Journal of the Neurological Sciences 320 (2012) 38–44

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Journal of the Neurological Sciences

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Increase in phosphorylation of PDK1 and cell survival after acute spinal cord injury

Yi Zhong a,1, Ye Huang a,1, Jianhua Cao a, Xiang Lu b, Meijiang Feng b, Gan Shen a,Aiguo Shen b, Xiaowei Yu a,⁎a Department of Orthopaedics, the Second Affiliated Hospital, Nanjing Medical University, Nanjing 210011, Chinab Department of Geriatrics, the Second Affiliated Hospital, Nanjing Medical University, Nanjing 210011, China

Abbreviations: PDK1, 3-Phosphoinositidedependentkinase B; PIP3, phosphoinositol 3,4,5-triphosphate; CNS, Cecord injury; SDS, Sodium dodecyl sulfate; PAGE, PolyacryBovine serum albumin; DAB, Diaminobenzidin; PBS, PhNeuronal nuclei; GFAP, Glial fibrillary acidic protein;phosphohydrolase; GAPDH, Glyceraldehyde-3-phosphate⁎ Corresponding author. Tel.: +86 25 58509849; fax:

E-mail address: yuxiaowei2011@gmail.com (X. Yu).1 These authors contributed equally to this work.

0022-510X/$ – see front matter © 2012 Elsevier B.V. Aldoi:10.1016/j.jns.2012.06.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 April 2011Received in revised form 23 February 2012Accepted 1 March 2012Available online 30 June 2012

Keywords:PDK1PhosphorylationPKBSpinal cord injuryRats

3-Phosphoinositidedependent protein kinase-1 (PDK1), which phosphorylates and activates a group ofkinases, plays important roles in cellular metabolism, growth, proliferation and survival. However, the func-tions of PDK1 in central nervous system (CNS) injury remain an enigma. To elucidate the expressions andpossible functions of PDK1 and its phosphorylation in CNS injury and repair, we performed an acute spinalcord injury (SCI) model in adult rats and detected the expression and localization of serine-241 phosphory-lated PDK1 (p-PDK1s241). Western blot and immunohistochemistry showed that serine-241 phosphorylatedPDK1 (p-PDK1s241) started increasing by 6 h after damage and peaked at 12 h, then declined to basal levelsby 3 days after injury. Immunohistochemical staining also revealed subcellular localization changes ofp-PDK1s241 staining between nucleus and cytoplasm after injury including neurons and glial cells. Doubleimmunofluorescence labeling suggested that p-PDK1s241 primarily localizes in neurons and oligodendro-cytes. It might also be expressed in other glial cells of spinal cord tissues within 2 mm from the epicenterat 12 h post-injury. Moreover, double staining indicated that p-PDK1s241 and active caspase-3 showed dif-ferent cellular distributions after SCI. Together with previous reports, we hypothesize that phosphorylationof PDK1 may be associated with cell survival and suggest PDK1 as a novel target for neuroprotection andfunctional repair in SCI.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Spinal cord injury (SCI) is a common neurologic trauma whichcauses serious tissue damage and permanent disability. The primaryinjury caused by shearing or compressive forces results in structuraldamage and leads to cell death. The secondary injury induced byinflammation and immunocyte activation leads to neuronal andoligodendroglial apoptosis, astrocyte proliferation/scar formation andmicroglial activation [1–3], which subsequently results in cell loss andpoor prognosis [4,5].

The 3-phosphoinositide-dependent protein kinase 1 (PDK1) wasfirst identified as a protein kinase to phosphorylate the T-loop ofPKBα (Thr308) [6]. It is a 63 kDa Ser/Thr kinase, which contains anN-terminal kinase domain and a C-terminal pleckstrin homology

protein kinase-1; PKB, proteinntral nervous system; SCI, Spinallamide gel electrophoresis; BSA,osphate Buffer solution; NeuN,CNPase, Cyclic nucleotide 3′-dehydrogenase.+86 25 58509994.

l rights reserved.

(PH) domain [6,7]. Previous studies showed that PDK1 and protein ki-nase B (PKB) bind to PIP3 and co-localize at the plasma membrane bytheir PH domain. The binding of PKB to PIP3 induces a conformationalchange and causes PDK1 to phosphorylate and activate PKB [8]. Thelatter respond to CNS trauma and play an important role in celldeath/survival process [9].

