ORIGINAL CONTRIBUTION The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases ␤-Amyloid Protein Levels Yuanlin Dong, MD; Guohua Zhang, MD, PhD; Bin Zhang, MD; Robert D. Moir, PhD; Weiming Xia, PhD; Edward R. Marcantonio, MD; Deborah J. Culley, MD; Gregory Crosby, MD; Rudolph E. Tanzi, PhD; Zhongcong Xie, MD, PhD Objective: To assess the effects of sevoflurane, the most Z-VAD decreased the effects of sevoflurane on apopto- commonly used inhalation anesthetic, on apoptosis and sis and A␤. Sevoflurane-induced caspase-3 activation was ␤-amyloid protein (A␤) levels in vitro and in vivo. attenuated by the ␥-secretase inhibitor L-685,458 and was potentiated by A␤. These results suggest that sevoflu- Subjects: Naive mice, H4 human neuroglioma cells, and rane induces caspase activation which, in turn, en- H4 human neuroglioma cells stably transfected to ex- hances ␤-site amyloid precursor protein–cleaving en- press full-length amyloid precursor protein. zyme and A␤ levels. Increased A␤ levels then induce further rounds of apoptosis. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor pro- Conclusions: These results suggest that inhalational an- tein were exposed to 4.1% sevoflurane for 6 hours. Mice esthetic sevoflurane may promote Alzheimer disease neu- received 2.5% sevoflurane for 2 hours. Caspase-3 acti- ropathogenesis. If confirmed in human subjects, it may vation, apoptosis, and A␤ levels were assessed. be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possess- ␤ Results: Sevoflurane induced apoptosis and elevated lev- ing excessive levels of cerebral A . els of ␤-site amyloid precursor protein–cleaving en- zyme and A␤ in vitro and in vivo. The caspase inhibitor Arch Neurol. 2009;66(5):620-631 XCESSIVE ␤-AMYLOID PRO- tween 1.2 and 1.6 for the association of pre- tein (A␤) accumulation is a vious general anesthesia/surgery and AD. major pathological hall- Moreover, the age of onset of AD has been mark of Alzheimer disease inversely correlated with cumulative expo- (AD).1,2 ␤-amyloid protein is sure to general anesthesia prior to 50 years Eproduced via serial proteolysis of the amy- of age.20,21 A recent study illustrated that pa- loid precursor protein (APP) by aspartyl pro- tients having coronary artery bypass graft tease ␤-site APP-cleaving enzyme (BACE; surgery with general anesthesia are at greater ␤-secretase) and ␥-secretase. ␤-site APP- risk for the emergence of AD than those hav- cleaving enzyme cleaves APP to generate a ing percutaneous transluminal coronary an- 99-residue membrane-associated C- gioplasty with local anesthesia.22 Though terminus fragment (APP-C99). This frag- there have been no conclusive studies to ment is further cleaved by ␥-secretase to re- strongly suggest an association between an- lease the 4-kDa A␤ and APP intracellular esthesia and AD, there have been studies domain.3-5 Increasing evidence suggests a suggesting that anesthetics such as isoflu- role for caspase activation and apoptosis in rane may promote AD neuropathogenesis AD neuropathogenesis.6-17 Recent studies in vitro and in vivo. A recent study showed suggested that caspase activation and apop- that an insult from a middle cerebral ar- tosis may enhance BACE levels to facilitate tery occlusion for 2 hours in rats caused tem- APP processing, leading to increases in A␤ porary increases in APP and A␤ staining in levels.15,18,19 a brain area near the ischemic region as well An estimated 200 million patients world- as long-term (up to 9 months) APP and A␤ Author Affiliations are listed at wide have surgery with anesthesia each year. deposits in a brain area distant from the is- the end of this article. Several studies showed an odds ratio of be- chemic region.23 These findings suggest that (REPRINTED) ARCH NEUROL / VOL 66 (NO. 5), MAY 2009 WWW.ARCHNEUROL.COM 620 ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 a transient insult, eg, ischemia or anesthesia with isoflu- chusetts). The temperature of the anesthetizing chamber was rane, could lead to secondary and persistent brain inju- controlled to maintain a mean (SD) rectal temperature in the ries. However, whether inhalation anesthetics other than animals of 37°C(0.5°C). Mean arterial blood pressure was mea- isoflurane can promote AD neuropathogenesis remains un- sured noninvasively using a tail cuff (Kent Scientific Corpora- known. We therefore set out to determine the effects of sevo- tion, Torrington, Connecticut) in the anesthetized mice. An- esthesia was terminated by discontinuing sevoflurane and placing flurane, currently the most commonly used inhalational an- animals in a chamber containing 100% O until 20 minutes af- esthetic, on caspase activation, apoptosis, APP processing, 2 ␤ ter the return of their righting reflex. They