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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Anesthesiology. Author manuscript; available in PMC Jun 1, 2011.
Published in final edited form as:
PMCID: PMC2877754
NIHMSID: NIHMS189924

Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Naïve and Alzheimer's Disease Transgenic Mice

Yan Lu, M.D., Ph.D.,1,# Xu Wu, M.D., Ph.D.,2,# Yuanlin Dong, M.D., M.S.,3 Zhipeng Xu, M.D., Ph.D.,4 Yiying Zhang, M.D.,5 and Zhongcong Xie, M.D., Ph.D.6,*

Abstract

Background

Recent studies have suggested that children having surgery under anesthesia could be at an increased risk for the development of learning disabilities, but whether anesthetics contribute to this learning disability is unclear. We therefore set out to assess effects of sevoflurane, the most commonly used inhalation anesthetic, on caspase activation, apoptosis, β-amyloid protein levels, and neuroinflammation in brain tissues of neonatal naïve and Alzheimer's disease (AD) transgenic mice.

Methods

Six-day-old naïve and AD transgenic [B6.Cg-Tg(amyloid precursor protein swe, PSEN1dE9)85Dbo/J] mice were treated with sevoflurane. The mice were euthanized at the end of the anesthesia and brain tissues were harvested, and were then subjected to Western blot, immunocytochemistry, ELISA and real-time polymerase chain reaction.

Results

Here we show for the first time that sevoflurane anesthesia induced caspase activation and apoptosis, altered amyloid precursor protein processing, and increased β-amyloid protein levels in the brain tissues of the neonatal mice. Furthermore, the sevoflurane anesthesia led to a greater degree of neurotoxicity in the brain tissues of the AD transgenic mice as compared to the naïve mice, and increased tumor necrosis factor-α levels only in the brain tissues of the AD transgenic mice. Finally, inositol 1,4,5-trisphosphate receptor antagonist 2-APB attenuated the sevoflurane-induced caspase-3 activation and β-amyloid protein accumulation in vivo.

Conclusion

These results suggest that sevoflurane may induce the neurotoxicity in neonatal mice. AD transgenic mice could be more venerable to such neurotoxicity. These findings should promote more studies to determine the potential neurotoxicity of anesthesia in animals and humans, especially in children.

Introduction

A recent study by Wilder et al. 1 investigated more than five thousand children and found that children who had early exposure to anesthesia were at increased risk for developing a learning disability. Furthermore, the risk for the development of a learning disability increases with longer cumulative duration of anesthesia exposure. Another pilot study by Kallkman et al. 2 has also found that children who had surgery and anesthesia at younger than two years of age could be at increased risk of developing deviant behavior later in life. These findings suggest that anesthesia may be a significant risk factor for later development of a learning disability and deviant behavior. However, these results cannot reveal whether anesthesia itself contributes to the learning disability and deviant behavior or whether the need for anesthesia is a marker for other unidentified factors that contribute to the development of learning disabilities and deviant behavior. Moreover, recent studies of twin pairs have suggested that there is no evidence for a causal relationship between anesthesia administration and later learning-related outcomes 3. Therefore, there is a need to study the effects of anesthetics, e.g., sevoflurane, on biochemical changes associated with cognitive dysfunction.

Several other studies have shown that the commonly used inhalation anesthetics, e.g., isoflurane and sevoflurane, may induce apoptosis in brain tissues of neonatal mice 4-6. However, the anesthesia-induced apoptosis is not the only cause of behavioral abnormalities 4. β-Amyloid protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) patients 7-9, is the hallmark feature of AD-associated dementia and learning/memory dysfunction [reviewed by 10-12]. In addition, neuroinflammation and elevation of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α have also been shown to be associated with AD-associated dementia and learning/memory dysfunction 13-15. However, the effects of inhalation anesthetics on caspase activation, apoptosis, Aβ levels, and neuroinflammation in neonatal mice remain largely to be determined. Furthermore, there have been no studies to compare the effects of anesthetics on apoptosis, Aβ accumulation and neuroinflammation between neonatal naïve mice and neonatal AD transgenic mice. Therefore, we set out to determine the effects of the most commonly used inhalation anesthetic, sevoflurane, on caspase activation, apoptosis, levels of Aβ, and pro-inflammatory cytokine TNF-α in the brain tissues of neonatal (six-day-old) mice, and to determine the underlying mechanisms. Moreover, we compared the neurotoxic effects of sevoflurane in neonatal naïve mice and neonatal AD transgenic mice [B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J].

