Introduction

Ketamine is frequently used in infants and toddlers as an anesthetic for surgery. It is a noncompetitive blocker of N-methyl-D-aspartate (NMDA) receptor ion channels. However, in recent years numerous animal studies in rodents have indicated that ketamine-induced neurodegereration in the delveloping brain [1-5]. Studies in vitro have also showed that ketamine administration induces neuronal apoptosis in culture condition [4,6,7]. These evidences call for caution with the use of ketamine in neonatal and pediatric anesthesia. Research on neuroprotective measures to prevent or ameliorate toxicity of NMDA antagonist such as ketamine, phencyclidine and MK-801 for the developing brain has been carried out. It has been reported that free radical scavengers [8], GABA mimetic agents (propofol, sodium thiopental) [9], cycloheximide and insulin-like growth factor Ⅰ [10] showed neuroprotective effect for NMDA antagonist induced injuries.

Erythropoietin (EPO) was first identified as a hematopoietic cytokine acting as a survival and differentiation factor [11]. It also has been reported that EPO displays efficient neuroprotective properties in a spectrum of different animal models. These extend from ischemia/hypoxia, excitotoxic paradigms, traumatic brain and spinal cord injury, and retina/optic nerve damage to inflammatory/auto-immunological diseases [12,13]. A recent study suggested a possible protective role for EPO in MK801-mediated brain damage by enhancing neurotrophin-associated signaling pathways [14]. EPO receptor expression is abundant in the embryonic, fetal, and adult brain in rats, mice, monkeys, and humans [15-18]. Once it is bound to its receptor, EPO initiates the downstream signaling pathways.

However, to date, the mechanisms that underlie the protective effect of EPO on the neurotoxicity of ketamine have not fully been understood. Here we investigated whether recombinant EPO (rEPO) can protect cultured cortical neurons against ketamine-induced apoptotic neurodegeneration. Moreover, we analyzed in detail the intracellular signal transduction cascades involved in EPO-dependent neuroprotection in this model. Our findings show that the Akt/GSK-3β/caspase-3 dependent pathway plays an important role in mediating the protection of EPO against cortical neuronal apoptosis induced by ketamine.

Materials and methods

Reagents

Recombinant Human Erythropoietin Injection was obtained from Shenyang Sunshine Pharmaceutical Co. Ltd. Ketamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), kinase inhibitor LY294002 were purchased from Sigma-Aldrich Inc. (St, Louis, MO, USA). The in situ cell death detection kit was from the Boehringer Mannheim, Co. (Mannheim, Germany). BCA kit and Enhanced chemiluminescence (ECL) were purchased from Pierce Chemical Company (Rockford, IL, USA). Phospho-Akt, and Akt antibodies were purchased from Cell Signal Technology Inc. (Beverly, MA, USA), phospho-GSK-3β and GSK-3β antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The Caspase-3 Fluorescent Assay kit was from R&D systems (Minneapolis, MN). Dulbecco’s modified Eagle’s medium (DMEM) and B27 supplement were purchased from Gibco BRL (Gaithersburg, MD, USA).

Cell Culture

Rat primary cortical neuronal cultures were prepared from the dissected cortical hemispheres of newborn rats (less than one day) under sterile condition. Dissociated neurons were supended in complete medium containing DMEM/F12, 10% fetal bovine serm, 10% horse serum, 100 U/ml penicillin, 100 U/ml streptomyclin sulfates, and counted in a hemocytometer. Approximately 10⁶ cells/ml per well were plated on poly-D-lysine coated 6 or 24 wells plates in medium and cultured in the incubator with 5% CO₂ at 37℃. Cell culture medium was changed with serum-free DMEM/F12+2% B27 supplement at 2 days, subsequent half of cell culture medium was replaced every 3 days. The purity of the cultures was confirmed using monoclonal antibodies against neuronal specific neurofilaments, 95% cells were positive for this marker. The neurons were cultured 1 week for experimental use.

