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. Author manuscript; available in PMC: 2014 Apr 2.
Published in final edited form as: Curr Alzheimer Res. 2011 Nov;8(7):741–752. doi: 10.2174/156720511797633223

The Potential Dual Effects of Anesthetic Isoflurane on Aβ-Induced Apoptosis

Zhipeng Xu 1,2,#, Yuanlin Dong 1,2,#, Xu Wu 1,2,3, Jun Zhang 1,2,4, Sayre McAuliffe 1,2, Chuxiong Pan 1,2,5, Yiying Zhang 1,2, Fumito Ichinose 6, Yun Yue 7, Zhongcong Xie 1,2,*
PMCID: PMC3973025  NIHMSID: NIHMS564197  PMID: 21244349

Abstract

β-amyloid protein (Aβ)-induced neurotoxicity is the main component of Alzheimer’s disease (AD) neuropathogenesis. Inhalation anesthetics have long been considered to protect against neurotoxicity. However, recent research studies have suggested that the inhalation anesthetic isoflurane may promote neurotoxicity by inducing apoptosis and increasing Aβ levels. We therefore set out to determine whether isoflurane can induce dose- and time-dependent dual effects on Aβ-induced apoptosis: protection versus promotion. H4 human neuroglioma cells, primary neurons from naïve mice, and naïve mice were treated with Aβ and/or isoflurane, and levels of caspase-3 cleavage (activation), apoptosis, Bcl-2, Bax, and cytosolic calcium were determined. Here we show for the first time that the treatment with 2% isoflurane for six hours or 30 minutes potentiated, whereas the treatment with 0.5% isoflurane for six hours or 30 minutes attenuated, the Aβ-induced caspase-3 activation and apoptosis in vitro. Moreover, anesthesia with 1.4% isoflurane for two hours potentiated, whereas the anesthesia with 0.7% isoflurane for 30 minutes attenuated, the Aβ-induced caspase-3 activation in vivo. The high concentration isoflurane potentiated the Aβ-induced reduction in Bcl-2/Bax ratio and caused a robust elevation of cytosolic calcium levels. The low concentration isoflurane attenuated the Aβ-induced reduction in Bcl-2/Bax ratio and caused only a mild elevation of cytosolic calcium levels. These results suggest that isoflurane may have dual effects (protection or promotion) on Aβ-induced toxicity, which potentially act through the Bcl-2 family proteins and cytosolic calcium. These findings would lead to more systematic studies to determine the potential dual effects of anesthetics on AD-associated neurotoxicity.

Keywords: Anesthesia, Alzheimer’s disease, isoflurane, apoptosis, β-Amyloid protein, dual effects, cytosolic calcium

INTRODUCTION

Alzheimer’s disease (AD) is an insidious and progressive neurodegenerative disorder, which accounts for the vast majority of dementia and is one of the greatest public health problems in the United States. AD is characterized by global cognitive decline, and robust accumulation of amyloid deposits and neurofibrillary tangles in the brain [reviewed in 1].

Aβ, the key component of senile plaques in AD patients [2–4], was first isolated from meningovascular amyloid deposits in AD and Down syndrome [2, 5]. Genetic evidence, confirmed by neuropathological and biochemical findings, indicates that excessive production and/or accumulation of Aβ play a fundamental role in the pathology of AD [reviewed by 6, 7]. Moreover, increasing evidence suggests a role for caspase activation and apoptosis in AD neuropathogenesis [reviewed in 8, 9]. Finally, Aβ has been reported to induce caspase activation and apoptosis [10–16].

The commonly used inhalation anesthetic isoflurane has recently been reported to induce caspase activation and apoptosis, affect APP processing, increase Aβ levels, and enhance Aβ aggregation [17–22, reviewed in 23]. However, other reports have suggested that isoflurane may protect against apoptosis [24–33]. The reason for these different findings of isoflurane is not clear.

Several studies have suggested that isoflurane may induce apoptosis by facilitating calcium release from endoplasmic reticulum (ER) [18, 22, 34, reviewed in 35]. Recent research works have illustrated that ER-associated cytosolic calcium level may be involved in protection of apoptosis as well [36]. Other studies have shown that a short duration of isoflurane treatment can increase anti-apoptotic factor Bcl-2 levels in rats [25], whereas a long duration of isoflurane treatment can decrease Bcl-2 levels in cultured cells [18].

Given these observations, the aim of our current study was to assess whether different isoflurane treatments, e.g., high concentration and/or long duration versus low concentration and/or short duration, can have different effects on levels of cytosolic calcium, pro-apoptotic factor Bax, anti-apoptotic factor Bcl-2, caspase-3 activation, and apoptosis in naïve H4 human neuroglioma cells (H4 naïve cells), primary neurons, and brain tissues of mice. The dual effects of isoflurane (promotion versus protection) on the Aβ-induced caspase-3 activation and apoptosis were observed in vitro and in vivo. The other cell lines and other apoptotic insults e.g., ischemia, which may have different responses to isoflurane treatments, were not investigated in the current experiments.

MATERIAL AND METHODS

Cell Lines

We employed H4 human neuroglioma cells (H4 naïve cells) in the experiments. All cell lines were cultured in DMEM (high glucose) containing 9% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.

