Neuroscience Letters 545 (2013) 17– 22

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Neuroscience Letters

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

JNK pathway may be involved in isoflurane-induced apoptosis in the hippocampi of neonatal rats

Yujuan Li a,∗,1, Fei Wang a,1, Chuiliang Liu b, Minting Zeng a, Xue Han a, Tao Luo c, Wei Jiang c, Jie Xu c, Huaqiao Wang c,∗∗

a Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China b Department of Anesthesiology, ChanCheng Central Hospital, Foshan 528031, China c Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China

h i g h l i g h t s

We investigated the effects of JNK pathway on isoflurane-induced neuroapoptosis. • SP600125 reduced isoflurane-induced apoptosis in the hippocampi of neonatal rats. • Isoflurane-induced activation of JNK and c-Jun was inhibited by SP600125. • SP600125 reversed isoflurane-induced decrease of Bcl-xL. • SP600125 maintained Akt activation.

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 14 February 2013 Received in revised form 30 March 2013 Accepted 1 April 2013

Keywords: Apoptosis Anesthetics volatile – isoflurane C-Jun N-terminal kinase Caspase-3 Hippocampus

Previous studies have demonstrated that isoflurane, a commonly used volatile anesthetic, can induce widespread apoptosis in the neonatal animal brains and result in persistent cognitive impairment. Isoflurane-induced cytosolic Ca2+ overload and activation of mitochondrial pathway of apoptosis may be involved in this neurodegeneration. The c-Jun N-terminal kinase (JNK) signaling can regulate the expression of the Bcl-2 family members that modulates mitochondrial membrane integrity. Therefore, we hypothesize that JNK signaling pathway activation contributes to isoflurane-induced apoptosis in the brain. In this study, Sprague-Dawley neonatal rats at postnatal day 7 were exposed to 1.1% isoflurane or (cid:2)g or the vehicle was intraventricularly air for 4 h. The JNK inhibitor SP600125 at 5 administered before the exposure. Neuronal apoptosis in the hippocampi of neonatal rats was detected by TUNEL 6 h after isoflurane or air exposure. The protein expression of phospho-JNK, phospho-c-Jun, and caspase-3 as well as the antiapoptotic protein Bcl-xL and Akt/glycogen synthase kinase (GSK)-3(cid:3) pathway was detected by Western blotting. Isoflurane significantly increased apoptotic cells in the hippocampal CA1, CA3, and DG regions. The JNK inhibitor SP600125 dose-dependently inhibited isoflurane-induced neuronal apoptosis and increase of caspase-3 and phospho-JNK. SP600125 also attenuated isoflurane- induced down-regulation of Bcl-xL and maintained the activated Akt level to increase the phosphorylation of GSK-3(cid:3) at Ser9. Our results indicate that JNK activation contributes to isoflurane-induced neuroapopto- sis in the developing brain. Maintaining Bcl-xL and Akt activation may be involved in the neuroprotective effects of SP600125.

(cid:2)g, 10

(cid:2)g, 20

(cid:2)g, 30

© 2013 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: Akt, protein kinase B; AP-1, activator-protein 1; Bcl-2, B cell lymphoma/lewkmia-2; CNS, central nervouse system; DG, dentate gyrus; GSK-3(cid:3), glycogen

synthase kinase 3(cid:3); i.c.v, intracerebroventricular; JNK, c-jun N-terminal kinase; PI3K, phosphatidylinositol 3 kinase; TUNEL, TdT-mediated dUTP nick end labeling.

∗

Corresponding author at: Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No. 107 Yanjiang West Road, Guangzhou 510120, China.

Tel.: +86 02081332060; fax: +86 02081332833. ∗∗

Corresponding author at: Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, No. 74 Zhongshan Second Road, Guangzhou

510080, China. Tel.: +86 02084111676; fax: +86 02084112545.

