HIF-1α expression in the hippocampus and peripheral macrophages after glutamate-induced excitotoxicity

Journal of Neuroimmunology 238 (2011) 12–18

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

HIF-1α expression in the hippocampus and peripheral macrophages after glutamate-induced excitotoxicity E. Vazquez-Valls a, M.E. Flores-Soto b, 1, V. Chaparro-Huerta b, 1, B.M. Torres-Mendoza b, c, G. Gudiño-Cabrera d, M.C. Rivera-Cervantes e, M. Pallas f, A. Camins f, J. Armendáriz-Borunda g, C. Beas-Zarate b, d,⁎ a

Unidad de Investigación Médica en Epidemiología Clínica, UMAE, Hospital de Especialidades, CMNO, IMSS, Mexico Lab. de Neurobiología Celular y Molecular, División de Neurociencias, CIBO, IMSS, Mexico c Instituto de Patología Infecciosa y Experimental, CUCS, U. de G., Mexico d Lab. de Regeneración y Desarrollo Neural, Instituto de Neurobiología, Departamento de Biología Celular y Molecular, CUCBA, Mexico e Lab. de Neurobiología Celular y Molecular, CUCBA, U. DE G., Mexico f Unitat de Farmacologia i Farmacognòsia i Institut de Biomedicina (IBUB), Centro de Investigación de Biomedicina en Red de Enfermedades Neurodegenerativas (CIBERNED), Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain g Instituto de Biología Molecular y Terapia Génica, CUCS, U. de G., Guadalajara Jalisco, Mexico b

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Article history: Received 4 March 2010 Received in revised form 30 May 2011 Accepted 1 June 2011 Keywords: Hippocampus Macrophages HIF-1 alpha VEGF EPO Excitotoxicity

a b s t r a c t Hypoxia-inducible factor-1 alpha (HIF-1α) is a master transcription factor that regulates the response to hypoxia and ischemia and induces the expression of various genes, including vascular endothelial growth factor (VEGF) and erythropoietin (EPO). This study shows the systemic response of increased HIF-1α, EPO, and VEGF mRNA and protein. In addition, VEGF expression was increased in neurons and over-expressed in glial cells in a model of neuroexcitotoxicity in the hippocampus, in which rats were neonatally exposed to high glutamate concentrations. Simultaneous increases in HIF-1α, EPO and VEGF mRNA in peritoneal macrophages were also observed. Our study is consistent with the hypothesis that these genes exert a protective effect in response to neurotoxicity. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Mammalian cells have developed an adaptive system allowing them to survive moderate or even severe hypoxia. This system involves an increase in the expression of genes encoding proteins responsible for the anaerobic production of ATP (Minet et al., 2001). During ischemia, the lack of oxygen activates the transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α), which is a master transcriptional regulator of the adaptive response to hypoxia (Huang and Bunn, 2003; Seta and Millhorn, 2004). HIF-1α binds to the core DNA sequence 5′-[AG]CGTG3′ that lies within the hypoxia response element of target gene promoters (Semenza, 2004). Hence, HIF-1α promotes transcription of a variety of genes that may help cells adapt to low oxygen levels. These genes include vascular endothelial growth factor (VEGF), erythropoietin

⁎ Corresponding author at: Lab. of Molecular and Cell Neurobiology, Neurosci Div., C.I.B.O., IMSS, Sierra Mojada #800, Col. Independencia, Guadalajara, Jalisco, 44340 Mexico. Fax: + 52 33 3618 1756. E-mail address: [email protected] (C. Beas-Zarate). 1 These co-authors worked like the first author. 0165-5728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.06.001