Materials and Methods

Animal treatments

The animal protocol was approved by Standing Committee on Animals at Massachusetts General Hospital (Boston, Massachusetts). Naïve mice [C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME)] and AD transgenic mice [B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J, (The Jackson Laboratory, Bar Harbor, ME)] were distinguished by genotyping. All animals (3 to 12 mice per experiment) were six days of age at the time of anesthesia and were randomized by weight and gender into experimental groups that received either 3% or 2.1% sevoflurane plus 60% oxygen for either six or two hours, and control groups that received 60% oxygen for six or two hours at identical flow rates in identical anesthetizing chambers. We chose this sevoflurane anesthesia because the recent study by Satomoto et al. 6 indicated that anesthesia with 3% sevoflurane plus 60% oxygen for six hours does not significantly alter blood gas and brain blood flow, which is consistent with our pilot studies. The mortality rate of the mice following the anesthesia with 3% sevoflurane plus 60% oxygen for six hours in the current studies was about 10-15%, which could be due to the higher than clinically relevant concentration of sevoflurane. We used this high concentration of sevoflurane anesthesia to illustrate the difference of sevoflurane-induced neurotoxicity between neonatal naïve and AD transgenic mice. Moreover, we also assessed the effects of anesthesia with 2.1% sevoflurane, a more clinically relevant concentration of sevoflurane (which did not cause the death of the mice), on the effects of caspase-3 activation and Aβ levels in the brain tissues of neonatal mice. Anesthetic and oxygen concentrations were measured continuously (Datex, Tewksbury, MA), and the temperature of the anesthetizing chamber was controlled to maintain the rectal temperature of the mice at 37 ± 0.5°C. In the interaction studies, Inositol triphosphate receptor (IP3R) antagonist 2-aminoethoxydiphenyl borate (2-APB) (5 and 10 mg/kg) was administered to the mice via intraperitoneal injection 10 minutes before the anesthetic was administered. 2-APB was first dissolved in dimethyl sulfoxide to 20 μg/μl, and then diluted with saline to 0.25 μg/μl (1:80 dilution) to 0.5 μg/μl (1:40 dilution).

Tissue preparation

Immediately after the sevoflurane anesthesia, the mouse was decapitated, and the brain cortex was harvested. The brain tissues were homogenized in an immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) plus protease inhibitors [(1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A), (Roche, Indianapolis, IN)]. The lysates were collected, centrifuged at 13,000 rpm for 15 min., and quantified for total proteins by using the bicinchoninic acid protein assay kit (Pierce, Iselin, NJ).

Western blots analysis

The harvested brain tissues were subjected to Western blots as described by Xie et al. 16. Briefly, 60 μg of each lysate was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride blots (Bio-Rad, Hercules, CA) using a semi-dry electrotransfer system (Amersham Biosciences, San Francisco, CA). The blot was incubated overnight at 4°C with primary antibodies, followed by washes and incubation with appropriate secondary antibodies, and visualized with a chemoluminescence system. A caspase-3 antibody (1:1,000 dilution; Cell Signaling Technology, Danvers, MA) was used to recognize caspase-3 fragment (17–20 kDa) resulting from cleavage at aspartate position 175 and full-length (FL) caspase-3 (35–40 kDa). TNF-α levels were recognized by antibody ab6671 (26 kDa, 1:1,000, Abcam, Cambridge, MA). The antibody to non-targeted protein β-Actin was used to control for loading differences in total protein amounts. The figures showing blots with caspase-3 fragment only are the same Western blots with extended exposure time during the development of the film. The signal of the Western blot band was detected using Molecular Imager VersaDoc MP 5000 System (Bio-Rad). The intensity of signals was analyzed using a Bio-Rad image program (Quantity One) and a National Institutes of Health Image Version 1.37v (National Institutes of Health, Bethesda, MD). We quantified Western blots using two steps. First, we used levels of β-Actin to normalize (e.g., determining the ratio of FL caspase-3 amount to β-Actin amount) the levels of proteins to control for loading differences in total protein amounts. Second, we presented changes in levels of proteins in the mice treated with sevoflurane as the percentage or fold of those in the mice treated with control condition. One hundred percent or one fold of change in protein levels in this article refers to control levels for comparison with experimental condition.

Immunoblot detection of Aβ

Brain samples were homogenized (150 mM NaCl with a protease inhibitor cocktail in 50 mM Tris, pH of 8.0) and centrifuged (65,000 rpm for 45 min), and then the supernatant was removed. The pellet was then resuspended by sonication and incubated for 15 minutes in homogenization buffer containing 1% sodium dodecyl sulfate. Following pelleting of insoluble material (18,000 rpm for 15 min), the sodium dodecyl sulfate-extract was electrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (4-12% Bis-Tris polyacrylamide gel from Invitrogen, Carlsbad, CA), blotted to polyvinylidene fluoride membrane, and probed with a 1:200 dilution of Aβ 6E10 (Convance, Berkeley, CA) 16,17.

