International Journal of Neuroscience, 122, 381–387, 2012Copyright © 2012 Informa Healthcare USA, Inc.ISSN: 0020-7454 print / 1543-5245 onlineDOI: 10.3109/00207454.2012.668726

17β-Estradiol Attenuates Neural Cell Apoptosis ThroughInhibition of JNK Phosphorylation in SCI Rats andExcitotoxicity Induced by Glutamate In Vitro

Wei Rong, Jun Wang, Xiaoguang Liu, Liang Jiang, Feng Wei, Hua Zhou, Xiaoguang Han,and Zhongjun Liu

Department of Orthopaedics, Peking University Third Hospital, Beijing, China

ABSTRACT

We investigated whether 17β-estradiol (E2) treatment could prevent the apoptosis of neural cells after spinalcord injury (SCI) and cultured cortical cells through inhibition of JNK (c-Jun N-terminal kinase) phosphorylation.SCI-induced rats were randomly divided into three groups: control, E2-treated, and sham-treated. Five rats fromeach group were sacrificed at 2, 4, 6, 12, or 24 h postinjury. Apoptotic neural cells were assessed using theTUNEL method. JNK phosphorylation was detected with immunohistochemistry. Cultured cortical cells werepretreated with E2 and the specific JNK inhibitor SP600125 and then treated with glutamate-induced cytotox-icity in vitro. Neuron viability was determined with an methyl thiazolyl tetrazolium (MTT) assay, morphology ofapoptotic cells was observed with 4′,6-diamidino-2-phenylindole (DAPI) staining, and JNK phosphorylation wasdetected using Western blot analysis. Treatment with E2 reduced neuron apoptosis and inhibited JNK phos-phorylation. Moreover, the number of apoptotic cells was correlated with JNK phosphorylation 24 h after therats suffered the SCI. Pretreatment with E2 significantly maintained neural cell viability, attenuated apoptosis,and inhibited JNK phosphorylation induced by glutamate in vitro. These neuroprotective effects of E2 on neuralcells were blocked by the co-administration of SP600125. Our results suggest that neuroprotection from E2 ispartially mediated by the inhibition of JNK phosphorylation.

KEYWORDS: apoptosis, JNK, spinal cord injury, 17β-estradiol

INTRODUCTION

Traumatic spinal cord injury (SCI) often results in acomplete loss of voluntary motor and sensory func-tion below the injury site. The long-term neurologi-cal deficits after spinal cord trauma may be due inpart to widespread apoptosis of neurons and oligoden-droglia in regions distant from and relatively unaffectedby the initial injury [1, 2]. The current paradigm forSCI pharmacotherapy promotes the use of antiapop-totic, anti-inflammatory agents [3]. Because apoptosisoccurs later and over a longer postinjury timeframe thannecrosis, pharmacological inhibition of apoptosis may

Received 16 November 2011.

This work was supported by the Nature Science Foundation of China(no. 58441-06).Address correspondence to Zhongjun Liu, M.D., Department ofOrthopaedics, Peking University Third Hospital, 49 North Garden Rd,Haidian District Beijing 100191, P.R. China. E-mail: ZJLiu@bjmu.edu.cn

offer substantial therapeutic advantages over traditionaltherapies [4]. Although the most widely recommendedpharmacotherapy is high-dose methylprednisolone ther-apy, this treatment does not significantly improvefunctional recovery and can result in serious adverseeffects [5].

In an in vitro study, toxic insults by glutamate in neu-ronal cell cultures were shown to mimic key compo-nents of central nervous system (CNS) injury, causingboth apoptosis and necrosis [6]. Two distinct pathwaysfor glutamate-induced cell death have been described:the excitotoxic pathway [7] and the oxidative pathway[8]. The excitotoxic pathway involves the overactivationof glutamate receptors that leads to both rapidly andslowly triggered cytotoxic events. The rapid effects in-volve the activation of the N-methyl-D-aspartate recep-tor (NMDAR), which leads to a large Ca2+ influx thatmay be detrimental to cell viability. The oxidative path-way involves the breakdown of the glutamate–cystine an-tiporter and a drop in glutathione levels, allowing for

381

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.