Measurement of Cell Viability

Cell viability was measured using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. The medium was incubated with 10 μl of 5 mg/ml MTT solution before the end of the experiment for 4 h at 37℃. Then the culture medium with MTT was removed and 200 μl dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 570 nm (540 nm as a reference) with a model 550-microplate reader. Cell viability was expressed as a percentage of the value in control cultures.

TUNEL assay for apoptotic DNA fragmentation

The DNA fragmentation of apoptotic cells were identified by the TUNEL method which permits the specific labeling of the 3’-OH end of DNA breaks with modified nucleotides by TdT. After incubation for up to 24 h, the cultures were washed with PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. Endogenous peroxidase was quenched by incubation with 0.3% (v/v) hydrogen peroxide in methanol for 30 min at room temperature and the cells further permeabilized with 0.1% Triton X-100 in 0.1% sodium acetate for 5 min at 4℃. Thereafter, the cells were labeled by incubation with the TUNEL reaction mixture for 60 min at 37℃ followed by labeling with peroxidase-conjugated anti-fluorescein anti-goat antibody (Fab fragment) for an additional 30 min. Subsequently, cells were incubated with diaminobenzidine substrate (DAB) to produce a dark brown precipitate. The TUNEL-positive cells were then photographed with an Olympus light microscope. The percentage of TUNEL-positive cells was estimated in 5 randomly selected fields. At least 200 cells were counted per condition, and each experiment was repeated in at least 3 different cultures.

Western blot analysis

After exposure to ketamine and /or EPO, cells were rinsed twice with cold PBS and lysed in buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100, 0.1% SDS, 50 mM sodium fluoride and 1 mM sodium vanadate) containing a protease inhibitor cocktail to obtain whole cell protein. Lysates were cleared by centrifugation and protein concentration was determined by BCA kit. Equal amounts of proteins were fractionated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% defatted milk in TBS-Tween (TBS-T) (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) and incubated with with anti-phosphospecific Akt, anti-Akt, anti-phosphospecific GSK-3b, anti-GSK-3b (all at 1 : 1000 dilution) overnight at 4°C. The signals were detected using goat anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL), then exposed to X-ray films (Fuji, Japan). Blots were quantified using analysis system (GDS8000, Ultra-Violet Products, UK). All data from three independent experiments were expressed as the ratio to optical density (OD) values of the corresponding controls for the statistical analyses.

Measurement of caspase-3 activity

The activity of caspase-3 was detected with the caspase-3 fluorometric assay kit (R&D systems, Minneapolis, MN) according to the instruction manual. This kit uses synthetic tetrapeptide DEVD-AFC as the substrate. In the presence of active caspase 3, the substrate is cleaved between DEVD and AFC, releasing the fluorogenic AFC, which is then detected by spectrofluorometry. The fluorogenic AFC reflects the activity of caspase-3. Cortical neurons were incubated in medium containing serum-free medium with ketamine and/or EPO. Caspase activities were measured at the indicated time points after ketamine and/or EPO treatment. After the incubation, cells were collected and lysed in a lysis buffer on ice for 10 minutes. The protein concentrations of the supernatant fluids were ascertained with the BCA kit. Samples containing 200 μg protein were mixed with the reaction buffer and DEVD-AFC substrate, followed by a 2 h incubation at 37⁰C. The fluorescence was measured at an excitation wavelength of 400 nm and emission wavelength of 505 nm with fluorometric reader. Experiments were run three times separately. A caspase-3-like activity was expressed as relative content against that in the cells incubated in the medium containing serum without ketamine.

Statistical analysis

Data are expressed as the mean ± S.D. Differences between the different groups were analysed by one-way analysis of variance (ANOVA) followed by the post-hoc Duncon’s test. A probability of P＜0.05 was considered to be statistically significant.