Quantification of Aβ Using a Sandwich Enzyme-linked Immunosorbent Assay

The Aβ42 and Aβ40 levels in the brain tissues of AD transgenic mice were measured by using Sandwich enzyme-linked immunosorbent assay (ELISA). The Human Aβ(1-42) ELISA kit or Human Aβ(1-40) ELISA kit (Wako, Richmond, VA) was used to detect levels of Aβ42 or Aβ40, respectively. The monoclonal antibody BAN50, the epitope of which is human Aβ(1–16), was coated on 96 well plate and acted as a capture antibody for the N-terminal portion of human Aβ42 or human Aβ40. Captured human Aβ42 or human Aβ40 was recognized by another antibody BC05 or BA27, which specifically detected the C-terminal portion of Aβ42 or Aβ40, respectively. The 96 plates were incubated overnight at 4°C with test samples and control, and then BC05 or BA27 was added. The plates were then developed with tetramethylbenzidine reagent, terminated by stop solution, and well absorbance was measured at 450 nm. Aβ42 and Aβ40 levels in the test samples were determined by comparing the results with signals from the controls using the standard curve. The mouse brain tissue samples were prepared by using the same method in the section of the Immunoblot detection of Aβ.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining assay

Mice were perfused transcardially with 0.1 M phosphate buffer with a pH of 7.4 followed by 4% paraformaldehyde in a 0.1M phosphate buffer immediately after the anesthesia with 3% sevoflurane plus 60% oxygen for six hours. Mouse brain tissues were removed and exposed to immersion fixation for 24 hours at 4°C in 4% paraformaldehyde, then 5μm paraffin-embedded sections were made from the brain tissues. TMR red kit (Roche, Palo Alto, CA) was used for TUNEL staining. Briefly, the brain sections were incubated in a permeabilization solution and then incubated with a TUNEL reaction mixture. Finally, the sections were incubated with 10 μg/ml Hoechst 33342 in a humidified dark chamber. Sections from the same brain areas between control group mice and the sevoflurane-treated mice were then analyzed in a mounting medium under a fluorescence microscope. The TUNEL-positive cells and total cells in five areas of the brain section from each of the mice in the experiments were counted under a 20× objective microscope lens by an investigator who was blinded to the experiments.

For the double immunocytochemistry staining to identify the cell type of the TUNEL positive cells, mouse brain tissues were quickly removed after the sevoflurane anesthesia and were put into a container with dry ice and ethanol, then were kept in -80°C freezer. 5 μm frozen sections were cut using a cryostat. Sections were fixed successively with 100% methanol at -20°C for 20 minutes, and incubated with permeabilization solution (7.8% gelatin and 1.25 ml 10% saponin in 500 ml phosphate buffered saline) for 30 minutes. Sections were incubated in 10% donkey serum in permeabilization solution for 30 minutes, then incubated with antibody for NeuN to identify neuron (1:500, mab 377, anti-mouse, Millipore, Billerica, MA), antibody for Glial fibrillary acidic protein to identify astrocyte (1:100, ab16997, anti-rabbit, Abcam), and antibody for Iba1 to identify microglia cell (1:100, ab5076, anti-goat, Abcam) at 4°C overnight. Then sections were exposed to secondary antibodies Alexa Fluor®488 goat anti-mouse Immunoglobulin G (1:1000, Invitrogen), Alexa Fluor®488 goat anti-rabbit Immunoglobulin G (1:1000, Invitrogen) and donkey anti-goat Immunoglobulin G-Cy2 (1:100, Jackson ImmunoResearch Inc., West Grove, PA), respectively, for one hour at 37°C in a dark chamber followed by TUNEL staining. Sections were counterstained with 10 μg/ml Hoechst 33342 at room temperature for 10 minutes. Finally, the sections were mounted and viewed immediately using a fluorescence microscope.

Reverse Transcriptase Polymerase Chain Reaction

Real-time reverse transcriptase polymerase chain reaction was carried out using the QuantiTect SYBR Green real-time polymerase chain reaction Kit (Qiagen, Valencia, CA). TNF-α messenger ribonucleic acid levels were determined and standardized with glyceraldehyde 3-phosphate dehydrogenase as an internal control. Primers of mouse TNF-α (ID No., QT00104006) and mouse lyceraldehyde 3-phosphate dehydrogenase (ID No., QT01658692) were purchased from Qiagen.

