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Sevoflurane Inhibits Glutamate-Aspartate Transporter and Glial Fibrillary Acidic Protein Expression in Hippocampal Astrocytes of Neonatal Rats Through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) Pathway

Wang, Wei MD, PhD; Lu, Rui MD; Feng, Da-yun MD, PhD; Zhang, Hui MD, PhD

doi: 10.1213/ANE.0000000000001238
Preclinical Pharmacology: Research Report

BACKGROUND: The mechanisms underlying general anesthesia-induced neurotoxicity are unclear. Astrocytes have been recognized as important contributors to neuronal development. Until now, the response of the astrocytes to neonatal general anesthetic exposure has been unreported.

METHODS: Postnatal day 7 rats received 2.5% sevoflurane for 6 hours. Expressions of glial fibrillary acidic protein (GFAP) and glutamate-aspartate transporter (GLAST) and phosphorylation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway were detected on days 1, 3, 7, and 14 after sevoflurane inhalation. In addition, cultured astrocytes were exposed to 2.5% sevoflurane for 2 hours and GFAP, GLAST expressions, and JAK/STAT phosphorylation were evaluated. Furthermore, we pharmacologically disrupted JAK/STAT signaling in vivo by treatment with the JAK/STAT inhibitor AG490 and in vitro by treatment with JAK inhibitor I to detect the consequent expression of GFAP and GLAST.

RESULTS: Sevoflurane induced a robust decrease of GFAP and GLAST expression in hippocampal tissue compared with sham control groups at 1 to 14 days after sevoflurane exposure. Immunohistochemistry showed colocalization of GFAP, GLAST, and pSTAT3 in the hippocampal CA1 region. Western blot analysis also revealed a significant decrease of pJAK1, pJAK2, and pSTAT3 in the sevoflurane group. In vitro study showed that GFAP, GLAST, pJAK1, pJAK2, and pSTAT3 expressions in cultured astrocytes were remarkably decreased at 24 to 48 hours after sevoflurane treatment. Either AG490 or JAK inhibitor I significantly decreased expressions of GFAP and GLAST in hippocampus or cultured astrocytes.

CONCLUSIONS: Astrocytic GLAST was inhibited by sevoflurane in the hippocampus of neonatal rats. Inactivation of the JAK/STAT pathway possibly contributes to this effect of sevoflurane. Astrocytic dysfunction induced by sevoflurane may contribute to its neurotoxicity in the developing brain.

Supplemental Digital Content is available in the text.Published ahead of print March 21, 2016

From the *State Key Laboratory of Military Stomatology, Department of Anesthesiology, School of Stomatology, and Department of Neurosurgery, Tangdu Hospital, the Fourth Military Medical University, Xi’an, China.

Accepted for publication January 15, 2016.

Published ahead of print March 21, 2016

Funding: This work was supported by the National Natural Science Foundation of China (nos. 81371265, 81071528, and 81271229).

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

Reprints will not be available from the authors.

Address correspondence to Hui Zhang, MD, PhD, State Key Laboratory of Military Stomatology, Department of Anesthesiology, School of Stomatology, The Fourth Military Medical University, No.145 West Changle Rd., Xi’an 710032, China. Address e-mail to zhanghuifmmua@yhoo.com.

General anesthetic exposure during the developmental stages of the brain can cause neuronal death in various mammalian species.1 Neuronal apoptosis and impaired synaptogenesis have been considered significant processes of general anesthetics’ neurotoxicity.2,3 It has been reported that sevoflurane may induce the biochemical changes that are associated with cognitive dysfunction in neonatal mice, which include apoptosis, Aβ accumulation, and neuroinflammation4; however, the underlying mechanisms are far from being clear. In recent decades, astrocytes emerged as increasingly important in the generation of neurons and functional synapses in the premature brain.5–7 Hypoactivated astrocytes probably result in interruption of neurogenesis and synaptogenesis during development. A disorder of astrocytes during development has been reported to be essential for several neuropsychological diseases.8 The detailed mechanism is unclear, but it is well accepted that astrocytes are capable of communicating with surrounding neurons in many ways, including release of trophic factors and neurotransmitters and modulation of extracellular glutamate levels.9 The role of general anesthetics in developing astrocytes, however, has not been reported.