PDK1 is a member of the AGC (protein kinase A, G, and C) familyof protein kinases, which also acts downstream of phosphoinositide3-kinase (PI3K) to regulate various cellular activities. Studies haveshown that PDK1 also phosphorylates several members of the AGCfamily, including p70 ribosomal protein S6 kinase (p70S6K) [10],serum and glucocorticoid-induced protein kinases (SGKs) [11], p90ribosomal protein S6 kinases (RSKs) [12] and several protein kinase C(PKC) isoforms [13]. Therefore, PDK1 plays a central role in regulatinga number of kinases that have been implicated in many cellular signal-ing pathways, including cell survival, proliferation and anti-apoptosis[14–16].

Previous studies confirmed that PDK1 is essential for the normaldevelopment and viability of organisms [17–19]. Mice embryoslacking PDK1 died at embryonic day 9.5, displaying multiple abnor-malities, including lack of somites, forebrain and neural crest derivedtissue [20]. Furthermore, The antiapoptotic effects of PDK1 have beenreported in non-neuronal cells [21,22] and inhibitory activity of PDK1was shown to inhibit tumor cell growth and to promote apoptosis

39Y. Zhong et al. / Journal of the Neurological Sciences 320 (2012) 38–44

[23,24]. However, the biological significance of PDK1 and its activa-tion in CNS injury is largely unknown.

In present study, we investigated the change of PDK1 phosphory-lation and the association of p-PDK1s241 with active caspase-3 andPKB. Our research is conducted to gain a better insight into the phys-iologic functions of PDK1 in the traumatic spinal cord.

2. Materials and methods

2.1. Animals and surgery

A total of 58 male Sprague–Dawley rats with an average bodyweight of 250 g (200–275 g) were used in this study (Table 1). Therats were deeply anesthetized with chloral hydrate (10% solution)and surgery was performed under aseptic conditions. Dorsal lam-inectomies at the level of the ninth thoracic vertebra (T9) were car-ried out. Rats in the injured group had their spinal cord compressedusing the NYU impactor device by dropping a 10 g weight and 2.0-mm-diameter rod from a height of 10 cm as previously described[25]. After SCI, the overlying muscles and skin were closed in layerswith 4-0 silk sutures and staples, respectively, rats were allowed torecover on a 30 °C heating pad. Animals were hydrated with 0.9%NaCl solution (2.0 cm3, s.c.). Bladders of injured rats were expressedtwice daily until complete recovery. The animals were housedunder a 12-h light/dark cycle in a pathogen-free area with free accessto water and food. The animals were sacrificed at 6 h, 12 h, 1 day,3 days, 5 days and 7 days post injury. Rats that did not receive surgerywere used as negative controls. All surgical interventions and postop-erative animal care were carried out in accordance with the Guide forthe Care and Use of Laboratory Animals (National Research Council,1996, USA) and were approved by the Chinese National Committeeto the Use of Experimental Animals for Medical Purposes, JiangsuBranch. All efforts were made to minimize the number of animalsused and their suffering.

2.2. Western blot analysis

The portion of spinal cord extending 5 mm rostral and 5 mm cau-dal to the epicenter of injury was removed and snap-frozen in liquidnitrogen. The samples were homogenized in lysis buffer (1% NP-40, 50 mmol/l Tris, pH 7.5, 5 mmol/l EDTA, 1% SDS, 1% sodiumdeoxycholate, 1% Triton X-100, 1 mmol/l PMSF, 10 mg/ml aprotinin,and 1 mg/ml leupeptin) and clarified by centrifuging for 20 min in amicrocentrifuge at 4 °C. After determination of its protein concentra-tion with the Bradford assay (Bio-Rad, Hercules, CA), the resultingsupernatant (50 μg of protein) was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separat-ed proteins were transferred to a polyvinylidine difluoride (PVDF)membrane (Millipore) by a transfer apparatus at 350 mA for 2.5 h.The membrane was then blocked with 5% nonfat milk and incubatedwith primary antibody against PDK1 (anti-rabbit, 1:1000; Cell Signal-ing Technology), p-PDK1s241 (anti-rabbit, 1:1000; Cell SignalingTechnology), PKB (anti-rabbit, 1:1000; Cell Signaling Technology),p-PKBt308 (anti-mouse, 1:1000; Cell Signaling Technology), activecaspase-3 (anti-goat, 1:1000; Santa Cruz) or GAPDH (anti-rabbit,1:1000; Santa Cruz). After incubating with the 800-nm infrared-

Table 1The number of animals used for each experiment.