(M. Zeng), hanmingxuan@rocketmail.com (X. Han), luotao20080808@163.com (T. Luo), jiangweijw@yahoo.com.cn (W. Jiang), xujie@mail.sysu.edu.cn (J. Xu), wanghq@mail.sysu.edu.cn (H. Wang).

E-mail addresses: yujuan 04@yahoo.com.cn

(Y. Li), 1995wangfei@sina.com.cn

(F. Wang),

lcl1204@yahoo.com.cn

(C. Liu), minting19@163.com

1 Both these authors contributed equally to this work.

0304-3940/$ – see front matter © http://dx.doi.org/10.1016/j.neulet.2013.04.008

2013 Elsevier Ireland Ltd. All rights reserved.

18

Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22

1. Introduction

Exposure to anesthetics has been associated with widespread apoptotic neurodegeneration in the developing brains and per- sistent cognitive in animals [5,16,23] Moreover, nociceptive stimuli, such as formalin subcutaneous injection or surgical incision, further augment apoptosis and cognitive impair- ment induced by anesthetics in developing rats [24]. Some clinical retrospective studies have found that anesthesia and surgery in children younger than 4 years increase their probability of devel- oping disabilities in reading, writing and mathematics learning [15,30]. These reports have led to concerns about the possible detrimental effects of anesthesia and sedation in the pediatric pop- ulation.

impairment

Isoflurane is a commonly used volatile anesthetic. Current stud- ies have suggested that isoflurane causes severe neuroapoptosis in both developing animal brains and primary neuronal cells [5,16,29]. Isoflurane induces neuronal apoptosis and degeneration via [Ca2+]i (cid:4)-aminobutyric acid (GABA)A overload through the opening of the receptor-mediated synaptic voltage-dependent calcium channels (VDCCs) and the excessive Ca2+ release from the endoplasmic reticulum via activation of inositol-1,4,5-trisphosphate (IP3) recep- tors [35,36]. Isoflurane-induced [Ca2+]i overload not only activates mitochondrial pathway of apoptosis [29,33], but also is linked to the activation of c-Jun N-terminal kinase (JNK) because the phosphory- lation of c-Jun is prevented by antagonizing IP3 receptors [5]. The regulation of mitochondrial membrane integrity and the release of apoptogenic factors from mitochondria are tightly controlled by the proteins of Bcl-2 family [34]. The JNK signaling plays a pivotal role in mediating neuronal apoptosis through direct regulation of the expression of Bcl-2 family members and activation of the acti- vator protein 1 (AP-1) transcription factor family member c-Jun [1] that provides indirect transcriptional regulation of the Bcl-2 family members [14,18], including down-regulation of the antiapoptotic proteins Bcl-2 and Bcl-xL [8,14].

(cid:2)g SP600125 or 12% DMSO only. The injection was performed 30 (cid:2)l as described before [6] under isoflurane anesthesia with a 5 microsyringe and 0.4 mm external diameter needle. The location of injection was 2.0 mm rostral, 1.5 mm lateral to the lambda and 2.0 mm deep to the skull surface of rats. The injection solution of (cid:2)l/min. The accuracy 5 of i.c.v. injection was verified by methylene blue in our preliminary experiments. All animals were sacrificed 6 h after termination of gas exposure and their hippocampi were used for Western blot- ting (n = 6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (n = 6).

