B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
ava i l ab l e a t www.sc i enced i r ec t . com
www.e l sev i e r . com/ loca te /b ra i n res
Research Report
Spatial–temporal expression of NDRG2 in rat brain after focalcerebral ischemia and reperfusion
Yan Lia,1, Lan Shenb,1, Lei Caic,1, Qiang Wanga,⁎, Wugang Houa, Feng Wanga, Yi Zenga,Gang Zhaod, Libo Yaob,⁎, Lize Xionga,⁎aDepartment of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, ChinabThe Institute of Molecular Biology and The State Key Laboratory of Cancer Biology, The Fourth Military Medical University,Xi'an 710032, Shaanxi Province, ChinacState Key Laboratory of Cancer Biology, Department of Gastrointestinal Surgery, Xijing Hospital of Digestive Diseases,The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, ChinadDepartment of Neurobiology, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
A R T I C L E I N F O
⁎ Corresponding authors. L. Xiong and Q.WangUniversity, Xi'an 710032, Shaanxi Province, ChLaboratory of Cancer Biology, Fourth Military
Article history:Accepted 8 January 2011Available online 15 January 2011
N-myc downstream regulated gene 2 (NDRG2) was reported to be widely expressed in thenervous system. However, the expression and potential role of NDRG2 in focal cerebralischemia brain remain unclear. Herein, we investigated spatial–temporal expression ofNDRG2 in the rat brain following transient focal cerebral ischemia. Male Sprague–Dawleyrats underwent a 120-min transient occlusion ofmiddle cerebral artery. Rats were killed andbrain samples were harvested at 4, 12, 24, and 72 h after reperfusion. Expression of NDRG2 inthe brain was determined by reverse transcriptase–polymerase chain reaction (RT-PCR),Western blot analysis and immunohistochemical staining. Cellular apoptosis was assessedby TUNEL staining. The results showed that NDRG2 was expressed on cells with anastrocytes-like morphology in ischemic penumbra. NDRG2 mRNA and protein expressionbegan to increase at 4 h after reperfusion and peaked at 24 h in the ischemic penumbra. Byusing immunofluorescence, NDRG2 signals were co-localized with GFAP-positiveastrocytes, and NDRG2 expression in astrocytes translocated from a cytoplasm to anuclear localization at 24 h after reperfusion. Double immunofluorescent staining forTUNEL andNDRG2 showed that someNDRG2 signals co-localizedwith TUNEL-positive cells,and that the apoptotic cells increased with enhancement of NDRG2-positive signals. Inconclusion, NDRG2 expression is up-regulated in ischemic penumbra following transientfocal cerebral ischemia. NDRG2 expression in astrocytes may play important pathologicalroles in cell apoptosis after stroke.
253B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
1. Introduction
Brain injury following stroke results from the complexinterplay of multiple pathways including excitotoxicity, acid-otoxicity, ionic imbalance, peri-infarct depolarization, oxida-tive and nitrative stress, inflammation and apoptosis, whichcontributes to cell death subsequent to cerebral ischemia andreperfusion (Doyle et al., 2008). Reduction of blood flow andhypoxia during stroke activates synthesis of potential dam-aging molecules and potentially protective molecules. Focalcerebral ischemia results in a densely ischemic core and a lessseverely ischemic penumbra (Astrup et al., 1977). The exis-tence of a penumbra implies that therapeutic salvage istheoretically possible after stroke. More recently, the ischemicpenumbra has been further defined as multiple molecularpenumbras, which provided a novel perspective for the studyof mechanisms of ischemic brain damage.
Human N-myc downstream regulated gene (NDRG) 2 wasfirst identified from a normal human brain cDNA library bysubtractive hybridization in our laboratory (GenBank acces-sion no. AF159092) (Yan-chun et al., 2001). AndNDRG2 belongsto the N-myc downstream regulated gene family that includesfour members: NDRG1, NDRG2, NDRG3, and NDRG4. TheseNDRG family members were reported to be involved in celldifferentiation and development (Lachat et al., 2002; Ohkiet al., 2002). Although NDRG2 is a candidate tumor-suppressor(Furuta et al., 2010), several studies suggest functions ofNDRG2 in the central nervous system. NDRG2 is highlyexpressed in the adult brain and is more abundant in graymatter than white matter (Nichols, 2003). NDRG2 is up-regulated at both the RNA and protein levels in Alzheimer'sdisease brains (Mitchelmore et al., 2004). Glucocorticoids alsoincreased NDRG2 mRNA in glia in the neurogenic zone(Nichols et al., 2005), while NDRG2 expression was decreasedin the rat frontal cortex after treatment with antidepressants(Takahashi et al., 2005). Although these data suggest thatNDRG2 might modulate glial cell function in the centralnervous system, the role of NDRG2 in the pathologic progres-sion of focal cerebral ischemia is unknown.