Group N 6 h 12 h 1 d 3 d 5 d 7 d

Western blot 5 5 5 5 5 5 5Immunohistochemistry 3 3 3 3 3 – –

Immunofluorescent 3 – 5 – – – –

labeled secondary antibody, the membranes were scanned using theOdyssey™ Infrared Imager (LI-COR Biosciences, Lincoln, NE).

2.3. Sections and immunohistochemistry

At the chosen time points the rats were sacrificed and perfusedthrough the ascending aorta with saline, followed by 4% paraformal-dehyde. After perfusion, spinal cords were removed and post-fixedin the same fixative for another 3 h, and they were replaced with20% sucrose for 2–3 days, then 30% sucrose for 2–3 days. After treat-ment with sucrose solutions, the tissues were embedded in O.C.T.compound. Then, 8-μm frozen cross-sections were made and stainingfollowing the standard immunostaining protocol. All sections werefirst blocked with 10% normal serum blocking solution (the same spe-cies as the secondary antibody) containing 3% (w/v) bovine serumalbumin, 0.1% Triton X-100, and 0.05% Tween-20 for 2 h at roomtemperature in order to avoid unspecific staining. Then the sectionswere incubated overnight at 4 °C with primary antibodies for anti-p-PDK1s241 (1:1000; Cell Signaling Technology), followed by incubationin biotinylated secondary antibody (Vector Laboratories, Burlingame,CA). Staining was visualized with DAB (Vector Laboratories). Cellswith strong or moderate brown staining were counted as positive,cells with no staining were counted as negative, while cells with weakstaining were scored separately.

2.4. Double immunofluorescent staining

Sections were firstly blocked with 10% normal serum blockingsolution species the same as the secondary antibody, containing 3%BSA and 0.1% Triton X-100 and 0.05% Tween-20 for 2 h at roomtemperature in order to avoid unspecific staining. Then the sectionswere incubated with both rabbit polyclonal primary antibodies forp-PDK1s241 (1:100; Cell Signaling Technology) and mouse polyclon-al primary antibodiesp-PKBt308 (1:100; Cell Signaling Technology),goat polyclonal primary antibodies for anti-active caspase-3 (1:200;Santa Cruz) or different markers as follows: NeuN (neuron marker,1:600; Chemicon), GFAP (astrocytes marker, 1:200; Sigma), CD11b(microglia marker, 1:50; Millipore) and CNPase (oligodendrocytesmarker, 1:100; Sigma). Briefly, sections were incubated with bothprimary antibodies overnight at 4 °C, followed by a mixture of FITC-and TRITC-conjugated secondary antibodies for 2 h at 4 °C. Finally,the stained sections were finally examined with a Leica fluorescencemicroscope (Germany).

2.5. Quantitative analysis

The number of p-PDK1s241-positive cells in the spinal cord 2 mmfrom the injury center was counted in a 500×500 μm measuringframe. For each animal, a measure was taken in a section throughthe dorsal horn, the lateral funiculus and the ventral horn. The cellcounts in the six sections were then used to determine the total num-ber of p-PDK1s241-positive cells per squared millimeter, measuredaround the lesion area for each animal. Cells double-labeled forp-PDK1s241 and the other phenotypic markers used in the experi-ment were quantified. Sections were double-labeled for p-PDK1s241and NeuN, GFAP, CD11b and CNPase. To identify the proportion ofeach phenotype marker-positive cells expressing p-PDK1s241, a min-imum of 200 phenotype marker-positive cells were counted in bothof the gray matter and white matter in each section, with the excep-tion that only gray matter was conducted for NeuN. Then double-labeled cells for p-PDK1s241 and phenotype markers were recorded.Six equally spaced sections per animal were sampled 2 mm to theepicenter.

40 Y. Zhong et al. / Journal of the Neurological Sciences 320 (2012) 38–44

2.6. Statistical analysis

All values are expressed as mean±SEM. One-way ANOVA followedby Tukey's post-hoc multiple comparison test was used for statisticalanalysis. P valuesb0.05 were considered statistically significant. Eachexperiment consisted of at least three replicates per condition.