382 W. Rong et al.

aberrant formation of neurotoxic reactive oxygen species(ROS).

Estrogen-mediated neuroprotection is well estab-lished; however, no single mechanism of action for thiseffect has been identified. In animal models, treatmentwith 17β-estradiol (E2) has been associated withimprovements in SCI [9], and gender differences havebeen noted in human clinical studies examining CNStrauma [10]. E2 also has antiapoptotic effects, whichmay contribute to the attenuation of damage followinga neurotoxic insult [11]; however, the mechanismsunderlying this type of E2 neuroprotection are notfully understood. Mitogen-activated protein kinases(MAPKs) are important intracellular molecules that areinvolved in the transduction of extracellular inflamma-tory stimuli and cell apoptosis [12, 13]. Various recentin vitro and in vivo studies have implicated MAPKactivation in the cascade of events associated with theoccurrence of neuronal damage after an SCI [14]. JNK(c-Jun N-terminal kinase) is a stress-activated memberof the MAPK family, and its activation has been stronglyimplicated in inflammatory responses, neurodegenera-tion, and apoptosis [15]. Recent evidence indicates thatthe JNK signaling pathway is also activated in neuronsafter tissue or nerve damage. In vivo delivery of sub-stance P (SP) or JNK antisense oligodeoxynucleotidesattenuates the expression of pro-apoptotic regulatorscaspase-3 and DP5 in SCI-induced rats [15]. However,whether E2 is able to regulate the JNK signaling pathwayremains unclear. Elucidating the relationship betweenthe E2 and the JNK signaling pathway will contributeto a better understanding of the protective effects of E2in female SCI patients and may ultimately provide abasis for targeted pharmacological intervention.

MATERIALS AND METHODS

Materials

E2, dimethylsulfoxide (DMSO), and methyl thi-azolyl tetrazolium (MTT) were purchased fromSigma-Aldrich (St. Louis, MO, USA). A TUNELApoptosis Detection kit was purchased from RocheDiagnostics (Mannheim, Germany). DMEM, 1 µg/ml4′,6-diamidino-2-phenylindole (DAPI), Neurobasalmedium, and B27 were obtained from Gibco InvitrogenCorporation (Carlsbad, CA, USA). SP was purchasedfrom Calbiochem (La Jolla, CA, USA). Glutamate(sodium salt) was purchased from Tocris Bioscience(Ellisville, MO, USA). Rabbit anti-phospho-JNK(anti-pJNK), anti-β-actin polyclonal antibody, andhorseradish peroxidase-conjugated goat antirabbitsecondary antibody were purchased from Santa CruzBiotechnology (Santa Cruz, CA, USA).

Animals

We used 55 adult female Sprague Dawley rats (weight200–220 g). All protocols were approved by the animalcare committee of the Peking University Third Hospital.To reduce the effect of the estrous cycle on blood levelsof estrogen, a bilateral ovariectomy was performed asdescribed previously [16], and the animals were allowedto recover from surgery for 7 days before further exper-imentation. An SCI was induced by a trained techni-cian using the modified weight-drop method [17]. Theexperimental protocol has been described in detail in aprevious publication [18]. After inducting the injury, theincision was closed in layers.