Results

EPO protected cultured rat cortical neurons from apoptosis-like cell death induced by ketamine

We first tested the neurotoxicity of ketamine in cortical neurons and cell viability was assessed by the MTT reduction assay (Fig.1). Compared with vehicle controls, ketamine at concentrations from 0.1 to 30 μM significantly decreased cell viability. Exposure to 30 µM ketamine for 24h resulted in the minimal survival of 23.3% of neurons. The neurotoxic effects of 10µM ketamine were significantly attenuated by EPO, when the cells were co-incubated with different concentrations (0, 0.3,1,3, or 10 U/ml) of EPO and ketamine for 24 h. Compared with the control group, the survival rate of neurons was 38.5±7%, 72.6±6%, 85.8±9%, 77.7±8% and 71.1±10% respectively when the cells were treated with the indicated concentrations of EPO for 24 h (Fig. 2). The results in Fig. 2 showed that EPO exhibited neuroprotective effects in a dose-dependent manner on the cytotoxicity induced by ketamine in neurons, and maximal rescue occurred at a concentration of 1 U/ml EPO.

Ketamine-induced cell death was further confirmed by the TUNEL assay, which was used to assess the morphological changes of apoptosis in neurons. As shown in Fig. 3A, in controls, no TUNEL-positive cells were seen. In contrast, the condensation and fragmentation of nuclei characteristic of apoptotic cells were evident in neurons treated with 10 µM ketamine for 24 h (Fig. 3B), which was also shown by quantitative assessment (Fig. 3E). In the cells which were co-treated with 1 U/ml EPO and ketamine at 10 µM for 24 h, the number of cells with nuclear condensation was significantly reduced (Fig. 3C and 3E), revealing that EPO can protect neurons against apoptosis induced by ketamine.

The neuroprotective effect of EPO involves PI3K pathway

In order to determine whether the PI3K signaling pathway was involved in the neuroprotective effect of EPO, we carried out experiments using LY294002, a specific inhibitor of PI3K. The survival rate of neurons was 81.7±7% after treatment with EPO for 24h. However, in the presence of LY294002 (10 μM), the survival rate of neurons was only 50.36±6%. These results indicated that LY294002 abolished the neuroprotection induced by EPO (Fig. 4). These results were also confirmed by TUNEL assay. After administration of 10µM ketamine, 1 U/ml EPO and 10µM LY294002 for 24 h, numerous darkly stained TUNEL-positive cells exhibiting typical nuclear condensation and fragmentation were observed (Fig.3D). Quantitative assessment indicated that the decreased percentage of TUNEL-positive cells induced by ketamine plus EPO was reversed by LY294002 administration (Fig.3E).

EPO increases the Akt and GSK3-β phosphorylation in ketamine -treated neurons

Considering that LY294002 abolished the neuroprotective effect of EPO, we analyzed the activation of Akt/PKB, a putative effector of PI3K involved in cell survival signaling. As shown in Fig. 5C, the level of pAkt was low in neurons under unchallenged condition. Ketamine reduced pAkt levels in the absence of EPO in a time-dependent manner (Fig. 5A). In contrast, enhanced levels of pAkt were detected 30 min after EPO was added to the culture alone, and levels remained elevated for at least 2 h (Fig. 5B). Two hour after the ketamine and EPO co-treatment, EPO markedly ameliorated this decrease of phosphorylated active forms of Akt induced by ketamine and even increased baseline levels of pAkt in neurons (Fig. 5C). To confirm the involvement of Akt further, we inhibited the upstream pathway that controls Akt activation. The PI3K inhibitor, LY294002 (10 μM) was added to neurons 1 h before EPO treatment. LY294002 thoroughly abolished EPO-induced phosphorylation of Akt, indicating that EPO activates Akt likely through PI3k (Fig. 5C). No alteration in the total amount of Akt was observed (representative Western blotting in Fig. 5).

