Effect of HypoxiaReoxygenation on Cell Viability and Expression and Secretion of Neurotrophic Factors NTFs in Primary Cultured Schwann Cells.код для вставкиСкачать
THE ANATOMICAL RECORD 293:865–870 (2010) Effect of Hypoxia/Reoxygenation on Cell Viability and Expression and Secretion of Neurotrophic Factors (NTFs) in Primary Cultured Schwann Cells HAO ZHU,1* FENG LI,1* WEN-JUAN YU,2 WEN-JIN WANG,1 LIU LI,1 LI-DAN WAN,1 YAN LE,1 AND WEN-LONG DING1 1 Department of Anatomy, Shanghai Jiao Tong University School of Medicine, Shanghai Jiao Tong University Institutes of Medical Sciences, Shanghai, China 2 Department of Psychiatry, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China ABSTRACT As the primary myelin-forming cells of the peripheral nervous system, Schwann cells (SCs) play a key role in the regeneration of injured peripheral nerves. However, hypoxia causes injury of SCs, as observed in peripheral neuropathies, including those caused by diabetes. So we investigated the effect of hypoxia/reoxygenation (H/R) on SCs in this study. To do so, SCs were cultured in hypoxic condition in vitro and then in normal condition for 24 hr; The effects H/R on SCs were evaluated by MTT (3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Hoechst staining, immunocytochemistry, western blotting, ELISA, and RT-PCR. H/R resulted in a signiﬁcant decrease in SCs survival and an increase in caspase-3 activity. H/R also reduced the mRNA level of BDNF (brain derived neurotrophic factor) and its secretion, but NGF mRNA level was elevated in these cells. These observations showed that H/R induces death of primary cultured SCs, and different mechanisms responsible for regulating NGF and BDNF C 2010 Wiley-Liss, Inc. expression. Anat Rec, 293:865–870, 2010. V Key words: Schwann cell; hypoxia/reoxygenation; caspase-3; neurotrophic factors Schwann cells (SCs) are the myelin-forming cells of the peripheral nervous system and play a key role in peripheral nerve regeneration. After peripheral nerves are transected, Wallerian degeneration takes place. SCs, together with macrophages remove necrotic tissue and myelin debris. Furthermore, SCs proliferate to form Büngner bands, which help the regrowing axons to elongate their growth cones in the direction of denervated targets. The whole regeneration process is completed when the regenerative axons are remyelinated by SCs. The nerve regeneration process is also regulated by neurotrophic factors (NTFs) synthesized directly or indirectly by SCs. In various pathological processes, SCs experience ischemia and hypoxia. Traumatic injury can damage blood supply of nearby nerve and SCs are both directly and indirectly induced to be exposed to hypoxia in diabetic neuropathy, contributing to SCs dysfunction. Besides, biC 2010 WILEY-LISS, INC. V ological activity of SCs seeded in grafts decays as time goes on and regeneration of nerve ﬁbers in the middle of Abbreviations used:DEPC, diethyl pyrocarbonate; DMSO, dimethyl sulfoxide; SCs, Schwann cells. Grant sponsor: Shanghai Leading Academic Discipline Project; Grant numbers: S30201; S30205; Grant sponsor: Shanghai Science and Technology Foundation of China; Grant number: 03BK15. y Authors contributed equally to this work. *Correspondence to: Prof. Wen-Long Ding, Prof. Department of Anatomy, Shanghai Jiao Tong University School of Medicine, No. 227 South Chongqing Road, Shanghai 200025, China E-mail: email@example.com. Received 12 October 2008; Accepted 8 December 2009 DOI 10.1002/ar.21105 Published online 23 February 2010 in Wiley InterScience (www. interscience.wiley.com). 