(EPO), several glycolytic enzymes, glucose transporters, cell cycle regulators, transferrin, heme oxygenase-1 and inducible nitric oxide synthase. Of these genes, VEGF and EPO may directly protect neurons from ischemic insults (Zhang et al., 2004). In several central nervous system (CNS) disorders, including epilepsy, stroke and hypoxic/ischemia events, excitotoxic processes occur over a short period of time. In these disorders, excess glutamate is released, with the main effect of over-stimulation of the NMDA receptor, promoting neuronal death and neurodegeneration (Xu et al., 2009). In addition, there is evidence in several neurodegenerative CNS diseases that indicates activation of immunological pathways as a result of neuroexcitotoxicity (Khan, 2004; Zipp and Aktas, 2006). Recently, the inflammatory process was described in a model of transient focal ischemia in which substantial microglia/macrophage activation and the presence of macrophages that promote both injury and repair at sites of CNS injury were observed (Moxon-Emre and Schlichter, 2010). Two classically activated “pro-inflammatory” or “alternatively activated” anti-inflammatory macrophage cell types that maintain the regenerative process after CNS damage have been described (Kigerl et al., 2009). Thus, the peripheral response of activated macrophages may promote

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CNS repair and limit secondary inflammation-mediated injury. However, macrophages also release factors that kill neurons (Gensel et al., 2009). These relationships between brain injury and a simultaneous pro-inflammatory response by macrophages are rarely considered together in the same experimental paradigm, in which the initial neuroexcitotoxicity in the brain and the peripheral macrophage response should be taken together. Therefore, the goal of this study was to evaluate the mRNA expression of HIF-1α, an important transcriptional factor that is over-expressed during ischemia and hypoxia, and two target genes of HIF-1α, EPO and VEGF, in the hippocampus of rats neonatally exposed to neuroexcitotoxicity by high glutamate concentrations. Expression of these genes was also measured in peripheral macrophages from the same rats. 2. Materials and methods 2.1. Animal preparation Pregnant Wistar rats were kept in separate cages under optimal environmental conditions, i.e. free access to water and food, with a 12– 12 h light–dark cycle, at temperatures ranging between 23 °C and 25 °C. Litters of newborn rats of both sexes were adjusted to eight per litter. For this study, only males that had been reared with their dams were used. Rats were studied on postnatal day (PD) 14. Newborn rats (n = 10) were given monosodium glutamate salt (MSG) (4 mg/g body weight) subcutaneously administered on PD 1, 3, 5, and 7, in line with the experimental model used previously by Beas Zárate et al. (2001, 2007). A group of animals (n = 10) treated with saline solution in a similar manner were used as controls (n = 10). At PD14, treated and control animals were used for macrophage extraction after being killed by decapitation, and the hippocampus was dissected out for molecular analysis. Other treated (n = 5) and control (n = 5) animals were used to examine the cell location of VEGF by immunohistochemistry. Animal care and handling were in accordance with Mexican General Health Laws (Official Newspaper, January 7, 1981) and the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80.23, revised 1996). All efforts were made to minimize the number of animals used and their suffering.

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Two micrograms of total RNA was reverse transcribed in 0.05 M Tris–HCl pH 8.3, 40 mM KCl, 7 mM MgCl2 buffer containing 0.05 U/μl RNAse inhibitor and 200 U/μl murine leukemia virus reverse transcriptase (M-MLV). The reactions were incubated for 10 min at 70 °C and then for 60 min at 37.5 °C. The reaction was then inactivated by incubating the samples at 95 °C for 10 min. cDNAs were used immediately or stored at − 20 °C. PCR was performed after defining the optimal conditions for detection of GAPDH in all the samples studied. Unless otherwise stated, the gene products were amplified in a PCR reaction containing 50 mM Tris–HCl, pH 9.0, 50 mM NaCl, 100 μM dNTPs and 1 U of Taq DNA polymerase. Amplification reactions were overlaid with light mineral oil, held at 95 °C for ‘hot-start’ PCR for 5 min and then run in an automated thermal cycler for the number of cycles specified. The thermal cycles were: 95 °C for 1 min, 60 °C for 1 min and 72 °C for 1.5 min, with a final extension of 5 min at 72 °C. We standardized a semi-quantitative PCR method based on the coamplification of HIF-1α (sense 5′-GAG ACC GCG GGC ACC GAT TCG CCA TGG-3′ and antisense 5′-TCG TCC TCC CCC GGC TTC TTA GGG TAC3′; product of 570 pb and 27 cycles), its target genes EPO (sense 5′GCC GCA GCA GCC AGG CGC GGA GAT GG-3′ and antisense 5′-GCT TGG GTC GCG TCT GGA GGC GAC AT-3′; product of 355 pb and 28 cycles) and VEGF (sense 5′-ATG AAC TTT CTG CTC TCT TGG GTG CA-3′ and antisense 5′-AAG CTG CCT CGC CTT GCA ACG CGA GT-3′; product of 387 pb and 26 cycles) and the constitutively expressed beta actin (sense 5′-CAC CAC AGC TGA GAG GGA AAT CGT GCG TGA-3′ and antisense 3′-ATT TGC GGT GCA CGA TGG AGG GGC CGG ACT-5′; product of 517 pb and 19 cycles), to adjust the PCR conditions in line with Lemus-Varela et al. (2010). PCR of the products was analyzed with 2.5% agarose gel electrophoresis. The intensity of the bands was determined by a video gel documentation and analysis system (Kodak computational system) and was evaluated semi-quantitatively with densitometry. The area corresponding to the band for each amplified PCR product was calculated automatically and normalized to the area represented by GAPDH. The data were initially expressed as arbitrary absorbance units, with less than 10% variation in the constitutive genes. The relevant amplified products were then expressed as intensity units relative to the control group.