(cid:2)l/rat was infused at a constant rate of 2.5

For Western blotting studies, rat pups were anaesthetized with isoflurane and then sacrificed by decapitation. Hippocampi of rats −80 ◦C until were isolated immediately on ice and then stored at used. Western blotting was performed as we have described pre- viously [20]. In brief, the protein concentrations of samples were determined using the BCA protein assay (Bio-Rad,Herts, UK). Sixty micrograms of each sample were subjected to Western blot analy- sis using the following primary antibodies: anti-cleaved caspase-3 at 1:2000 dilution, anti-phospho-JNK at 1:2000 dilution, anti-JNK at 1:2000 dilution, anti-phospho-c-Jun at 1:1000 dilution, anti- phospho-Akt (Ser 473) at 1:2000 dilution, anti-Akt at 1:5000 dilution, anti- phospho-GSK-3(cid:3) (Ser 9) at 1:2000 dilution, anti- GSK-3(cid:3) at 1:2000 dilution, anti-Bcl-xL at 1:2000 dilution and anti-(cid:3)-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology Company, USA. Images were scanned by an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of phospho-JNK, phospho-Akt and phospho-GSK-3(cid:3) were normalized to their total JNK, Akt and GSK-3(cid:3) from the same sam- ples. The band signals of other interesting proteins were normalized (cid:3)-actin and the results in each group were normalized to those of to that of corresponding control group.

Protein kinase B (Akt), a serine/threonine kinase, also plays a prominent role in regulating neuronal survival. Once Akt is acti- vated, it inhibits apoptosis through inactivating Bad and glycogen synthase kinase 3(cid:3) (GSK-3(cid:3)) by phosphorylation [21,25]. Recent studies show that there is a potential crosstalk between JNK and Akt signaling, Akt signaling is involved in the apoptotic effect of JNK [10,31]. SP600125, a selective JNK inhibitor [2], has showed neu- roprotective effects in several neurodegenerative diseases [17,26]. Whether JNK signaling pathway contributes to isoflurane-induced neuroapoptosis remains underdetermined. In this study, we inves- tigated the effects of SP600125 on isoflurane-induced neuronal apoptosis and the expression of the antiapoptotic proteins Bcl-xL and Akt in the hippocampi of neonatal rats.

For TUNEL studies, rat pups were anaesthetized with isoflu- rane and perfused transcardially with 4% paraformaldehyde. Their (cid:2)m thickness. brains were paraffin embedded and sectioned at 6 (cid:2)m apart) As we described before [19], four or five sections (200 for each animal at the same plane of the hippocampus were chosen for detecting apoptosis using TUNEL fluorescent method (Promega, Madision, WI, USA). The slides were protected from direct light during experiment. Hoechst was used to stain nuclei. The TUNEL positive cells in CA1, CA3 and dentate gyrus (DG) areas of hippocam- pus were analyzed immediately with NIS-Elements BR imaging processing and analysis software (Nikon Corporation, Japan). The densities of the TUNEL positive cells in CA1, CA3 and DG were cal- culated by dividing the number of TUNEL positive cells by the area of that brain region.

2. Materials and methods

All animal procedures were in compliance with the NIH Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-sen University. Seven-day-old (P7) Sprague-Dawley rat pups (Guangdong Medical Laboratory Animal ± 3 g were exposed to 1.1% isoflu- Co, China) with body weight at 16 rane (about 0.5 MAC in P7 rats [22]) for 4 h to induce neuronal apoptosis, or to air in a temperature-controlled chamber as we described before [21]. The concentrations of anesthetic gas, oxy- gen and carbon dioxide (CO2) in the chamber were measured by a gas analyzer (Datex-Ohmeda, Madison, WI).

Four doses of SP600125 (Selleck Chemicals LLC, Houston, TX, (cid:2)g) or 12% dimethyl sulfoxide (DMSO) as USA) (5, 10, 20 or 30 the vehicle were administered by intracerebroventricular (i.c.v.) injection 15 min before isoflurane exposure. Some rats received

±

SEM. The Graphpad Prism 4.0 soft- ware was used to conduct the statistical analyses. A two-tailed P value of less than 0.05 was considered statistically significant. One way ANOVA with Newman–Keuls Multiple Comparison Test was used when data was normally distributed and had equal variances. Otherwise, non-parametric test with Dunn’s Multiple Comparisons was used to compare the density of TUNEL positive cells as well as the relative protein abundance data among groups in Western blots.