3. Results

3.1. The expression changes of phosphorylated PDK1 after SCI

Western blot analysis revealed that p-PDK1s241 expressionstarted to increase by 6 h after injury compared with controls. Thep-PDK1s241 expression reached its peak at 12 h after SCI. The highlevel expression of p-PDK1s241 declined to the basal level at 3 daysafter SCI. Total PDK1 expression did not change at all time points(Fig. 1).

3.2. The staining changes of phosphorylated PDK1 after SCI

To identify the distribution of p-PDK1s241 after SCI, we performedimmunohistochemistry staining in the spinal cord. Low expressionof p-PDK1s241 was observed in gray and white matters in negativecontrols (Fig. 2A). While at 12 h after injury, p-PDK1s241 stainingwas obviously increased at 2 mm around the epicenter, mainly inthe white matter and the ventral horn of the gray matter (Fig. 2B).Notably, -PDK1s241 was expressed in both cytoplasm and nucleusin uninjured spinal cord (Fig. 2D, G), however, when rats were sub-jected to SCI, the stainings of p-PDK1s241 were predominantly ex-pressed in cytoplasm including neurons and glial cells (Fig. 2E, H).At 3 days after contusion, the upregulated p-PDK1s241 immuno-activity decreased to the basal level (Fig. 2C, F, H). Besides, PDK1immuno-activity had no alterations (data are not shown). Therefore,the changes of p-PDK1s241 expression showed by immunostainingwere consistent with the Western blot results (Fig. 2J).

Fig. 1.Western blot analysis of p-PDK1s241 and PDK1 expression after SCI. p-PDK1s241 prot6 h after SCI, reached a peak at 12 h (Pb0.01) and reduced thereafter. On the other hand, thgraphs (relative optical density) of the intensity of staining of p-PDK1s241 to PDK1 and GAdifferent from the control group.

3.3. The co‐localization of phosphorylated PDK1 with differentphenotype-specific markers in the rat spinal cord after SCI

To further confirm the cellular localization of p-PDK1s241, doublelabeling immunofluorescent staining was performed with cell-specificmarkers at 3 days after SCI . In the intact spinal cord, p-PDK1s241was mainly expressed in neurons and oligodendrocytes and scarcelyexpressed in microglia and astrocytes (Fig. 3D, h, l, p). After SCI, it wasobserved a significant increase of p-PDK1s241 immuno-activity in neu-rons (Fig. 3A–C) and oligodendrocytes (Fig. 3E–G). It was also observedthat p-PDK1s241 was co-expressed with some astrocytes (Fig. 3I–K)and microglia (Fig. 3M–O), as determined by GFAP and CD11b doubleimmunostaining. To investigate the proportion of cells expressingp-PDK1s241, cell counting was performed in naive and 12 h group.The results showed that p-PDK1s241 expression was increased signifi-cantly in neurons, oligodendrocytes, astrocytes and microglia at 12 hafter SCI (Fig. 3S).

3.4. Phosphorylation of PDK1 was not involved in the cell apoptosis afterSCI

Caspase-3 is known as a marker of apoptosis, we thus examinedthe expression of active Caspase-3 to investigate the apoptosis afterSCI. Western blot indicated that active caspase-3 occurred at 6 h andreach its peak at 3 days (Fig. 4). Furthermore, we performed doubleimmunofluorescent staining with active caspase-3 and p-PDK1s241 toexamine the co-localization of p-PDK1s241 and activated caspase-3in the injured spinal cord at 12 h post injury. No co-localization wasobserved between p-PDK1s241-positive cells and active caspase-3-positive cells (Fig. 5).

3.5. Association between PDK1 and PKB in spinal cord after SCI

Since p-PDK1s241 phosphorylation and activation of PKB, whichare involved in cell survival after SCI, we further examined the

ein level was low in the normal spinal cords, Western blot showed a prominent increasee expression of PDK1 was no different among the time points tested (A). QuantificationPDH, PDK1 to GAPDH at each time point (B, C, D). *Pb0.05 and **Pb0.01 significantly

Fig. 2. Immunohistochemical expression of p-PDK1s241 in the peripheral area after SCI. Histological sections of naive (A, D and G), 12 h (B, E andH) and 3 days after injury (C, F and I)were incubated with p-PDK1s241 antibody. p-PDK1s241 staining was observed mainly in the ventral horn and white matter. In the control group, p-PDK1s241 staining was both incytoplasm and nuclei (D, G), while 12 h after injury the stainings of p-PDK1s241 were increased and mainly in cytoplasm (E, H). At 3 days after the contusion, p-PDK1s241 immu-noactivity decreased to the basal level (F, I). Scale bars: 200 μm (A–C) and 20 μm (D–I). Quantitative analysis of p-PDK1s241 and PDK1 positive cells in naive and injured spinal cord(g). * indicates Pb0.01 significantly different from the control group. Error bars represent S.E.M.

