Pediatric Anesthesia ISSN 1155-5645

O R I G I N A L A R T I C L E

Effects of sevoﬂurane on the expression of tau protein mRNA and Ser396/404 site in the hippocampus of developing rat brain Zhi-yong Hu1, Hai-yan Jin1, Li-li Xu2, Zhi-rui Zhu1, Yi-lei Jiang1 & Robert Seal3

1 Department of Anesthesiology, The Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Department of Anesthesiology, The Second Afﬁliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China 3 Department of Anesthesia and Pain Medicine, University of Alberta and Stollery Children’s Hospital, Edmonton, AB, Canada

Keywords phosophorulation; tau; sevoﬂurane; neonatal

Summary

Correspondence Zhi-yong Hu, Department of Anesthesiology, The Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Email: huzhiyong777@126.com

Section Editor: Andrew Davidson

Accepted 18 August 2013

doi:10.1111/pan.12263

Background: General anesthesia induces a transient hyperphosphorylation of tau protein that is associated with neurotoxicity in neonatal rats, but the mechanism remains unknown. The current study sought to investigate the effects of sevoﬂurane on the levels of tau phosphorylation at phosphor- Ser396/404 and total tau mRNA in the hippocampus of neonatal rats. Materials and Methods: Thirty-six 7-day-old rats were randomly exposed for 6 h to either 3% sevoﬂurane (S) or air (NC) as a placebo. They were sacri- ﬁced at 1, 7 and 14 days after the anesthesia, respectively, and thus assigned to S1d, S7d, S14d, NC1d, NC7d, and NC14d groups (n = 6). Their brain tissues were harvested and then subjected to histopathologic, Western blot and real- time polymerase chain reaction analysis. Results: Microtubule cytoskeletons were arranged in neat parallel rows in rats exposed only to air, whereas the microtubules were arranged in a disor- derly and intermittent (nonparallel) fashion in rats exposed to sevoﬂurane. The levels of tau mRNA in the S1d and S7d groups were signiﬁcantly higher than those in the NC1d and NC7d groups. There was no signiﬁcant differ- ence in the levels of tau mRNA between the S14d and NC14d groups. The levels of tau protein at Ser404 in the S1d, S7d, and S14d groups were signiﬁ- cantly higher than those in NC1d, NC7d, and NC14d groups. The levels of tau protein at Ser396 in the S1d, and S7d groups were signiﬁcantly higher than those in the NC1d, and NC7d groups, while there was no signiﬁcant difference in the levels of tau protein at Ser396 between the S14d group and the NC14d group, respectively. Conclusion: In rat hippocampus, sevoﬂurane was associated with microtubu- lar disarray as well as increased levels of tau mRNA and excessive phosphor- ylation of tau protein at Ser396 and Ser404. This implicates that sevoﬂurane may induce neurotoxicity.

Introduction

In humans, anesthetic agents sometimes have to be administered during the brain growth spurt period that occurs between the third trimester and the age of approx- imately 2 years. This time period is equivalent to the ﬁrst week after birth in mice and rats. Recently, it has been demonstrated in rodents that neonatal administration of

anesthetics induced widespread neurodegeneration and severe deﬁcits in spatial learning tasks (1,2). The underly- ing mechanism is not fully understood. To minimize risks, the risk of anesthesia in neonates, it is necessary to undertake further study to assess the effects of anesthet- ics on the developing nervous system.

(2,2,2-triﬂuoro-1-[triﬂuoromethyl]ethyl ﬂuoromethyl ether) is one of the most frequently used

Sevoﬂurane

1138

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Z. Hu et al.

Effects of sevoﬂurane on the expression of tau protein

volatile anesthetics for the induction and maintenance of general anesthesia during surgery because of its low blood–gas partition coefﬁcient and low pungency. In infants and children, these properties convey the beneﬁt of rapid induction and recovery as well as less irritation to the airway. Sevoﬂurane has been shown to enhance GABAA receptors (3) and to block NMDA receptors, although more research is necessary to better character- ize its effects on NMDA receptors (4).

drawn from the same litters, so that each experimental condition had its own group of littermate controls. All animals were kept in standard animal cages under con- ventional housing conditions (12-h light-dark cycle, 22°C), with ad libitum access to food and water. All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Labora- tory Animals, formulated by the Ministry of Science and Technology of China (8).