2.2. Isolation of peritoneal macrophages 2.4. Western blot for HIF-1α Male Wistar rats (280–300 g) obtained from Bioterio CIBO were anesthetized with ether. Peritoneal macrophages were harvested by two peritoneal washes of the abdominal cavity with 40 ml of phosphate buffered saline (PBS) containing 1 U/ml heparin at 37 °C. Cell suspension was centrifuged (280 ×g; 7 min); and cells were resuspended in PBS containing 1% bovine serum albumin (BSA). Aliquots of about 1 × 10 6 cells were plated in plastic Petri dishes (Nunc, Denmark) at 37 °C in 5% CO2 in air for 2 h. Non-adherent cells were decanted and eliminated; and adherent cells were washed twice and harvested. Adherent macrophages were incubated in ice-cold 0.02% EDTA/PBS for 10 min. The resulting adherent population consisted of N95% peritoneal macrophages. After centrifugation, the cells were resuspended in PBS with BSA. These cells were viable, as indicated by trypan blue exclusion. The concentration of macrophages was determined by a hemocytometer and adjusted to 500,000 cells/ml. 2.3. RNA extraction and analysis of HIF-1α, EPO and VEGF with RT-PCR Total RNA was isolated from peritoneal macrophages or hippocampal tissue, according to the method of Chomczynski and Sacchi (1987). Briefly, cells were homogenized in Trizol chloroform, and total RNA was precipitated overnight from the aqueous phase with isopropanol at 4 °C. The quantity and integrity of the RNA were routinely tested by determining the A260/280 ratio and the RNA was viewed on ethidium bromide-stained 1% formaldehyde agarose gels.

Total protein extracts were prepared by homogenization of the hippocampal tissue obtained from control and MSG-treated animals in a sodium dodecyl sulfate (SDS) buffer (20% glycerol, 4.0% SDS, 125 mM Tris–HCl, 10% mercaptoethanol, pH 6.8). Protein concentrations were determined according to Lowry's method (Lowry et al., 1951), using BSA as the standard. The samples were boiled for 3 min at 100 °C in SDS buffer, and 50 μg of protein was separated in each lane of a 10% polyacrylamide gel by electrophoresis at 200 mA. The proteins were subsequently electroblotted onto nitrocellulose membranes (Hybond TM-C pure, Amersham Pharmacia Biotech), and the membranes were then incubated for 1 h at room temperature with a blocking solution containing 10 mM Tris, pH 7.4, 150 mM NaCl, 0.01% Tween 20, 2% BSA and 2% skimmed milk. The membranes were then incubated overnight at 4 °C with different monoclonal antibodies against HIF-1α (1:1000; Chemicon Int. Inc.). Following incubation with anti-rabbit IgG biotinylated secondary antibody generated in horse (1:2000; Vector Laboratories Vectastain, Burlingame, CA, USA), proteins were made visible by 3, 3′-diamino-benzidine oxidation. Proteins were analyzed by Kodak Digital Science 1D software (ver. 3.0.2., Eastman Kodak Co. Rochester, NY), which permits the total intensity of the entire spot area to be evaluated, and then expressed as arbitrary units. Tissue from five control (n = 5) and five experimental (n = 5) animals was analyzed, and calculations were done in duplicate.

