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Volatile Anesthetics May Not Induce Significant Toxicity to Human Neuron-Like Cells

Lin, Daowei, MD*,†; Feng, Chenzhuo, PhD*; Cao, Minghui, MD; Zuo, Zhiyi, MD, PhD*

doi: 10.1213/ANE.0b013e3181fdf69d
Neuroscience in Anesthesiology and Perioperative Medicine: Research Reports

BACKGROUND: In vitro experiments and in vivo animal studies suggest detrimental effects of volatile anesthetics including isoflurane on brain cells. It is not clear whether volatile anesthetics can cause human brain cell injury.

METHODS: The SH-SY5Y cells, a human neuroblastoma cell line, were induced to differentiate into terminal neuron-like cells. These differentiated cells and the HCN-2 cells, a human cortical neuronal cell line, were exposed to 2% to 5% isoflurane, 6% sevoflurane, or 12% desflurane for 48 hours at 37°C. Lactate dehydrogenase (LDH) release and the expression of caspase 3, synaptophysin, and drebrin were then measured.

RESULTS: Exposure of the differentiated SH-SY5Y and HCN-2 cells to 2% to 4% isoflurane did not increase LDH release and the expression of caspase 3 whose activation leads to apoptosis. The expression of synaptophysin, a synaptic protein, and drebrin, a dendritic spine protein, in the differentiated SH-SY5Ycells was also not affected by 2% to 4% isoflurane. Exposure to 6% sevoflurane or 12% desflurane did not affect LDH release from differentiated SH-SY5Y cells. However, 5% isoflurane significantly increased LDH release from those cells.

CONCLUSIONS: Our results suggest that volatile anesthetics at clinically relevant concentrations do not cause human neuron-like cell injury. Isoflurane also may not alter the quantity of dendritic spines and synapses in these human cells.

Published ahead of print October 21, 2010

From the *Department of Anesthesiology, University of Virginia, Charlottesville, Virginia; and Department of Anesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.

Study funding information is provided at the end of the article.

The authors report no conflicts of interest.

Address correspondence and reprint requests to Zhiyi Zuo, MD, PhD, Department of Anesthesiology, University of Virginia Health System, 1 Hospital Dr., PO Box 800710, Charlottesville, VA 22908-0710. Address e-mail to

Accepted August 27, 2010

Published ahead of print October 21, 2010

Volatile anesthetics are the most frequently used general anesthetics in clinical practice. Although they are relatively safe and easy to use, several lines of evidence have suggested that these anesthetics may cause detrimental effects on brain cells.

Postoperative cognitive decline (POCD) has been shown to occur after cardiac and noncardiac surgery.13 It has been suggested that the duration of general anesthesia is a risk factor for short-term POCD.1 However, the contribution of general anesthetics to POCD in humans is not yet defined. In contrast, animal studies have provided strong evidence to suggest that volatile anesthetics can cause neurotoxicity. Anesthetic exposure for 2 hours caused significant learning and memory impairments in rats.4 The volatile anesthetic isoflurane increased the expression of activated caspase 3, an important mediator for cell apoptosis, and β-amyloid peptide (Aβ) production in the mouse brain.5 Aβ has been proposed to be a mediator for the development of Alzheimer disease, the most common form of dementia in adult humans.6

Each year in the United States, many millions of patients have surgery under general anesthesia. Because volatile anesthetics are used in the majority of patients under general anesthesia, it is imperative to define the effects of volatile anesthetics on human brain cells. It is especially important to determine whether the volatile anesthetic– induced brain cell injury shown in animals or their cultured cells occurs also in human brain cells. To address this issue, we used retinoic acid–induced neuron-like cells differentiated from human SH-SY5Y cells and HCN-2 cells, a human cortical neuronal cell line, in this study. In addition to monitoring their survival, we also determined the expression of synaptophysin and drebrin, proteins expressed in the synapses and dendritic spines of neurons, respectively. The expression of synaptophysin and drebrin has been used for quantification of synapses and dendritic spines.7,8

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Cell Culture

SH-SY5Y cells, a human neuroblastoma cell line, were obtained from the American Type Culture Collection (Manassas, VA). As we described previously,9 these cells were cultured in Dulbecco's modified Eagle medium/Ham's F-12 nutrient mixture (1:1) containing 10% fetal bovine serum. The culture medium was changed every 2 days. The cells were kept in a humidified incubator containing 95% air/5% CO2 at 37°C and subcultured when they were 70% to 80% confluent.