Tau protein is a highly soluble microtubule-associated protein (MAP). In humans, these proteins are mostly found in neurons compared with nonneuronal cells. One of tau’s main functions is to modulate the stability of axonal microtubules. Hyperphosphorylation of the tau protein (tau inclusions, pTau) can result in the self- assembly of tangles of paired helical ﬁlaments (PHFs) and straight ﬁlaments, which are involved in the patho- genesis of Alzheimer’s disease (AD) and other tauopa- thies. Several other studies (5,6) have shown that sevoﬂurane may induce apoptosis in the brain tissues of neonatal mice. It has also been associated with increased tau phosphorylation through speciﬁc kinase activation and with spatial memory deﬁcits. These data support a correlation between exposures to anesthetic agents and cognitive decline. Tau is hyperphosphorylated in PHFs, and speciﬁc phosphorylation sites have been implicated in the loss of tau’s association with the membrane cortex during AD disease state, including Ser 199/202, Thr 231, and Ser 396/404. Over-activation of proline-directed kinases such as cyclin-dependent kinase 5(CDk5) and glycogen synthase kinase 3(GSK3) has been implicated in the aberrant phosphorylation of tau at the proline- directed site (7). The hippocampal formation is essential for the processing of episodic memories for autobio- graphical events that happen in unique spatiotemporal contexts. Distinct regions, layers, and cells of the hippo- campal formation exhibit different proﬁles of structural and molecular development during early postnatal life.

Anesthesia treatment

Our research protocol was approved by the institutional animal research review board of Zhejiang University (Zju201301-1-02-021). Thirty-six 7-day-old male rats were allocated by computer-generated random numbers to a 6 h exposure in an anesthesia chamber with either 3% sevoﬂurane (H20100586; Abbott, Chicago, IL, USA) plus 60% oxygen (group S) or air as a normal control (group NC). Sevoﬂurane was delivered into the chamber by an agent-speciﬁc vaporizer. All anesthetized rats breathed spontaneously and underwent heart rate monitoring. As well, body temperature was monitored with a rectal probe and maintained between 36.0°C and 37.0°C by means of a heating pad. Rats were sacriﬁced at 1, 7 and 14 days following exposure, respectively, and were thus assigned to sevoﬂurane group (S1d, S7d, S14d groups, n = 6 in each) and normal control group (NC1d, NC7d, NC14d groups, n = 6 in each). Their brains were removed immediately after death and then frozen in dry ice and stored at (cid:1)70°C until used.

Arterial blood gas analysis

To determine the adequacy of ventilation, arterial blood was sampled immediately after removal from the mater- nal cage (0 h) or at the end of anesthesia (6 h) by obtain- ing a single sample (100 ll) from the left carotid artery using a 24 gauge SURFLO (Terumo, Tokyo, Japan) catheter. Bicarbonate concentration (millimoles per liter), oxygen saturation (%), pH, paCO2 (mmHg), and paO2 (mmHg) were measured immediately after blood collection, using a Nova Biomedical blood gas apparatus (ABL800; Radiometer, Copenhagen, Denmark).

In this investigation, we examined the roles of Cdk5 and GSK3 in tau hyperphosphorylation in neonatal rat hippocampus induced by sevoﬂurane. We also assessed the effect of sevoﬂurane on the levels of tau phosphory- lation at phosphor-Ser396/404 and tau mRNA in the hippocampus of neonatal rat.

Materials and methods

Examination of microtubule structure by electron microscopy

Animals

The hippocampus was removed and cut into 1 mm3 fragments. These were then ﬁxed in 2.5% glutaraldehyde for 2 hours followed by 1% osmium tetroxide (pH 7.3–7.4) for 1–2 h. After ﬁxation, the samples were rinsed with buffer for 20 min, dehydrated, soaked, and

Thirty-six neonatal male Wistar rats aged 7 days were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) (SYXK(zhe)2005-0072). A balanced number of control and experimental animals were

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

1139

14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Z. Hu et al.