Fig. 1 – NDRG2 expression in the ischemic penumbra by immunsection at a level of striatum at 24 h after reperfusion, the includepenumbra. (B) NDRG2 expression (brown signal) in the identicalpenumbra and (C) in the ischemic penumbra after ischemia and
We previously demonstrated that NDRG2 is expressed inastrocytes in different brain regions, including cerebral cortex,olfactory bulb, midbrain, hippocampus, and thalamus, withhigh levels in the midbrain and thalamus (Shen et al., 2008),and NDRG2 was also reported to be detected in astrocytes butnot neurons (Okuda et al., 2008). In the present study, usingtransient focal cerebral ischemia induced by middle cerebralartery occlusion (MCAO), we demonstrated that NDRG2mRNAand protein were up-regulated at 24 h after reperfusion.Further, NDRG2 was localized in astrocytes and apoptosis-positive cells, suggesting a potential role for NDRG2 in cellularapoptosis induced by ischemia.
2. Results
2.1. Immunohistochemical detection of NDRG2 in theischemic penumbra
As shown in Fig. 1, NDRG2 immunoreactions were detected incells with an astrocytes-like morphology. A pattern of weakcytoplasm cell staining with anti-NDRG2 was observed in thesham-operated group (Fig. 1B), while a strong cytoplasm andnuclear staining with anti-NDRG2 was detected in ischemicpenumbra at 24 h after reperfusion (Fig. 1C).
2.2. Temporal expression of NDRG2 in the ischemicpenumbra
NDRG2 mRNA expression in ischemic penumbra brain tissuebegan to increase at 4 h after reperfusion, and reached a peakat 24 h (Fig. 2A). Consistent with RT-PCR data, a 43 kDa bandcorresponding to NDRG2 protein increased at 4 h afterreperfusion, and then peaked at 24 h (Fig. 2B) where it wassignificantly up-regulated compared to the sham-operatedgroup (*P<0.05; Fig. 2C). However, protein expression of NDRG2in the ischemic penumbra was similar to the sham-operatedgroup at 72 h after reperfusion (Fig. 2C).
ohistochemical staining. (A) Representative TTC-stainedd angle area between two white lines indicates the ischemicipsilateral region after sham operation to the ischemicreperfusion. Bars=20 μm.
Fig. 2 – Temporal expression of NDRG2 in the ischemic penumbra by RT-PCR andWestern blot analysis. (A) NDRG2mRNA leveland (B) protein expression in the ischemic penumbra at 4, 12, 24, and 72 h after reperfusion, and in the sham-operated group.(C) Relative density of NDRG2 protein versus GAPDH protein expression was plotted. Data are means±SD (n=5 at each timepoint in each group).
254 B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
2.3. Cellular location of NDRG2 in the ischemic penumbra
NDRG2 protein (green) was co-localized with GFAP protein(red) in both sham-operated and ischemic brains, supportingan astrocytic localization. Consistent with immunohisto-chemical evidence, NDRG2 expression was higher in theischemic penumbra compared to the sham-operated groupat 24 h after reperfusion (Fig. 3). Furthermore, NDRG2 proteinexpression was weak in the nucleus and strong in thecytoplasm in the sham-operated group (Fig. 4A–D). However,as shown in Fig. 4E–H, the NDRG2 protein expression was
Fig. 3 – Immunofluorescent double-labeling staining of NDRG2 asham-operated group (A–C) and 24 h after reperfusion group (D aco-localization of NDRG2 and GFAP (white arrowheads). Bars=20
strong in nucleus and the NDRG2 protein (red) was co-localized with DAPI staining (blue) at 24 h after reperfusion,indicating that NDRG2 expression in astrocytes translocatedfrom a cytoplasm to a nuclear localization after stroke.
