Brain Genomic Response following Hypoxia and Re-oxygenation in the Neonatal Rat
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m204619200
ISSN1083-351X
AutoresMyriam Bernaudin, Yang Tang, Melinda Reilly, Edwige Petit, Frank R. Sharp,
Tópico(s)Cancer-related molecular mechanisms research
ResumoHypoxic preconditioning (8% O2, 3 h) produces tolerance 24 h after hypoxic-ischemic brain injury in neonatal rats. To better understand the ischemic tolerance mechanisms induced by hypoxia, we used oligonucleotide microarrays to examine genomic responses in neonatal rat brain following 3 h of hypoxia (8% O2) and either 0, 6, 18, or 24 h of re-oxygenation. The results showed that hypoxia-inducible factor (HIF)-1- but not HIF-2-mediated gene expression may be involved in brain hypoxia-induced tolerance. Among the genes regulated by hypoxia, 12 genes were confirmed by real time reverse transcriptase-PCR as follows: VEGF,EPO, GLUT-1, adrenomedullin,propyl 4-hydroxylaseα, MT-1,MKP-1, CELF, 12-lipoxygenase,t-PA, CAR-1, and an expressed sequence tag. Some genes, for example GLUT-1, MT-1,CELF, MKP-1, and t-PA did not show any hypoxic regulation in either astrocytes or neurons, suggesting that other cells are responsible for the up-regulation of these genes in the hypoxic brain. These genes were expressed in normal and hypoxic brain, heart, kidney, liver, and lung, with adrenomedullin,MT-1, and VEGF being prominently induced in brain by hypoxia. These results suggest that a number of endogenous molecular mechanisms may explain how hypoxic preconditioning protects against subsequent ischemia, and may provide novel therapeutic targets for treatment of cerebral ischemia. Hypoxic preconditioning (8% O2, 3 h) produces tolerance 24 h after hypoxic-ischemic brain injury in neonatal rats. To better understand the ischemic tolerance mechanisms induced by hypoxia, we used oligonucleotide microarrays to examine genomic responses in neonatal rat brain following 3 h of hypoxia (8% O2) and either 0, 6, 18, or 24 h of re-oxygenation. The results showed that hypoxia-inducible factor (HIF)-1- but not HIF-2-mediated gene expression may be involved in brain hypoxia-induced tolerance. Among the genes regulated by hypoxia, 12 genes were confirmed by real time reverse transcriptase-PCR as follows: VEGF,EPO, GLUT-1, adrenomedullin,propyl 4-hydroxylaseα, MT-1,MKP-1, CELF, 12-lipoxygenase,t-PA, CAR-1, and an expressed sequence tag. Some genes, for example GLUT-1, MT-1,CELF, MKP-1, and t-PA did not show any hypoxic regulation in either astrocytes or neurons, suggesting that other cells are responsible for the up-regulation of these genes in the hypoxic brain. These genes were expressed in normal and hypoxic brain, heart, kidney, liver, and lung, with adrenomedullin,MT-1, and VEGF being prominently induced in brain by hypoxia. These results suggest that a number of endogenous molecular mechanisms may explain how hypoxic preconditioning protects against subsequent ischemia, and may provide novel therapeutic targets for treatment of cerebral ischemia. hypoxia-inducible factor-1 glyceraldehyde-3-phosphate dehydrogenase quantitative one-step reverse transcriptase reverse transcriptase vascular endothelial growth factor erythropoietin expressed sequence tag tissue-type plasminogen activator c-Jun N-terminal kinase mitogen-activated protein metallothionein-1 MAP kinase phosphatase-1 glucose transporter-1 cell adhesion regulator-1 Impaired oxygen (hypoxia) or reduced blood flow (ischemia) to the brain is a major cause of morbidity and mortality in the perinatal period, often resulting in cognitive impairment, seizures, and other neurological disabilities. Although hypoxia-ischemia animal models have increased our understanding of the processes leading to cell death, there are still no pharmacological treatments available to reduce cell death in ischemic neonatal brain. Interestingly, cells can be protected when a non-injurious hypoxic stress is performed several hours or days before a lethal hypoxic-ischemic stress (preconditioning). This phenomenon is called tolerance. Ischemic tolerance can be achieved in brain by several preconditioning sublethal stresses such as hypoxia (1Gidday J.M. Fitzgibbons J.C. Shah A.R. Park T.S. Neurosci. Lett. 1994; 168: 221-224Crossref PubMed Scopus (304) Google Scholar, 2Ota A. Ikeda T. Abe K. Sameshima H. Xia X.Y. Xia Y.X. Ikenoue T. Am. J. Obstet. Gynecol. 