Treatment With E2

SCI-induced rats were randomly divided into threegroups: control, 4.0 mg/kg E2-treated, and sham-treated. All drugs were delivered by tail vein injection30 min postinjury. E2 was dissolved in the smallest vol-ume of DMSO possible to reduce the amount of vehi-cle delivered (0.4 ml/kg). Both the control group andthe sham group received equal volumes of DMSO atthe same time points. Five rats from each experimen-tal group were sacrificed at 2, 4, 6, 12, and 24 h aftersurgery. The postinjury euthanized rats were perfusedtranscardially with phosphate-buffered saline (PBS) fol-lowed by 4% paraformaldehyde, and a 1-cm length ofspinal cord encompassing the injury site was then re-moved. The samples were transferred to a 30% sucrosesolution, and upon sinking, they were then blocked inoptimal cutting temperature (OCT) compound (SakuraFinetek USA, Torrance, CA, USA), with tissue blockscut into 8-µm sections on a cryostat. The sections werethen used in a TUNEL assay and immunohistochemicalstaining.

TUNEL Assay

The sections obtained as described above were ana-lyzed according to the manufacturer’s instructions. TheTUNEL-labeled positive cells were visualized by stain-ing with diaminobenzidine (DAB) for 3 min followed byhematein staining. Each section was counted indepen-dently by two observers under a light microscope (LeicaDM 4000B, Wetzlar, Germany). Five images from dif-ferent parts of each section were analyzed; the number ofpositive cells in each image was counted visually in orderto determine the positive rate, and the average numberfor the section was then calculated.

Immunohistochemical Staining

The sections were rinsed with PBS, blocked withbovine serum (10%) in PBS for 30 min, and incubated

International Journal of Neuroscience

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.

E2 Attenuate Neural Cell Apoptosis Through Inhibition of pJNK 383

overnight at 4◦C with rabbit anti-pJNK (1:200) as theprimary antibody. Subsequently, the sections were incu-bated with secondary antibodies. Finally, immunoreac-tivity was visualized by staining with DAB for 3 min. Inthe negative control experiments, the primary antibodywas substituted with PBS to ascertain the specificity ofantibody staining. Each section was counted using themethod described earlier.

Primary Cortical Cell Cultures

Primary cortical neuron cultures were obtained fromday 15 to 17 pregnant Sprague Dawley rats as previ-ously described [19] and then plated at 1 × 106 cells/mlin 96-well plates or 22-mm dishes precoated with poly-D-lysine. Cortical cells were maintained in Neurobasalmedium supplemented with 0.5 mM L-glutamine and2% B27 supplement. Cortical cells were maintained at37◦C in humidified air containing 5% CO2, with half themedium replaced every 3–4 days. Experiments were re-alized on pure neuronal cultures, which were generallyobtained 7–10 days after seeding.

Cell Treatments

To investigate the neuroprotective effects of E2, cellswere organized into four treatment groups: control(Con), 26-h treatment with 10 nM E2 (E2), 24-h treat-ment with 500 µM glutamate (GLU), and 2-h pre-treatment with E2 followed by 24-h co-treatment with500 µM glutamate (E2+GLU). The control cells un-derwent a procedure similar to that of the experimentalgroups but were treated with unadulterated DMEM.

To determine whether the JNK protein may beinvolved in the neuroprotective effects of E2, SP(E2+GLU+SP) was used. In each experiment, SP(100 nM; solubilized in DMSO) was added 30 min be-fore E2 treatment and co-administered with glutamatefor an another 24 h.

MTT Assays

Cortical neurons were plated in 96-well plates for 7 days.After various treatments, the cell viability was deter-mined by an MTT assay. A 10-µl MTT solution (5 mg/ml in PBS, pH 7.4) was added to each well at a final con-centration of 0.5 mg/ml, and the plates were incubatedfor an additional 4 h at 37◦C. The medium was subse-quently replaced by 150 µl of DMSO per well to dis-solve the formazan crystals. Absorbance at 570 nm wasmeasured using a Thermo Scientific Varioskan Flash(Thermo Fisher Scientific, Rockford, IL, USA). The re-sults are presented as a percentage of the control value.