2.4. Cellular NDRG2 co-localization with apoptotic cells
As shown in Fig. 5, TUNEL and DAPI staining showed that littleapoptotic cells was observed throughout the shamhemisphere,while the number of apoptotic cells in the ischemic penumbrawas increased at 24 h after reperfusion. Furthermore, double
nd GFAP. NDRG2 expression (green) and GFAP (red) in thend E). The merged yellow images in C and F indicateμm.
Fig. 4 – Cellular localization of NDRG2 in astrocytes by immunofluorescent labeling. In the sham-operated group, NDRG2 (red)was expressed in the cytoplasm of astrocytes (green) (A–D). In the ischemia–reperfusion group, NDRG2 (red) wasmainly locatedin nucleus (blue) and weak in cytoplasm (E–H). DAPI (blue) was used to stain nucleus. Themerged purple images in H indicatesco-localization of NDRG2, GFAP and DAPI (white arrowheads). Bars=10 μm.
255B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
immunofluorescence for TUNEL andNDRG2 demonstrated thatsomeTUNEL-positive cells were co-localized in NDRG2-positivecells in the ischemic penumbra after ischemia and reperfusion.
3. Discussion
About 80% of all strokes are ischemic. Once vessel occlusionhas occurred, a volume of functionally impaired but structur-ally intact tissue surrounds the ischemic core. This tissue isknown as the ischemic penumbra and is the target fortherapeutic interventions since its salvage is associated withneurological improvement and recovery (Weinstein et al.,2004). Within the ischemic penumbra, a cascade of neuro-chemical events begins with energy depletion followed bydisruption of ion homoeostasis, release of glutamate, calciumchannel dysfunction, release of free radicals, membranedisruption, inflammatory changes, and necrotic and apoptotic
Fig. 5 – Immunofluorescent staining of TUNEL, NDRG2, and DAPIsham-operated group (A–D) and 24 h after reperfusion group (E–Hco-localization of NDRG2 and TUNEL (white arrowheads). Bar=20
cell death triggering (Hata et al., 2000; Sun et al., 2010). Inanimal models of stroke, these cascades can be arrested atvarious points, which form the basis of neuroprotectivetherapies (Hossmann, 1994; Ginsberg, 2003). Thus, it isimportant to characterize the molecular events of thedynamic penumbra for finding novel neuroprotectivestrategy.
NDRG2 is involved in hypoxia response and its expressionwasmarkedly up-regulated in several tumor cell lines exposedto hypoxic conditions (Wang et al., 2008a). Although previousevidence suggest that NDRG2 may play potential function inthe central nervous system, the expression and role of NDRG2in the pathophysiology of focal cerebral ischemia remainunclear. In the present study we characterized the expressionof NDRG2 in ischemic penumbra after reperfusion by immu-nohistochemistry. We found a significant increase of NDRG2expression in ischemic penumbra after reperfusion comparedto sham-operated group. The level of NDRG2 mRNA and
. TUNEL (green), GFAP (red), and DAPI (blue) staining in the). The merged yellow images in C and G indicateμm.
256 B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
protein also began to increase in ischemic penumbra at 4 hafter reperfusion and peaked at 24 h after reperfusion. Therapid change of NDRG2 expression in ischemic penumbraafter reperfusionmade it plausible to deduce that NDRG2maybe involved in cell damage of ischemic penumbra.
It is believed that astrocytes are critical determinant instroke pathophysiology since besides coupling to neuron,astrocytes link vascular signal to brain. Therefore, astrocytesmay modulate ischemic brain damage depending upon theseverity and duration of ischemia. It is well established thatastrocytes play an important role in ischemic brain injury, andexhibit some of the earliest and most dramatic cellularresponses (Petito and Babiak, 1982). Astrocytes impact onneuron survival during the acute ischemic period by modu-lating glutamate excitotoxicity, releasing erythropoietin toblock neural death, attenuating oxidative neuronal injury(Swanson et al., 2004). In the current study, we found thatNDRG2 was predominantly expressed in astrocytes in bothsham-operated and stroke animals, as previously reported(Okuda et al., 2008). Interestingly, a high expression of NDRG2in reactive astrocytes was detected in ischemic penumbra.Both GFAP, which is commonly used as astrocytes marker(Barnett et al., 2010), and NDRG2 expression peaked at 24 hafter reperfusion, suggesting a role for NDRG2 in astrocytesactivation. Furthermore, our results proved that NDRG2mRNAand protein level returned to the normal level in ischemicpenumbra at 72 h after reperfusion, suggesting that NDRG2may influence neuronal survival in the post-ischemic period.Astrocytes also secrete several angiogenic and neurotrophicfactors that are important for vascular and neuronal regener-ation after stroke (Swanson et al., 2004). Thus, it is possiblethat NDRG2 is involved in the astrocyte response in ischemiccerebral injury, suggesting that NDRG2maymodulate glial cellfunction to affect neuronal fate after cerebral ischemia.