Data are presented in mean

3. Results

Our preliminary experiments for arterial blood gas monitoring showed that the neonatal rats had no hypoglycemia and acidosis during isoflurane exposure. Neuronal apoptosis in the hippocam- pal CA1, CA3 and DG regions of P7 rats were detected by TUNEL (Fig. 1). Isoflurane increased the number of apoptotic cells by 498% in CA1 (P < 0.01), 214% in CA3 (P < 0.001) and 217% in DG (P < 0.001)

Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22

Fig. 1. JNK/SAPK inhibitor SP600125 inhibited the increase of isoflurane-induced TUNEL positive cells in the hippocampi of P7 rats. Representative images of TUNEL in the (cid:2)m. (C) Quantification of TUNEL hippocampal CA1 region (A) and CA3 region (B). Green staining indicated TUNEL-positive cells, blue staining indicated nuclear. Scan bar = 50 (cid:2)g SP600125; Iso: isoflurane. **P < 0.01, ***P < 0.001, vs. group DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001, positive cells in the hippocampal CA1, CA3 and DG regions. SP30: 30 vs. group SP30; (cid:2)P < 0.05, (cid:2)(cid:2)(cid:2)P < 0.001, vs. group Iso + DMSO. Mean ± SEM, n = 6/group. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

(cid:2)g as compared to sham controls. The JNK inhibitor SP600125 at 30 inhibited the increase of isoflurane-induced neuronal apoptosis by 84% in CA1 (P < 0.05), by 84% in CA3 (P < 0.001) and 71% in DG (P < 0.05). In addition, we detected the change of cleaved caspase- 3 protein expression in the hippocampus (Fig. 2). Isoflurane with or without DMSO increased the expression of cleaved caspase-3 by 174.6% (P < 0.001) or 187.2% (P < 0.001), respectively. SP600125 dose-dependently decreased the expression of cleaved caspase-3. (cid:2)g, inhibited isoflurane-induced All doses of SP600125, except for 5 increase of cleaved caspase-3 by 71.3% (P < 0.01), 84.5% (P < 0.001) and 95.5% (P < 0.001), respectively (Fig. 2B). SP600125 alone neither increased the expression of cleaved caspase-3 nor the number of apoptotic cells in hippocampus.

Isoflurane increased phospho-JNK at 46 kd by 38.7% (P < 0.05) as (cid:2)g inhibited phospho- compared to sham controls. SP600125 at 30 rylation of the 46 kd (P < 0.001) and 54 kd (P < 0.01) JNK (Fig. 2C). In accord with JNK activation, isoflurane increased the expression of phospho-c-Jun by 47.1% (P < 0.001) and decreased the expression of (cid:2)g significantly Bcl-xL protein by 40.4% (P < 0.05). SP600125 at 30 reversed the isoflurane-induced expression change of phospho-c- Jun (P < 0.01) (Fig. 3A and B) and Bcl-xL (P < 0.05) (Fig. 3C and D).

SP600125 attenuated this inhibition by 70.0% (P < 0.05) (Fig. 3E and F). Isoflurane did not significantly influence the protein expression of phospho-GSK-3(cid:3), while isoflurane combined with SP600125 sig- nificantly increased its expression compared with control (P < 0.05) (Fig. 3G and H).

4. Discussion

Isoflurane is a commonly used volatile anesthetic during human surgery. Previous studies have demonstrated that it increases neuroapoptosis and induces long-term cognitive dysfunction in developing animals [5,16,29]. The present study demonstrates for the first time the neuroprotective effect of the JNK inhibitor SP600125 against neurodegeneration induced by isoflurane, as evi- denced by diminishing isoflurane-induced activation of caspase-3 and formation of apoptotic cells in the hippocampi of neonatal rats. SP600125 significantly inhibited isoflurane-induced increase of phosphorylation of JNK and c-Jun, downregultion of Bcl-xL, and decrease of Akt activation, which may be involved in its neuropro- tective effects.