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2.5. Immunohistochemistry for VEGF The following primary antibodies were used for immunohistochemistry: rabbit anti-VEGF (Chemicon 07-1376), an IgG, used at 1:500 dilution; monoclonal mouse anti-GFAP (clone G-A-5; Chemicon MAB3402), an IgG used at 1:500 dilution; monoclonal mouse antiNeuN (Chemicon MAB377) used at 1:500 dilution; Secondary goat antimouse IgG and goat anti-rabbit IgG, conjugated to Alexa 488 (Invitrogen A21121) or Alexa 594 (Invitrogen A11012), at 1:1000 dilution. For immunohistochemistry, animals were anesthetized with an overdose of sodium pentobarbital (Nembutal, Abbott, Chicago, IL) and perfused intracardially with heparinized physiological saline (50 ml), followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3 (PB; 100 ml). The brain was rapidly removed and post-fixed in the same fixative for 8 h, followed by 16 h in a solution of 25% sucrose in PB. Coronal sections were cut in a cryostat and collected on gelatincoated slides (10-m sections). Sections were preincubated for 30 min in a solution of 2% NGS in PBS, pH 7.4, containing 0.1% Triton X-100. After washing once in PBS, the sections were incubated with primary antibody solution containing 1% NGS for 16–24 h at 4 °C. After three washes in PBS, the sections were incubated for 45 min at room temperature with the secondary antibodies, goat anti-mouse IgG or goat anti-rabbit IgG, mounted with glycerol/PBS and photographed on the Olympus BX51 photo-microscope, fitted with a fluorescence attachment and appropriate single, double or triple filters. 2.6. Statistical analysis The results obtained were analyzed by BIOSTAT software; and onetailed analysis of variance (ANOVA) was used to assess the significance of differences between the average values of the groups. The graphs were plotted with Slide writer software.

Fig. 2. Upper panel: microphotograph representative HIF-1α protein expression in the hippocampus at PD14 in control (CTL) rats and rats neonatally exposed to glutamate as monosodium salt (MSG). Lower panel: semiquantitative analysis of HIF-1α protein. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

known to regulate the expression of hundreds of genes, including EPO and VEGF. Thus, using RT-PCR, we followed those genes and found that MSG increased the expression of EPO and VEGF in the hippocampus of rats exposed to excitotoxicity when compared with the control group (Figs. 3 and 4). 3.2. VEGF cell co-location in the hippocampus

Excitotoxicity induced by neonatal administration of glutamate produced an increase in HIF-1α mRNA levels in the hippocampus of rats 6 days after the last dose, corresponding to 14 days of age, when compared to the control group (Fig. 1). In addition, Western blotting showed that corresponding protein levels also rose, with combined over-expression of HIF-1α in response to excitotoxicity by glutamate, when compared with the control group (Fig. 2). HIF-1α is widely

To characterize the cell location of VEGF over-expressed by neurotoxicity phenomena, an immunohistochemical study of hippocampus slices was performed, using NeuN and GFAP antibody to identify neuronal cells and glial cells, respectively. The presence of NeuNpositive neurons and GFAP-positive glial cells in the hippocampus was assessed by analysis with a microscope equipped with an epifluorescence system. The fluorescein molecules coupled to the secondary antibody allowed the identification of neurons and glia via their characteristic morphology. This immunofluorescent label was strongly visible in tissues obtained from control and experimental rats (Figs. 5 and 6). VEGF was expressed mainly in non-neuronal cells (arrows Fig. 5A and C), with no evidence it was present in the neuronal body under

Fig. 1. HIF-1α mRNA expression in the hippocampus of PD14 rats. The upper panel shows RT-PCR results for HIF-1α in control (CTL) rats and rats neonatally exposed to glutamate as monosodium salt (MSG) administered at 1, 3, 5, and 7 days postnatal. Semiquantitative analysis of the mRNA expression is shown in the lower panel. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