High-Content Imaging Nuclear CondensationAssay

To further determine whether glutamate inducesprocesses of necrosis or apoptosis in neural cells, weanalyzed the presence of chromatin condensation andnuclear fragmentation with fluorescence microscopy,using the DNA-binding fluorescent dye DAPI. Aftervarious treatments, the cells were fixed for 15 minin 4% paraformaldehyde and were then rinsed withPBS. The nuclei were then stained with DNA-bindingfluorescent dye DAPI, and plates were rinsed againin PBS, sealed, and stored in darkness at 4◦C priorto being analyzed on an ArrayScan VTI high-contentimager (Thermo Scientific, Waltham, MA, USA).After staining with DAPI, the normal cells showednuclei with a homogeneous chromatin distribution asindicated by low-intensity DAPI labeling. In contrastto normal cells, the nuclei of apoptotic cells generallydisplay highly condensed chromatin that is uniformlystained by DAPI. This pattern can take the form ofcrescents around the periphery of the nucleus, orthe entire nucleus can appear to be one or a groupof featureless, bright spherical beads. Each plate wascounted independently by two observers. The imagerwas programmed to count DAPI-stained nuclei with anarea greater than 74 µm2. Images were captured using a20× objective, and eight fields per well were analyzed.

Western Blot Assays

After the experimental treatment, the total proteinwas isolated, subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and transferred toa nitrocellulose membrane. The blots were first stainedwith pJNK antibodies (1:500) overnight at 4◦C andthen stained with horseradish peroxidase-conjugatedgoat antirabbit (1:5000) for 1 h at room temperature.The signal was detected with the Odyssey InfraredImaging System (LI-COR Biosciences, Lincoln, NE,USA) and analyzed using Odyssey software, and β-actinwas used as the internal control. Data are presented asthe mean ± SD of the percentage ratio of the control.

Statistical Analysis

Statistical analyses were performed using SPSS 13.0.All data are expressed as the mean ± SD. A correla-tion analysis was performed using a Pearson correlationcoefficient. Statistical comparisons among the groupswere performed using a one-way analysis of variance(ANOVA) followed by Tukey’s post hoc test. Differenceswere considered statistically significant at p < .05.

C© 2012 Informa Healthcare USA, Inc.

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.

384 W. Rong et al.

FIGURE 1. Representative photos in the spinal cord from E2-treated rats 24 h postinjury. (A)Spinal cord sections stained by HE, immunohistochemical staining of pJNK, and photographs ofTUNEL staining in the spinal cord. The number of TUNEL-positive cells statistically decreasedin the E2-treated group. These photos indicate the square areas of HE. Scale bar = 10 µm; (B)average number of apoptotic cells and (C) pJNK-positive cells (∗p <.05 vs. control group; ∗∗p <

.01 vs. control group).

RESULTS

E2 Treatment Prevented Neural CellApoptosis After SCI In Vivo

Acute SCI induces extensive apoptotic neuron and glialcell death [20]. As E2 has been shown to exert neuropro-tective effects on CNS neurons exposed to a variety ofinsults [21], we used TUNEL method to assess whetherE2 could reduce apoptotic cell death after SCI. In thepresent study, TUNEL-positive neural cells were sparsein the sham group [see Figure 1(A)]. However, the num-ber of apoptotic cells was significantly reduced 24 h afterinjury in the E2-treated group compared with the con-trol group (p < .05). The average numbers of positive

cells in the control group, the E2-treated group, and thesham group were 11.93 ± 2.78, 9.40 ± 1.57, and 1.30 ±0.82, respectively [see Figure 1(B)]. Treatment with E2appeared to attenuate the number of apoptotic cells re-sulting from SCI at 24 h after the injury.

E2 Treatment Inhibited JNK PhosphorylationAfter SCI In Vivo

After SCI, JNK phosphorylation increased gradually ina time-dependent manner (see Figure 2). No specificimmunopositive staining was detected in the controlgroup. At 12 and 24 h postinjury, the average numberof pJNK-positive cells had decreased significantly in the

International Journal of Neuroscience

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.