were then returned and levels of BACE and A in H4 human neuroglioma cells to individual home cages until they were humanely killed. Mice as well as in naive mice. were killed by decapitation 6, 12, and 24 hours after sevoflu- rane anesthesia. The brain was removed rapidly and the pre- METHODS frontal cortex was dissected out and frozen in liquid nitrogen for subsequent processing to determine caspase activation and levels of FL-APP, APP-C99, APP-C83, BACE, and A␤. CELL LINES BRAIN TISSUE LYSIS AND PROTEIN We used H4 human neuroglioma cells (naive H4 cells) and H4 AMOUNT QUANTIFICATION human neuroglioma cells stably transfected to express full- length (FL)–APP (H4-APP cells). All cell lines were cultured in The harvested brain tissues were homogenized on ice using im- Dulbecco Modified Eagle Medium (high glucose) containing 9% munoprecipitation buffer (10mM Tris-HCl; pH, 7.4; 150mM heat-inactivated fetal calf serum, 100-Us/mL penicillin, 100- NaCl; 2 mM EDTA; 0.5% Nonidet P-40) plus protease inhibi- µg/mL streptomycin, and 2mM L-glutamine. Stably transfected tors (1-µg/mL aprotinin, 1-µg/mL leupeptin, 1-µg/mL pep- H4 cells were additionally supplemented with 200- µg/mL G418. statin A). The lysates were collected, centrifuged at 12 000 rpm for 10 minutes, and quantified for total proteins by BCA pro- CELL TREATMENT tein assay kit. The cells were treated with 21% oxygen, 5% carbon dioxide, WESTERN BLOT ANALYSIS and 4.1% sevoflurane (2 minimum alveolar concentration) for 6 hours, during which time the cells were incubated in serum- The cells and brain tissues were harvested at the end of the ex- free cell culture media, as described by Xie et al.24 21% O ,5% 2 periment and were subjected to Western blot analysis, as de- CO , and 4.1% sevoflurane were delivered from an anesthesia 2 scribed by Xie et al.25 Antibodies A8717 (1:2000; Sigma, St Louis, machine to a sealed plastic box in a 37°C incubator containing Missouri), C66 (1:1000; generous gift of Dora Kovacs, PhD, at 6-well plates seeded with 1 million cells in 1.5-mL cell culture Massachusetts General Hospital and Harvard Medical School), media. A Datex infrared gas analyzer (Puritan-Bennett, Tewks- and anti–␤-actin (1:5000; Sigma) were used to visualize FL- bury, Massachusetts) was used to continuously monitor the con- APP (110 kDa), APP-C83 (12 kDa), APP-C99 (10 kDa), and centrations of delivered CO ,O, and sevoflurane. In the in- 2 2 ␤-actin (42 kDa), respectively. A caspase-3 antibody (1:1000; teraction studies, the cells were treated with Z-VAD (100µM), Cell Signaling Technology Inc, Beverly, Massachusetts) was used A␤40 (7.5µM) plus A␤42 (7.5µM), and L-685,458 (0.5µM) 1 to recognize the caspase-3 fragment (17-20 kDa) resulting from hour before the treatment with 4.1% sevoflurane. Control con- cleavage at asparate position 175 and caspase-3 FL (35-40 kDa). ditions included 5% CO plus 21% O , which did not affect 2 2 Rabbit polyclonal anti–BACE1 antibody ab2077 (1:1000; Ab- caspase-3 activation, cell viability, APP processing, or A␤ gen- cam, Cambridge, Massachusetts) was used to detect the pro- eration (data not shown). tein levels of BACE (65 kDa). The quantification of Western blots was performed in 2 steps, as described by Xie et al.25 Briefly, CELL LYSIS AND PROTEIN the intensity of signals was analyzed by using an image pro- AMOUNT QUANTIFICATION gram from the National Institutes of Health (NIH ImageJ; Bethesda, Maryland). First, we used levels of ␤-actin to nor- Cell pellets were detergent-extracted on ice using immunopre- malize (eg, determining ratio of FL-APP amount to ␤-actin cipitation buffer (10mM Tris-hydrochloride [HCl]; pH, 7.4; amount) the levels of FL-APP, APP-C83, APP-C99, FL- 150mM sodium chloride [NaCl]; 2mM EDTA; 0.5% Nonidet caspase-3, caspase-3 fragment, BACE, and A␤ to control for the P-40) plus protease inhibitors (1-µg/mL aprotinin, 1-µg/mL leu- loading differences in total protein amounts. Second, we pre- peptin, and pepstatin A). The lysates were collected, centri- sented the changes in the levels of FL-APP, APP-C83, APP- fuged at 12 000 revolutions per minute (rpm) for 10 minutes, C99, FL-caspase-3, caspase-3 fragment, A␤, and BACE in the and quantified for total proteins by the BCA (bicinchoninic acid) cells or animals treated with sevoflurane, Z-VAD, A␤, and protein assay kit (Pierce, Iselin, New Jersey). L-685,458 as the percentage of those in the cells or animals treated with controls. We refer to 100% caspase-3 activation, ␤ MOUSE ANESTHESIA AND TREATMENT FL-APP, APP-C83, APP-C99, A , and BACE in this article as control levels for the purpose of comparison with experimen- The animal protocol was approved by the Standing Commit- tal conditions. tee on Animals at Massachusetts General Hospital.