Effects of sevoﬂurane on the expression of tau protein

embedded. An ultra-thin slicer was used to cut slices of 1–10 lm thickness that were then stained and viewed under electron microscopy (model CM10; Philips, Ein- dhoven, captured through a CCD camera (model C4742-95; Hamamatsu, Bridgewater, NJ, USA) and Advantage CCD Camera System software (Advanced Microscopy Techniques Corporation, Danvers, MA, USA).

the SYBR Green PCR signal was conﬁrmed by melting curve analysis. Acquired data were analyzed by LIGHTCY- CLE 2000 software 3.5 (Roche). The Ct value of each gene was normalized against that of GAPDH. Tau pri- mer sequences were as follows: tau-sense 5′ACC CCG CCA GGA GTT TGA C-3′, tau-antisense 5′-GAT CTT CGC CCC CGT TTG-3′ 244 bp, GAPDH-sense 5′- CTA CAA TGA GCT GCG TGT GGC-3′, GAPDH- antisense 5′-CAG GTC CAG ACG CAG GAT GGC-3′ 207 bp.

the Netherlands).

Images were

Western blotting analysis for tau pSer396 and pSer404

All data are expressed as mean (cid:3) SD. SPSS 12.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Numerical data including Oxygen saturation, PaO2, PaCO2, pH, and the levels of tau phosphorylation at phosphor-Ser396/404 and tau mRNA between groups were analyzed by the Student’s t-test, and intragroup numerical data were analyzed by repeated measures ANOVA. Statistical signiﬁcance was accepted as P < 0.05.

For the Western blot analysis, samples (80 lg protein) were prepared using neonatal rat hippocampal tissue. These were mixed with sample buffer, separated by 10% SDS-PAGE and electroblotted to a nitro cellulose mem- brane. The membrane was blocked for 1 hour at room temperature with blocking solution (5% nonfat milk in Tris-buffered saline with Tween 20 [TBST]). Blots were then incubated overnight at 4°C with the speciﬁc rat monoclonal antibodies anti-pSer396 (sc-101815) and anti-pSer40 (sc-12952) (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or b-actin anti- body (Santa Cruz Biotechnology). The samples were then washed three times and incubated with a horserad- ish peroxidase-labeled second antibody rabbit anti-rat IgG (1 : 2000 dilution; GE Healthcare, Shanghai, China) for 1 h at room temperature prior to visualiza- tion with a chemiluminescence detection technique (Su- perSignal West Pico Chemiluminescent Substrates; Pierce Biotechnology, Rockford, IL, USA). Densitomet- ric techniques were performed to quantify the protein band absorbance (GEL-PRO ANALYZER software; Bio-Rad Laboratories, Hercules, CA, USA) and expressed as rel- ative densitometric units of the corresponding control.

Results

The results of arterial blood gas analysis

To assess the effects of selected anesthetics on the devel- oping brain, we exposed the rats to sevoﬂurane for 6 h. There were no signs of metabolic or respiratory distress. Oxygen saturation, PaO2, PaCO2, and pH did not differ signiﬁcantly comparing with the control animals exposed to air for 6 h (Table 1).

The observation of microtubes by electron microscopy

The microtubule cytoskeleton was arranged in neat rows and parallel to each other in the NC group, whereas the microtubules were arranged in a disorderly and inter- mittent fashion and were not parallel to each other in group S (Figure 1).

Tau assay and quantitative real-time PCR

Total RNA was isolated from sevoﬂurane group and control group. Hippocampus neurons using the RNA- easy mini kit (Takara, Dalian, China) according to the manufacture’s instruction. First-strand cDNA was syn- thesized from 5 lg of total RNA using the Super Script III ﬁrst-strand synthesis kit (Takara) and random hex- amer system (Roche, Shanghai, China). Quantiﬁcation of the target genes was performed with Power SYBR Green PCR master mix kit (ABI, Carlsbad, CA, USA) in Bio-Rad MX3000P real-time PCR system according to the manufacturer’s instructions. Triplicate quantita- tive reverse transcription PCRs were carried out for each sample. The PCR ampliﬁcation cycles were as follows: initial denaturation at 95°C for 15 min, followed by 40 cycles with denaturation at 95°C for 20 s, and annealing-extension at 60°C for 35s. The speciﬁcity of

The expression of tau mRNA in neonatal rat hippocampus tissues

The levels of tau mRNA in the S1d and S 7d groups were signiﬁcantly higher than those in the NC1d and NC7d groups(P < 0.05). There was no signiﬁcant difference in the levels of tau mRNA between S14d and NC14d groups (P > 0.05; Figure 2).