When Akt is activated (i.e., phosphorylated), it phosphorylates and inactivates GSK-3β. In respect to GSK-3β, a reduction of pAkt reduces the phosphorylation of GSK-3β to pGSK-3β. Therefore, GSK-3β phosphorylation was also evaluated after 0.5, 1 and 2 h after ketamine or EPO treatment. As shown in Fig.6A, phosphorylation of GSK-3β at Ser9 was inhibited following ketamine-treated. The time course of decreased phosphorylation of GSK-3β is in parallel with the inactivation of Akt (Fig. 5A, 6A). Concurrent increase in Akt and GSK-3 phosphorylation in neurons treated by EPO is showed in Fig. 5B and Fig. 6B. LY294002 pretreatment abolished phosphorylation of both Akt and GSK-3β by EPO (Fig. 5C, 6C). These data indicate that GSK-3β was the major substrate of Akt contributing to the EPO-mediated neuroprotection against neurotoxicity of ketamine.

Akt pathway prevented apoptosis by blocking caspase-3-like protease activity in ketamine-treated neurons

To estimate further the contribution of the Akt pathway to the caspase activity that induces DNA fragmentation, caspase-3-like protease activity was measured with or without LY294002. As is shown in Fig. 7, we found that ketamine induced a time dependent increase in caspase-3-like proteinase activities. EPO significantly decreased caspase-3-like activities induced by ketamine toxicity. To estimate further the contribution of the Akt pathway to the caspase activity that induces DNA fragmentation, caspase-3-like protease activity was measured with or without LY294002. Consistent with the critical role of PI3K in mediating EPO-induced neuroprotection, the addition of the PI3K inhibitor, LY 294002 (10 μM), reversed the effect of EPO on caspase-3 activation in neurons (Fig. 7). These results indicate caspase-3 activation as a downstream event after Akt signaling.

Discussion

Ketamine plays an important role in pediatric anesthesia. Recent studies on anesthetics have shown that clinically relevant doses of ketamine, a non-competitive NMDA receptor antagonist, trigger massive and widespread apoptotic neurodegeneration in the immature rat brain [19-21].

EPO has a potential for protection and repair following injury to the developing brain [22]. Recent research reported that EPO treatment of the neonatal rat provided a marked inhibition of neuronal apoptosis induced by the NMDA receptor antagonist MK801 [14]. However, to date, the molecular mechanisms that underlie the protective effect of erythropoietin on the neurotoxicity of ketamine have not been fully understood. In order to explore this subject, we studied the effects of EPO on ketamine induced neurotoxicity.

In the present study, we observed a significant toxic effect of ketamine treatment at concentrations of 10 or 20 μM compared with control cultures. Ketamine administration produced a dose-related increase in neurotoxicity. Thus, the cortical neuron culture model in our research does mimic the previous animal studies in vivo [2,4]. We demonstrated that EPO prevented the degeneration of neurons induced by ketamine. As we and others have previously found in various cell culture models of neuronal cell death [23-25], the survival-promoting action of EPO follows a bell-shaped dose-response curve. Increasing EPO concentration in the cell culture medium above an optimal dose resulted in a decline of the cell rescue rate. Several studies have confirmed this particular dose-response behavior of EPO in vivo [26,27], which may caused by high concentration of EPO, which induced a rapid down-regulation of EPO receptor, failing to transmit EPO-mediated signals to the neurons [26]. Thus, these phenomena emphasize optimised dosing in further experimental research.

Phosphoinositide 3-kinases (PI3K) are agonist-activated lipid signaling enzymes that initiate signaling cascades that play a critical role in a variety of cellular processes most commonly associated with growth and survival [28]. Activation of PI3K protects cells from apoptosis and has emerged as a master regulator of cell survival. Pro-survival actions of PI3K occur through activation of the anti-apoptotic effector Akt [29]. The NMDA receptor has a recognized role in a variety of neuronal physiological and pathological processes. Activation of Akt by low (physiological) levels of NMDA receptor activation may function to promote neuronal survival [30]. Ketamine, an antagonist of NMDA receptor, inhibited Akt phosphorylation (Fig. 5A).