1998; 179: 1075-1078Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 3Vannucci R.C. Towfighi J. Vannucci S.J. J. Neurochem. 1998; 71: 1215-1220Crossref PubMed Scopus (128) Google Scholar), ischemia itself (4Kitagawa K. Matsumoto M. 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Sharp F.R. Ann. Neurol. 2000; 48: 285-296Crossref PubMed Scopus (397) Google Scholar, 14Jones N.M. Bergeron M. J. Cereb. Blood Flow Metab. 2001; 21: 1105-1114Crossref PubMed Scopus (204) Google Scholar,16Bernaudin M. Nedelec A.S. Divoux D. MacKenzie E.T. Petit E. Schumann-Bard P. J. Cereb. Blood Flow Metab. 2002; 22: 393-403Crossref PubMed Scopus (329) Google Scholar). Indeed, hypoxic preconditioning induces expression of HIF-1α and its target genes in neonatal (14Jones N.M. Bergeron M. J. Cereb. Blood Flow Metab. 2001; 21: 1105-1114Crossref PubMed Scopus (204) Google Scholar) and adult brain (16Bernaudin M. Nedelec A.S. Divoux D. MacKenzie E.T. Petit E. Schumann-Bard P. J. Cereb. Blood Flow Metab. 2002; 22: 393-403Crossref PubMed Scopus (329) Google Scholar). In addition, desferrioxamine and cobalt chloride, two agents that activate HIF-1 (17Ehleben W. Porwol T. Fandrey J. Kummer W. Acker H. Kidney Int. 1997; 51: 483-491Abstract Full Text PDF PubMed Scopus (79) Google Scholar), also induce tolerance against hypoxia-ischemia in neonatal rat brain (13Bergeron M. Gidday J.M., Yu, A.Y. Semenza G.L. Ferriero D.M. Sharp F.R. Ann. Neurol. 2000; 48: 285-296Crossref PubMed Scopus (397) Google Scholar). HIF-1 is an important transcription factor regulating gene expression in response to hypoxia. Moreover, HIF-1 target genes such aserythropoietin (EPO) and vascular endothelial growth factor (VEGF) protect the brain against ischemia (11Bernaudin M. Marti H.H. Roussel S. Divoux D. Nouvelot A. MacKenzie E.T. Petit E. J. Cereb. Blood Flow Metab. 1999; 19: 643-651Crossref PubMed Scopus (679) Google Scholar, 18Hayashi T. Abe K. Itoyama Y. J. Cereb. Blood Flow Metab. 1998; 18: 887-895Crossref PubMed Scopus (302) Google Scholar, 19Sadamoto Y. Igase K. Sakanaka M. Sato K. Otsuka H. Sakaki S. Masuda S. Sasaki R. Biochem. Biophys. Res. Commun. 1998; 253: 26-32Crossref PubMed Scopus (292) Google Scholar, 20Sakanaka M. Wen T.C. Matsuda S. Masuda S. Morishita E. Nagao M. Sasaki R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4635-4640Crossref PubMed Scopus (902) Google Scholar). This suggests that HIF-1 might be involved in the establishment of ischemic tolerance in brain. However, it is possible that there are other adaptive mechanisms underlying this protection. Understanding changes in gene expression in brain following exposure to hypoxia could reveal new mechanisms of ischemic tolerance. Accordingly, we exploited DNA microarray technology to investigate the brain genomic response of neonatal rat to hypoxia (3 h, 8% O2). Previous studies have analyzed the gene expression after hypoxia in fish (21Gracey A.Y. Troll J.V. Somero G.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1993-1998Crossref PubMed Scopus (494) Google Scholar), PC12 pheochromocytoma cells (22Beitner-Johnson, D., Seta, K., Yuan, Y., Kim, H., Rust, R. T., Conrad, P. W., Kobayashi, S., and Millhorn, D. E. (2001)7, 273–281.Google Scholar, 23Seta K.A. Kim R. Kim H.W. Millhorn D.E. Beitner-Johnson D. J. Biol. Chem. 2001; 276: 44405-44412Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), glioblastoma cells (24Lal A. Peters H., St Croix B. Haroon Z.A. Dewhirst M.W. Strausberg R.L. Kaanders J.H. van der Kogel A.J. Riggins G.J. J. Natl. Cancer Inst. 2001; 93: 1337-1343Crossref PubMed Scopus (268) Google Scholar), and Hep3B hepatocarcinoma cells (25Scandurro A.B. Weldon C.W. Figueroa Y.G. Alam J. Beckman B.S. Int. J. Oncol. 2001; 19: 129-135PubMed Google Scholar). Therefore, this is the first in vivo study of the genomic response to hypoxia in neonatal rat brain and in vertebrates in general. All animal experiments were performed in strict accordance with National Institutes of Health guidelines, and animal protocols were approved by the University of Cincinnati Animal Research Committee. Sprague-Dawley rats (Harlan, Indianapolis, IN) were acclimated to the animal quarters at least 3 days prior to study. The animals, maintained on a 12-h light/dark cycle, were given food and water ad libitum. In addition, all experimental and control animals were housed in the same room prior to