For experiments, the SH-SY5Y cells were plated at a density of 1 × 105 cells/cm2 in 12-well plates for lactate dehydrogenase (LDH) release assay or 100-mm dishes for Western blotting. One day after plating, cells were incubated in neurobasal medium, supplemented with B-27 supplement (GIBCO, Carlsbad, CA) and L-glutamine (500 μM; Nacalai Tesque, Inc., San Diego, CA). Retinoic acid (10 μM; Sigma, St. Louis, MO) was added to the medium for 3 days to induce SH-SY5Y cells to differentiate into a homogenous population of cells with neuronal morphology.10

HCN-2 cells, a cortical neuronal cell line, were purchased from the American Type Culture Collection. These cells were cultured in Dulbecco's modified Eagle medium with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 10% fetal bovine serum. The medium was changed every 2 to 3 days. The cells were maintained in a humidified incubator containing 95% air/5% CO2 at 37°C. They were plated in 12-well plates at a density of 5 × 104 cells/cm2 for LDH assay.

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Exposure to Volatile Anesthetics

Volatile anesthetic exposure was performed as we described previously.11 Briefly, fresh culture medium was pregassed with 95% air/5% CO2 through an isoflurane vaporizer set at 0%, 2%, 3%, 4%, or 5%, a sevoflurane vaporizer set at 6%, or a desflurane vaporizer set at 12% for 30 minutes at 37°C. After replacing the culture medium with this freshly prepared and pregassed medium, the differentiated SH-SY5Y cells or HCN-2 cells were placed in an airtight chamber. The chamber was then gassed with 0%, 2%, 3%, 4%, or 5% isoflurane, 6% sevoflurane, or 12% desflurane in the carrying gases (95% air/5% CO2) for 15 minutes. Volatile anesthetic concentrations in the gases exiting from the chamber were monitored with a Datex™ infrared analyzer (Capnomac, Helsinki, Finland) and reached the target concentrations at approximately 3 minutes after the onset of gassing. The chamber was sealed and the incubation lasted for 48 hours at 37°C. At the end of incubation, volatile anesthetic concentrations in the gases from the chamber were confirmed to be at the target concentrations by the infrared analyzer. Our previous experiments showed that the aqueous isoflurane concentration at 37°C stayed at approximately 415 and 620 μM, respectively, when 2% and 3% isoflurane was delivered to the chamber.11

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LDH Assay

LDH activity was determined using an LDH cytotoxicity detection kit as done previously.12 Briefly, the incubation solution harvested from the 12-well plates at the end of experiments was centrifuged at 13,000 rpm for 10 minutes and 100 μL of the cell-free supernatant was transferred to 96-well plates. The supernatant was incubated with the same amount of reaction mixture. LDH activity was determined by a colorimetric assay with the absorbance wavelength at 492 nm and the reference wavelength at 655 nm in a spectrophotometer (Bio-Rad Laboratories, Hercules, CA). Background absorbance from the cell-free buffer solution was subtracted from all absorbance measurements. After removal of the buffer from 12-well plates, 1% triton X-100 lysing solution was applied to the remaining cells. The percentage of LDH released to incubation buffer in total LDH was calculated: spontaneously released LDH in the buffer/(spontaneously released LDH in the buffer + intracellular LDH released by triton X-100).

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Western Blot Analysis

After the isoflurane exposure period, the differentiated SH-SY5Y cells were scraped from 100-mm dishes. The samples were homogenized in lysing buffer containing 50 mM Tris (pH 7.4), 140 mM NaCl, 1% triton X-100, 0.1% sodium dodecyl sulfate, 30 μM MG132, and protease inhibitor mixture (complete; Roche Applied Science, Indianapolis, IN). Homogenates were centrifuged at 4°C for 30 minutes at 13,000 rpm. The supernatant was used for Western blotting. Protein content was estimated using a Bio-Rad Protein Assay Kit. Thirty micrograms proteins per lane were electrophoresed on a 15% polyacrylamide gel and then blotted onto a polyvinylidene difluoride membrane. After blocking with 5% nonfat milk, the membranes were incubated with the following primary antibodies: mouse monoclonal antisynaptophysin antibody (1:15,000; catalog number: MAB329; Millipore Corp., Billerica, MA), rabbit polyclonal antidrebrin antibody (1:1000; catalog number: D3816; Sigma), goat polyclonal anticaspase 3 antibody (1:200; catalog number: SC-1225; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and mouse monoclonal anti–β-actin polyclonal antibody (1:2000; catalog number: A2228; Sigma). Appropriate secondary antibodies were used, and protein bands were visualized using a Genomic and Proteomic Gel Documentation (Gel Doc) System from Syngene (Frederick, MD). The protein band intensities of caspase 3, synaptophysin, and drebrin were normalized by the corresponding band intensities of β-actin from the same samples. The results from cells under various experimental conditions were then normalized by those of the corresponding control cells.