Statistics

Given the potential presence of background caspase-3 activation and apoptosis in brain tissues of neonatal mice, we did not use absolute values to describe changes in caspase-3 activation and apoptosis. Instead, caspase-3 activation and cell apoptosis were presented as a percentage or fold of those in the control group in the naïve mice or the AD transgenic mice. One hundred percent or one fold caspase-3 activation or apoptosis refers to control levels for the purposes of comparison to experimental condition. We presented changes in levels of caspase-3 activation, apoptosis, levels of Aβ and TNF-α in treated mice as percentages or folds of those in mice in the control condition. Data were expressed as mean ± S.D. The number of samples varied from 3 to 12, and the samples were normally distributed. A randomization table generated using a computer random number generator and stored in a Microsoft Excel spreadsheet was used to randomize animals to conditions. ANOVA or t-test was used to compare the differences from the control group. Only a single measurement of each outcome value was collected from each experiment animal. As a result no repeated measurement was involved in the analysis. Post hoc adjustment for multiple comparisons was conducted using the Bonferroni method. P-values less than 0.05 (* or #) and 0.01 (** or ##) were considered statistically significant. The significance testing was two-tailed, and we have used SAS software (Cary, NC) to analyze the data.

Results

Sevoflurane induced caspase-3 activation and amyloid precursor protein (APP) processing in the brain tissues of neonatal mice

We have previously reported that the commonly used inhalation anesthetic, sevoflurane, can cause neurotoxicity by inducing apoptosis and enhancing Aβ levels in vitro and in brain tissues of adult naïve mice 18. But the effects of sevoflurane on apoptosis, Aβ accumulation, and neuroinflammation in neonatal mice remain largely to be determined. Furthermore, the comparison of these effects between neonatal naïve mice and AD transgenic mice has not been done. We therefore set out to determine and compare the effects of sevoflurane on apoptosis, Aβ accumulation, and neuroinflammation in neonatal (six-day-old) naïve (C57BL/6J) and AD transgenic [B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J] mice.

Caspase-3 activation is one of the final steps of cellular apoptosis 19. We therefore assessed the effects of sevoflurane on caspase-3 activation in brain tissues of neonatal naïve mice by quantitative Western blots analyses. The six-day-old neonatal naïve mice were treated with 3% sevoflurane plus 60% oxygen for six hours, the brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis by which caspase-3 antibody was used to detect both caspase-3 fragment (17 – 20 kDa) and FL-caspase-3 (35 - 40 kDa). Caspase-3 immunoblotting showed visible increases in protein levels of caspase-3 fragment following the sevoflurane anesthesia as compared to the control condition (Figure 1A). The blot with caspase-3-fragment only is the same Western blot with extended exposure time during the development of the film. Quantification of the Western blot, by determining the ratio of cleaved (activated) caspase-3 fragment (17 – 20 kDa) to FL-caspase-3 (35 - 40 kDa), revealed that the sevoflurane anesthesia led to a 242% increase in caspase-3 cleavage (activation) as compared to the control condition (Figure 1B) (P = 0.009).

Figure 1
Anesthesia with 3% sevoflurane for six hours induces caspase-3 activation and APP processing in the brain tissues of neonatal naïve mice

Given that the sevoflurane-induced caspase activation and apoptosis may lead to alterations in amyloid precursor protein (APP) processing in vitro 18, we asked whether sevoflurane can also alter APP processing in the brain tissues of neonatal mice. APP immunoblotting showed visible decreases in protein levels of APP-C83 and APP-C99 following the anesthesia with 3% sevoflurane for six hours as compared to control condition (Figure 1C). The quantification of the Western blot, by determining the ratio of APP-C-terminal fragments [APP-C83 fragment (10 kDa) and APP-C99 fragment (12 kDa)] to APP-FL (110 kDa)], revealed that the sevoflurane anesthesia led to a 45% and 33% decrease in the ratio of APP-C83 to APP-FL (Figure 1D, P = 0.0199) and APP-C99 to APP-FL (Figure 1E, P = 0.0471), respectively, as compared to the control condition in the brain tissues of neonatal naïve mice. These results suggest that sevoflurane can alter the APP processing by decreasing the levels of APP-C-terminal fragments (APP-C83 and APP-C99).

Next, we asked whether the anesthesia with same concentration (3%) of sevoflurane but for a shorter treatment time can also induce caspase-3 activation in the brain tissues of neonatal naïve mice. We found that the anesthesia with 3% sevoflurane for two hours did not increase caspase-3 activation (Figure 2A and 2B): 100% versus 128%, P = 0.074. These findings suggest that the commonly used inhalation anesthetic sevoflurane can induce caspase-3 activation in the brain tissue of neonatal mice in a time-dependent manner.