Disruption in glutamate homeostasis is thought to be a factor in the pathogenesis of many neurological diseases.10 Glutamate-aspartate transporter (GLAST; excitatory amino acid transporter 1) is one of the most abundant glutamate transporters.11 It is responsible for taking up the bulk of synaptically released glutamate and hence preventing glutamate spilling over to other synapses. GLAST is primarily expressed on astrocytic membrane, and its abnormalities contribute to a number of central nervous system (CNS) diseases or disorders.12 It has been reported in adult mice that isoflurane produces small but statistically significant inhibition of glutamate uptake,13 while another study indicated that isoflurane increases EAAT3 activity.14 It is still unclear, however how general anesthetic exposure during earlier life stages influences astrocytic functions. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is important in astrocyte function and contributes to several CNS pathologies.15 In the early postnatal stage, inactivation of the JAK/STAT pathway is associated with GLAST dysfunction after hypoxic insult.16 It remains unknown whether this pathway is involved in general anesthetic-induced neurotoxicity.

Sevoflurane is an inhalational anesthetic commonly used in clinical practice. In the present study, we sought to examine the influence of sevoflurane on astrocytic GLAST expression together with JAK/STAT signaling changes. We used both in vivo (postnatal day 7 [P7] Sprague-Dawley rat pups) and in vitro (primary hippocampal astrocyte culture) systems. Our hypothesis was that sevoflurane exposure decreases GLAST, glial fibrillary acidic protein (GFAP), and JAK-STAT expressions in developing rat brains. Disruption to this pathway causes astrocytic dysfunction and may contribute to sevoflurane neurotoxicity.

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METHODS

Animal Model

All protocols were approved by the Animal Use and Care Committee for Research and Education of the Fourth Military Medical University, Xi’an, China. Sprague-Dawley rat pups (P7) were randomly divided into 3 groups: naive (no treatment was given), sevoflurane + 30% oxygen group, and 30% oxygen group. In the sevoflurane group, pups received 2.5% sevoflurane in 30% oxygen balanced with nitrogen in an anesthesia chamber via a calibrated flowmeter and a vaporizer (Datex-Ohmeda, Madison, WI) for 6 hours. Pups in the 30% oxygen control group received 30% oxygen balanced with nitrogen in an anesthesia chamber for 6 hours. Rectal temperature was monitored and was kept at 37.0 ± 0.5°C with a heating lamp throughout the experiment. Pulse oximetry via the tail was monitored continuously (MouseOx; Starr Life Sciences, Oakmont, PA). After 6 hours of sevoflurane exposure, the chamber was flushed with 30% oxygen until the rats recovered. The pups were then wrapped with bedding material from their home cage and returned to their natural litter where they remained. At 1, 3, 7, and 14 days after the experiment, rats were killed and their brains were harvested for Western blot analysis and immunohistochemical staining. Separate pups (n = 12 in sevoflurane treatment group and n = 12 in sham group) were used for transcardial arterial blood gas analysis (ABL800; Radiometer, Copenhagen, Denmark). Samples were obtained at 2 periods: immediately after separation from mother rats (0 hour) or at the end of the experiment (6 hours). Rats were immediately killed by decapitation and not used for further experiment.

To test the effect of JAK2 inhibitor AG490 (Sigma, St. Louis, MO) on GLAST expression, AG490 was dissolved in dimethylsulfoxide (DMSO) and intraperitoneally administered (10 mg/kg) once daily for 3 days (from P7 to P9). Rats were killed on P10, and Western blot analysis combined with immunohistochemistry was performed to detect GLAST expression in the hippocampus.

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Primary Astrocyte Culture and Treatment

Primary hippocampal astrocyte culture was performed according to our previous protocol.17 Briefly, hippocampal cortices were removed from P0 or P1 rats and were dissected. After enzymatic digestion, the dissociated cells were resuspended in Dulbecco modified Eagle’s medium supplemented with fetal bovine serum (10%), penicillin (200 IU), and streptomycin (0.1 mg/mL). Cells were then seeded in 75-cm2 flasks and cultured for 10 to 14 days at 37°C in a humidified incubator (5% CO2/95% air). The flasks were shaken at 220 rpm (24 hours, 37°C) after 6 days to produce purified astrocytes. Astrocyte-defined medium was produced in the following formulation: Dulbecco modified Eagle’s medium supplemented with transferrin (50 μg/mL), fibronectin (1.5 μg/mL), sodium selenite (5.2 ng/mL), heparin sulfate (0.5 g/mL), basic fibroblast growth factor (5 ng/mL), epidermal growth factor (10 ng/mL), and insulin (5 μg/mL). Astrocytes with a purity of 95% were used in the following experiments.