Fig. 3. Upper panel: Erythropoietin (EPO) mRNA levels in three different samples from the hippocampus of control (CTL) and monosodium glutamate salt (MSG)-treated rats. Lower panel: semiquantitative analysis of the RT-PCR results. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

3. Results 3.1. HIF-1α expression in the hippocampus

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3.3. HIF-1α expression in peripheral macrophages To examine whether neuroexcitotoxicity produced a systemic response, we examined the mRNA levels of HIF-1α, EPO and VEGF, using RT-PCR in peripheral macrophages from the same rats neonatally treated with MSG. We observed an increase in HIF-1α, EPO and VEGF mRNA levels in peripheral macrophages from rats exposed to excitotoxicity, compared with the control group (Figs. 7, 8 and 9, respectively), suggesting an innate immunological response to excitotoxicity induced by high glutamate administration and apparently originating in the initial brain injury. 4. Discussion

Fig. 4. Upper panel: Vascular Endothelial Growth Factor (VEGF) mRNA levels from the hippocampus of control rats (CTL) and rats exposed to neurotoxicity induced with monosodium glutamate salt (MSG) treatment. Lower panel: semiquantitative analysis of RT-PCR results. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

normal conditions in the hippocampus (Fig. 5B). VEGF was mostly expressed in well-characterized GFAP-positive glial cells around the hippocampal neuronal bodies, as well as in the stratus radians and around the vascular endothelial cells in the control group (star Fig. 5D, E and F). However, the over-expression of VEGF induced by neuroexcitoxicity suggests that VEGF-immunopositivity appears to be present in and around some neuronal bodies (arrows Fig. 6A, B and C). Additionally, VEGF was also expressed in GFAP-positive glial cells (arrows Fig. 6D, E and F), but apparently less expressed in or around vascular endothelial cells (star Fig. 6D, E and F). It could represent an initial protective mechanism, mainly in the neuronal response against an excitotoxic event.

There are several pathological conditions following brain damage and states of several neurodegenerative diseases that are associated with a prolonged inflammatory response, including the secondary phase of the injury causing acute neurotoxicity (Castellani et al., 2008; Sayre et al., 2008). However, little is known about the temporal and spatial relationships between inflammation and brain damage. In this study, HIF-1α, an important transcriptional factor that is overexpressed during ischemia and hypoxia, and two target genes, EPO and VEGF, were measured in the hippocampus and peripheral macrophages of rats neonatally exposed to neuroexcitotoxicity induced by high glutamate concentrations. Results of this study showed an increase in HIF-1α expression in both the hippocampus (100%) and peripheral macrophages (400%), indicating a potentially important involvement of macrophages in the immune response to neuroexcitotoxicity induced by glutamate. Consistent with this observation, several reports have shown that glutamate toxicity acts via two distinct pathways: an excitotoxic pathway in which glutamate receptors are hyperactivated and an oxidative pathway in which cysteine uptake is inhibited, resulting in glutathione depletion,

Fig. 5. Vascular Endothelial Growth Factor (VEGF) immunoreactivity in neonatal Control Group of rat brain sections. Coronal brain sections at the level of hippocampus were double immunostained with rabbit anti-VEGF and monoclonal anti NeuN (A–C) or anti-GFAP (D–F); Secondary antibodies were coupled to alexa 488 (green) or 594 (red). VEGF was mainly expressed in astrocytes double-staining whit anti-VEGF and anti GFAP (arrow). Yellow indicates colocalization of two markers in the same cell. Note that VEGF was expressed around the neuronal bodies (arrowhead). Neurons were identify with NeuN expression. Scale bar 50 μ.