Primary Neurons

The protocol was approved by the Massachusetts General Hospital Standing Committee on Animals (Boston, Massachusetts) on the Use of Animals in Research and Teaching. Naïve mice with a gestation stage of day 15 were euthanized with carbon dioxide. We then performed a cesarean section to pull out the embryos and decapitate them in a 100-mm dish of phosphate-buffered saline. We placed the head on the top of a 100-mm dish and dissected out the cortices, removed the meninges, and placed the cortices into another 100-mm dish of phosphate-buffered saline. The neurons were dissociated by trypsinization and trituration. The dissociated neurons were resuspended in serum-free B27/neurobasal medium and were placed into six well plates with a confluent rate of 50%. Seven to ten days after the harvest, the neurons were exposed to isoflurane. The B27/neurobasal medium (Invitrogen, Carlsbad, CA) has shown excellent long-term viability of primary neurons. The glial cell growth at five days is reduced to less than 0.5% for a nearly pure neuronal population in the medium [37]. The 0.5 mM L-glutamine and 25 μM glutamate added to the B27/neurobasal medium can reduce glial cell growth.

Treatments for cells and primary neurons. The cells and primary neurons were treated with 2% isoflurane for six hours or 30 minutes (high concentration and long or short duration treatment) or 0.5% isoflurane for six hours or 30 minutes (low concentration and long or short duration treatment). Control conditions included 5% CO2 plus 21% O2 for six hours or 30 minutes, respectively, which did not affect caspase-3 activation and apoptosis (Data not shown). Treatment with 7.5 uM Aβ40 plus 7.5 uM Aβ42 for six hours was used to induce caspase-3 activation and apoptosis in the cells or neurons as previously described [20].

Lysis of Cells or Neurons and Protein Amount Quantification

The pellets of the cells or the neurons were detergent-extracted on ice using immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Non-idet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A). The lysates were collected, centrifuged at 12,000 × g for 10 min, and quantified for total proteins using the bicinchoninic acid protein assay kit (Pierce, Iselin, NJ).

ICV Injection of Aβ and Mice Anesthesia

Eight-month-old C57/BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were used in the experiments. All surgical procedures were performed with care to minimize pain and discomfort. The mice were anesthetized with an intraperitoneal (i.p.) injection of 1% avertin (10ml/kg body weight). Avertin has not been reported to interact with isoflurane on caspase activation and apoptosis. A Hamilton syringe was placed perpendicular to the skull at the following coordinate: anteroposterior, 0.5 mm; mediolateral, 1 mm; dorsoventral, 2.0 mm relative to bregma as described before [38] with modification. 250 ng human Aβ42 or DMSO (dissolved in artificial cerebrospinal fluid to make a working solution of 5 ul) was slowly injected at a rate of 0.5 ul/min; the needle was left undisturbed for 2 minutes before removal. The burr hole was covered by bone wax and the scalp was sutured. Then the mice received 1.4% isoflurane in 100% oxygen for 2 hours or 0.7% isoflurane in 100% oxygen for 30 minutes. The control group received 100% oxygen at an identical flow rate for either 2 hours or 30 minutes in an identical chamber. The mice breathed spontaneously, the concentration of isoflurane, O2 and CO2 were measured continuously (Datex, Tewksbury, MA). The temperature of the anesthetizing chamber was controlled to maintain rectal temperature of the animals at 37 ± 0.5°C. The isoflurane anesthesia did not significantly affect blood pressure or blood gas of mice [21]. Anesthesia was terminated by discontinuing isoflurane and placing animals in a chamber containing 100% oxygen until 20 minutes after the return of the righting reflex. They were then returned to individual home cage until sacrifice. Mice were euthanized by decapitation six hours after the anesthesia. The brain was removed rapidly and prefrontal cortex was dissected out and frozen in liquid nitrogen for subsequent processing for the determination of caspase-3 fragment levels.