866 ZHU ET AL. a conduit is usually poorer than that at both ends after graft transplantation (Guenard et al., 1992; Yank et al., 1999), which may be associated with SCs exposure to local hypoxia induced by ischemia. How does hypoxia/reoxygenation (H/R) affect SCs? Thus, it is very important to make it clear effects of H/R on SCs and mechanisms of action. In this study, we examined cell survival, caspase-3 activity, expression, and secretion of NTFs after SCs were exposed to H/R. probed with rabbit anti-active caspase-3 (Stressgen Bioreagents, Victoria, BC, Canada). Cells were then incubated with peroxidase-conjugated goat anti-rabbit IgM or IgG (1:200) for immunocytochemistry. The results of immunostaining were examined with a Leica DM2500 microscope using a DDC 2/3 camera and analyzed with RS IMAGE ProTM Version4.5 (Roper Scientiﬁc, Trenton, NJ). Western Blot Analysis MATERIALS AND METHODS Primary Cultures of Schwann Cells SCs cultures were obtained utilizing our previous method (Zhu et al., 2008). Brieﬂy, sciatic nerves and brachial plexus from 2- to 3-day-old Sprague-Dawley rats were harvested and digested with 0.12% collagenase and 0.25% trypsin (Sigma, St. Louis, MO) at 37 C for 10–13 min. Twenty-four hours after plating, cells were treated with cytosine arabinoside (5 lg/mL; Sigma, St. Louis, MO) for 3 days to eliminate proliferating ﬁbroblasts. Following this treatment, the culture medium was replaced with fresh medium supplemented with forskolin (4 lM; Sigma) and basic ﬁbroblast growth factor (bFGF; 20 ng/ mL; R&D Systems, Minneapolis, MN) to allow for Schwann cell proliferation. After 8 days, SCs were detached with 0.25%EDTA- trypsin (Sigma) and passaged. The culture medium was changed three times per week. Hypoxia/Reoxygenation Hypoxic conditions were induced according to our previous method (Zhu et al., 2008). Brieﬂy, the incubation chamber was ﬁlled with a mixture of 5% CO2/92% N2 and monitored by an oxygen analyzer (Shanghai Instrument Co, Shanghai, China) with an oxygen sensitive metal electrode. The chamber was sealed and placed at 37 C in a humidiﬁed incubator for 15 min when the concentration of oxygen was lower than 1%. Subsequent to the hypoxic treatment, the medium was replaced with fresh medium and cells were then cultured for 24 h under normoxic conditions (95% air, 5% CO2). Three replicates were used in each experiment. Evaluation of Apoptotic Cells Cultured cells were incubated for 15 min with Hoechst 33342 (10 lg/mL) under dim light, and then observed under an epiﬂuorescence microscope. Cells with small nuclei, high ﬂuorescence intensity due to chromatin condensation or nuclear fragmentation were considered as apoptotic cells. The number of high-ﬂuorescent condensed or fragmented nuclei was counted using a Leica DM2500 microscope. Five different ﬁelds were counted per dish. Results are expressed as percentage of condensed or fragmented nuclei relative to the total number of nuclei counted per dish for each experimental condition. The number of dead and apoptotic cells in each group was expressed as percentage of total cell number. Immunocytochemistry Twenty-four hours following hypoxia treatment, cultured cells were ﬁxed with 4% paraformaldehyde and To conﬁrm the results of active caspase-3 immunocytochemistry, we detected active caspase-3 using Western blot analysis. SCs were washed with PBS and lysed with a lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium ﬂuoride, 4 lg/mL leupeptin, 1 lg/mL aprotinin, and 100 lg/ mL PMSF; all from Sigma). After incubation on ice for 30 min, cell lysates were then clariﬁed by centrifugation at 16,000 g for 10 min at 4 C and the supernatant was saved for protein analysis and Western blot. Total protein concentration was determined by using a commercially available kit based on the bicinchoninic acid (BCA) method. For Western blot analysis, 20 lg of the cell extract was electrophoresed on a 12% SDS/polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk in trisbuffered saline-0.1% Tween 20 (TBS-T), washed with TBS–T and incubated overnight with anti-active caspase-3 (1:500) and anti-GAPDH antibody (1:2000, Sigma). The membrane was washed and then incubated with secondary antibody conjugated with peroxidase (1:1000) in 5% nonfat dry milk in TBS–T buffer for 1 h. Detection was carried out using an enhanced chemiluminescence detection kit (Pierce). MTT Assay The MTT (3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay was used to study the effect of H/ R on SC survival. Cells were planted in a 96-well plate and cultured for 24 h after hypoxia. 