To investigate whether Akt/GSK signaling is involved in the antiapoptotic effect of SP600125, we measured proteins expres- sion of phospho-Akt and phospho-GSK-3(cid:3). Isoflurane inhibited the expression of phospho-Akt protein by 55.2% (P < 0.001), while

Isoflurane, when used in low doses or for short periods, induces small to moderate increases in [Ca2+]i by activating ryanodine and IP3 receptor in endoplasmic reticulum of neuron, which trig- gers important survival signals including phosphorylation of Akt and Bcl-2 families. These signals play very important roles in the

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Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22

Fig. 2. SP600125 dose-dependently inhibited isoflurane-induced increase of caspase-3 and phosphorylation of JNK in the hippocampi of P7 rats. (A) Representative Western (cid:2)g SP600125; blots of caspase-3, phospho-JNK and JNK; (B and C) the quantitative analysis of cleaved caspase-3 (B) and phospho- JNK (C). Con: control; Iso: isoflurane; SP5: 5

(cid:3)

(cid:2)g SP600125; SP20: 20

(cid:2)g SP600125; SP30: 30

(cid:2)g SP600125. *P < 0.05, ***P < 0.001, vs. group Con; #P < 0.05, ##P < 0.01, vs. group DMSO;

SP10: 10 (cid:2)(cid:2)P < 0.01, (cid:2)(cid:2)(cid:2)P < 0.001, vs. group Iso + DMSO; (cid:2)P < 0.05, (cid:2)(cid:2)P < 0.01, (cid:2)(cid:2)(cid:2)P < 0.001, vs. group Iso. Mean

P < 0.05, vs. group SP30;

±

SEM, n = 6/group.

neuroprotection of isoflurane preconditioning against ischemia or hypoxia in rat brain [3,4]. While when isoflurane is used in high doses or for long periods, especially if developing neurons are exposed to isoflurane, it will induce [Ca2+]i overload and results in neuronal apoptosis.

The JNK signaling pathway is implicated in neuronal apo- ptosis such as ischemia/reperfusion and ethanol [9,11–13]. In the present study, our results suggest that JNK signaling also involves in isoflurane- induced neuronal apoptosis. The JNK pathways include nuclear pathway and non-nuclear pathway [11,12]. Activated JNK phos- phorylates nuclear substrate, the transcription factor c-Jun, which leads to increase of AP-1 transcription activity to modulate tran- scription of genes related to apoptosis. On the other hand, activated JNK regulates the activation of non-nuclear substrates including Bcl-2 family members [11,12]. Our current results showed that SP600125 pretreatment prevented isoflurane-induced increase of phosphorylation of JNK and c-Jun as well as increase of caspase- 3, which suggest that activated JNK nuclear pathway is involved in isoflurane-induced neuronal apoptosis. Sevoflurane, another inhaled anesthetic, also leads to an increase of phospho-JNK and apoptosis in neonatal rat brain [27,28]. However, SP600125 did not

triggered by

several brain

injury

stimuli,

attenuate sevoflurane-induced apoptosis [27], which indicates that isoflurane and sevoflurane may induce neuroapoptosis in develop- ing brain by different mechanisms.

The antiapoptotic protein Bcl-xL is widely expressed in the central nervous system (CNS), which enhances cell survival by maintaining mitochondrial membrane integrity and inhibits cytochrome c release [34]. Anesthesia cocktail containing isoflu- rane, nitrous oxide (N2O) and midazolam can downregulate Bcl-xL expression to induce neurotoxicity in developing rat brains [33]. In this context, we observed that isoflurane alone also caused a decreased expression of Bcl-xL in the hippocampi of P7 rats, and that this decrease was blocked by SP606125, thus preventing the mitochondrial membrane alteration and neuronal apoptosis. This result is in agreement with previous studies that suggest that JNK signaling promotes apoptosis possibly via transcriptional regula- tion of Bcl-2 family gene, including Bcl-xL [8,14,18]. Our results indicate that inhibition of Bcl-xL expression is a critical step in the isoflurane-induced apoptosis pathway and that this effect is dependent on JNK activation.