Western Blots Analysis

The cells, neurons, or brain tissues of the mice were harvested at the end of the experiments and were subjected to Western blots analyses as described by Xie et al. [20]. A caspase-3 antibody (1:1,000 dilution; Cell Signaling Technology, Inc. Beverly, MA) was used to recognize the caspase-3 fragment [(17–20 kDa in vitro) and 12 kDa in vivo as previously demonstrated 39], which results from cleavage at the asparate position 175 and caspase-3 FL (35 – 40 kDa). Bax antibody (1:1,000 dilution; Cell Signaling Technology, Inc. Beverly, MA) was used to recognize Bax (20 kDa). Bcl-2 antibody (1:1,000 dilution; Cell Signaling Technology, Inc.) was used to recognize Bcl-2 (28 kDa). Antibody to non-targeted protein β-Actin (42 kDa, 1:5,000, Sigma, St. Louis, MO) was used to control for loading differences in total protein amounts. Each band in the Western blot represents an independent experiment. We have averaged the results from three to ten independent experiments. The intensity of signals in each Western blot was analyzed using the National Institute of Health image program (National Institute of Health Image 1.62, Bethesda, MD). We quantified the Western blots using two steps. First, we used levels of β-Actin to normalize (e.g., determine ratio of the amount of FL-caspase-3 to the amount of β-Actin) levels of Bax, Bcl-2, and caspase-3 to control for any loading differences in total protein amounts. Second, we presented changes in levels of Bax, Bcl-2, and caspase-3 in treated cells or mice as percentages of those in cells or mice from the control condition.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining-TMR red kit (Roche Diagnostics, Mannheim, Germany) was used for TUNEL staining. Specifically, cells or neurons were grown on coverslips overnight in an incubator. The cells or neurons were fixed in 4% paraformaldehyde for 30 minutes following the treatment with isoflurane plus Aβ or the control conditions. The coverslips were washed with phosphate buffered saline and were incubated with a permeabilization solution (0.2% TritonX - 100 in 0.1% sodium citrate) at 4 °C for 10 minutes, and then with a TUNEL reaction mixture for one hour at 37 °C in a humidified dark chamber. The negative control was prepared by incubating the coverslips in label solution only. The coverslips for the positive controls were incubated with DNase I recombinant. Finally, the coverslips were incubated with 10 μg/ml Hoechst 33342 in a humidified dark chamber for 10 minutes. Samples were then analyzed in a mounting medium under a fluorescence microscope. The TUNEL-positive cells were counted manually in five randomly selected areas under a 20X objective microscope lens by an investigator who was blinded to the experiments. The positive TUNEL cells were expressed as the fold of the number of nuclei. One fold of TUNEL positive cells in the figure refers to the control levels for the purposes of comparison to experimental conditions.

Cytosolic Calcium Measurement

Cytosolic calcium levels were determined as described by Wei et al. [34]. Specifically, naïve H4 cells were loaded with Fura-2 (Molecular probe, Eugene, OR), perfused with Tyrode buffer, and [Ca2+]i transients were recorded as changes in Fura-2 ratio (340/380 nm) using a spectrofluoroscope system (Ionoptix, Milton, MA). The cells were exposed to 0.57 mM isoflurane (2%) or 0.14 mM isoflurane (0.5%) solution for 100 seconds.

Statistics

Given the presence of background caspase-3 activation and apoptosis in cells cultured in serum free media, we did not use absolute values to describe changes in caspase-3 activation and apoptosis. Instead, changes in caspase-3 activation and apoptosis were presented as a percentage of those of the control group. 100% caspase-3 activation or cell viability refers to control levels for purposes of comparison to experimental conditions. Data were expressed as mean ± S.D. The number of samples varied from 3 to 10, and the samples were normally distributed. We used a two-tailed t-test to compare the difference between the experimental groups. P-values less than 0.05 (* or #) and 0.01 (** or ##) were considered statistically significant.

RESULTS

Low Concentration and Short Duration of Isoflurane Treatment Attenuates the Aβ-Induced Apoptosis In Vitro and In Vivo

We previously reported that the common inhalation anesthetic isoflurane can induce caspase activation and apoptosis in vitro [19, 20, 40] and in vivo [21]. However, many other studies have shown that isoflurane may protect against apoptosis [24–33]. One of the reasons for this discrepancy could be that different isoflurane treatments (e.g., different duration and concentration) may have different effects on caspase activation and apoptosis. Thus, we set out to determine the effects of low (0.5%) and high (2%) concentrations of isoflurane with long (six hours) and short (30 minutes) treatment times on the Aβ-induced apoptosis in vitro and in vivo.

Since caspase-3 activation is one of the final steps of cellular apoptosis [41], we first assessed the effects of isoflurane plus Aβ on caspase-3 activation by quantitative Western blots analyses. The H4 naïve cells were treated with 7.5 uM Aβ40 plus 7.5 uM Aβ42 for 6 hours, or the Aβ treatment plus 0.5% isoflurane for the first 30 minutes then the Aβ treatment alone for the rest of the remaining 5.5 hours. The cells were harvested at the end of the experiment and were subjected to Western blot analysis. Caspase-3 immunoblotting revealed that the Aβ treatment induced caspase-3 activation Fig. (1A) as evidenced by increased ratios of cleaved (activated) caspase-3 fragment (17 kDa) to full-length (FL) (35 – 40 kDa) caspase-3. The treatment with 0.5% isoflurane for 30 minutes Fig. (1A) attenuated the Aβ-induced caspase-3 activation. Quantification of the Western blots, based on the ratio of caspase-3 fragment to FL caspase-3, revealed that the Aβ treatment led to caspase-3 activation as compared to control condition: 100% versus 167% (* P = 0.043), and the 0.5% isoflurane treatment attenuated the Aβ-induced caspase-3 activation: 167% versus 79% (# P = 0.028). These findings suggest that a low concentration and a short duration of isoflurane treatment may attenuate the Aβ-induced caspase-3 activation in the H4 naïve cells.

Fig. 1.