10 lL of MTT stock solution (10 mg/mL) (Sigma) was then added to the remaining medium and the cultures were incubated for another 4 h at 37 C. The medium was discarded after the incubation and the insoluble dark blue formazan was dissolved in 100 lL of DMSO and quantiﬁed at 570 nm with a reference wavelength of 630 nm using a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA). Survival of the control group was deﬁned as 100% and that of H/R group was expressed as percentage of control group. Reverse Transcriptase–Polymerase Chain Reaction (RT–PCR) Twenty-four hours following hypoxia treatment, the total RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA). RNA was reverse transcribed in a ﬁnal volume of 20 lL, containing 1 lg of total RNA, 50 pM oligodT, 10 lM of each deoxyribonucleoside triphosphate (dNTP), 20 units RNasin and 10 units AMV Reverse Transcriptase (Promega), 4 lL of 5 Reverse Transcriptase buffer with DEPC H2O (TaKaRa, Dalian, 867 THE EFFECT OF HYPOXIA/REOXYGENATION ON SCHWANN CELLS TABLE 1. Nucleotide sequence for oligonucleotide primers and size of the expanded PCR products Gene NGF BDNF GAPDH Primer forward reverse forward reverse forward reverse Sequence 0 0 5 TCCACCCACCCAGTCTTCCA3 50 GCCTTCCTGCTGAGCACACA30 50 AGCTGAGCGTGTGTGACAGT30 50 TCCATAGTAAGGGCCCGAAC30 50 CACCACCATGGAGAAGGCC30 50 GATGGATGCCTTGGCCAGG30 Product (bp) Gene bank 343 V00836 252 X55573 190 M32599 China) supplied by the manufacturer. Two separate negative control reactions, either without RNA or without AMV, were run in parallel. PCR was performed using 2 lL of synthesized cDNA with 0.125 lL Taq polymerase (TaKaRa, China), 2 lL of dNTPs, 0.5 lL of each primer, 2.5 lL of PCR buffer, supplied by the company, and DEPC H2O added to give a total reaction volume of 25 lL. All common components were added to a master mix and then aliquoted. The cycling conditions were as follows: Initial denaturation at 95 C for 5 min followed by 28 cycles of: 95 C for 1 min, 55 C for 1 min, 72 C for 2 min, and a ﬁnal extension at 72 C for 5 min. GAPDH was used as the loading control. Primers were designed as shown in Table 1. Quantiﬁcation of Neurotrophic Factors by ELISA Twenty-four hours after hypoxia treatment, NTFs concentrations in cellular supernatants were measured using commercially available assays according to the manufacture’s directions: BDNF Emax Immunoassay (Promega Corp, Madison, WI) for BDNF, and rat b-NGF ELISA kit (R&D Systems) for NGF. Brieﬂy, the monoclonal antibody (mAb) was added to each well of a 96-well plate and incubated overnight at 4 C. The following additions to each well were carried out sequentially: samples and BDNF standards in duplicate (incubated 2 h at room temperature); anti-human BDNF pAb (incubated 2 h at room temperature); anti-IgY HRP (incubated 1 h at room temperature); TMB solution (incubated 20 min at room temperature). The plate was washed with Tris buffered saline with 0.05% Tween 20 (TBST) buffer each time. Finally, stop solution was added and absorbance was read at 450 nm on a microplate reader (Bio-Rad Laboratories) within 30 min. The same procedure was carried out for NGF with following reagents: goat anti-ratb-NGF, biotinylated goat antiratb-NGF, and streptavidin-HRP. NTFs concentrations were calculated based on a standard curve. Statistical Analysis One-way ANOVA followed by a Newman Keuls’ multiple comparison tests was used to compare control and treated groups with P < 0.05 being considered statistically signiﬁcant. RESULTS H/R Induces SCs Apoptosis Hoechst33342, a bis-benzimidazole dye, was used to measure SCs apoptosis. It could penetrate the plasma membrane and stain DNA in cells without permeabiliza- Fig. 1. H/R induced apoptosis of SCs. (A) Hoechst 33342 staining was performed to visualize the extent of programmed cell death. Condensed or fragmented nuclei were considered as apoptotic cells. Arrows indicated condensed nuclei and arrowheads indicated fragmented nuclei. Bar: 20 lm. (B) Quantiﬁcation of SCs death after H/R. Data (mean SEM) were summarized from three independent experiments, ** p < 0.01 vs. control group. tion. In contrast to the nuclei of normal cells, those of apoptotic cells have highly condensed chromatin stained uniformly by Hoechst 33342 (Fig.1). We found that H/R could induce up to 