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Fig. 6. Vascular Endothelial Growth Factor (VEGF) immunoreactivity in the hippocampus of neonatal rats exposed to neurotoxicity induced with glutamate as monosodium salt treatment (Experimental group). Coronal brain sections were double immunostained with rabbit anti-VEGF and monoclonal anti NeuN (A–C) or anti-GFAP (D–F); Secondary antibodies were coupled to alexa 488 (green) or 594 (red). VEGF was expressed into and around the neuronal cells (arrowhead). Additionally, VEGF was also expressed in astrocytes double-staining whit anti-VEGF and anti GFAP (arrow). Scale bar 50 μ.

oxidative stress and cell degeneration (Gras et al., 2006). Both events are usually present in the CNS. In vitro and in vivo studies have demonstrated that microglia and brain macrophages activate the expression of transporters and enzymes of the glutamate cycle (Gras et al., 2006; Pacheco et al., 2007; Sayre et al., 2008). Therefore, this peripheral response may be part of a common pathway linking the inflammatory process and neuroexcitotoxicity, because immune

mechanisms may influence progressive CNS damage during primary neurodegeneration (Zipp and Aktas, 2006; Castellani et al., 2008). Recently, macrophage activation was shown to modulate glutamate metabolism as a protective mechanism against glutamate neurotoxicity in the brain (Gras et al., 2006; Porcheray et al., 2006). Thus, in our excitotoxicity model, HIF-1α over-expression may be part of this protective response against brain neuroexcitotoxic damage.

Fig. 7. HIF-1α mRNA levels in peritoneal macrophages from the same control (CTL) animals used to study this gene in the hippocampus, as well as rats neonatally treated with monosodium glutamate salt (MSG). Upper panel: RT-PCR results for HIF-1α. Lower panel: semiquantitative analysis of mRNA expression. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

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Fig. 8. Upper panel: Erythropoietin (EPO) mRNA levels in peritoneal macrophages from control (CTL) rats and rats neonatally exposed to neuroexcitotoxicity induced with monosodium glutamate salt (MSG). Lower panel: semiquantitative analysis of mRNA expression. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

Pro-inflammatory cytokines such as TNF-alpha and interleukine-1 beta are mediators of brain inflammation and are produced by neurons and non-neuronal cells (Chaparro-Huerta et al., 2002, 2005, 2008; Kigerl et al., 2009; Toyooka and Fujimura, 2009). Inflammatory responses also include the formation of reactive oxygen species (ROS) and nitrogen species such as nitric oxide (NO) (Brune and Zhou, 2007; Castellani et al., 2008). Recent results have confirmed that NO, an important mediator produced by activated macrophages (Thomas et al., 2008), stabilizes the HIF-1α protein, thus attenuating its ubiquitination during normoxia (for review, see Dehne and Brune, 2009). In addition, TNF-alpha appears to activate HIF-1α via multiple pathways (De Ponti et al., 2007; Dehne and Brune, 2009). Previous results from our group showed an increase in pro-inflammatory cytokine production, neuronal death and glial and microglial reactivity after neurotoxicity induction by high glutamate concentrations (Chaparro-Huerta et al., 2002, 2005, 2008; Martinez-Contreras et al., 2002). Hence, the increase in HIF-1α expression may also be due to an increase in the production and release of pro-inflammatory cytokines during excitotoxicity induced by high glutamate. Our results also showed an increase in EPO and VEGF levels (100%) in the hippocampus, whereas in peripheral macrophages the increase

Fig. 9. Vascular Endothelial Growth Factor (VEGF) mRNA levels in peritoneal macrophages. Upper panel: VEGF mRNA levels in control (CTL) rats and rats neonatally exposed to monosodium glutamate salt (MSG). Lower panel: semiquantitative analysis of mRNA expression. Results represent the mean ± S.E.M. of five experiments (n = 5 animals) performed in duplicate. Statistically significant at *p b 0.001 compared to CTL.