Fig. 1

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Low concentration and short duration of isoflurane treatment attenuates the Aβ-induced caspase-3 activation and apoptosis in H4 naïve cells and in brain tissues of naïve mice. A. Aβ treatment (lanes 3 and 4) induces caspase-3 cleavage (activation) by increasing caspase-3 fragment levels and decreasing FL-caspase-3 levels as compared to the control condition (lanes 1 and 2) in Western blots. 0.5% isoflurane (lanes 7 and 8) attenuates the Aβ-induced caspase-3 activation as compared to Aβ treatment (lanes 3 and 4) alone. B. Quantification of the Western blots shows that Aβ treatment (gray bar) induces caspase-3 activation, assessed by quantifying the ratio of caspase-3 fragment to FL-caspase-3, as compared to that of the control condition (white bar). 0.5% isoflurane (striped bar) attenuates the Aβ-induced caspase-3 activation as compared to Aβ treatment (gray bar). C. The Aβ treatment (column 3) increases the amount of TUNEL positive cells (apoptosis) as compared to the control conditions (column 1) in H4 naïve cells. Treatment with Aβ plus 0.5% isoflurane (column 4) decreases the amount of TUNEL positive cells as compared to the Aβ treatment alone (column 3). D. Quantification of the TUNEL image shows that the Aβ treatment (gray bar) increases the amount of TUNEL positive cells (apoptosis) as compared to control conditions (white bar) in H4 naïve cells. The treatment with Aβ plus 0.5% isoflurane induces fewer TUNEL positive cells (striped bar) as compared to Aβ treatment (gray bar) alone in H4 naïve cells. E. The Western Blots show that 0.7% isoflurane (lanes 4 to 6) attenuates the Aβ-induced caspase-3 activation by decreasing levels of caspase-3 fragment as compared to Aβ treatment alone (lanes 1 to 3) in vivo. F. Quantification of the Western blots shows that 0.7% isoflurane (black bar) attenuates the Aβ-induced caspase-3 activation (white bar).

Given that caspase-3 activation alone may not represent apoptotic cell damage [42], we also assessed the effects of the Aβ treatment and the Aβ treatment plus 0.5% isoflurane on cellular apoptosis using TUNEL studies. We found that the Aβ treatment increased TUNEL positive cells (apoptosis) as compared to the control condition Fig. (1C and ID): 1 fold versus 6.92 fold (** P = 0.001), and 0.5% isoflurane attenuated the Aβ-induced apoptosis: 6.92 fold versus 3.74 fold (## P = 0.003). These findings suggest that a low concentration and a short duration of isoflurane treatment may attenuate the Aβ-induced apoptosis.

Finally, we determined the in vivo relevance of these in vitro findings. 250 ng Aβ42 was administrated to the mouse brain through intraeerebroventricular (ICV) injection to create an in vivo model of Aβ-induced caspase-3 activation as evidenced by the fact that the Aβ treatment increased the levels of activated (cleaved) caspase-3 fragment Fig. (IE). Then, the Aβ-treated mice were exposed to 0.7% isoflurane or a control condition for 30 minutes. The brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis. Caspase-3 immunoblotting showed that the treatment of 0.7% isoflurane for 30 minutes attenuated the Aβ-induced increases in the levels of caspase-3 fragment as compared to control condition Fig. (IE). The quantification of the Western blot showed that the anesthesia with 0.7% isoflurane for 30 minutes decreased the Aβ-induced caspase-3 activation in the brain tissues of naïve mice as compared to that of the control condition: 100% versus 22%, * P = 0.014 Fig. (IF). These in vivo findings have further illustrated that a low concentration and a short duration of isoflurane treatment can attenuate the Aβ-induced neurotoxicity.

High Concentration and Long Duration of Isoflurane Treatment Potentiates the Aβ-Induced Apoptosis In Vitro and In Vivo

Next, we asked whether a high concentration and a long duration of isoflurane treatment may have different effects on the Aβ-induced apoptosis. We thus assessed the effects of a treatment with 2% isoflurane for six hours on the Aβ-induced apoptosis in H4 naïve cells. We found that the treatment with 7.5 uM Aβ40 plus 7.5 uM Aβ42 for six hours induced caspase-3 activation Fig. (2A and 2B) and the treatment with 2% isoflurane for six hours potentiated the Aβ-induced caspase-3 activation: 272% versus 323%, # P = 0.04. We further found that Aβ led to apoptosis as compared to the control condition in the TUNEL studies: one fold versus 6.21 fold, ** P = 0.002. Finally, the treatment with 2% isoflurane for six hours potentiated the Aβ-induced apoptosis: 7.91 fold versus 6.21 fold, # P = 0.01. These results suggest that a high concentration and a long duration of isoflurane treatment may potentiate the Aβ-induced apoptosis.

Fig. 2.