31.7 4.5% SCs to apoptosis, which were consistent with the results obtained by MTT reduction assay. H/R Reduces SCs Viabilities The effect of H/R on SCs injury was investigated by MTT assay and trypan blue staining. Cells were incubated in normal condition for 24 h after exposure to hypoxia. Cell viability was assessed by MTT reduction assay. We found that H/R resulted in a signiﬁcant decrease in SCs survival (MTT: 64 4.7%) (Fig. 2). This result was further veriﬁed by cell count with a hemocytometer. Trypan blue staining was employed to identify dead cells, and cell survival rate was determined before ELISA assay. There were (9.86 0.42) 105 cells in the control group, and only (6.56 0.28) 105 in the H/R group. Thus, the number of viable cells in the H/R group was 66.5 5.4% of that in the control group. This was consistent with the results obtained by MTT assay. 868 ZHU ET AL. Fig. 2. Effects of H/R on viabilities of SCs examined by MTT assay. SCs were exposed to hypoxia for 15 min, then incubated in normal condition for 24 h. Data are collected from three independent experiments and expressed as mean SEM. ** p < 0.01 vs. control group. H/R Activates Caspase-3 in SCs We investigated the possible mechanism of H/R induced cell death by studying the activation of the initiator and the executioner (caspase-3) in the apoptotic cascade. The level of the active caspase-3 in H/R SCs was examined by immunocytochemistry and western blot. As shown in Fig. 3, the grade of caspase-3 was higher in H/R group than in control group. Treatment of H/R increased intracellular level of active caspase-3 nearly 3.3-fold of control level in SCs. In Figure 4, western blot analysis further conﬁrmed H/R induced caspase-3 to be activated in SCs. Fig. 3. Microscopic ﬁndings of immunoreactivity of active caspase3 in SCs. (A) SCs were exposed to hypoxia for 15 min, then incubated in normal condition for 24 h. Extensive positive cellular labeling of caspase-3 was seen in H/R-induced SCs. Only mild positive staining was shown in control group. Bar: 100 lm. (B) The grade of caspase-3 in the H/R group was higher than in control group. Data (mean SEM) were summarized from three independent experiments. ** p < 0.01 vs. control group. H/R Enhances the mRNA level of NGF and Reduces BDNF mRNA in SCs We studied the effect of H/R on the biological activities of SCs by examining NGF and BDNF mRNA through expression RT-PCR. RT-PCR revealed that H/R induced a signiﬁcant reduction of BDNF mRNA in SCs, which was about 75% of normal level; whereas the level of NGF mRNA was elevated to 1.27 fold of control after H/R (Fig. 5). H/R Decreases BDNF Secretion in SCs To test whether H/R treatment also inﬂuences NTFs secretion, we examined secreted proteins through ELISA assay. ELISA assay revealed that NGF and BDNF concentrations in cellular supernatant of H/R group were only 2.97 0.29 and 6.0 1.3 (pg/mL), respectively, both lower than that of control, in which it was 4.3 0.6, and 29.8 2.7 (pg/mL) (Fig. 6). However, H/R leads to large amount of cell death, which might confuse the result of ELISA assay, as the concentration of NTFs in the supernatant correlates with cell number. To eliminate this effect, we counted the cell number in two groups (data described above: cell number within H/R group was only 66.5 5.4% of the control group) and adjusted the results obtained by ELISA assay by cell number. Adjusted data was named reactive values. In Fig. 4. Western blot analysis of activated caspase-3 in SCs exposed to H/R. SCs were exposed to hypoxia for 15 min, then incubated in normal condition for 24 h. H/R induced caspase-3 to be activated in SCs. this way, secreted NTFs levels were compared based on identical cell number. Adjusted NGF and BDNF concentrations were 4.48 0.34 and 9.2 1.9 (pg/mL) respectively in H/R group. Compare of this reactive value demonstrated that H/R signiﬁcantly reduced BDNF secretion (P < 0.01), but had no effect on NGF secretion (Fig. 6). DISCUSSION The present study indicates that H/R is able to reduce SCs viabilities. Our results from MTT assay, trypan blue, and Hoechst 33342 staining provided the ﬁrst evidence that H/R results