Cell signal transduction pathways frequently activate thetranscription of specific exocytosis, cellular differentiationand apoptosis. Activation of certain transcription factors leadsto the translocation of molecules from the cytoplasm to itsnucleus. Our results demonstrated that NDRG2 was translo-cated from the cytoplasm to the nucleus in astrocytes afterstroke, indicating that ischemia and hypoxia lead to NDRG2up-regulation and nuclear translocation in astrocytes. It wasreported that NDRG2 expression in A549 cells was up-regulated under hypoxia conditions with concomitant trans-location of NDRG2 from the cytoplasm to the nucleus (Wang etal., 2008a). Thus, it is possible to deduce that when cells werein quiescent condition, NDRG2 resided in cytoplasm withinactive form, but after cells were activated by stimulatingfactors (ischemia), NDRG2 was translocated from the cyto-plasm to the nucleus to produce biological effects. Ourfindings provided a novel clue to study the function ofNDRG2 nuclear translocation after stroke.
There is strong evidence that apoptosis contributes to celldeath of ischemic brain tissue (Hirt et al., 2005). NDRG2 is verysimilar to NDRG1, which has been linked to apoptosis(Weinstein et al., 2004), and was also reported to be anapoptosis-induced factor. In order to assess the role of NDRG2in cellular apoptosis, we detected a relationship betweenNDRG2 expression and cellular apoptosis in the brain follow-ing ischemia–reperfusion. Our results showed that with the
increase of NDRG2 positive cells, TUNEL-positive cells alsoincreased in ischemic penumbra after reperfusion, indicatingthat NDRG2 may be involved in cell apoptosis during stroke.Importantly, we also found that NDRG2 expressed in someapoptotic cells, suggesting that NDRG2 expression may beassociated with the process of cell apoptosis induced byischemia. Together, these data suggest that NDRG2 expressionmight be involved in the physiological and pathogenic cellapoptosis in the rat cerebral ischemia. However, the effect ofNDRG2 in astrocytes apoptosis induced by ischemia is stillunknown.
A previous study reported that NDRG2 is a novel hypoxia-inducible factor-1 (HIF-1) target gene necessary for hypoxia-induced apoptosis in A549 cells (Wang et al., 2008a), whileNDRG2 was also shown to act as downstream of HIF-1 topromote radioresistance via suppression of radiation-inducedBax expression in human cervical cancer Hela cells (Lin et al.,1998). HIF-1 plays an important role in stroke by regulatinggenes such as erythropoietin andVEGF (Bernaudin et al., 2002).Thus, it is possible that NDRG2 acts as HIF-1 target gene toregulate ischemia-induced apoptosis. The role of NDRG2 inastrocytes apoptosis induced by ischemia forms the basis ofour future studies.
In conclusion, our findings suggest that the up-regulationof NDRG2 in astrocytes of ischemic penumbra is an importantfeature of cerebral ischemia and reperfusion. NDRG2 expres-sion in astrocytes may play important pathological roles incell apoptosis after stroke. To explore more precisely role ofNDRG2 and its pathways in cerebral infarction and brainplasticity after the injury requires additional investigations.
4. Experimental procedures
4.1. Experimental groups
The experimental protocol was approved by the EthicsCommittee for Animal Experimentation and was conductedaccording to the Guidelines for Animal Experimentation of theFourth Military Medical University. We made efforts tominimize the number of animals used and their suffering. Atotal of 40 adult male Sprague–Dawley rats (250–300 g) wererandomly assigned to one of the following five groups (n=8,each group): sham-operated group, ischemia/reperfusion (I/R)4 h, 12 h, 24 h, and 72 h groups. The rats in Sham groupreceived the same surgery without middle cerebral arteryocclusion (MCAO). The rats in I/R groups were subjected to120 min MCAO, then were killed at 4, 12, 24, and 72 h groupsafter reperfusion.