Fig. 2

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High concentration and long duration of isoflurane treatment potentiates the Aβ-induced caspase-3 activation and apoptosis in H4 naïve cells and brain tissues of naïve mice. A. Aβ treatment (lanes 4 and 6) induces caspase-3 activation as compared to control condition (lanes 1 to 3) in Western blots analysis. 2% isoflurane (lanes 10 to 12) potentiates the Aβ-induced caspase-3 activation as compared to Aβ treatment alone (lanes 4 to 6). B. Quantification of the Western blots shows that Aβ treatment (gray bar) induces caspase-3 activation as compared to control condition (white bar). 2% isoflurane (striped bar) potentiates the Aβ-induced caspase-3 activation as compared to Aβ treatment (gray bar). C. The Aβ treatment (column 3) increases the amount of TUNEL positive cells (apoptosis) as compared to the control conditions (column 1). 2% isoflurane plus the Aβ treatment (column 4) induces a greater increase in the TUNEL positive cells as compared to the Aβ treatment alone (column 3). D. Quantification of the TUNEL image shows that the Aβ treatment (gray bar) increases TUNEL positive cells (apoptosis) as compared to the control conditions (white bar). The treatment with Aβ plus 2% isoflurane induces a greater amount of TUNEL positive cells (striped bar) as compared to Aβ treatment (gray bar) alone. E. 1.4% isoflurane (lanes 4 to 6) potentiates the Aβ-induced caspase-3 activation by increasing the levels of caspase-3 fragment as compared to Aβ treatment alone (lanes 1 to 3) in the Western blots. F. Quantification of the Western blots shows that 1.4% isoflurane (black bar) potentiates the Aβ-induced caspase-3 activation (white bar).

The in vivo relevance studies showed that the anesthesia with 1.4% isoflurane for 2 hours potentiated the Aβ-induced increases in the levels of caspase-3 fragment in the brain tissues of mice as compared to that of the control condition Fig. (2E and 2F): 100% versus 168%, * P = 0.046. Taken together, these in vitro and in vivo results have revealed that the common inhalation anesthetic isoflurane may induce dose- and time-dependent dual effect, protection versus promotion, on the Aβ-induced neurotoxicity.

It should be emphasized that we only observed the dual effects of isoflurane in the naïve H4 cells and primary neurons in the current experiments. It is possible that isoflurane may show different effects in other cell lines. Moreover, we only demonstrated that isoflurane promoted or protected the Aβ-induced caspase activation and apoptosis with a dose- and time-dependent manner in the current experiments. It is also possible that isoflurane may not show such dual effects towards to other apoptotic insults, e.g., ischemia.

The effects of low concentration plus long duration or high concentration plus short duration isoflurane treatment on Aβ-induced caspase-3 activation.

Next, we asked whether the isoflurane treatment with either low concentration (0.5%) plus long duration (six hours) or high concentration (2%) plus short duration (30 minutes) can affect the Aβ-induced caspase-3 activation. As expected, the treatment with 7.5 uM Aβ40 plus 7.5 uM Aβ42 for six hours induced caspase-3 activation in the H4 naïve cells Fig. (3A and 3B): 225% versus 100%, P = 0.005. Whereas the treatment with 0.5% isoflurane for six hours alone did not induce caspase-3 activation (P = 0.284), this isoflurane treatment attenuated the Aβ-induced caspase-3 activation: 227% versus 80%, ## P = 0.002 Fig. (3A and 3B). Moreover, we were able to show that the treatment with 2% isoflurane for 30 minutes can promote the Aβ-induced caspase-3 activation: 174% versus 266%, ## P = 0.002 Fig. (3C and 3D), whereas the treatment with 2% isoflurane alone for 30 minutes did not induce the caspase-3 activation (P = 0.536). These results suggest that the dual effects of isoflurane (promotion versus protection) on the Aβ-induced caspase-3 activation may be dependent on concentration, but not duration.

Fig. 3.

Fig. 3

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The effects of isoflurane treatment with low concentration plus long duration or high concentration plus short duration on the Aβ-induced caspase-3 activation in H4 naïve cells. A. Aβ treatment (lanes 4 to 6) induces caspase-3 cleavage (activation) by increasing caspase-3 fragment levels and decreasing FL-caspase-3 levels as compared to the control condition (lanes 1 to 3) in Western blots. The treatment with 0.5% isoflurane for six hours (lanes 7 to 9) attenuates the Aβ-induced caspase-3 activation as compared to Aβ treatment (lanes 4 to 6) alone. B. Quantification of the Western blots shows that Aβ treatment (gray bar) induces caspase-3 activation as compared to control condition (white bar). The treatment with 0.5% isoflurane for six hours (striped bar) attenuates the Aβ-induced caspase-3 activation as compared to that of Aβ treatment alone (gray bar). C. Aβ treatment (lanes 4 to 6) induces caspase-3 activation as compared to the control condition (lanes 1 to 3) in Western blots. Treatment with 2% isoflurane for 30 minutes (lanes 7 to 9) promotes the Aβ-induced caspase-3 activation as compared to the Aβ treatment alone (lanes 4 to 6). D. Quantification of the Western blots shows that Aβ treatment (gray bar) induces caspase-3 activation as compared to the control condition (white bar). Treatment with 2% isoflurane for 30 minutes (striped bar) promotes the Aβ-induced caspase-3 activation as compared to the Aβ treatment alone (gray bar).