41Y. Zhong et al. / Journal of the Neurological Sciences 320 (2012) 38–44

phosphorylation of PKB with Western blot analysis. Our resultsshowed that p-PKBt308 increased after injury, reached the highestlevel at 12 h, and then declined to the baseline 3 days after SCI(Fig. 6). These results are consistent with the change of p-PDK1s241after SCI. Furthermore, double immunofluorescent staining showedthat p-PDK1s241 and p-PKBt308 were co-localized in the neuronsand glial cells near the damaged area at 12 h after SCI (Fig. 7), indicat-ing that PDK1 may regulate apoptosis via PKB after SCI.

4. Discussion

In the current study, we demonstrated the phosphorylation levelof PDK1 at s241 was significantly increased during the early stages ofSCI. Studies showed that p-PDK1s241 slightly expressed in neuronsand glial cells in the normal spinal cord. While at 12 h after injury, wefound that p-PDK1s241 positive neurons or oligodendrocytes were sig-nificantly increased. Meanwhile, we observed subcellular localization

changes of p-PDK1s241 between nucleus and cytoplasm after injury.Furthermore, active caspase-3 positive cells did not co-localize withp-PDK1s241 positive cells after SCI. In addition, double immunofluores-cence staining indicated that p-PDK1s24 co-expressed with p-PKBt308in neurons and glial cells. These results showed important evidences tothe biological functions of PDK1 and its phosphorylation in regulatingcell apoptosis after SCI.

It has been demonstrated that PDK1 activates members of the AGCprotein kinase by autophosphorylation at Ser241, which is mediatedby an intermolecular reaction [26,27]. We found the protein levelof p-PDK1s241 was up-regulated and reached its peak at the 12 h,then declined to basal levels at 3 days after injury. Besides, immuno-histochemistry analysis revealed subcellular localization changes ofp-PDK1s241 immunochemical staining between nucleus and cyto-plasm after injury. It was reported that nuclear translocation maysequestrate PDK-1 from activation of the cytosolic signaling path-ways and regulating PDK-1-mediated cell signaling and function

Fig. 3. Double immunofluorescence staining for p-PDK1s241 and cells markers in adult rat spinal cord after SCI. The tissue from naive or injured spinal cord was sectioned andimmunostained with p-PDK1s241 and different cell markers. In the adult rat spinal cord within 2 mm distance from the lesion site at 12 h after SCI, transverse sections labeledwith p-PDK1s241 (red) and different cell markers (green), such as NeuN, GFAP, CD11b and CNPase. The yellow color visualized in the merged images represented co‐localizationof p-PDK1s241with different phenotype-specificmarkers. Co‐localizations of p-PDK1s241with different phenotype-specificmarkers in the negative group are shown (D, H, L and P).(M, N) as negative controls. The photomicrograph shows control examples without the primary antibody. Quantitative analysis of different phenotype-specific markers positive cellsexpressing p-PDK1s241 (%) in naive spinal cord and 12 h after SCI (O). (*) indicate significant difference at Pb0.05 compared with naive group. Scale bars: 20 μm.

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[28]. These findings suggest that PDK1 might play a role in early stageof SCI.

A number of studies have provided that caspase-3 activation andapoptotic cell death can be detected from hours to weeks after injury[29,30]. Caspase-3 activation plays a key role in the regulation of ap-optosis. Studies have demonstrated that caspase-3 activation could betriggered by the release of cytochrome c from the mitochondria. Cyto-chrome c then interacts with Apaf-1 (apoptosis protease activatingfactor-1) to remote the activation of caspase-9, an upstream activatorof caspase-3 [30]. In this research, we found that the active caspase-3protein increased and reached the highest point at the 3rd day afterinjury. Additionally, the p-PDK1s241 positive cells did not co‐localizewith active caspase-3 positive cells at 12 h after SCI, suggesting thatp-PDK1s241 positive cells are not associated with apoptotic death.