The expression of tau protein Ser396 site in neonatal rat hippocampus issues

The levels of tau protein at Ser396 in S1d, and S7d groups were signiﬁcantly higher than those in NC1d, and NC7d groups (P < 0.05). There were no signiﬁcant difference

1140

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Z. Hu et al.

Effects of sevoﬂurane on the expression of tau protein

Table 1 Arterial blood gas analysis. Neonatal exposure to 3% sevoﬂurane does not induce signiﬁcant cardiorespiratory dysfunction. Analysis of arterial blood gas revealed no signiﬁcant differences in any of the measured parameters between rats exposed for 6 h to sevoﬂurane and control rats exposed to air for 6 h (t-test, all P values > 0.05)

Arterial blood gas

PaO2, mmHg

PaCO2, mmHg

pH

SaO2

Time, h

98.6 (cid:3) 0.3 99.3 (cid:3) 0.7 98.6 (cid:3) 0.4 98.2 (cid:3) 0.8

98.6 (cid:3) 8.5 99.1 (cid:3) 10.2 98.7 (cid:3) 6.3 98.2 (cid:3) 12.5

39.9 (cid:3) 5.7 40.2 (cid:3) 3.9 41.2 (cid:3) 4.4 42.5 (cid:3) 7.2

7.41 (cid:3) 0.03 7.40 (cid:3) 0.04 7.43 (cid:3) 0.06 7.42 (cid:3) 0.03

Exposed to air for 6 h (n = 18) Exposed to 3% sevoﬂurane for 6 h (n = 18)

0 6 0 6

PaCO2, arterial carbon dioxide tension; PaO2, arterial oxygen tension; SaO2, arterial oxygen saturation.

(b)

(a)

(c)

(d)

Figure 1 Electron microscopic examination of hippocampal neurons from 7-day-old rats exposed for 6 h in an anesthesia chamber to either 3% sevoﬂurane in 60% oxygen or air. Anesthesia caused dis- array of microtubule in rat hippocampus. Neonatal rats (7 day) were exposed to air (a) or 3% sevoﬂurane plus 60% oxygen (b–d) for 6 h, then the hippocampal tissues were taken and examined by electron microscopy. After exposure to air for 6 h (a), the microtubules were

arranged in neat parallel rows. After exposure to 3% sevoﬂurane plus 60% oxygen for 6 h (b–d), the microtubules were arranged in a disor- derly and intermittent fashion and were not in parallel with each other (b), or became disrupted, indistinct and have lost the normal order of arrangement (c,d). Arrows indicate the microtubule structures. Scale bars: 0.2 lm. (magniﬁcation 960 000).

and tau mRNA in neonatal rat hippocampus following the administration of 3% sevoﬂurane for 6 h. We found that sevoﬂurane induced increased levels of tau mRNA at 1 and 7 days as well as producing excessive phosphor- ylation of tau protein at Ser404 at 1, 7, and 14 days and Ser396 at 1 and 7 days in neonatal rat hippocampus. These results suggest that sevoﬂurane may induce neuro- toxicity in neonatal rats. This is consistent with recent evidence that different types of anesthetic agents, includ- ing sevoﬂurane, promote tau phosphorylation (6). Furthermore, in our study on electron microscopy, we observed that hippocampal neuron microtubules were signiﬁcantly changed and became disorganized follow- ing sevoﬂurane exposure.

in the levels of tau protein at Ser396 between the S14d and the NC14d groups, respectively (P > 0.05; Figure 3).

The expression of tau protein Ser404site in neonatal rat hippocampus tissues

The levels of tau protein at Ser404 in S1d, S7d, and S14d groups were signiﬁcantly higher than those in NC1d, NC7d, and NC14d groups. (P < 0.05; Figure 4).

Discussion

In this study, we investigated the effects of sevoﬂurane on the levels of tau phosphorylation at phosphor-Ser396/404

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

1141

14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Z. Hu et al.

Effects of sevoﬂurane on the expression of tau protein

We chose sevoﬂurane anesthesia because a recent study by Satomoto et al. (9) indicated that anesthesia with 3% sevoﬂurane plus 60% oxygen for 6 h does not signiﬁcantly alter blood gas and brain blood ﬂow. At

the same time, we hypothesized that this high concentra- tion of sevoﬂurane anesthesia would be sufﬁcient to demonstrate changes in tau phosphorylation at phos- phor-Ser396/404 and tau mRNA in the brain tissues of neonatal rats.