For sevoflurane treatment, dissociated astrocytes were placed in airtight, humidified modular chambers connected to a sevoflurane calibrated vaporizer (Datex-Ohmeda) that delivered 2.5% sevoflurane mixed with 5% carbon dioxide/95% air carrier gas at 12 L/min. After a 15-minute equilibration, the chambers were sealed and kept at 37°C. Astrocytes were treated with sevoflurane or control carrier gas for 2 hours. The chamber was flushed with control carrier gas. Astrocytes were then cultured continuously for 48 hours for further experiments. To test the effect of JAK inhibitor I (2-[1,1-dimethylethyl]-9-fluoro-3, 6-dihydro-7H-benz[h]-imidazo[4,5-f] isoquinoline-7-1; EMD Chemicals, Gibbstown, NJ) on GLAST expression, primary astrocytes (without sevoflurane treatment) were incubated with JAK inhibitor I (1 μM) for 24 hours. JAK inhibitor I was dissolved in DMSO. In the control group, astrocytes were treated with DMSO.

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Immunofluorescent Histochemistry and Cytochemistry

For immunohistochemistry, rats were perfused with chilled cold normal saline followed by 0.1 M phosphate buffer (PB, pH 7.3) containing 4% paraformaldehyde under anesthesia with chloral hydrate (350 mg/kg intraperitoneally). The brain was then removed and postfixed in the same fixative for 4 hours and then cryoprotected for 24 hours at 4°C in 0.1 M PB containing 30% sucrose. Serial coronal sections (30 μm) were obtained subsequently via a Leica cryostat (Leica CM1800, Heidelberg, Germany), collected serially into 3 dishes, each of which contained a complete serial section. For immunohistochemistry, cells were fixed in 4% paraformaldehyde in PB at room temperature (RT) for 10 minutes. Cells or sections were rinsed in 0.01 M phosphate-buffered saline (PBS; pH 7.3) 3 times (10 minutes each), blocked with 2% goat serum in 0.01 M PBS containing 0.3% Triton X-100 for 1 hour at RT. The sections were incubated overnight at 4°C with the primary antibodies: mouse anti-GFAP (a marker for astrocytes) (1: 5000; Millipore, Billerica, MA) mixed with goat anti-GLAST (1: 200; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit anti-pSTAT3 (1: 100; Cell Signaling Technology, Beverly, MA). Cells were incubated with the abovementioned 3 kinds of primary antibodies for triple immunofluorescence cytochemistry. The sections were then washed 3 times in 0.01 M PBS (10 minutes each) and then incubated for 2 hours at RT with the corresponding secondary antibody: Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500; Invitrogen, Carlsbad, CA) and Alexa Fluor 594-conjugated donkey anti-goat IgG (1:500; Invitrogen) or Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1: 500; Invitrogen). The cells were incubated for 2 hours at RT with Alexa Fluor 488-conjugated donkey anti-mouse IgG (1: 500; Invitrogen), Alexa Fluor 594-conjugated donkey anti-goat IgG (1: 500; Invitrogen), and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (1: 500; Invitrogen). Images were obtained using a confocal laser microscope (FV1000; Olympus, Tokyo, Japan), and digital images were captured with FluoView 1000 (Olympus).

Figure 1

Figure 1

Counting of astrocytes was performed according to previous reports. Five nonadjacent sections from each animal in identical fields from serial sections were selected randomly for analyzing number of astrocytes. The number of GFAP-positive cells was analyzed in stratum radiatum of the peripyramidal cell layer of CA1. GFAP-positive cells were counted within a 400 μm × 400 μm frame. Estimates of the number of astrocytes were obtained using a nucleator probe (blue point). The number was recorded when the “cross-point” was between the cells and white line. By contrast, when the cross-point was between the cells and the red line, the cell was not recorded (Fig. 1). Cell number was calculated and averaged across the 5 sections.