in the injury of primary cultured SCs. Previous studies have shown that most cells of mammals can not continue to meet the energy demands THE EFFECT OF HYPOXIA/REOXYGENATION ON SCHWANN CELLS 869 Fig. 5. The in vitro effects of H/R on mRNA level of NGF and BDNF in SCs demonstrated by RT-PCR analysis. SCs were exposed to hypoxia for 15 min, then incubated in normal condition for 24 h. Values of densitometric analysis are mean SEM of three independent experiments, *p < 0.05 vs. control group. Fig. 6. Effect of H/R on secretion of NGF (A) and BDNF (B) by hypoxic SCs in vitro. SCs were exposed to hypoxia for 15 min, then incubated in normal condition for 24 h. NTF levels in the cellular supernatants were examined by ELISA assay. The left bars (examined) display secretion level of NTFs in two groups, and the right bars (reac- tive) display secretion level after cell number calibration (measured value adjusted to cell number). Data are collected from three independent experiments and expressed as mean SEM. **p < 0.01 vs. control group. of active ion-transporting systems after exposure to hypoxia, leading to exhaustion of fermentable substrate, catastrophic membrane failure and cell death (Boutilier, 2001). In peripheral neuropathy, caspase-3 was shown to be activated in SCs. The studies of injury mechanisms in SCs revealed that apoptosis is associated with the level of caspase-3 activation (Iida et al., 2004; Wang et al., 2005). Caspase-3 is considered as a key initiator and executioner in the apoptotic pathway and mediates the cleavage of itself, other downstream caspases and other caspase substrates (Nunez et al., 1998). Caspase-3 and Bcl-2 mutually adjust and jointly participate in apoptosis (Nunez et al., 1998). Bcl-2 regulates the activation of key caspases and also serves as a caspase substrate (Soilu-Hänninen et al., 1999). The ﬁnding of H/R activating caspase-3 in SCs suggests that H/R might promote the execution of the apoptotic program, similar to the mechanism in the study that the activity and the expression of caspase-9 and caspase-3 are increased during hypoxia in the cerebral cortex of newborn piglets (Nunez et al., 1998). Our results showed that H/R increased the mRNA level of NGF, whereas had an adverse effect on mRNA level and protein secretion of BDNF in SCs,. In addition, although not signiﬁcant, there is also a trend of increase in NGF secretion. These results indicated that H/R is able to enhance the biological activities of SCs through different effects on NTFs, and H/R induced different mechanisms responsible for regulating NGF and BDNF expression. This study is similar to Meyer’s research in 1992. They demonstrated that there were marked increases in NGF and BDNF mRNA in the non-neuronal cells of the transected nerve. However, the time-course and spatial pattern of both expression are distinctly different. There is a marked rapid increase in NGF mRNA, whereas a continuous slow increase of BDNF mRNA starting after day 3 postlesion and reaching maximal levels 3 weeks–4 weeks later. Rapid increased NGF might be correlated with protecting injured neurons and SCs and stimulating SCs retrodifferentiation and proliferation. Subsequent increased BDNF might play an important role in inducing SCs migration and expression of myeline proteins. So SCs are likely to promote self survival against H/R-induced damage through autocrine production of NGF. In response to hypoxia, mammalian cells activate adaptive mechanisms and express a 870 ZHU ET AL. variety of gene products to avert necrotic or apoptotic cell death (Oka et al., 2007). To SCs exposed to hypoxia, they upregulated NGF to meet urgent requirements of sustaining survival, and downregulated BDNF, a nonurgent requirement, in short term. The phenomenon that H/R stimulates survival and proliferation is also seen in other cells (Lou et al., 1997; Sung et al., 2007). The effect of H/R on SCs might be associated with hypoxiainducible factor-1 (HIF-1). HIF-1 is a transcription factor that regulates the adaptive response to hypoxia in mammalian cells (Chavez and LaManna, 2002). For example, HIF-1 regulates the expression of erythropoietin (Epo) and vascular endothelial growth factor (VEGF), basic ﬁbroblast growth factor (bFGF), which have neuroprotective effect (Taouﬁk et al., 2008; Liu et al., 2007). Upregulation of HIF-1 in Schwann cells is caused by ischemic damage or hypoxia (Oka et al., 2007; Keilhoff et al., 2008), which shows HIF-1 may have a role in protecting Schwann cells against hypoxia. In addition, although the results of RT-PCR analysis and ELISA examination for NGF had an accordant trend of increase, RT-PCR results showed that H/R induced a signiﬁcant increase in the mRNA level of NGF. The reason might be that RT-PCR analysis, based on a single cycle number, is not a particularly accurate measure and may demonstrate a change trend. In conclusion, hypoxia has been documented as an integral part of many pathological states including ischemic diseases (neoplasia, vascular malformations), neurodegenerative diseases, chronic inﬂammatory diseases, and tumor progression. Exposure to H/R activates caspase-3 and reduces SCs viabilities, on the other hand, improves SCs survival and biological activities by autocrine NGF. Regulation of SCs survival and death is mediated by the two ways after H/R. Further studies are in progress in our institute to investigate the mechanisms, by which H/R induces SCs injury. LITERATURE CITED Boutilier RG. 2001. Mechanisms of cell survival in hypoxia and hypothermia. J Exp Biol 204:3171–3181. Chavez JC, LaManna JC. 2002. Activation of hypoxia-inducible factor-1 in the rat cerebral cortex after transient global ischemia: potential role of insulin-like growth factor-1. J Neurosci 22:8922–8931. Guenard V, Kleitman N, Morrissey TK, Bunge RP, Aebischer P. 1992. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci 12:3310–3320. Iida H, Schmeichel AM, Wang Y, Schmelzer JD, Low PA. 2004. Schwann cell is a target in ischemia-reperfusion injury to peripheral nerve. Muscle Nerve 30:761–766. Keilhoff G, Schild L, Fansa H. 2008. Minocycline protects Schwann cells from ischemia-like injury and promotes axonal outgrowth in bioartiﬁcial nerve grafts lacking Wallerian degeneration. Exp Neurol 212:189–200. Liu Y, Lu JB, Chen Q, Ye ZR. 2007. Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF-induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes. Neurosci Bull 23:221–228. Lou Y, Oberpriller JC, Carlson EC. 1997. Effect of hypoxia on the proliferation of retinal microvessel endothelial cells in culture. Anat Rec 248:366–373. Meyer M, Matsuoka I, Wetmore C, Olson L, Thoenen H. 1992. Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J Cell Biol 119:45–54. Nunez G, Benedict MA, Hu Y, Inohara N. 1998. Caspases: the proteases of the apoptotic pathway. Oncogene 17:3237–3245. Oka N, Kawasaki T, Mizutani K, Sugiyama H, Akiguchi I. 2007. Hypoxia-inducible factor 1alpha may be a marker for vasculitic neuropathy. Neuropathology 27:509–515. Soilu-Hänninen M, Ekert P, Bucci T, Syroid D, Bartlett PF, Kilpatrick T. 1999. Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a Bcl-2-independent pathway. J Neurosci 19:4828–4838. Sung SM, Jung DS, Kwon CH, Park JY, Kang SK, Kim YK. 2007. Hypoxia/reoxygenation stimulates proliferation through PKC-dependent activation of ERK and AKT in mouse neural progenitor cells. Neurochem Res 32:1932–1939. Taouﬁk E, Petit E, Divoux D, Tseveleki V, Mengozzi M, Roberts ML, Valable S, Ghezzi P, Quackenbush J, Brines M, Cerami A, Probert L. 2008. TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury. Proc Natl Acad Sci USA 105:6185–6190. Wang Y, Schmeichel AM, Iida H, Schmelzer JD, Low PA. 2005. Ischemia-reperfusion injury causes oxidative stress and apoptosis of Schwann cell in acute and chronic experimental diabetic neuropathy. Antioxid Redox Signal 7:1513–1520. Yank Q, Qiu YF, Shen XY, Liu CD. 1999. Studies on the inﬂuences of hypoxia on morphology and migration of the cultured Schwann cells and anti-hypoxia effect of EGb 761. Chin J Neuroanat 15:273–276. Zhu H, Wang WJ, Ding WL, Li F, He J. 2008. Effect of panaxydol on hypoxia-induced cell death and expression and secretion of neurotrophic factors (NTFs) in hypoxic primary cultured Schwann cells. Chem Biol Interact 174:44–50.