting in additional rounds of apoptosis and Aβ generation.
Wei et al38
showed that treatment with 4.1% sevoflurane for 24 hours did not induce cell death in rat PC12 pheochromocytoma cells and primary cortical neurons. In addition, many studies have suggested that sevoflurane can protect cells from cytotoxicity.39-47
However, many other studies have suggested that sevoflurane may induce cytotoxic effects.27-32
This discrepancy could be owing to the use of different cell lines, eg, rat kidney cells vs human neural-derived cells, and the duration and concentration of sevoflurane exposure in these studies. Future studies need to assess the effects of sevoflurane on apoptosis, APP processing, and Aβ levels with different concentrations and durations. A recent study by Wei et al48
showed that isoflurane inhibited the cytotoxicity induced by isoflurane itself. These findings suggest that isoflurane could have neuroprotective effects through induction of endogenous neuroprotective mechanisms, eg, preconditioning, while different concentrations of isoflurane with different exposure times could cause inherent neurotoxic effects. We have postulated that sevoflurane may also have dual effects on cytotoxicity. Future studies are necessary to further test this hypothesis.
The exact molecular mechanisms by which sevoflurane induces caspase activation and apoptosis, alters APP processing, and increases Aβ levels are unknown. A recent study showed that caspase activation can reduce levels of the golgi-associated, γ-adaptin ear containing adenosine diphosphate–ribosylation factor binding protein 3 (GGA-3), a protein involved in BACE degradation.15
We therefore hypothesized that sevoflurane induces caspase activation, which then reduces GGA-3 levels. The reduced GGA-3 levels will result in accumulation of BACE; the increased BACE will finally increase Aβ levels by facilitating amyloidogenic processing of APP. Our in vivo findings that sevoflurane induces caspase-3 activation at 6 or 12 hours after anesthesia but enhances Aβ levels at a later times (eg, 12 and 24 hours after anesthesia) further support this hypothesis.
Sevoflurane might also affect APP processing and Aβ generation through energy inhibition. Velliquette et al49
reported that insulin, 2-deoxyglucose, 3-nitropropionic acid, and kainic acid can induce acute energy inhibition to enhance levels of BACE and Aβ in wild-type and AD transgenic (Tg2576) mice. A recent neuroimaging study showed that sevoflurane blocked emotional memory in humans and suppressed cerebral metabolism, as evidenced by the fact that sevoflurane induced a 17% reduction of the cerebral metabolic rate of glucose use in human brains.50
Future studies will be necessary to determine whether a sevoflurane-induced increase in levels of BACE and Aβ is dependent on sevoflurane-induced changes in glucose use or GGA-3 levels.
A recent study by Groen et al23
showed that an insult from a 2-hour occlusion of the middle cerebral artery increased levels of APP and Aβ in axons at the corpus callosum and in neurons at the border of the ischemic region. Moreover, this transient insult caused persistent APP and Aβ deposits in the thalamic nuclei (ventroposterior lateral and ventroposterior medial nuclei) that eventually developed into dense plaque-like deposits 9 months after the initial insult.23
This secondary and persistent brain harm could be due to axonal damage of the thalamic neurons, leading to retrograde degeneration,51
damage from vasogenic edema and some noxious substance,52
and brain ischemia15
have been shown to induce caspase activation and apoptosis, which then enhance levels and activities of BACE to facilitate APP processing and to increase Aβ generation. Based on the study by Groen et al,23
it is possible that exposure to sevoflurane for 2 hours not only induces transient injuries (eg, caspase activation and apoptosis, increases in levels of BACE and Aβ), but could also lead to longer-term neurodegeneration and Aβ accumulation in other brain regions. Future studies will be necessary to address the potential longer-term effects of sevoflurane on AD neuropathogenesis in the mouse brain to test this hypothesis.
The limitation of the current study is that there is currently no satisfactory way to extrapolate the findings of caspase activation and Aβ metabolism in cultured cells and in the mouse brain to the human brain. Furthermore, some of these changes are moderate, and the measured effects on the mouse brain tissue are not long-lasting. Collectively, these findings do not present any direct evidence that inhalation of anesthetic sevoflurane can cause irreversible harm to the human brain. Determination of the in vivo relevance of sevoflurane on AD neuropathogenesis in the human brain will be necessary before we can conclude that anesthetic sevoflurane facilitates or exacerbates AD neuropathogenesis in humans.
Collectively, our studies have illustrated that sevoflurane can induce caspase activation and apoptosis, alter APP processing, and increase Aβ levels, key changes associated with AD neuropathogenesis, in vitro and in vivo. Moreover, these studies have defined the underlying molecular pathways. As can be seen in , sevoflurane can induce caspase activation and apoptosis, which then increase the levels and activities of BACE, leading to elevated Aβ levels. Finally, increased Aβ levels can further potentiate caspase activation and apoptosis, resulting in subsequent rounds of apoptosis and Aβ generation following sevoflurane treatment. We would like to emphasize that though our current findings suggest that sevoflurane may induce key aspects of AD neuropathogenesis in vitro and in vivo, the in vivo relevance of these effects in humans remains unclear. Nonetheless, our current findings should ultimately help to facilitate the design of safer anesthetics and improved anesthesia care for patients, especially elderly individuals and patients with AD.
Figure 7 Hypothetical pathway by which sevoflurane induces apoptosis and β-amyloid protein (Aβ) generation. Sevoflurane induces caspase-3 activation and apoptosis. Caspase activation and apoptosis, in turn, increase β-site APP-cleaving (more ...)