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was only around 40%. In addition, VEGF cell location was confined to a presence mainly in the non-neuronal cell, as in glia and vascular endothelial cells under normal conditions (Fig. 5); but it was overexpressed in both hippocampal neurons and non-neurons and was apparently less expressed in the vascular endothelial cells under neuro-excitotoxicity conditions (Fig. 6). This result may indicate that VEGF could promote neuronal neuroprotective mechanisms under pathological conditions such as hypoxia, ischemia, epilepsy and in some neurodegenerative diseases like Alzheimer's, Parkinson's etc., in which excitotoxicity is a common mechanism of neuronal death. Recent studies support this idea, in that a neuroprotective and vascular regeneration effect of VEGF used as post-ischemic treatment has been demonstrated (Zhao et al., 2011) and a significant enhancement of dopaminergic differentiation, reducing the loss of dopaminergic neurons in the injured substantia nigra by means of a recombinant protein for the VEGF-based gene therapy model, has also been shown (Tian et al., 2006; Xiong et al., 2011). There is a growing body of evidence indicating that VEGF can act directly on neurons and plays its role as a neuroprotective factor via multiple mechanisms, mainly in neurodegenerative diseases (Drew et al., 2001; Lunn et al., 2009; Rosenstein et al., 2010; Manoonkitiwongsa, 2011). VEGF may modulate neurogenesis in constitutive neurogenic and non-neurogenic regions of adult mammalian brains (Wang and Sun, 2007; Rosenstein et al., 2010; Manoonkitiwongsa, 2011). Another mechanism involved in this neuronal VEGF neuroprotective effect may be via phosphatidylinositol-3 kinase/Akt activation (PI3K/Akt) (Fujiki et al., 2010) and by dual activation of ERK-1/2 and Akt pathways (Kilic et al., 2006). Recently, it was also shown that pre-treatment with VEGF protects motor neurons against excitotoxicity by inducing higher GluR2 levels that block calcium influx into neurons (Bogaert et al., 2010). In addition, VEGF also exerts a neuroprotective effect against status epilepticus-induced cell loss in the rat hippocampus (Vezzani, 2008; Nicoletti et al., 2010) and against chronic glutamate excitotoxicity in the rat spinal cord by activating the PI3-K/Akt signal transduction pathway (Tolosa et al., 2008). However, the role of VEGF in peripheral macrophages is still unknown, although it was recently reported that its upregulation in human macrophages is involved in angiogenesis that stimulates cardiac repair (Ernens et al., 2010). However, these classic target genes (EPO and VEGF) may be more relevant in the CNS than in macrophages, as the erythropoietic and angiogenic pathways may constitute a more important protective response in the brain than in macrophages. It has been widely demonstrated that both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) express EPO following ischemic injury, and this response is known to ameliorate damage to the brain (Weidemann et al., 2009). EPO has also been shown to have a neuroprotective effect that attenuates the inflammatory response by reducing brain edema, blood brain barrier permeability and apoptotic cells in the injured rat brain (Chen et al., 2007). Thus, high EPO levels expressed in peripheral macrophages may constitute part of a protective response to brain inflammation induced by excitotoxicity and consequently neuroprotective adaptations in various neurodegenerative diseases (Drew et al., 2001). Additional experiments will be necessary to explore the role of VEGF in macrophages associated with neuronal injury. In summary, VEGF and EPO over-expression in the CNS during neuroexcitotoxicity induced by high glutamate concentrations may be a consequence of high HIF-1α levels. This, together with these target genes, may indicate an important neuronal inflammatory response and stimulate survival mechanisms mainly in the neuronal cells, which suggests that these genes may be interesting initial markers of the degree of neuronal damage under brain injury conditions and/or in some neurodegenerative diseases. Additional experiments will be needed to establish the specific neuronal mechanism involved in this VEGF and EPO neuroprotection, which could be an interesting

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therapeutic target to combat the neuronal damage seen in neurodegenerative diseases. Finally, these results support the hypothesis that reduced expression of genes in the HIF-1α neuroprotective pathway may contribute to a poor prognosis following neuroexcitotoxicity. Acknowledgments This study was supported by grants from Spain's “Ministerio de Educación y Ciencia” SAF2009-13093, the “Fondo de Investigación Sanitaria”, and the “Instituto de Salud Carlos III” PI080400 and PS09/01789 (FEDER FOUNDS). 610RT0405 from Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED). We would like to thank the “Generalitat de Catalunya” for supporting the research groups (2009/SGR00853) and the “Fundació la Marató TV3” (063230). We wish to thank the Language Assessment Service of the University of Barcelona for revising the manuscript. References Beas Zárate, C., Rivera-Huizar, S.V., Martinez-Contreras, A., Feria-Velasco, A., Armendariz-Borunda, J., 2001. 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