4.2. Transient focal cerebral ischemia
Focal cerebral ischemia was induced by MCAO using theintraluminal filament technique in the rats as described in ourprevious studies (Wang et al., 2008b; Wang et al., 2009). Briefly,the rats were anesthetized with oxygen/isoflurane mixture(98:2%) during surgical preparation. The right MCA wasoccluded by an insertion of a 3-0 nylon monofilament suture(EthiconNylon Suture, Ethicon Inc., Japan) with its tip rounded
257B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
by heating near a flame through the common carotid artery.Achievement of ischemia was confirmed by monitoringregional cerebral blood flow through a laser Doppler flowme-try (PeriFlux 5000, Perimed AB, Jarfalla, Sweden). Animals thatdid not show a cerebral blood flow reduction of at least 70%were excluded from the experimental group, as well asanimals that died after ischemia induction. Reperfusion wasaccomplished by withdrawing the suture after 120 min ofischemia. The incision sites were infiltrated with 0.25%bupivacaine hydrochloride. Rectal and temporalis muscletemperature wasmaintained at 37.0±0.5 °C by surface heatingor cooling during surgery until rats recovered from anesthesia.During the MCAO, PaO2 and PaCO2 were maintained atphysiological levels. The animals were sacrificed at 4, 12, 24,and 72 h post-reperfusion or sham operation, and theischemic penumbra dissected according to well establishedprotocols in rodent models of unilateral proximal MCAO(Ashwal et al., 1998) (Fig. 2A).
4.3. TTC staining
2,3,5-Triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO,USA) staining was performed to verify successful MCAOmodeland todefinepenumbral regions. The animalwas sacrificed andthe brain was dissected out. The brain was sliced into 2 mmthick coronal sections fromthe frontal pole to a level of striatumusing a Brain Matrix (Muromachi Kikai, Tokyo, Japan) andstained with TTC solution for 10min at 37 °C and fixed in 10%formalin overnight. The sections were photographed with acamera. Thereafter, slices were divided into two zones, i.e.,infarction core and penumbra region in the ischemia ipsilateralhemisphere according to the TTC staining pattern (Fig.1A). Eachhemisphere was cut longitudinally, from dorsal to ventral at2 mm from the midline to exclude medial brain structures thatwere supplied primarily by the anterior cerebral artery. And atransverse diagonal incision at approximately the 2 o'clockposition separated the core from the penumbra.
For reverse transcriptase RT-PCR analysis 100 mg tissues (n=5eachgroup)werecollected immediatelyandtotalRNAwas isolatedfrom each sample using TRIZOL reagent (Invitrogen, Carlsbad, CA,USA) and then quantified. Two micrograms of total RNA wasreverse-transcribedusingreverse transcriptase (Promega,Madison,WI, USA) according to the manufacturer's instructions. All PCRexperiments were performed using Taq polymerase (Promega)with the following primers: 5′-TTGCTACCCTAACCTTGACC-3′ and5′-TCCCGTTCGACTTTCTTTT-3′ for rat NDRG2, and 5′-GCAAATT-CAACGGCACAGTCAAGG-3′ and 5′-ATCACGCCACAGCTTTCCA-GAGG-3′ for rat glyceraldehyde-3-phosphate dehydrogenase(GAPDH) control. The PCR products were resolved on a 1% agarosegel containing ethidium bromide and bands were visualized usingultraviolet light (Cai et al., 2009).
4.5. Western blot analysis
For Western blot analysis 100 mg tissues (n=5 each group)were lysed in modified radioimmunoprecipitation assaybuffer. Protein concentration was measured by bicinchoninic
acid (BCA) protein assay (Pierce, Rockford, IL, USA). Proteinswere separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to Hybond ECLnitrocellulose membranes (Amersham Bio-sciences, LittleChalfont, Buckinghamshire, UK). Anti-NDRG2 mouse mono-clonal antibody (1:1000; Abnova Corporation, Taipei, Taiwan)and anti-GAPDH rat monoclonal antibody (1:500; Boster,Wuhan, China) were used for immunoblotting. To visualizeprimary antibody-bound proteins, secondary antibodies con-jugated to IRDye800 (1:20000; Rockland Inc., Gilbertsville, PA,USA) and an Odyssey infrared imaging system (LI-COR Inc., StLincoln, NE, USA) were used.