Low Concentration and Short Duration of Isoflurane Treatment Attenuates the Aβ-induced Decrease in the Bcl-2/Bax Ratio in Primary Neurons from Naïve Mice

Given that isoflurane may induce dual effects to either promote or protect the Aβ-induced neurotoxicity, we next investigated the potential underlying molecular mechanisms. Bcl-2 protein family members, including Bcl-2 and Bax, can regulate apoptosis by modulating outer mitochondrial membrane permeability [43, 44]. The pro-apoptotic protein Bax can promote a sustained and complete calcium release from the ER, and therefore cause an extreme/sustained elevation of cytosolic calcium levels, leading to apoptosis. In contrast, the anti-apoptotic protein Bcl-2 can induce an unregulated ER calcium leak, and therefore cause a mild/temporary elevation of cytosolic calcium levels, leading to the attenuation of apoptosis [45, 46]. Therefore, we set out to determine the effects of Aβ or Aβ plus isoflurane treatments on the levels of Bax, Bcl-2, and the ratio of Bcl-2 to Bax in the primary neurons from naïve mice.

We were able to show that the treatment with 7.5 uM Aβ40 plus 7.5 uM Aβ42 for six hours decreased Bcl-2 levels Fig. (4A) and increased Bax levels Fig. (4B) in the primary neurons, which can be attenuated by treatment with 0.5% isoflurane for 30 minutes Fig. (4A and 4B). The quantification of the Western blot illustrated that the Aβ treatment decreased the Bcl-2/Bax ratio: 100% versus 40% (** P = 0.0006), and the treatment with 0.5% isoflurane for 30 minutes protected the Aβ-induced reduction in the Bcl-2/Bax ratio: 40% versus 68% (## P = 0.004) Fig. (4C). These results suggest that Aβ may induce apoptosis by reducing the Bcl-2/Bax ratio, and a low concentration and a short duration of isoflurane treatment may attenuate the Aβ-induced apoptosis by mitigating the Aβ-induced reduction in the Bcl-2/Bax ratio.

Fig. 4.

Fig. 4

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Low concentration and short duration of isoflurane treatment attenuates the Aβ-induced decrease in the Bcl-2/Bax ratio in primary neurons from naïve mice. A. Aβ treatment (lanes 3 and 4) decreases Bcl-2 levels as compared to the control condition (lanes 1 and 2) in Western blots. Treatment of Aβ plus 0.5% isoflurane (lanes 5 and 6) increases the Bcl-2 levels as compared to the Aβ treatment alone (lanes 3 and 4). B. Aβ treatment (lanes 3 and 4) increases Bax levels as compared to the control condition (lanes 1 and 2) in Western blots. Treatment of Aβ plus 0.5% isoflurane (lanes 5 and 6) also increases the Bax levels as compared to the control condition (lanes 1 and 2). C. Quantification of the Western blots shows that Aβ treatment (black bar) decreases Bcl-2/Bax ratio as compared to control condition (white bar). The treatment with Aβ plus 0.5% isoflurane (gray bar) increases Bcl-2/Bax ratio as compared to the Aβ treatment alone (black bar).

High concentration and long duration of isoflurane treatment potentiates the Aβ-induced decrease in the Bcl-2/Bax ratio in primary neurons from naïve mice.

Given that a high concentration and a long duration of isoflurane treatment can potentiate the Aβ-induced apoptosis, we next assessed whether such isoflurane treatment can also potentiate the Aβ-induced decrease in the Bcl-2/Bax ratio. Immunoblotting of Bcl-2 and Bax showed that the treatment with 7.5 uM Aβ40 plus 7.5 uM Aβ42 decreased levels of Bcl-2 Fig. (5A) and increased Bax levels Fig. (5B) in the primary neurons from naïve mice. The treatment with 2% isoflurane for six hours potentiated the Aβ-induced changes on the levels of Bcl-2 and Bax. The quantification of the Western blots showed that the Aβ treatment decreased the Bcl-2/Bax ratio: 100% versus 47% (** P = 0.003), and the treatment with the 2% isoflurane for six hours potentiated the Aβ-induced reduction in the Bcl-2/Bax ratio: 47% versus 20% (## P = 0.006) Fig. (5C). These results have suggested that Aβ may induce apoptosis by reducing the Bcl-2/Bax ratio. A high concentration and a long duration of isoflurane treatment may promote the Aβ-induced apoptosis by potentiating the Aβ-induced reduction in the Bcl-2/Bax ratio

Fig. 5.

Fig. 5

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High concentration and long duration of isoflurane treatment promotes the Aβ-induced decrease in the Bcl-2/Bax ratio in primary neurons from naïve mice. A. Aβ treatment (lanes 3 and 4) decreases Bcl-2 levels as compared to the control condition (lanes 1 and 2) in Western blots. Treatment with 2% isoflurane plus Aβ (lanes 5 and 6) decreases the Bcl-2 levels as compared to the control condition (lanes 1 and 2) and Aβ alone (lanes 3 and 4). B. Aβ treatment (lanes 3 and 4) increases Bax levels as compared to the control condition (lanes 1 and 2) in Western blots analysis. Treatment of Aβ plus 2% isoflurane (lanes 5 and 6) also increases the Bax levels as compared to the control condition (lanes 3 and 4). C. Quantification of the Western blots shows that Aβ treatment (black bar) decreases Bcl-2/Bax ratio as compared to the control condition (white bar). The treatment with Aβ plus 2% isoflurane (gray bar) further decreases Bcl-2/Bax ratio as compared to the Aβ treatment alone (black bar).