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Western Blot

The protocols for Western blot were according to our previous report.18 After brain removal, both sides of the hippocampus were dissected and then homogenized in sodium dodecyl sulfate sample buffer with a mixture of proteinase and phosphatase inhibitors (Sigma-Aldrich). The homogenate was centrifuged at 4°C for 15 minutes at 1000g. The procedures for primary cell culture were similar to tissue protocols. The cytoplasmic and nuclear protein extracts were prepared with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, Rockford, IL) according to the manufacturer’s instructions. The protein concentration of supernatant was quantified. About 30 µg of sample was denatured in Laemmli sample loading buffer (Bio-Rad, Hercules, CA) at 99°C for 5 minutes and loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels with standard Laemmli solutions (Bio-Rad). The proteins were electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore). After being blocked in the solution containing Tris-buffered saline with 0.02 % Tween-20 and 3% nonfat milk for 1 hour, the membrane was incubated overnight with one of the following primary antibodies: mouse anti-GFAP (1:5000; Millipore), goat anti-GLAST (1:500; Santa Cruz), rabbit anti-JAK-1 (1:1000; Cell Signaling), rabbit anti-pJAK-1 (1:1000; Cell Signaling), rabbit anti-JAK-2 (1:1000; Cell Signaling), rabbit anti-pJAK-2 (1:1000; Cell Signaling), mouse anti-STAT3 (1:1000; Cell Signaling), and rabbit anti-pSTAT3 (1:1000; Cell Signaling). The immunoblots were then reacted with the horseradish peroxidase–conjugated secondary antibody (anti-rabbit 1:3000; Amersham Pharmacia Biotech Inc., Piscataway, NJ). All reactions were detected by enhanced chemiluminescence (Amersham) and then developed onto films. Similar size of square was drawn around each band to measure the density, and the background near that band was subtracted. The membranes were then reprobed with mouse anti-β-actin (1:5000; Millipore) and then horseradish peroxidase-conjugated secondary antibody (anti-mouse 1:5000; Amersham). Target protein levels were normalized against β-actin levels and expressed as fold changes relative to the control group.

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Statistical Analysis

Sample sizes were n = 6 for Western blotting and immunohistochemistry in each time point (at 1, 3, 7, and 14 days after experiment) of sevoflurane and sham groups, respectively. For blood gas analysis, n = 6 at 0 hour and 6 hours after experiment in sevoflurane and sham groups, respectively. In the JAK2 inhibitor experiment, 12 rats were divided into 2 groups: AG490 group (n = 6) and DMSO control group (n = 6).

The residuals were tested with the Shapiro-Wilk method (Supplemental Digital Content 1, http://links.lww.com/AA/B387) and met normality. The Levene test for equality of variances was performed, and results are shown in Supplemental Digital Content 2 (http://links.lww.com/AA/B388). Analysis of variance (ANOVA) was used for 2 group comparisons. The Dunnett test was used for post hoc multiple comparisons in ANOVA according to equal variances. Blood gas analysis data were analyzed with 2-way ANOVA. All data are presented as mean ± SEM. A P value <0.05 was considered statistically significant. All analyses were performed using Prism for Macintosh 5.0 (GraphPad Software Inc., San Diego, CA).

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RESULTS

Sevoflurane Did Not Influence Ventilation or Oxygenation

Table 1

Table 1

To examine the effect of sevoflurane exposure, we exposed P7 rats to 2.5% sevoflurane for 6 hours. Hypoxia is thought to affect GLAST expression.16 To assess the respiratory function of rat pups during anesthesia, we performed blood gas analysis in rats before and after sevoflurane inhalation. Control samples were obtained from rat pups exposed to 30% oxygen during the same period. Blood gas data including pH (sevoflurane versus sham: P = 0.22), arterial oxygen saturation (sevoflurane versus sham: P = 0.41), arterial oxygen tension (sevoflurane versus sham: P = 0.34), and arterial carbon dioxide tension (sevoflurane versus sham: P = 0.36) did not differ significantly from the sham control group (Table 1). In addition, all pups’ color was pink and their breathing was normal throughout the exposure period, indicating that there was no hypoxic episode during exposure.

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Sevoflurane Decreased GFAP and GLAST Expression in Rat Hippocampus