Figure 2
Anesthesia with 3% sevoflurane for two hours does not induce caspase-3 activation in the brain tissues of neonatal naïve mice

Finally, we investigated whether lower concentration of sevoflurane with same treatment time (six hours) can induce caspase-3 activation in the brain tissues of neonatal mice. We were able to show that the anesthesia with 2.1% sevoflurane for six hours induced caspase-3 activation in the brain tissues of neonatal naïve mice (Figure 3A and 3B): 100% versus 183%, P = 0.002, and of neonatal AD transgenic mice (Figure 3C and 3D): 100% versus 178%, P = 0.045. These results suggest that certain length of treatment time (e.g., six hours) of sevoflurane anesthesia may be needed before we can observe the sevoflurane-induced caspase activation in vivo.

Figure 3
Anesthesia with 2.1% sevoflurane for six hours induces caspase-3 activation in the brain tissues of neonatal naïve and AD transgenic mice

Sevoflurane induced a greater degree of caspase-3 activation in neonatal AD transgenic mice

We next asked whether the same sevoflurane anesthesia can also induce caspase-3 activation in the brain tissues of neonatal AD transgenic mice. The APPswe/PSEN1dE9 mouse is a particularly aggressive AD transgenic mouse model generated with mutant transgenes for APP (APPswe: KM594/5NL) and presenilin 1 (dE9: deletion of exon 9) 20. We therefore assessed and compared the effects of the anesthesia with 3% sevoflurane for six hours on caspase-3 activation in brain tissues of six-day-old naïve and AD transgenic mice. Caspase-3 immunoblotting showed visible increases in protein levels of caspase-3 fragment following the sevoflurane anesthesia as compared to the control condition (Figure 4A) in both neonatal naïve (lane 1 versus lane 2) and AD transgenic mice (lane 3 versus lane 4). The blot with caspase-3-fragment only is the same Western blot with extended exposure time during the development of the film. Moreover, caspase-3 immunoblotting showed that the sevoflurane anesthesia induced a more visible increase in the band of caspase-3 fragment in the neonatal AD transgenic mice than that in the neonatal naive mice. We quantified the Western blot using fold change. One fold in the manuscript referred to the ratio of activated (cleaved) caspase-3 fragment to FL-caspase-3 in control group of either the naïve mice or the AD transgenic mice. The quantification of the Western blot revealed that the sevoflurane anesthesia induced caspase-3 activation in the brain tissues of both neonatal naïve mice (one fold versus 2.13 folds, P = 0.003) and AD transgenic mice (one fold versus 2.45 folds, P = 0.001) (Figure 4B). Moreover, the sevoflurane anesthesia induced a greater degree of caspase-3 activation in the brain tissues of the neonatal AD transgenic mice than that in the neonatal naïve mice: 2.13 folds versus 1.48 folds, P = 0.008 (Figure 4B). These findings suggest that sevoflurane may cause a greater degree of neurotoxicity in neonatal AD transgenic mice than that in neonatal naïve mice.

Figure 4
Anesthesia with 3% sevoflurane for six hours induces a greater degree of caspase-3 activation in the brain tissues of neonatal AD transgenic mice than that in neonatal naïve mice

Sevoflurane induced more TUNEL positive cells in neonatal AD transgenic mice

Given that caspase-3 activation alone may not represent apoptotic cell damage 21, we also assessed effects of 3% sevoflurane plus 60% oxygen for six hours on cellular apoptosis using TUNEL study. We quantified the TUNEL positive cells using fold change. One fold in the manuscript referred to the ratio of TUNEL positive cells to the total cells in control group of either the naïve mice or the AD transgenic mice. We found that the sevoflurane anesthesia increased TUNEL positive cells (apoptosis) as compared to the control condition in brain tissues of neonatal naïve mice (Figure 5A and 5B; one fold versus 2.08 folds; P = 0.0001) and neonatal AD transgenic mice (Figure 5A and 5B; one fold versus 2.45 folds; P = 0.0004). Consistent with the findings in caspase-3 activation studies (Figure 4), the sevoflurane anesthesia induced more TUNEL positive cells (apoptosis) in the brain tissues of the neonatal AD transgenic mice than those in the neonatal naïve mice: 2.45 folds versus 2.08 folds, P = 0.0012 (Figure 5A and 5B). Furthermore, the double immunocytochemistry staining indicated that the majority of the TUNEL positive cells in the brain tissues of the neonatal AD transgenic mice following the sevoflurane anesthesia were neurons (NeuN staining), but not microglia cells (Iba1 staining) or astrocytes (Glial fibrillary acidic protein staining) (Figure 5C). Taken together, these findings suggest that sevoflurane can induce apoptosis in the neurons of brain of neonatal mice. Theses findings also suggest that the neonatal AD transgenic mice are more vulnerable to such sevoflurane-induced neurotoxicity.