The tau protein is the taylorism end product of selective montaging from a single gene designated microtubule-associated in humans (10). Its primary function is to regulate the stabilization of axonal microtubules, and it has two means of dominating microtubule stability: isoforms and phosphorylation. Moreover, hyperphosphoryla- tion of the tau protein (tau inclusions, p-tau) can lead to the self-assembly of tangles of paired helical ﬁla- ments and straight ﬁlaments involved in the pathogen- esis of Alzheimer’s disease and other tauopathies (11). In other neurodegenerative diseases, the deposition of assemblages substantial in certain tau isoforms has been found. When misfolded, this otherwise extraordi- narily soluble protein can constitute exceedingly insol- uble assemblages resulting in a few neurodegenerative diseases. Recent research (5) suggests that tau protein

(MAPT)

protein

tau

Figure 2 The expression of tau mRNA (mean (cid:3) SD). After exposure to 3% sevoﬂurane in 60% oxygen for 6 h, the levels of tau RNA in the S1d and S7d groups were signiﬁcantly higher than those in the NC1d and NC7d groups (P < 0.05). There was no signiﬁcant difference in the levels of tau mRNA between S14d and NC14d groups (P > 0.05). There were 6 rats in each group at each time point. *P < 0.05 compared with the control group.

(a)

(b)

respectively (P > 0.05). *P < 0.05, compared with the control group. (a) The expression of p-tau protein Ser396 site by Western blot analy- sis; S, sevoﬂurane group; NC, control group. (b) Quantitative expres- sion of p-tau protein Ser396 site. The data are expressed as mean (cid:3) SD (ratio to b-actin). There were six rats from each group at each time point.

Figure 3 The expression of p-tau protein Ser396 site in neonatal rat hippocampus tissues. After exposure to 3% sevoﬂurane in 60% oxygen for 6 h, the levels of tau protein at Ser396 in the S1d and S7d groups were signiﬁcantly higher than those in the NC1d and NC7d groups (P < 0.05). There were no signiﬁcant differences in the levels of tau protein at Ser396 between the S14d and the NC14d groups,

(a)

(b)

Figure 4 The expression of p-tau protein pSer404 in neonatal rat hip- pocampus tissues. After exposure to 3% sevoﬂurane plus 60% oxy- gen for 6 h, the levels of tau protein at Ser404 in S1d, S7d, and S14d groups were signiﬁcantly higher than those in NC1d, NC7d, and NC14d groups (P < 0.05). *P < 0.05, compared with the control group. (a)

The expression of p-tau protein Ser404 site by western blot analysis; S, sevoﬂurane group; NC, control group. (b) quantitative expression of p-tau protein Ser404 site. The data are expressed as mean (cid:3) SD (ratio to b-actin).There were six rats from each group at each time point.

1142

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Z. Hu et al.

Effects of sevoﬂurane on the expression of tau protein

might be discharged extracellularly by an exosome- based mechanism in Alzheimer’s disease and in anes- thetic neurotoxicity in neonatal animals with similar geriatric neuroapoptosis. Lunardi et al. found that anesthesia caused long-lasting ultrastructural dis- array in the subicular neuropil and mitochondria of 21-day-old rats. Head et al. (13) showed that isoﬂura- ne signiﬁcantly decreased the number of synapses in the hippocampus compared with baseline in postnatal day (PND) 5 mice. Our electron microscopic ﬁnding in hippocampal neurons of microtubular disorganiza- tion supports the ﬁnding of Planel et al. (14). They found that exposure to isoﬂurane at clinically relevant doses led to increased levels of phospho-tau, increased insoluble aggregated forms of tau and detachment of tau from microtubules. these It microtubular structure changes may destroy the stabil- ity of microtubules, damage axonal transport and eventually led to neuroapoptosis.