To examine the effects of sevoflurane on astrocytes in the hippocampus of the immature brain, we examined the expression of the astrocyte-specific marker GFAP at 1, 3, 7, and 14 days after sevoflurane treatment on P7 rats. Immunofluorescent histochemistry showed a significant difference in astrocyte morphology or GFAP distribution (Fig. 1, A–D). The number of GFAP-positive astrocytes in the hippocampal CA1 region was significantly reduced at postexperimental day 3 (10.4 ± 1.7 vs 14.3 ± 2.6; P = 0.031) and day 7 (15.1 ± 1.6 vs 18.6 ± 1.5; P = 0.037) (Fig. 1, E and F). Western blot analysis in the hippocampus revealed a significant decrease of GFAP expression (Fig. 1G). At 1 day after anesthesia, GFAP was decreased, however, not significantly, compared with the sham control group (0.78 ± 0.12 vs 1.09 ± 0.34-fold of naive 1 day control, n = 6, P = 0.68). At 3 days and 1 week, sevoflurane but not 30% oxygen significantly reduced GFAP expression (3 days: 1.25 ± 0.12 vs 1.90 ± 0.22-fold, n = 6, P = 0.016; 7 days: 1.46 ± 0.16 vs 2.42 ± 0.35-fold n = 6, P = 0.028). At 2 weeks after treatment, GFAP expression was still lower than sham control group, although the difference was not significant (P = 0.067).

Figure 2

Figure 2

We then detected GLAST expression. Immunofluorescent double staining showed that GLAST was completely colocalized with GFAP (Fig. 2, A–C), suggesting that this glutamate transporter is highly expressed in astrocytes. GLAST intensity was decreased in the hippocampal CA1 region at 3 days after sevoflurane exposure compared with sham or naive rats (Fig. 2, D–F). Western blot analysis indicated that sevoflurane induced a robust decrease of GLAST expression compared with sham control groups at the same period. The decrease was significant throughout the whole observation window: 1 day (0.65 ± 0.09-fold of sham 1 day control, n = 6, P = 0.016), 3 days (1.35 ± 0.43 vs 0.58 ± 0.17-fold, n = 6, P = 0.031), 7 days (1.67 ± 0.35 vs 0.94 ± 0.13-fold, n = 6, P = 0.022), and 14 days (1.89 ± 0.24 vs 1.21 ± 0.25-fold, n = 6, P = 0.027) (Fig. 2, G–H). These data indicated that sevoflurane could downregulate astrocytic GLAST expression in the developing brain.

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Phosphorylation of Astrocytic JAK-STAT3 Was Inhibited After Sevoflurane Treatment

Figure 3

Figure 3

A previous study suggested that the JAK/STAT pathway is important in astrocyte maturation because the onset of GFAP expression depends on STAT3 phosphorylation and astrocytic GLAST expression. We sought to determine whether activation level changes in the JAK/STAT signaling pathway also occurred after sevoflurane treatment. Double immunostaining showed that phosphorylated STAT3 (pSTAT3) was highly expressed on astrocytes (Fig. 3, A–F). Different from that of GLAST expression, pSTAT3 was concentrated in the center of astrocytes, suggesting its nucleus localization. Compared with oxygen controls, pSTAT3 immunoreactivity was much lower in the sevoflurane-treated group (Fig. 3, A–F). Western blot analysis revealed a significant decrease of pJAK1, pJAK2, and pSTAT3 in the sevoflurane group compared with the corresponding sham groups (Fig. 3, G–H). On day 3 after sevoflurane treatment, pJAK1, pJAK2, and pSTAT3 were 2.45 ± 0.35 vs 1.24 ± 0.21-fold (n = 6, P = 0.017), 1.55 ± 0.31 vs 0.93 ± 0.23-fold (n = 6, P = 0.029), 2.46 ± 0.24 vs 0.79 ± 0.13-fold, respectively (n = 6, P = 0.013). However, levels of total JAK1, JAK2, and STAT3 were unchanged after sevoflurane inhalation (Fig. 3, G–H). These results indicated that sevoflurane reduces JAK/STAT signaling in the neonatal hippocampus with a time course similar to the reduction in GFAP and GLAST expression.

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Sevoflurane Reduced GLAST Expression in Primary Astrocytes

Figure 4

Figure 4

We further confirmed the abovementioned data in a cell culture model. Primary astrocyte cultures were exposed to sevoflurane for 2 hours. At 3 periods (6, 24, and 48 hours) after sevoflurane treatment, astrocytes were harvested and preceded to immunocytochemistry and Western blot. Triple immunofluorescence histochemistry showed colocalization of GFAP with GLAST and pSTAT3. GFAP and GLAST had a similar subcellular distribution, whereas pSTAT3 was limited to the nucleus (Fig. 4, A–H). Immunoreactivities of GFAP, GLAST, and pSTAT3 were all significantly weakened compared with that of control group 24 hours after sevoflurane treatment (Fig. 4, A–H). Western blot also showed a significant decrease in GFAP and GLAST protein expression, as well as in pJAK1, pJAK2, and pSTAT3 levels at 24 to 48 hours after sevoflurane treatment (Fig. 4, I–K). However, in the sham groups, expressions of GFAP, GLAST, and pJAK-STAT were unchanged at 6, 24, or 48 hours (data not shown). These in vitro data were consistent with our findings in an animal study.