Figure 5
Anesthesia with 3% sevoflurane for six hours induces more TUNEL positive cells in the brain tissues of neonatal AD transgenic mice than in neonatal naïve mice

Sevoflurane enhanced Aβ levels in neonatal naïve and AD transgenic mice

Sevoflurane has been shown to induce apoptosis, which then leads to Aβ accumulation in vitro and in the brain tissues of adult mice18. Given that sevoflurane can induce apoptosis, alter APP processing in the brain tissues of neonatal mice, we next asked whether sevoflurane can also enhance Aβ levels in brain tissues of these neonatal mice. The harvested brain tissues were subjected to Western blot analysis, by which antibody 6E10 was used to detect Aβ levels as described in our previous studies 16. Aβ immunoblotting revealed that the anesthesia of 3% sevoflurane for six hours caused visible increases in Aβ levels in the Western blot as compared to control condition (Figure 6A). Quantification of the Western blot revealed that the sevoflurane anesthesia increased Aβ levels in both neonatal naïve mice: 100% versus 401%, P = 0.023, and AD transgenic mice: 287% versus 491%, P = 0.042, as compared to control condition (Figure 6B). Note that the baseline Aβ level in the brain tissues of the neonatal AD transgenic mice was higher than those in the neonatal naïve mice: 100% versus 287%, P = 0.009. Furthermore, ELISA sandwich identified that the sevoflurane anesthesia increased Aβ42 levels: 100% versus 233%, P = 0.007 (Figure 6C), but not Aβ40 levels (Figure 6D), in the brain tissues of neonatal AD transgenic mice, but not neonatal naïve mice (data not shown). In addition, we were able to show that the anesthesia with 2.1% sevoflurane for six hours also increased levels of Aβ42 in the brain tissues of neonatal AD transgenic mice (data not shown). Taken together, these results suggest that sevoflurane may specifically increase Aβ42 levels in brain tissues of neonatal mice.

Figure 6
Anesthesia with 3% sevoflurane for six hours increases Aβ levels in the brain tissues of neonatal naïve and AD transgenic mice

IP3R antagonist 2-aminoethoxydiphenyl borate (2-APB) attenuated the sevoflurane-induced caspase-3 activation and the increases in Aβ levels

The underlying mechanism by which inhalation anesthetics induce apoptosis and enhance Aβ accumulation is largely unknown. Several studies have shown that the inhalation anesthetic isoflurane may elevate cytosolic calcium levels, leading to apoptosis 22,23. We therefore asked whether the sevoflurane-induced apoptosis and Aβ accumulation in the neonatal mice are also associated with IP3R. For this purpose, we assessed the effects of the IP3R antagonist 2-APB on the sevoflurane-induced caspase-3 activation and Aβ accumulation in the brain tissues of neonatal naïve mice. As can be seen in Figure 7A, anesthesia with 3% sevoflurane for six hours led to caspase-3 activation as compared to control condition. 2-APB 5 mg/kg (lanes 6 to 8) and 10 mg/kg (lanes 4 and 5) attenuated the sevoflurane-induced caspase-3 activation in a dose-dependent manner. Quantification of the Western blot showed that the sevoflurane anesthesia induced caspase-3 activation: 100% versus 356%, P = 0.002 (Figure 7B). The IP3R antagonist 2-APB attenuated the sevoflurane-induced caspase-3 activation in a dose-dependent manner, 5 mg/kg 2-APB (gray bar): 356% versus 149%, P = 0.001; 10 mg/kg 2-APB (net bar): 356% versus 115%, P = 0.005. Moreover, we were able to show that 2-APB also attenuated the sevoflurane-induced increases in Aβ levels (Figure 7C and 7D), 304% versus 157%, P = 0.042. These findings suggest that IP3R may be involved in the sevoflurane-induced caspase activation, apoptosis and Aβ accumulation.

Figure 7
2-APB attenuates the sevoflurane-induced caspase-3 activation and Aβ accumulation in the brain tissues of neonatal naïve mice

Sevoflurane increased TNF-α levels in neonatal AD transgenic mice

Increasing evidence suggests that neurons and microglia cells can produce inflammatory mediators including pro-inflammatory cytokine TNF-α 24. TNF-α is a death-inducing cytokine, which can induce both apoptosis and necrosis through receptor-interacting protein 3, a protein kinase 25. We therefore assessed the effects of sevoflurane on neuroinflammation by determining TNF-α levels in brain tissues of neonatal naïve and AD transgenic mice following the anesthesia of 3% sevoflurane for six hours. We were able to show that the sevoflurane anesthesia increased protein levels (Figure 8A and 8B, 100% versus 219%, P = 0.001) and messenger RNA levels (Figure 8C, P = 0.002) of TNF-α levels in the brain tissues of the neonatal AD transgenic mice but not in the brain tissues of the neonatal naïve mice (Figure 8D, 8E and 8F) in current experiments. These results suggest that sevoflurane may increase the TNF-α levels by enhancing its generation in the brain tissues of the neonatal AD transgenic mice, leading to neuroinflammation.