of nontransgenic mice. Some phosphorylation sites have been linked to speciﬁc aspects of tau pathology such as the sequestration of normal tau, the inhibition of tau MT binding, and the promotion of tau aggre- gation. Planel et al. (14) also demonstrated that in JNLP3 mice, a mouse model of tauopathy expressing P301L mutant tau that exposure to 1.3% isoﬂurane for 4 h increased tau phosphorylation at the AT8, CP13(Ser202), pS262, MC6, and PHF-1 epitopes. Recently, Tan et al. (19) conﬁrmed these results in rats and demonstrated that 1.5% isoﬂurane for 2 h resulted in tau hyperphosphorylation at the Thr205 and Ser396 epitopes in the hippocampus and attrib- uted this effect to anesthesia-induced hypothermia. Our study showed that tau protein at Ser404 was excessively hyperphosphorylated after and 14 days, and at Ser396 after 1 and 7 days in neonatal rat hippocampus following sevoﬂurane anesthesia. Therefore, consistent with the ﬁndings of Van der Jeugd, sevoﬂurane may lead to neuronal apoptosis in the developing rodent brain through tau hyperphosph- orylation at the Ser396 and Ser404 sites. (20).

(12)

1,

is probable that

7,

The hippocampus is involved in learning and in con- solidation of explicit memories from short-term memory to cortical memory storage for the long term; its precise role in memory storage remains an active area of research and is beyond the scope of this research. Recent investigations have shown both that activation of a mutant tau gene in mice results in neuronal loss, brain atrophy, and memory impairment; and that inhibition of the mutant tau gene leads to cessation of neuronal loss, decreased atrophy of brain, and improvement in memory (15). Increased tau phosphorylation following anesthesia has also been observed with sodium pento- barbital, ketamine, or urethane (16). Yan et al. (5) examined the effects of sevoﬂurane on caspase-3 activa- tion and Ablevels in the brain tissues of neonatal mice and concluded sevoﬂurane may induce neurotoxicity. Our research showed that in the newborn rats sevoﬂura- ne induced increased levels of tau mRNA at 1 and 7 days followed by a decline by day 14. This suggests that a mechanism of sevoﬂurane-induced neurotoxicity could occur through activation of mutant tau genes.

This study has some limitations. Firstly, a 6-h-long exposure to sevoﬂurane in a 7-day-old rat pup has potentially small clinical relevance. Secondly, we did not correlate our ﬁndings with testing for learning and mem- ory defects. Thirdly, our observations of phosphoryla- tion of tau protein and microtubular disarray are suggestive, but not conclusive about the possible mecha- nisms for sevoﬂurane neurotoxicity. Finally, compara- bility of our work with those of others is confounded by differences in experimental animal ages, test parameters, and the dose, duration and method of administration of sevoﬂurane. Future studies will help elucidate these mechanisms.

In conclusion, we have shown that sevoﬂurane, the most commonly used neonatal general anesthetic, can induce an increase in the levels of tau mRNA as well as excessive phosphorylation of tau protein at Ser396 and Ser404 in neonatal rat hippocampus. As well, we observed electron microscopic evidence of microtubular disarray in the hippocampus of sevoﬂurane-exposed rats. These ﬁndings suggest that sevoﬂurane anesthesia (up to 3%) may be neurotoxic in neonatal rats. These ﬁndings should support the need for further studies to determine the potential neurotoxicity of sevoﬂurane anesthesia in the developing brain of animals and humans.

Phosphorylation of tau is adjusted by a host of kin- ases, containing PKN, a serine/threonine kinase, which phosphorylates tau, giving rise to destruction of microtubule organization. Because the hyperphosph- orylated tau was situated at the PHF-1(S396/S404) and threonine (T) T231 positions (17) in the intraneu- the PHF-1(S396/ roﬁbrillary tangles S404) was chosen to as the marker of sevoﬂurane- induced neurotoxicity in our study. Planel et al. (18) observed anesthesia induced by chloral hydrate, sodium pentobarbital, or isoﬂurane resulted in a robust hyperphosphorylation of tau at PHF-1 (Ser396/Ser404) epitopes in the brain

stage,

(NFT)

short-term (30–60 min)

that

Acknowledgments

This work was the Ministry of Education, Zhejiang, China (Y201017446),

supported by the project of

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

1143

14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Z. Hu et al.

Effects of sevoﬂurane on the expression of tau protein

the Bureau of (Y201121392) and the project of Chinese Medicine, Zhejiang, China (2011ZA067) and the Project of Medical Technology, Zhejiang, China (2013ZDA011), (2013KYB193). The Authors wish to thank the Key Laboratory for Diagnosis and Therapy of Neonatal Diseases and the Key Laboratory of

Reproductive Genetics, Zhejiang University, Ministry of Education, Zhejiang, China, for their support.