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Disruption of JAK-STAT3 Signaling Mimicked the Effect of Sevoflurane on GLAST In Vivo and In Vitro

Figure 5

Figure 5

To determine the role of the JAK/STAT signaling pathway in modulating GLAST expression in the developing brain, we treated neonatal rats that had not been exposed to sevoflurane with AG490 (JAK/STAT inhibitor). Astrocyte culture also was treated with JAK inhibitor I, with DMSO as vehicle control. Compared with DMSO-treated animals, AG490 administration significantly downregulated levels of pSTAT3 in the hippocampus of P10 rats (Fig. 5, A–C). In addition, STAT3 phosphorylation levels were remarkably decreased in cultured astrocytes after 24-hour treatment with the JAK inhibitor I (Fig. 5, A–C). The aforementioned results confirmed the pharmacological effect of JAK inhibitors. Western blot data showed that both GFAP and GLAST expressions were reduced proportionally after JAK inhibitor treatment in vivo and in vitro. In the hippocampus of rats receiving AG490, GFAP and GLAST expression levels were 0.75 ± 0.17 (P = 0.026) and 0.32 ± 0.06 (P = 0.014) folds of DMSO control. In cultured astrocytes treated with JAK inhibitor I, GFAP and GLAST expression levels were 0.45 ± 0.12 (P = 0.004) and 0.35 ± 0.08 (P = 0.003) folds of DMSO control (Fig. 5, A–C). Conversely, levels of JAK1, JAK2, and STAT3 were not affected (data not shown). Immunofluorescent histochemical and cytochemical studies also confirmed that after treatment with AG490 or JAK inhibitor I, immunoreactivities of GFAP and GLAST were obviously weakened compared with DMSO control (Fig. 5, D–Q). These results indicated that pharmacological disruption of JAK/STAT signaling is correlated with a decrease in GLAST expression. Under conditions of sevoflurane exposure, inactivated JAK/STAT signaling and inhibited GLAST expression may be causally linked.

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DISCUSSION

Our in vivo and in vitro data indicated that GFAP and GLAST were decreased significantly after sevoflurane treatment. In addition, the JAK/STAT signaling pathway in the developing rat brain was impaired by sevoflurane exposure. We thus conclude that sevoflurane induced astrocytic damage and GLAST dysfunction possibly through the JAK/STAT pathway.

The growing use of general anesthetics has become a matter of great concern with the evidence that anesthesia may cause neurodevelopmental deficits in children.19–21 However, the underlying cellular and molecular mechanisms are still waiting for in-depth study. Mounting evidence suggests that neurons could respond actively after general anesthetic exposure. Neuronal apoptosis and inhibited neurogenesis, as well as impaired synaptogenesis, have been observed after general anesthesia.22–24 Although in this study, we found that astrocyte numbers and GFAP expression were both decreased after sevoflurane exposure, our data suggest that the global mechanisms by which general anesthetics influence development may be through reduced numbers and/or weakened functions of cell types other than neurons.

Astrocytes are the most abundant cells in the brain that support nutrients, recycle neurotransmitters, and maintain homeostasis.5 In CNS injury and other neurological diseases, reactive astrogliosis (hyperactivated), as well as morphological and functional changes, was observed. Although in some other conditions and in the present study, astrocytes presented a “hypoactivation” state,25,26 the role of sevoflurane in developing astrocytes has not yet been reported. However, in other conditions, general anesthetics including sevoflurane significantly inhibited astrocytic activity. In a rat model of intracerebral hemorrhage, 2.2% sevoflurane but not fentanyl combined with dehydrobenzperidol and midazolam evoked not only the delay of activation of astrocytes but also a decrease in GFAP level.27 In a recent study, calcium imaging in awake mice showed that 3 commonly used anesthetic combinations markedly and selectively suppressed calcium transients in neocortical astrocytes.28

Another laboratory in our university also has reported that sevoflurane significantly inhibited N-myc downstream-regulated gene-2 expression in cerebral ischemic conditions.29 N-myc downstream-regulated gene-2 is a transcription factor that is specifically expressed on astrocytes and essential for astrocytic proliferation.29 Together with these data, we conclude that sevoflurane may affect astrocytic activity in many conditions, and its role during neurodevelopment should be carefully observed. It should be pointed out that in the immunohistochemistry experiment of our study, rats were anesthetized with chloral hydrate and quickly perfused. As a hypnotic sedative, chloral hydrate may influence neuronal activity; however, no data have been reported on the effect of chloral hydrate on astrocytic function, especially in this “short-acting” condition.