4.6. Immunohistochemical staining
Rats (n=3 each group) were anesthetized and transcardiallyperfused with physiologic saline followed by 4% paraformalde-hyde (PFA) in order to remove the blood and fix the brain tissue.Thebrainswere stored in 4%PFA for 24 h, and the tissuewas thendehydrated and embedded in paraffin. Paraffin sections weredewaxed in xylene, dehydrated through descending concentra-tions of ethanol, immersed in 0.3% H2O2–methanol for 10min,and incubatedwith 10%normal goat serum for 1 h. SectionswereincubatedwithNDRG2 antibody (1:200) in PBS at 4 °C overnight ina moist box, and then with biotinylated goat anti-mouse IgG(1:300; Sigma, USA) at room temperature for 2 h. The sectionswere detected using a streptavidin–peroxidase complex. Thebrowncolor indicative of peroxidaseactivitywasdevelopedusing0.1% 3, 3-diaminobenzidine (DAB; Sigma) in PBS with 0.05% H2O2
for 3minat roomtemperature. The sectionswere counterstainedwith hematoxylin.
4.7. Immunofluorescent double-labeling staining
Paraffin sections were deparaffinized in xylene and rehydrated.The sections were incubated with proteinase K (10 μg/mL) for15min at 37 °C for antigen retrieval, and nonspecific antibody-binding sites were blockedwith 1% bovine serum albumin in PBS(1% BSA–PBS). The sections were incubated with anti-NDRG2mouse monoclonal antibody (1:200) and anti-GFAP rabbit mono-clonal antibody (1:300; DakoCytomation, Glostrup, Denmark) in1%BSA–PBSovernightat4 °C.Thesectionswere thenwashedTBSand incubated with anti-mouse FITC tagged secondary antibody(1:200; CWBIO, Peking, China) and anti-rabbit CY3 taggedsecondary antibody (1:200; CWBIO, Peking, China) for 2 h atroom temperature. At last, DAPI (1 ng/μL) was used to stainnucleus. The sections were mounted with 50% glycerol forexamination under a fluorescence microscope.
4.8. TUNEL and NDRG2 immunofluorescent double-labeling
Apoptosis was quantified using a commercially availablefluorescent terminal deoxynucleotidyl transferase nick-endlabeling (TUNEL) kit, in accordance with the manufacturer'sprotocol (Roche Diagnostics Corporation, Indianapolis, IN,USA). The sections were also incubated with anti-NDRG2mouse monoclonal antibody (1:200) and anti-mouse CY3tagged secondary antibody (1:200), and then stained withDAPI (1 ng/μL). The sections were mounted with 50% glycerolfor examination under a fluorescence microscope.
258 B R A I N R E S E A R C H 1 3 8 2 ( 2 0 1 1 ) 2 5 2 – 2 5 8
4.9. Statistical analysis
All data are presented as means±SD and were analyzed usingANOVA. Statistical analyses were performed by using SPSSsoftware (version 11.1; SPSS, Chicago, IL). P<0.05 was consid-ered statistically significant.
Acknowledgments
This work was supported by grants from the National NaturalScience Foundation of China for distinguished young scholars(no. 30725039), the Major Program of National Natural ScienceFoundation of China (no. 30930091), and the National NaturalScience Foundation of China (nos. 30873326, 81072888,81000563, 30600314 and 30830054), and the National KeyBasic Research and Development Program (no. 2010CB529705).
Appendix A. Supplementary data
Supplementary data to this article can be found online atdoi:10.1016/j.brainres.2011.01.023.
Bernaudin, M., Nedelec, A.S., Divoux, D., MacKenzie, E.T., Petit, E.,Schumann-Bard, P., 2002.Normobaric hypoxia induces toleranceto focal permanent cerebral ischemia in association with anincreased expression of hypoxia-inducible factor-1 and its targetgenes, erythropoietin and VEGF, in the adult mouse brain. J.Cereb. Blood Flow Metab. 22, 393–403.