E2 Attenuate Neural Cell Apoptosis Through Inhibition of pJNK 385

FIGURE 2. E2 treatment inhibitedthe expression of pJNK at differ-ent times in SCI. The expression ofpJNK was determined by immuno-histochemical staining. At 12 and 24h postinjury, the number of pJNK-positive cells was significantly less inthe E2 group than that in the controlgroup (∗p < .05 vs. control group; ∗∗p< .01 vs. control group).

E2 group compared with the control group (p < .05).At 24 h postinjury, the mean numbers of positive cells inthe control group, the E2-treated group, and the shamgroup were 18.73 ± 2.88, 11.73 ± 2.55, and 3.00 ±1.00, respectively [see Figure 1(B)]. These data suggestthat E2 therapy can significantly inhibit JNK phospho-rylation after SCI. Additionally, a correlation analysis in-dicated that the number of apoptotic cells was correlatedwith the rate of JNK phosphorylation (Pearson correla-tion = 0.428, p = .018).

E2 Treatment Reduced Glutamate ToxicityThrough JNK Phosphorylation In Vitro

To determine whether the activation of the JNK pro-tein is a component of E2 neuroprotection, cells werepretreated with SP. In the E2+GLU+SP group, neuro-protection elicited by E2 was blocked by the addition of100 nM; SP. The addition of SP alone did not signifi-cantly affect cell viability in the presence or absence ofglutamate [see Figure 3(A)], in accord with the resultsof a previous study [22].

After incubation with glutamate, chromatin conden-sation was evident in cells, as revealed by intense DAPIlabeling showing a typical pattern of nuclear fragmen-tation in apoptotic nuclei. The results of DAPI staininganalysis, shown in Figure 3(B), revealed that pretreat-ment with E2 significantly inhibited neural cell apopto-sis compared with the GLU group (p < .05).

E2 Treatment Maintained Neural Cell Viabilityand Inhibited JNK Phosphorylation In Vitro

To evaluate the neuron viability after the treatment withglutamate alone or co-treatment with 17β-estrogen and

FIGURE 3. E2 attenuated neural cell apop-tosis through inhibition of pJNK expressionin vitro. (A) E2 cytoprotection measured us-ing the MTT assay. MTT assay results arereported as absorbance at 570 nm. (B) Theresults of DAPI staining analyzed on neuroncells. The neuroprotection elicited by estrogenis blocked by the addition of 100 nM SP (#p <

.05 vs. GLU group; ##p < .01 vs. GLU group;&p < .05 vs. E2+GLU group).

glutamate, the MTT assay was used. The results of theMTT assay for cell viability are shown in Figure 4(A).Compared with the control group, treatment with E2alone did not cause a significant change in cell viabil-ity. However, treatment with 500 µM glutamate de-creased cell viability by approximately 33.21% (p <

.01), while the percentage of living cells was 89.98%in the E2+GLU group. These results demonstrate thattreatment with E2 significantly attenuated glutamate-induced cell death (p < .05, compared with the GLUgroup). These results are in agreement with observa-tions from previous research [19].

The results of the Western blot assay detailed JNKphosphorylation in the different treatment groups. Den-sitometric analysis showed the relative expression ofJNK phosphorylation in the control group, E2 group,GLU group, and E2+GLU group to be 45.01 ± 4.19,41.42 ± 3.96, 68.11 ± 7.96, and 47.87 ± 3.87, respec-tively [see Figure 4(B)]. These results suggest that treat-ment with glutamate promotes JNK phosphorylation,while pretreatment with E2 can effectively inhibit thisglutamate-induced JNK phosphorylation.

C© 2012 Informa Healthcare USA, Inc.

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.