Figure 8
Anesthesia with 3% sevoflurane for six hours increases TNF-α levels in the brain tissues of neonatal AD transgenic mice

Discussion

Several studies have suggested that anesthesia may be a significant risk factor in children for the later development of learning disabilities and/or deviant behavior 1,2. However, it is still possible that the need for anesthesia is a marker for other unidentified factors, rather than anesthesia itself, that contributes to the development of the learning disability and/or deviant behavior. Thus, it is important to assess the effects of sevoflurane, the most commonly used inhalation anesthetic (especially in pediatric patients), on the biochemical changes that are associated with cognitive dysfunction in neonatal mice, which include apoptosis, Aβ accumulation, and neuroinflammation.

We have shown in the current studies that anesthesia with 3% or 2.1% sevoflurane for six hours, but not 3% sevoflurane for two hours, can induce caspase activation and apoptosis, alter APP processing, and increase Aβ levels in the brain tissues of the neonatal naïve and the AD transgenic mice. These findings suggest that sevoflurane can induce caspase activation and apoptosis with a time-dependent manner. The sevoflurane anesthesia may specifically induce apoptosis in neurons and increase Aβ42 levels. Moreover, the sevoflurane anesthesia may induce a greater degree of caspase activation and apoptosis in the brain tissues of the neonatal AD transgenic mice [B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J] than that in the neonatal naïve mice. Finally, the sevoflurane anesthesia can induce neuroinflammation by increasing pro-inflammatory cytokine TNF-α in the brain tissues of the AD transgenic mice, but not in the brain tissues of the naïve mice. Collectively, these findings suggest that the sevoflurane anesthesia may lead to neurotoxicity by inducing apoptosis and neuroinflammation and by increasing Aβ levels in the brain tissues of the neonatal mice, and the overexpression of AD genes and/or elevated Aβ levels in the AD transgenic mice could potentiate such neurotoxicity. Pending on human studies, these findings raise novel concerns regarding the use of sevoflurane, the mostly commonly used inhalation anesthetic, in individuals with increased Aβ burden, including patients with Down's syndrome, the unaffected carriers of APP or presenilin gene mutations, and the late-onset AD risk factor, APOE-ε4, that increase Aβ accumulation in the brain. The future studies should include further characterization of the sevoflurane-induced neurotoxicity in animals and humans, as well as determination of the causative link between sevoflurane anesthesia, apoptosis and Aβ levels.

Consistent with our in vitro studies 18, the sevoflurane anesthesia can reduce the levels of APP-C-terminal fragments including APP-C83 and APP-C99. Given that APP-C83 and APP-C99 are metabolized by γ-secretase [reviewed in 10,12], these findings suggest that sevoflurane may increase the activity of γ-secretase, leading to reductions in the levels of APP-C83 and APP-C99. Future studies will include the systematically investigating the effects of sevoflurane and other anesthetics on the levels of γ-secretase components, e.g., PS1, nicastrin, PEN-2, and APH 26-30, and the γ-secretase activity 31”.

The mechanisms by which the sevoflurane anesthesia can only induce neuroinflammation in the brain tissues of AD transgenic mice, but not neonatal naïve mice, in the current experiment are not well understood. Both Aβ accumulation and neuroinflammation are important parts of AD neuropathogenesis, and they can potentiate each other's neurotoxicity [32,33; reviewed in 11]. It is therefore conceivable that the higher baseline levels of Aβ in the AD transgenic mice can facilitate the effects of sevoflurane on increasing TNF-α levels, leading to apparent neuroinflammation in the current experiment. Future studies should include systematic assessment of dose- and time-dependent effects of sevoflurane on levels of TNF-α and other pro-inflammation cytokines (e.g., Interleukin-6) in both naïve and the AD transgenic mice to further test this hypothesis.

Even though the baseline Aβ levels in the brain of B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J mice were higher than those in the brain of the naïve mice, the sevoflurane anesthesia did not lead to significantly greater increases of Aβ levels in the brain tissues of the AD transgenic mice than the naïve mice. This could be due to the ceiling effects of the sevoflurane-induced increases in Aβ levels. It is also possible that sevoflurane may enhance Aβ levels through non-apoptosis pathway. A recent study34 has shown that cellular stress induced by glucose deprivation can lead to increases in levels of β-secretase (the enzyme to generate Aβ) and Aβ through phosphorylation of the translation initiation factor eIF2α independent of caspase activation and apoptosis. The future studies should include the determination of whether anesthetics can also increase Aβ generation through this translation mechanism.