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Statistical Analysis

Data are expressed as means ± SD (n ≥ 9 for each experimental condition). Results were analyzed by 1-way analysis of variance followed by the Tukey test for post hoc analysis after confirmation of normal distribution of the data or by Kruskal-Wallis analysis of variance on ranks followed by the Dunn test when the data were not normally distributed. A P value <0.05 was considered statistically significant.

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Incubation of the differentiated SH-SY5Y cells or the HCN-2 cells with 2% to 4% isoflurane for 48 hours did not change the LDH release from those cells (Fig. 1). These results suggest that those isoflurane exposures did not induce cell plasma membrane injury to the cells. Similarly, exposure of the differentiated SH-SY5Y cells to 6% sevoflurane or 12% desflurane also did not change the LDH release (Fig. 2). However, incubation of the differentiated SH-SY5Y cells with 5% isoflurane for 48 hours significantly increased LDH release (extracellular LDH was 17.4% ± 11.1% and 23.5% ± 13.5%, respectively, of the total LDH for control or 5% isoflurane group; n = 24, P = 0.013), suggesting that isoflurane at extremely high concentrations caused cell injury.

Figure 1

Figure 1

Figure 2

Figure 2

As another variable to quantify possible detrimental effects of isoflurane on human neuron-like cells, we used an anticaspase 3 antibody in a Western blotting study. Per company data, this antibody can bind to the procaspase 3 and its fragments at 20 kDa and 17 kDa. These fragments are subunits of activated caspase 3.13 We detected a protein band at 32 kDa with this antibody in the differentiated SH-SY5Y cells. This molecular weight corresponds to that of procaspase 3. Isoflurane exposure did not significantly change the expression of this procaspase 3 protein (Fig. 3). We could not detect any protein bands corresponding to the 20- and 17-kDa subunits under control and isoflurane exposure conditions. Isoflurane exposure also did not change the expression of synaptophysin and drebrin in the differentiated SH-SY5Y cells (Fig. 3).

Figure 3

Figure 3

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Volatile anesthetics have been frequently used in clinical practice for many decades. However, there is a growing concern in recent years that these drugs can have detrimental effects on the brain. The learning and memory functions of middle-aged rats and old rats were impaired after a 2-hour exposure to clinically relevant concentrations of general anesthetics.4 It has also been shown that isoflurane increases activated caspase 3 expression and Aβ production in mouse brain.5 Volatile anesthetics at clinically relevant concentrations can also induce Aβ oligomerization in vitro,14 a process that is considered to be important for the development of Alzheimer disease. These studies suggest that volatile anesthetics can cause cell injury and functional impairment of brain cells. Consistent with this suggestion, it has been proposed that anesthesia may contribute to the acute phase of POCD,1 a well established clinical syndrome. However, in clinical studies, it has been very difficult to separate anesthesia from other factors, such as surgery and the underlying diseases for which surgery is required. Also, patients who received general anesthesia or regional anesthesia were not different in cognitive functions assessed at 3 months or 6 months after surgery.1517 Thus, it is not known whether general anesthesia/anesthetics can cause brain cell injury in humans. We showed here that 2% to 4% isoflurane did not cause significant injury to human neuron-like cells and did not increase the expression of the activated caspase 3 in these cells. Desflurane and sevoflurane at high concentrations also did not cause significant cell injury as assessed by LDH release. These results suggest that volatile anesthetics at clinically relevant concentrations may not cause a direct damaging effect on these cells. Consistent with our results, incubation of the differentiated SH-SY5Y cells with 2.5% isoflurane for 16 hours did not cause injury to these cells as assessed by the trypan blue exclusion, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining analysis, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.18 However, these cells could have been injured by isoflurane in our study because isoflurane at a very high concentration (5%) increased LDH release from them.

We used human neuron-like cultures because it is almost impossible to determine anesthetic-induced human neuronal injury in vivo or in primary human neuronal cultures. We used 2 cell lines in this study to make it less likely that the observed noninjury effect is specific to 1 cell line.