To clarify the signaling pathway probably involved in the neuroprotection by EPO observed in our model, we explored the PI3K pathway. We used LY294002, an inhibitor of PI3K. Results from Cell viability, TUNEL assay, and Western blots strongly suggest that the PI3K signaling pathway is involved in the neuroprotection by EPO observed in our model of neuron degeneration.

Following this pathway, GSK-3b is a substrate of Akt [31]. The main regulatory mechanism of these enzymes is by phosphorylation: Akt is activated while GSK-3b is inhibited by phosphorylation [32]. In present study, ketamine enchanced GSK-3b phosphorylation in cultured neurons (Fig. 6A), which is consistent with previous report that activation of GSK-3 was involved in ketamine-induced apoptosis in rat cortical neurons [6]. We presented convincing evidence that treatment of EPO activates Akt, resulting in the inhibition of GSK-3b activity in the anti-apoptotic process. In agreement with a previous report showing that GSK-3b activity is suppressed by EPO in erythroid progenitors [33], our data demonstrated EPO was able to increase the level of the inactive form of GSK-3b in parallel with the activation of Akt. We also showed that the PI3K inhibitor, LY294002, blocked EPO-induced phosphorylation of GSK-3b. These results indicate that the protective effect of EPO on apoptosis is also mediated by PI3K/Akt-dependent GSK-3b phosphorylation.

Caspase-3 is thought to be activated during the final step of the proapoptotic signaling pathway in many cells. In the present study, we confirmed that EPO-mediated Akt signaling effects in regulating neuronal death are executed by inhibition of the caspase-3 activity. Previous studies in neuronal apoptosis induced by ketamine have showed that the increased activity of GSK-3 precedes activation of caspase-3 [6,10]. Consistent with the critical role of Akt/GSK-3b in mediating EPO induced neuroprotection, our results indicated that the addition of LY 294002 reversed the effect of EPO on caspase-3 activation. Thus, the activation of caspase-3 is a downstream event after Akt/GSK-3b signaling.

In conclusion, these data demonstrate that EPO exerted protection against apoptosis induced by ketamine in neurons. We observed that LY294002, an inhibitor of PI3K, reversed EPO’s neuroprotective effect. In addition, EPO induces phosphorylation of two proteins that leads to activation of Akt and inhibition of GSK-3b. EPO also prevented the decrease in phosphorylation Akt and GSK-3b induced by ketamine. Our results identified the PI3K/Akt and GSK-3b signaling pathway as the one responsible for protecting neurons from apoptosis induced by ketamine. Our results also implicate caspase-3 activation as a critical downstream signaling effector in EPO/PI3K/Akt/GSK-3b signaling. The results from the present study may help in designing clinical studies on the potentially beneficial role for treatment of ketamine neurotoxicity in youngsters.

Fig. 6 Ketamine-induced down regulation of pGSK-3β in neurons is counteracted by EPO treatment. The bars represent the mean + SD of the ratio to OD values of the loading control (total GSK-3beta). A representative blot is shown. (A) Ketamine (10 μM) time-dependently induced down regulation of pGSK in neurons. * P<0.05 vs. 0 h. (B) The average levels of pGSK-3β in neurons were increased at all time points in the presence of EPO (1 U/ml). * P<0.05, ** P<0.01 vs. 0 h. (C) EPO-induced phosphophorylation of GSK-3β in neurons after exposure to EPO (1 U/ml) and/or ketamine (10 μM) for 2 h was suppressed by PI3K inhibitor LY294002 (10 μM), which was added to medium 1 h before EPO and ketamine treatment. * P<0.05, ** P<0.01 vs. control; # P<0.05 vs. ketamine; + P<0.05 vs. ketamine plus EPO.