These results indicated that PDK1 may play an essential role in theregulation of anti-apoptosis or cell survival after SCI.

Our results have shown the expression of p-PKBt308 reached thehighest point at 12 h after injury.Meanwhile, we detected co‐localizationof p-PKBt308 and p-PDK1s241 both in glial cells and neurons. Since bothphosphorylation sites (Ser 473 and Thr 308) need to be phosphorylatedto fully activation of PKB, so this evidence suggests that PDK1may direct-ly phosphorylate Thr 308 and indirectly phosphorylate Ser 473 of PKB[31]. PKB plays a key role in cell survival signaling. Once activated, PKBpromotes cell survival and prevents apoptosis by inactivating severaltargets, such as Bcl-2-associated death protein (Bad) [32], forkheadtranscription factor (FKHR) [33], glycogen synthase kinase-3 (GSK-3)[34] and caspase-9 [35]. So we speculate that the survival effects ofPDK1 may be attributed to its role as an activator of PKB. Besides, PDK1

Fig. 4. Western blot analysis of active caspase-3 in the adult rat spinal cord after SCI.The 19 kDa active caspase-3 expression increased gradually after injury and reachedits peak at 3 days after injury, then maintained for 7 days. Quantification graphs (rela-tive optical density) of the intensity of staining of active caspase-3 to GAPDH at eachtime point. *Pb0.01 significantly different from the naive group.

Fig. 6.Western blot analysis of p-PKBt308 and PKB in the adult rat spinal cord after SCI.p-PKBt308 was significantly increased and reached its peak at 12 h after SCI. On theother hand, the expression of PKB was no different among the time points tested (A).GAPDH was used as an internal control. Quantification graphs (relative optical density)of the intensity of staining of p-PKBt308 and PKB to GAPDH (B, C). *Pb0.05 and**Pb0.01 significantly different from the negative group.

43Y. Zhong et al. / Journal of the Neurological Sciences 320 (2012) 38–44

can phosphorylate numerous of other kinases, the anti-apoptosis effectsof PDK1may also involve other PDK1 targets. There are evidences show-ing that PDK1 also activates mitogen-activated protein kinase (MAPK)pathway which plays a key roles in the regulation of cell survival[36,37]. Based on previous reports and our experimental results, we con-sider that up-regulation of phosphorylated PDK1 may possibly “delay”the deleterious consequences of neuronal and oligodendroglia deathsafter SCI. However, it still requires more results to confirm whether up-regulation of PDK1 contributes to cell survival after SCI.

In summary, we found that phosphorylation of PDK1 at serine 241was temporarily up-regulated in the spinal cord adjacent to theimpact site after SCI. Our results suggest that the phosphorylationof PDK1 might mediate cell survival after SCI. These data mayprovide a novel strategy for the treatment of SCI. However, we only

Fig. 5. Double immunofluorescent staining for p-PDK1s241 and active caspase-3 in spinal copositive cells were observed (A and D, red) and fewer active caspase-3 positive cells (B andno co‐localization between p-PDK1s241 positive cells and active caspase-3 positive cells (C

investigated the expression and location of phosphorylated PDK1 inthis study, further study about the function and mechanism of PDK1should be performed in the central nervous system injury.

rd after SCI. In the border zone adjacent to the epicenter at 12 h after SCI, p-PDK1s241E, green) were also observed in the same area. However, the overlapped image showedand F). Scale bars: 20 μm.

Fig. 7. Double immunofluorescence staining for p-PDK1s241 with p-PKBt308 in spinal cord after injury. In adult rat spinal cord 2 mm border of epicenter at 12 h after SCI, horizontalsections labeled with p-PDK1s241 (red) and p-PKBt308 (green) are shown in the gray matter (A–C) and white matter (D–F). p-PDK1s241 was largely co‐localized with p-PKBt308.Scale bars: 20 μm.

44 Y. Zhong et al. / Journal of the Neurological Sciences 320 (2012) 38–44

Conflict of interest

No conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Founda-tion of China (No. 81071487, No. 81071480, No. 31070723 and No.81070275); Natural Science Foundation of Jiangsu Province, China(No. BK2009449); Social Development Program of Jiangsu Province,China (No. BE2010744); and Medical Scientific Research Foundationof Jiangsu Province, China (No. H201008).

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