Isoflurane Induces a Dose-Dependent Alteration in Cytosolic Calcium Levels

Our previous studies have shown that the isoflurane may regulate cytosolic calcium levels, leading to caspase activation and apoptosis [22]. We, therefore, asked whether different concentrations of isoflurane treatment may cause different effects on the cytosolic calcium levels. We were able to show that 0.5 and 2% isoflurane treatment led to a mild and robust elevation of cytosolic calcium levels, respectively Fig. (6A), in the H4 naïve cells. The amplitude graph Fig. (6A) represents a single experiment and the bar graph Fig. (6B) was used for a better demonstration of the isoflurane-induced changes in cytosolic calcium levels. Quantification of the image showed that the 0.5% isoflurane treatment led to 139% increase in cytosolic calcium levels, whereas the 2% isoflurane treatment led to 229% increase in cytosolic calcium levels Fig. (6B). These results have suggested that low concentrations of isoflurane treatment may induce a mild elevation in cytosolic calcium levels, whereas high concentrations of isoflurane treatment may cause a robust elevation in cytosolic calcium levels.

Fig. 6.

Fig. 6

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Isoflurane induces a dose-dependent alteration in cytosolic calcium levels. A. 2% isoflurane treatment induces a robust increase in the fura-2 ratio (340nm/380nm) in H4 naïve cells. Treatment with 0.5% isoflurane induces a mild increase in the fura-2 ratio (340nm/380nm) in H4 naïve cells. The amplitude graph here represents a single experiment. B. Quantification of the amplitude suggests that the 2% isoflurane treatment leads to a 229% increase in cytosolic calcium levels, and the 0.5% isoflurane treatment leads to a 139% increase in cytosolic calcium levels. The bar graph represents a single experiment to better demonstrate the isoflurane-induced changes in cytosolic calcium levels.

Collectively, these findings have suggested that a low concentration of isoflurane treatment (e.g., 0.5% isoflurane) may attenuate the Aβ-induced reduction in Bcl-2/Bax ratio and cause a mild elevation of cytosolic calcium levels, leading to the protection of Aβ-induced neurotoxicity. In contrast, treatment with a high concentration isoflurane (e.g., 2% isoflurane) may potentiate the Aβ-induced reduction in Bcl-2/Bax ratio and cause a robust elevation of cytosolic calcium levels, leading to promotion of Aβ-induced neurotoxicity Fig. (7).

Fig. 7.

Fig. 7

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The hypothesized pathway of the dual effects of isoflurane on Aβ-induced neurotoxicity. Low concentration isoflurane treatment and high concentration isoflurane treatment may induce different effects on the ratio of Bcl-2/Bax and cytosolic calcium levels, leading to either promotion or protection of the Aβ-induced neurotoxicity.

DISCUSSION

Our previous studies have shown that the common inhalation anesthetic isoflurane can induce caspase activation, apoptosis, and Aβ accumulation in vitro [19, 20, 40] and in vivo [21]. However, other reports have suggested different findings [24–33]. This difference could be due to different treatments of isoflurane, e.g., different concentration and duration of treatment. We therefore set out to compare the effects of high concentration isoflurane treatment versus low concentration treatment on the Aβ-induced caspase-3 activation and apoptosis.

We have found that the treatment with 2% isoflurane for six hours can promote the Aβ-induced caspase-3 activation Fig. (2), whereas the treatment with 0.5% isoflurane for 30 minutes can protect the Aβ-induced caspase-3 activation Fig. (1) in naïve H4 cells. In the in vivo studies, we first identified that ICV administration of 250 ng of Aβ42 can induce more robust caspase-3 activation as compared to control condition six hours (versus two or 12 hours) after the administration (Supplemental Data). Then, we have shown that the anesthesia with 1.4% isoflurane for two hours promotes Fig. (2), whereas the anesthesia with 0.7% isoflurane for 30 minutes protects Fig. (1), this Aβ-induced caspase-3 activation in the brain tissues of mice. In addition, we have shown that the treatment with 0.5% isoflurane for six hours can protect the Aβ-induced caspase-3 activation; and the treatment with 2% isoflurane for 30 minutes can promote the Aβ-induced caspase-3 activation in the H4 naïve cells. Taken together, these results suggest that different isoflurane treatments may have different effects on caspase activation and apoptosis. Moreover, it seems that the dual effects (promotion versus protection) of isoflurane on Aβ-induced toxicity are dependent on concentration rather than duration because the treatment with 2% and 0.5% isoflurane for either six or 30 minutes promotes and protects the Aβ-induced caspase-3 activation, respectively, in our experiments Fig. (3).

Other studies support our findings. Wei et al. [47] showed that a treatment with isoflurane (0.6%, 1.2%, and 2.4%) for one hour inhibited the neurotoxicity induced by 2.4% isoflurane for 24 hours in vitro in a dose-dependent manner. These findings suggest that short duration of exposure to isoflurane may attenuate the isoflurane-induced neurotoxicity via induction of endogenous neuroprotective mechanisms (protection), while long duration of exposure to isoflurane induces neurotoxicity directly by its inherent neurotoxic effects [47]. In another study, Lee et al. [48] showed that isoflurane provided postconditioning effects on brain ischemia in vitro and in vivo. The studies further showed that 2% isoflurane provided postconditioning effects with 20- and 30-minute treatment times, but the postconditioning effects of 2% isoflurane disappeared when the isoflurane treatment time was increased to 60 minutes Fig. (3A) in [48]. Similarly, treatments with 1.5% and 2.0% isoflurane for 30 minutes provided the postconditioning effects, but these postconditioning effects disappeared when the isoflurane concentration was increased to 2.5% and 3.0% Fig. (3B) in [48]. These results support our findings that isoflurane may have concentration-dependent dual effects (promotion and protection) on the Aβ-induced caspase activation and apoptosis.