Conﬂict of interest

No conﬂicts of interest declared.

References

15 Santacruz K, Lewis J, Spires T et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science 2005; 309: 476–481.

7 Shahani N, Brandt R. Functions and mal- functions of the tau proteins. Cell Mol Life Sci 2002; 59: 1168–1680.

1 Jevtovic-Todorovic V, Hartman RE, Izumi Y et al. Early exposure to common anes- thetic agents causes widespread neurodegen- eration in the developing rat brain and persistent learning deﬁcits. J Neurosci 2003; 23: 876–882.

8 The Ministry of Science and Technology of the People s Republic of China. Guidance Suggestions for the Care and Use of Labora- tory Animals. 2006-09-30.

16 Holscher C, van Aalten L, Sutherland C.

Anaesthesia generates neuronal insulin resis- tance by inducing hypothermia. BMC Neuro- sci 2008; 9: 100.

2 Fredriksson A, Ponte′n E, Gordh T et al. Neonatal exposure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegenera- tion and persistent behavioral deﬁcits. Anesthesiology 2007; 107: 427–436.

9 Satomoto M, Satoh Y, Terui K et al.

17 Augustinack JC, Schneider A, Mandelkow EM et al. Speciﬁc tau phosphorylation sites correlate with severity of neuronal cytopa- thology in Alzheimer’s disease. Acta Neuro- pathol 2002; 103: 26–35.

Neonatal exposure to sevoﬂurane induces abnormal social behaviors and deﬁcits in fear conditioning in mice. Anesthesiology 2009; 110: 628–637.

10 Sultan A, Nesslany F, Violet M et al.

Nuclear tau, a key player in neuronal DNA protection. J Biol Chem 2011; 286: 4566–4575.

3 Nishikawa K, Harrison NL. The actions of sevoﬂurane and desﬂurane on the gamma- aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subun- its. Anesthesiology 2003; 99: 678–684. 4 Hollmann MW, Liu HT, Hoenemann CW

18 Planel E, Richter KE, Nolan CE et al. Anes- thesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia. J Neurosci 2007; 27: 3090–3097.

11 Iqbal K, Liu F, Gong CX et al. Tau in

Alzheimer disease and related tauopathies. Curr Alzheimer Res 2010; 7: 656–664. 12 Lunardi N, Ori C, Erisir A et al. General

19 Tan W, Cao X, Wang J et al. Tau hyper-

et al. Modulation of NMDA receptor func- tion by ketamine and magnesium, part II: interactions with volatile anesthetics. Anesth Analg 2001; 92: 1182–1191.

phosphorylation is associated with memory impairment after exposure to 1.5% isoﬂurane without temperature maintenance in rats. Eur J Anaesthesiol 2010; 27: 835–841.

Anesthesia causes long-lasting disturbances in the ultrastructural properties of develop- ing synapses in young rats. Neurotox Res 2010; 17: 179–188.

5 Lu Y, Wu X, Dong Y et al. Anesthetic sevo- ﬂurane causes neurotoxicity differently in neonatal na€ıve and Alzheimer disease trans- genic mice. Anesthesiology 2010; 112: 1404–1416.

20 Van der Jeugd A, Ahmed T, Burnouf S et al. Hippocampal tauopathy in tau transgenic mice coincides with impaired hippocampus- dependent learning and memory, and attenuated late-phase long-term depression of synaptic transmission. Neurobiol Learn Mem 2011; 95: 296–304.

13 Head BP, Patel HH, Niesman IR et al. Inhi- bition of p75 Neurotrophin receptor attenu- ates Isoﬂurane-mediated neuronal apoptosis in the neonatal central nervous system. Anes- thesiology 2009; 110: 813–825.

6 Le Freche H, Brouillette J, Fernandez-

14 Planel E, Bretteville A, Liu L et al. Accelera- tion and persistence of neuroﬁbrillary pathol- ogy in a mouse model of tauopathy following anesthesia. FASEB J 2009; 23: 2595–2604.

Gomez FJ et al. Tau phosphorylation and sevoﬂurane anesthesia: an association to postoperative cognitive impairment. Anes- thesiology 2012; 116: 779–787.

1144

© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144