Disruption in glutamate homeostasis is thought to be a factor in the pathogenesis of many neurological diseases.10 To prevent the initiation of glutamate-induced neurotoxicity, glutamate transporters have evolved to maintain low extracellular levels of glutamate. One of these transporters, GLAST, was shown to be predominant in astrocytes,30 which also has been confirmed by our double-labeling study. Our results show significant changes in the expression of GLAST after sevoflurane anesthesia. These data are suggestive that changes in astrocytic glutamate uptake and the concentration of extracellular glutamate likely occur in the developing hippocampus after sevoflurane inhalation. An increased concentration of extracellular glutamate causes excitatory neurotoxicity and consequent neuronal death and/or apoptosis.31 This could enhance the direct influence of general anesthetics on developing neurons and induce extensive neuronal injury after anesthesia.32

In addition, a normal level of extracellular glutamate is also important for oligodendrocytes development. It is reported that overactivation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors and subsequent Ca2+ influx could damage oligodendrocytes progenitors.33 Interruption of oligodendrocytes maturation may result in myelination disorders and related neurological consequences.34 Inhaled anesthetic-induced alterations of glutamate transporter expression also have been observed in other studies. It is reported that isoflurane induced increased hippocampal glutamate and an upregulation of GLAST in the hippocampus of aged rats with learning/memory impairment.35 In this condition, a GLAST increase is thought to be responsive to the elevation of glutamate. Another study observed that in an in vitro model of oxygen-glucose deprivation, sevoflurane reduced the decrease in glutamate uptake.36 Explanations for these variations in different conditions are still in need of further study. Astrocyte proliferation is very active, and sevoflurane could probably interrupt this process and induce downregulation of GLAST on astrocytic membrane in the developing brain but not the aged brain or in oxygen-glucose deprivation conditions.

The JAK/STAT pathway is essential for astrocyte proliferation, maturation, and active response to insults.37,38 It has been observed that GFAP transcription could be regulated by STAT3 phosphorylation in the developing brain.39 Disruption of STAT3 in primary astrocyte induces oxidative stress,40 suggesting the essential role of the STAT3 pathway for the survival of astrocytes. In the present study, STAT3 phosphorylation was robust in hippocampal astrocytes in the early postnatal stages. After sevoflurane anesthesia, JAK/STAT phosphorylation is inhibited in a similar pattern as GFAP and GLAST. Astrocyte numbers were reduced after sevoflurane inhalation, indicating that astrocytic proliferation could be inhibited by sevoflurane. In addition, GLAST-mediated astrocytic functions could also be suppressed by sevoflurane. These data suggest that sevoflurane exposure inhibits astrocyte maturation and function via JAK/STAT inactivation, although other cell signaling pathways including mitogen-activated protein kinase are also possible.41

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CONCLUSIONS

GLAST was inhibited by sevoflurane in the hippocampus of neonatal rats. Inactivation of the JAK/STAT pathway possibly contributes to this effect of sevoflurane. Our data may thus extend the previous knowledge that astrocytic dysfunction may be also a contributor to the neurotoxicity of general anesthetics in the developing brain. Furthermore, JAK/STAT inactivation and GLAST downregulation could be new targets for treating general anesthetic-induced neurotoxicity in the developing brain.

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DISCLOSURES

Name: Wei Wang, MD, PhD.

Contribution: This author helped in designing the study and conducted most of the experiments with some other authors.

Attestation: Wei Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Rui Lu, MD.

Contribution: This author helped in designing the study, conducting experiments, and in statistical analysis.

Attestation: Rui Lu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Da-yun Feng, MD, PhD.

Contribution: This author helped in conducting experiments and preparing the manuscript.

Attestation: Da-yun Feng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Hui Zhang, MD, PhD.

Contribution: This author designed the study and wrote the manuscript and is also the archival author of this manuscript.

Attestation: Hui Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.

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