Dendritic spines and synapses are unique structures of neurons and the fundamental structures for interneuronal communication and learning and memory functions.19,20 Anesthetic effects on learning and memory functions could be attributable to their effects on dendritic spines and synapses. Our results showed that isoflurane did not affect the expression of synaptophysin and drebrin, proteins used to quantify synapses and dendritic spines, respectively, in studies.7,8 These results suggest that isoflurane does not have a significant effect on the quantity of dendritic spines and synapses in these human cells. A previous study showed that exposure of primary rat neuronal culture to isoflurane decreased drebrin expression and that exposure of 7-day-old mice to isoflurane decreased synaptic density.8 Interestingly, a recent study showed that volatile anesthetics increased dendritic spine density in rats when they were exposed to isoflurane at 16 days old.21 The reason for these different findings is that 7-day-old rodents are in peak time of synaptogenesis and 16-day-old rodents are not. This difference may change their vulnerability/responsiveness to volatile anesthetics.22 However, it is not clear whether age-dependent changes of synaptic vulnerability to volatile anesthetics occur in humans.

Our results do not suggest a significant detrimental effect of volatile anesthetics on human neuron-like cells. Consistent with this suggestion, the role of general anesthesia/anesthetics per se in POCD has not yet been established. Although 2 early studies suggest that general anesthesia may contribute to the development or speed up the development of Alzheimer disease,23,24 the majority of studies have not shown a relationship between general anesthesia/surgery and Alzheimer disease.2528 Our recent study suggests that spine surgery under general anesthesia may not increase the risk for Alzheimer disease.29 In addition, a recent study did not show that noncardiac surgery was a risk for long-term cognitive decline after surgery.30 Finally, anesthetic toxicity on developing brain has been a research focus because of the concern that brain in the peak synaptogenesis phase may be vulnerable to anesthetics. A recent study showed that children exposed to 1 surgery under general anesthesia before 4 years old did not have learning deficits but children subjected to >1 surgery under anesthesia had higher chances of learning deficits later in their lives.31 However, another recent study using genetically identical twins showed that anesthesia and surgery exposure before 3 years old did not cause learning and memory deficits. Rather, the need for anesthesia and surgery early in life is a marker of vulnerability for developing learning problems later.32 Thus, additional studies are needed to define the potential detrimental anesthetic effects, such as their roles in POCD, and the possible molecular mechanisms/structure changes for these effects in humans.

Our studies have limitations. We used human neuron-like cell cultures. These cells are obviously different from in situ human neurons. It is also impossible to provide cell cultures with an environment identical to what the in situ human neurons have, such as local pH, oxygen and electrolyte concentrations, and the support and interaction from other cells. Thus, our cell cultures may have a lower vulnerability to volatile anesthetics than in situ human neurons. However, mouse neuronal cultures were clearly as vulnerable to isoflurane as in situ mouse neurons,8 suggesting the usefulness of cell cultures in anesthetic toxicity studies. Also, although we used 2 neuron-like cell lines, it is still possible that our findings are cell line specific. This issue can be resolved when additional human neuron-like cell lines are identified and are available. Finally, our results have relatively large standard deviations, although the corresponding standard errors of means are rather small. Large standard deviations can become a resource to obscure drug effects. However, LDH release data from cells exposed to 2% to 4% isoflurane, 6% sevoflurane, or 12% desflurane have relatively small standard deviations and the means under these volatile anesthetic exposure conditions are very similar to those under control conditions. These findings increase our confidence on the conclusion that volatile anesthetics at clinically relevant concentrations do not affect LDH release from human neuron-like cell cultures. Our Western blotting data have relatively large variability. This phenomenon can be attributed to variations in cell conditions (various sets of cells were used in the experiments) and blotting conditions in various experiments. To reduce the effects of variation in experimental conditions on final results, we normalized the data of caspase 3, synaptophysin, and drebrin by that of β-actin in the same samples and then normalized the results of samples treated with 2% to 4% isoflurane to those of the corresponding control. These normalization processes should have increased the possibility to detect isoflurane effects on the expression of caspase 3, synaptophysin, and drebrin proteins.

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Supported by a grant (R01 GM065211 to ZZ) from the National Institutes of Health, Bethesda, MD, a grant from the International Anesthesia Research Society (2007 Frontiers in Anesthesia Research Award to ZZ), Cleveland, OH, and the Robert M. Epstein Professorship endowment, University of Virginia.

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All 4 authors participated in study design. Drs. Lin and Feng conducted the study. Dr. Lin performed initial data analysis and drafted the Methods section. Dr. Zuo performed the final data analysis and prepared the manuscript.

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