Prosurvival pathways, such as Akt, may be inactivated dur- ing the apoptotic process [25,32]. Our experiments showed that isoflurane inhibited Akt phosphorylation while SP600125

Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22

Fig. 3. SP600125 prevented isoflurane-induced increase of phospho-c-Jun and decrease of BcL-xL, and maintained activated Akt level and increased phosphorylation of GSK-3(cid:3) (G); (B, D, F and H) the quantitative analysis of phospho-c-Jun (B), Bcl-xL (D), phospho-Akt (F) and phospho-GSK-3(cid:3) (cid:2)g SP600125. *P < 0.05, ***P < 0.001, vs. group DMSO; (cid:2)P < 0.05, (cid:2)(cid:2)P < 0.01, vs. group Iso + DMSO. Mean

in hippocampus of P7 rats. (A, C, E and G) Representative Western blots of phospho-c-Jun (A), Bcl-xL (C), phospho-Akt (E) and phospho-GSK-3(cid:3)

(H). Iso: isoflurane; SP30: 30

±

SEM, n = 6/group.

maintained the level of activated Akt. This result is supported by the evidence that phospho-GSK-3(cid:3) at Ser9 (inactivated form), one of the Akt phosphorylation site, is increased. This result is in agreement with previous studies that show that there is poten- tial crosstalk between JNK and Akt signaling [10,31]. It is possible that SP600125 maintain the mitochondrial membrane integrity by increasing Bcl-xL expression, thus prevents [Ca2+]i overload induced by isoflurane. However, it should be noted that isoflu- rane inhibited the expression of phospho-Akt but did not influence the expression of phospho-GSK-3(cid:3) at Ser9. The possible reason for this discrepancy may include activation of other intracellular mechanisms as a response to isoflurane, which consequently could also regulate the phosphorylation of GSK-3(cid:3). In our other exper- iments, we found isoflurane also activated p38 mitogen-activated protein kinase. A recent study suggests p38 can increase the

phosphorylation of GSK-3(cid:3) at Ser 9 [7]. Thus, it is possible that phospho-GSK-3(cid:3) stays unchangeable due to the effects of isoflu- rane on Akt and p38. This effect on Akt may explain, in part, the antiapoptotic effects of SP600125 against isoflurane-induced neu- ronal cell apoptosis in developing rat brain. Our experiments only observed short-time effects of SP600125 in the hippocampi of neonatal rats, additional studies are needed to test the impact of SP600125 on isoflurane-induced cognitive dysfunction.

5. Conclusions

In conclusion, our results are consistent with the hypothesis that the JNK inhibitor SP600125 potentially attenuates isoflurane- induced neuroapoptosis in the hippocampi of developing rats by inhibiting isoflurane-induced JNK activation, the subsequent JNK

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Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22

dependent activation of c-Jun, suppression of Bcl-xL expression, and activation of caspase-3. In addition, maintenance of activated Akt may also be involved in the antiapoptotic effect of SP600125. Our findings suggest that JNK signaling pathway activation is cru- cial for isoflurane-induced neuroapoptosis.

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Acknowledgments

The study was supported by NSFC (30700787) and the GNSF (S2011010004558) to Yujuan Li; NSTFG (2008B030301320 and 2010B031600070), and STFG (2012J410076) to Huaqiao Wang.

[18] C. Li, G. Xing, M. Dong, L. Zhou, J. Li, G. Wang, D. Zou, R. Wang, J. Liu, Y. Niu, Beta- asarone protection against beta-amyloid-induced neurotoxicity in PC12 cells via JNK signaling and modulation of Bcl-2 family proteins, Eur. J. Pharmacol. 635 (2010) 96–102.

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