386 W. Rong et al.

FIGURE 4. Effects of E2 on neural cell via-bility and the expression of pJNK in vitro. (A)E2 cytoprotection measured using the MTTassay. MTT assay results are reported as ab-sorbance at 570 nm. (B) The expression ofpJNK protein was detected by Western blotanalysis. β-actin was used as a loading control.Densitometry of the Western blots to show op-tical density (OD) values, relative to control(∗∗p < .01 vs. control group; #p < .05 vs.GLU group).

DISCUSSION

In the present study, we found that after inducing SCI inrats, treatment with E2 significantly reduced the numberof apoptotic cells and inhibited JNK phosphorylation. Inaddition, the number of apoptotic cells was correlatedwith the JNK phosphorylation rate 24 h after SCI. Pre-treatment with E2 significantly maintained neural cellviability, inhibited neural cell apoptosis, and attenuatedthe JNK phosphorylation induced by glutamate in vitro.This neuroprotective effect of E2 was blocked by a co-administration of SP.

Acute SCI induces extensive necrotic and apoptoticcell death of neurons and glia [20]. Necrotic cell deathoccurs following an actual mechanical disruption of cellsas well as from overwhelming cellular injury that may oc-cur during the secondary injury cascade. This can ren-der the cellular membrane ineffective in the presenceof swelling of subsequent cells and a significant inflam-matory response provoked by lysis. Necrotic cell death

occurs outside the realm of cellular control and thusoccurs passively. In contrast, apoptosis is a continuousphysiological process to facilitate noninflammatory, pro-grammed cell death [23]. Apoptosis mechanisms playan integral role in many biological events, and balancedapoptosis is crucial to ensuring good health. The stim-ulus responsible for the apoptotic signal may be derivedfrom the surrounding cellular environment, the internalmetabolism of the cell, or its genome. The occurrence ofapoptosis following acute SCI could prove to be an im-portant phenomenon to consider in efforts toward im-proving neuronal cell survival [24].

Apoptotic cells in the injured spinal cord were ob-served 6 h after SCI, with levels increasing gradually at12 h until reaching a maximal level 3 days after the in-jury [25]. Estrogen-mediated neuroprotection has beennoted in vitro in neurons and neuronal cell lines undera variety of stress conditions [26].

JNK has been suggested to play a critical role in nu-merous cellular activities [27]. JNK is thought to in-duce apoptosis via both transcription-dependent andtranscription-independent mechanisms. In addition, themitochondrion is a primary target of pro-apoptotic sig-naling by JNK [28]. The present results demonstratethat the expression of pJNK increases gradually in atime-dependent manner and that JNK phosphorylationhas a significant positive correlation with the number ofapoptotic cells present 24 h after an injury. However,treatment with E2 inhibited JNK phosphorylation andattenuated neural cell apoptosis, suggesting that neuro-protection from E2 is partially mediated by the inhibi-tion of JNK phosphorylation.

Abundant evidence has established E2 as a neu-roprotective factor that is important across the lifespan [29], but the mechanisms underlying E2 neuro-protection are not fully understood. The activation ofthe JNK signaling pathway has been closely linked toa variety of apoptotic stimuli, whereas the inhibitionof this pathway provides protection against neuronalapoptosis in multiple paradigms, including glutamateneurotoxicity [30]. Our results demonstrate that signif-icant activation of JNK in SCI tissues occurs 24 h afteran injury in rats. JNK is de novo expressed in neuralcells in vitro. The inhibition of JNK phosphorylationby both the pharmacological inhibitor SP and by E2could attenuate the expression of pJNK and neural cellapoptosis induced by glutamate. We have found thatthe neuroprotection elicited by E2 is partially blockedby the addition of SP. However, additional experimentsdesigned to study the mechanisms of JNK pathwayregulation by E2 in more detail are necessary.

In conclusion, our results show that neuroprotectionby E2 is partly mediated by the inhibition of JNK phos-phorylation in rats with an SCI. These data contributeto the understanding of the mechanisms behind the

International Journal of Neuroscience

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.