Cai, L., Li, Y., Liu, F., Zhang, W., Huo, B., Zheng, W., Ding, R., Guo, J.,Zhao, Q., Dou, K., 2009. The influence of ADAR1's regulation onlymphocyte cell function during rejection. Mol. Biol. Rep. 37,2703–2709.
Furuta, H., Kondo, Y., Nakahata, S., Hamasaki, M., Sakoda, S.,Morishita, K., 2010. NDRG2 is a candidate tumor-suppressor fororal squamous-cell carcinoma. Biochem. Biophys. Res.Commun. 391, 1785–1791.
Ginsberg, M.D., 2003. Adventures in the pathophysiology of brainischemia: penumbra, gene expression, neuroprotection: the2002 Thomas Willis Lecture. Stroke 34, 214–223.
Hata, R., Maeda, K., Hermann, D., Mies, G., Hossmann, K.A., 2000.Evolution of brain infarction after transient focal cerebralischemia in mice. J. Cereb. Blood Flow Metab. 20, 937–946.
Nichols, N.R., 2003. Ndrg2, a novel gene regulated by adrenalsteroids and antidepressants, is highly expressed in astrocytes.Ann. NY Acad. Sci. 1007, 349–356.
Nichols, N.R., Agolley, D., Zieba, M., Bye, N., 2005. Glucocorticoidregulation of glial responses during hippocampalneurodegeneration and regeneration. Brain Res. Brain Res. Rev.48, 287–301.
Ohki, T., Hongo, S., Nakada, N., Maeda, A., Takeda, M., 2002.Inhibition of neurite outgrowth by reduced level of NDRG4protein in antisense transfected PC12 cells. Brain Res. Dev.Brain Res. 135, 55–63.
Okuda, T., Kokame, K., Miyata, T., 2008. Differential expressionpatterns of NDRG family proteins in the central nervoussystem. J. Histochem. Cytochem. 56, 175–182.
Petito, C.K., Babiak, T., 1982. Early proliferative changes inastrocytes in postischemic noninfarcted rat brain. Ann. Neurol.11, 510–518.
Shen, L., Zhao, Z.Y., Wang, Y.Z., Ji, S.P., Liu, X.P., Liu, X.W., Che,H.L., Lin, W., Li, X., Zhang, J., Yao, L.B., 2008.Immunohistochemical detection of Ndrg2 in the mousenervous system. NeuroReport 19, 927–931.
Sun, M., Zhao, Y., Gu, Y., Xu, C., 2010. Neuroprotective actions ofaminoguanidine involve reduced the activation of calpainand caspase-3 in a rat model of stroke. Neurochem. Int. 56,634–641.
Swanson, R.A., Ying, W., Kauppinen, T.M., 2004. Astrocyteinfluences on ischemic neuronal death. Curr. Mol. Med. 4,193–205.
Takahashi, K., Yamada, M., Ohata, H., Momose, K., Higuchi, T.,Honda, K., Yamada, M., 2005. Expression of Ndrg2 in the ratfrontal cortex after antidepressant and electroconvulsivetreatment. Int. J. Neuropsychopharmacol. 8, 381–389.
Wang, L., Liu, N., Yao, L., Li, F., Zhang, J., Deng, Y., Liu, J., Ji, S., Yang,A., Han, H., Zhang, Y., Zhang, J., Han, W., Liu, X., 2008a. NDRG2is a new HIF-1 target gene necessary for hypoxia-inducedapoptosis in A549 cells. Cell. Physiol. Biochem. 21, 239–250.
Wang, Q., Gou, X., Xiong, L., Jin, W., Chen, S., Hou, L., Xu, L., 2008b.Trans-activator of transcription-mediated delivery of NEP1-40protein into brain has a neuroprotective effect against focalcerebral ischemic injury via inhibition of neuronal apoptosis.Anesthesiology 108, 1071–1080.
Wang, Q., Peng, Y., Chen, S., Gou, X., Hu, B., Du, J., Lu, Y.,Xiong, L., 2009. Pretreatment with electroacupunctureinduces rapid tolerance to focal cerebral ischemia throughregulation of endocannabinoid system. Stroke 40,2157–2164.
Yan-chun, D., Li-bo, Y., L., X.-p., 2001. Exploring a new genecontaining ACP like domain in human brain and expression ofit in E. coli. Prog. Bichem. Biophys. 28, 72–76.