Nevertheless, the ELISA studies showed that the sevoflurane anesthesia enhanced levels of Aβ42, but not Aβ40, in the brain tissues of neonatal AD transgenic mice, but not neonatal naïve mice. These findings suggest that the sevoflurane anesthesia may lead to a greater degree of Aβ accumulation in the brain tissues of the AD transgenic mice than that in the naive mice. However, it is still possible that the ELISA kit used in the current experiment was not sensitive enough to detect non-human Aβ levels in the brain tissues of neonatal naïve mice.

The IP3 receptor, located in the endoplasmic reticulum membrane, regulates release of calcium from the endoplasmic reticulum to the cytoplasm [35, reviewed in 36]. We have found that the IP3R antagonist 2-APB 37 can attenuate the sevoflurane-induced caspase-3 activation and Aβ accumulation in the neonatal naïve mice. These results have suggested that sevoflurane may act on IP3R to affect calcium homeostasis, leading to apoptosis and Aβ accumulation. Moreover, these findings imply that 2-APB may be able to prevent or reduce the sevoflurane-induced neurotoxicity. However, it is expected that IP3 antagonism may lead to many other effects. Therefore, a specific effect of IP3 antagonism on caspase-3 activation and Aβ accumulation cannot be made from the results in current studies. Future studies should include assessing the effects of other IP3 antagonists, e.g., xestospongin C, on the sevoflurane-induced caspase activation and Aβ accumulation to further test this hypothesis.

One caveat of the current study is that we did not measure blood gas in each of the mice following the anesthesia with 3% sevoflurane plus 60% oxygen for six hours. This is because the same sevoflurane anesthesia has been shown not to significantly alter blood gas and brain blood flow 6, which is consistent with our pilot studies (See table, Supplemental Digital Content, which is a table listing the values of mice blood gas measured in this study). In addition, the findings that anesthesia with 3% sevoflurane plus 60% oxygen for two hours does not induce casase-3 activation further suggest that it is the sevoflurane, but not physiological changes (e.g., alterations in oxygen, carbon dioxide or pH in blood), that causes the neurotoxicity. However, it is still possible that the combination of sevoflurane and the anesthesia-induced hypoxia and/or acidosis induces the neurotoxicity in some mice in the present studies.

There is currently no satisfactory way to extrapolate the findings of apoptosis, Aβ accumulation, and neuroinflammation in the mouse brain to the human brain. Thus, the findings from the current studies do not present any direct evidence that inhalation anesthetic sevoflurane can cause harm to the human brain. Determination of the in vivo relevance of sevoflurane on neurotoxicity in the human brain and the longitudinal learning/memory studies will be necessary before we can conclude that anesthetic sevoflurane can cause neurotoxicity in humans.

In conclusion, we have shown that sevoflurane, the most commonly used inhalation anesthetic, can induce caspase activation and apoptosis, alter APP processing and increase Aβ levels in the brain tissues of neonatal naïve and AD transgenic mice. Importantly, more severe apoptosis and Aβ accumulation, as well as neuroinflammaion may occur in the brain tissues of the neonatal AD transgenic mice as compared to the neonatal naïve mice. These findings suggest that sevoflurane may cause neurotoxicity in neonatal mice, and that the overexpression of mutated AD genes, i.e., presenilin 1 and APP, and/or elevated Aβ levels in the AD transgenic mice may potentiate such neurotoxicity. We have further found that the sevoflurane-induced neurotoxicity may be associated with IP3R, and 2-APB, one of the IP3R antagonists, may attenuate the sevoflurane-induced neurotoxicity. Given the findings that anesthesia could be a risk factor for the development of a learning disability in children and that sevoflurane is used extensively in pediatric patients, these current findings will hopefully lead to further studies to determine the potential neurotoxicity of sevoflurane, including confirmation studies in humans.

Supplementary Material

Supp1

Acknowledgments

This study was supported by National Institutes of Health grants (Bethesda, Maryland) (K08 NS048140, R21 AG029856 and R01 GM088801); American Geriatrics Society Jahnigen Award (New York, NY); Investigator-Initiated Research Grant from Alzheimer's Association (Chicago, IL) to Dr. Zhongcong Xie. The cost of anesthetic sevoflurane was provided by the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School. The authors would like to thank Chuxiong Pan, M.D., M.S. and Jun Zhang, M.D., Ph.D., the research fellows in the Department of Anesthesia, Critical Care and Pain Medicine in Massachusetts General Hospital and Harvard Medical School, for the technical support and scientific discussion.

Footnotes

Summary Statement: Sevoflurane induces apoptosis, Aβ accumulation, and neuroinflammation in brain tissues of neonatal naïve and Alzheimer's disease transgenic mice with a greater degree of neurotoxicity in the Alzheimer's disease transgenic mice.

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