E2 Attenuate Neural Cell Apoptosis Through Inhibition of pJNK 387

neuroprotective effects of E2 and has potential thera-peutic relevance for the treatment of acute SCI. Furtherexperiments are needed to study the long-term effectsand specific mechanisms of JNK pathway regulationby E2.

Declaration of interest: The authors report no con-flict of interest. The authors alone are responsible for thecontent and writing of this paper.

REFERENCES

1. Shuman SL, Bresnahan JC, Beattie MS. Apoptosis of microgliaand oligodendrocytes after spinal cord contusion in rats. J Neu-rosci Res. 1997;50(5):798–808.

2. Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS.Apoptosis and delayed degeneration after spinal cord injury inrats and monkeys. Nat Med. 1997;3(2):240.

3. Beattie MS. Inflammation and apoptosis: linked therapeutictargets in spinal cord injury. Trends Mol Med. 2004;10(12):580–3.

4. Yakovlev AG, Faden AI. Caspase-dependent apoptotic path-ways in CNS injury. Mol Neurobiol. 2001;24(1–3):131–44.

5. Pereira JE, Costa LM, Cabrita AM, Couto PA, Filipe VM, Ma-galhaes LG, Fornaro M, Di Scipio F, Geuna S, Maurıcio AC,Varejao AS. Methylprednisolone fails to improve functional andhistological outcome following spinal cord injury in rats. ExpNeurol. 2009;220(1):71–81.

6. Mize AL. Estrogen receptor-mediated neuroprotection from ox-idative stress requires activation of the mitogen-activated proteinkinase pathway. Endocrinology. 2003;144(1):306–12.

7. Michaels RL, Rothman SM. Glutamate neurotoxicity in vitro:antagonist pharmacology and intracellular calcium concentra-tions. J Neurosci. 1990;10(1):283–92.

8. Murphy TH, Miyamoto M, Sastre A, Schnaar RL, CoyleJT. Glutamate toxicity in a neuronal cell line involves inhibi-tion of cystine transport leading to oxidative stress. Neuron.1989;2(6):1547–58.

9. Sribnick EA, Wingrave JM, Matzelle DD, Ray SK, Banik NL.Estrogen as a neuroprotective agent in the treatment of spinalcord injury. Ann NY Acad Sci. 2003;993:125–33.

10. Bayir H, Marion DW, Puccio AM, Wisniewski SR, Janesko KL,Clark RS, Kochanek PM. Marked gender effect on lipid perox-idation after severe traumatic brain injury in adult patients. JNeurotrauma. 2004;21(1):1–8.

11. Das A, Smith JA, Gibson C, Varma AK, Ray SK, BanikNL. Estrogen receptor agonists and estrogen attenuate TNF-alpha-induced apoptosis in VSC4.1 motoneurons. J Endocrinol.2011;208(2):171–82.

12. Genovese T, Melani A, Esposito E, Mazzon E, Di Paola R,Bramanti P, Pedata F, Cuzzocrea S. The selective adeno-sine A2A receptor agonist CGS 21680 reduces JNK MAPKactivation in oligodendrocytes in injured spinal cord. Shock.2009;32(6):578–85.

13. Esposito E, Genovese T, Caminiti R, Bramanti P, MeliR, Cuzzocrea S. Melatonin reduces stress-activated/mitogen-activated protein kinases in spinal cord injury. J Pineal Res.2009;46(1):79–86.

14. Gao YJ, Ji RR. Activation of JNK pathway in persistent pain.Neurosci Lett. 2008;437(3):180–3.

15. Fan J, Xu GX, Nagel DJ, Hua Z, Zhang N, Yin G. A modelof ischemia and reperfusion increases JNK activity, inhibits theassociation of BAD and 14-3-3, and induces apoptosis of rabbitspinal neurocytes. Neurosci Lett. 2010;473(3):196–201.