It has been reported that Bcl-2 protein family members, including Bcl-2 and Bax, can regulate apoptosis by modulating outer mitochondrial membrane permeability [43, 44]. The pro-apoptotic protein Bax can promote a sustained and complete calcium release from the ER, and therefore cause an extreme/sustained elevation of cytosolic calcium levels, leading to apoptosis. In contrast, the anti-apoptotic protein Bcl-2 can induce an unregulated ER calcium leak, and therefore cause a mild/temporary elevation of cytosolic calcium levels, leading to the attenuation of apoptosis [45, 46]. The anti-apoptotic protein Bcl-2 can also antagonize Bax by forming heterodimers that prevent the oligomerization of Bax, leading to inhibition of apoptosis [49]. In addition, both Bax and Bcl-2 can compete with each other for interaction with Inositol 1,4,5-Triphosphate Receptor (IP3R), resulting in either an extreme/sustained or mild/temporary elevations of cytosolic calcium levels [reviewed by [50].

In the current studies, we have first found that Aβ can decrease the ratio of Bcl-2 to Bax by decreasing the levels of Bcl-2 and increasing the levels of Bax Figs. (4 and 5). Then, we have found that the low concentration (0.5%) and short duration (30 minutes) of isoflurane treatment can mitigate this Aβ-induced reduction in the Bcl-2/Bax ratio Fig. (4), but the high concentration (2%) and long duration (6 hours) of isoflurane treatment can potentiate this Aβ-induced reduction in the Bcl-2/Bax ratio Fig. (5). Furthermore, we have found that high concentration (2%) isoflurane treatment can induce a robust increase, whereas low concentration (0.5%) isoflurane treatment can only cause a mild increase, in cytosolic calcium levels Fig. (6).

Taken together, these findings suggest that a mild/temporary elevation of cytosolic calcium levels following a lower concentration of isoflurane treatment could provide cytoprotection via up-regulating the host protection response [51–53]. However, an extreme/sustained elevation in cytosolic calcium levels following a high concentration of isoflurane can induce a caspase 12-, caspase 9- and caspase 3-associated form of apoptosis [18, 34, 54, 55, reviewed in 56] Fig. (7).

The mechanisms by which Aβ and isoflurane affect the levels of Bcl-2 and Bax are largely unknown. Recent studies by Zhang et al. [57] have shown that isoflurane can affect mRNA levels of Bcl-2 and Bax. Therefore, it is possible that Aβ and isoflurane may regulate the generation of Bcl-2 and Bax by affecting the gene expression of Bcl-2 and Bax, leading to the alterations in the ratio of Bcl-2/Bax. The future studies will include the assessment of the effects of Aβ and isoflurane on the gene expression of Bcl-2 and Bax, as well as other Bcl-2 protein family members.

Isoflurane may affect the cytosolic calcium levels by acting on NMDA receptors, IP3R and Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) [22, 34; reviewed in 35]. Therefore, the future studies will also include the investigation of the effects of different isoflurane treatments on the levels of NMDA receptors, IP3R, and SERCA, which may further elucidate the molecular mechanisms of the dual effects of isoflurane on Aβ-induced neurotoxicity.

One limitation to the current study is that we only investigated the potential dual effects of isoflurane on caspase-3 activation and apoptosis in naïve H4 cells and primary neurons of naïve mice treated with Aβ. It is possible that isoflurane may not show such dual effects in other cell lines and to other apoptotic insults, e.g., ischemia.

In conclusion, we have found that different isoflurane treatments could have different effects on the Aβ-induced neurotoxicity. Specifically, a low concentration isoflurane treatment may protect the Aβ-induced caspase-3 activation and apoptosis, whereas a high concentration isoflurane treatment may promote the Aβ-induced caspase-3 activation and apoptosis. Furthermore, different concentration and duration exposures of isoflurane may lead to different effects on the Aβ-induced reduction in Bcl-2/Bax ratio and cytosolic calcium levels, leading to the potential dual effects on the Aβ-induced neurotoxicity: protection and promotion. These findings should facilitate future studies to determine whether inhalation anesthetics may dose- and time-dependently induce neurotoxic and neuroprotective effects, which may eventually lead to safer anesthesia care for patients.

Acknowledgments

FUNDING

This research was supported by K08NS048140, R21AG029856 and R01 GM088801 (National Institutes of Health), Jahnigen Career Development Award (American Geriatrics Society), Investigator Initiated Research Grant (Alzheimer’s Association) (to Z. X.), R01GM079360 (National Institutes of Health) (to FI), and China National Science Foundation Oversea young scholar collaboration research award NSF30928026 (to Y.Y. and Z. X.).

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