16. Mohamed MK, Abdel-Rahman AA. Effect of long-termovariectomy and estrogen replacement on the expression ofestrogen receptor gene in female rats. Eur J Endocrinol.2000;142(3):307–14.

17. Perot PL Jr., Lee WA, Hsu CY, Hogan EL, Cox RD, GrossAJ. Therapeutic model for experimental spinal cord injury inthe rat: I. Mortality and motor deficit. Cent Nerv Syst Trauma.1987;4(3):149–59.

18. Han X, Yang N, Xu Y, Zhu J, Chen Z, Liu Z, Dang G, SongC. Simvastatin treatment improves functional recovery after ex-perimental spinal cord injury by upregulating the expression ofBDNF and GDNF. Neurosci Lett. 2011;487(3):255–9.

19. Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM.The mitogen-activated protein kinase pathway mediates estro-gen neuroprotection after glutamate toxicity in primary corticalneurons. J Neurosci. 1999;19(7):2455–63.

20. Yune TY, Kim SJ, Lee SM, Lee YK, Oh YJ, Kim YC, Markelo-nis GJ, Oh TH. Systemic administration of 17 beta-estradiolreduces apoptotic cell death and improves functional recoveryfollowing traumatic spinal cord injury in rats. J. Neurotrauma.2004;21(3):293–306.

21. Garcia-Segura LM, Azcoitia I, DonCarlos LL. Neuroprotectionby estradiol. Prog. Neurobiol. 2001;63(1):29–60.

22. Chen RW, Lu XC, Yao C, Liao Z, Jiang ZG, Wei H, GhanbariHA, Tortella FC, Dave JR. PAN-811 provides neuroprotectionagainst glutamate toxicity by suppressing activation of JNK andp38 MAPK. Neurosci Lett. 2007;422(1):64–7.

23. Williams GT, Smith CA, McCarthy NJ, Grimes EA. Apop-tosis: final control point in cell biology. Trends Cell Biol.1992;2(9):263–7.

24. Lou J, Lenke LG, Ludwig FJ, O’Brien MF. Apoptosis as amechanism of neuronal cell death following acute experimen-tal spinal cord injury. Spinal Cord. 1998;36(10):683–90.

25. Nakahara S, Yone K, Sakou T, Wada S, Nagamine T, NiiyamaT, Ichijo H. Induction of apoptosis signal regulating kinase 1(ASK1) after spinal cord injury in rats: possible involvementof ASK1-JNK and-p38 pathways in neuronal apoptosis. J Neu-ropath Exp Neurol. 1999;58(5):442–50.

26. Liu T, Jin H, Sun QR, Xu JH, Hu HT. Neuroprotective effectsof emodin in rat cortical neurons against beta-amyloid-inducedneurotoxicity. Brain Res. 2010;1347:149–60.

27. Lin A. Activation of the JNK signaling pathway: breaking thebrake on apoptosis. BioEssays. 2003;25(1):17–24.

28. Nakamura M, Nakakimura K, Matsumoto M, Sakabe T. Rapidtolerance to focal cerebral ischemia in rats is attenuated byadenosine A1 receptor antagonist. J Cereb Blood Flow Metab.2002;22(2):161–70.

29. Samantaray S, Sribnick EA, Das A, Thakore NP, Matzelle D,Yu SP, Ray SK, Wei L, Banik NL. Neuroprotective efficacy ofestrogen in experimental spinal cord injury in rats. Ann NYAcad Sci. 2010;1199:90–4.

30. Perrella J, Bhavnani BR. Protection of cortical cells by equineestrogens against glutamate-induced excitotoxicity is mediatedthrough a calcium independent mechanism. BMC Neurosci.2005;6:34.

C© 2012 Informa Healthcare USA, Inc.

Int J

Neu

rosc

i Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f Q

ueen

slan

d on

04/

21/1

3Fo

r pe

rson

al u

se o

nly.