and at the conclusion of the study. Neocortical cultures of neurons from rat embryos at 14–15 days of embryonic development (Sprague-Dawley rats, CERJ, Le Genest Saint-Isle, France) were prepared as described (26Bernaudin M. Bellail A. Marti H.H. Yvon A. Vivien D. Duchatelle I. Mackenzie E.T. Petit E. Glia. 2000; 30: 271-278Crossref PubMed Scopus (261) Google Scholar). Cultures were allowed to grow in a serum-free, chemically defined medium: Dulbecco's modified Eagle's medium plus N-2 (Invitrogen) for 3 days in a humidified 5% CO2 incubator at 37 °C before use. Neocortical cultures of astrocytes were prepared from neonatal (1–3-day-old) rats (Sprague-Dawley CERJ) as described (26Bernaudin M. Bellail A. Marti H.H. Yvon A. Vivien D. Duchatelle I. Mackenzie E.T. Petit E. Glia. 2000; 30: 271-278Crossref PubMed Scopus (261) Google Scholar). Cultures were allowed to grow in a humidified 5% CO2 incubator at 37 °C to confluency (15–20 days) before use in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen). Hypoxia was induced by placing neuron and astrocyte cultures in a modular incubator chamber (Anaerobic Chamber, Plas Labs, Lansing, MI) gassed with a mixture consisting of 4.5% CO2 and 95.5% N2. The growth medium was replaced by the same medium (without serum for astrocytes) that had been equilibrated with a gas mixture consisting of 4.5% CO2 and 95.5% N2for 30 min before bathing the cells. The hypoxia was maintained for 3 h, i.e. the duration known to induce tolerancein vivo (1Gidday J.M. Fitzgibbons J.C. Shah A.R. Park T.S. Neurosci. Lett. 1994; 168: 221-224Crossref PubMed Scopus (304) Google Scholar, 2Ota A. Ikeda T. Abe K. Sameshima H. Xia X.Y. Xia Y.X. Ikenoue T. Am. J. Obstet. Gynecol. 1998; 179: 1075-1078Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 3Vannucci R.C. Towfighi J. Vannucci S.J. J. Neurochem. 1998; 71: 1215-1220Crossref PubMed Scopus (128) Google Scholar, 12Gidday J.M. Shah A.R. Maceren R.G. Wang Q. Pelligrino D.A. Holtzman D.M. Park T.S. J. Cereb. Blood Flow Metab. 1999; 19: 331-340Crossref PubMed Scopus (207) Google Scholar). Immediately after hypoxia, total RNA was prepared from cultured cells as described below. Male and female rats at postnatal day 6 (P6) (Sprague-Dawley Harlan) were used. The hypoxia chamber (Reming Bioinstruments, Redfield, NY) consists of four identical Plexiglas chambers, each 16 liters in size, that are arranged adjacent to one another. The inlet O2, CO2, and N2 are fixed using gas tanks of appropriate concentrations and valves to monitor inlet gas concentrations. The exiting gas is continuously monitored for O2, CO2, and N2 as well. These data are monitored and stored for each experiment. Rat pups were placed in cages inside these large Plexiglas chambers through which 8% O2 and 92% N2 were circulated for a period of 3 h. This degree and duration of hypoxia is not known to produce any injury in brain and has been shown to protect the newborn brain from subsequent hypoxic-ischemic injury (1Gidday J.M. Fitzgibbons J.C. Shah A.R. Park T.S. Neurosci. Lett. 1994; 168: 221-224Crossref PubMed Scopus (304) Google Scholar, 2Ota A. Ikeda T. Abe K. Sameshima H. Xia X.Y. Xia Y.X. Ikenoue T. Am. J. Obstet. Gynecol. 1998; 179: 1075-1078Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 3Vannucci R.C. Towfighi J. Vannucci S.J. J. Neurochem. 1998; 71: 1215-1220Crossref PubMed Scopus (128) Google Scholar, 12Gidday J.M. Shah A.R. Maceren R.G. Wang Q. Pelligrino D.A. Holtzman D.M. Park T.S. J. Cereb. Blood Flow Metab. 1999; 19: 331-340Crossref PubMed Scopus (207) Google Scholar). Control animals from the same littermates were placed in one of the four chambers and exposed to ambient oxygen (approximately 21% O2) for the same duration. After 3 h of hypoxia or normoxia, all animals were returned to their dams for 0, 6, 18, or 24 h (re-oxygenation period). After the different re-oxygenation periods, rat pups (at P6 or P7) were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), and their brains were removed for RNA or protein isolation (Fig. 1). In addition, total RNA was also prepared from different organs (heart, kidney, liver, and lung) after 3 h of hypoxia and from controls. After 3 h of hypoxia or normoxia followed by different periods of re-oxygenation (0, 6, 18, or 24 h), rat pups were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), and their brain (whole brain hemisphere), heart, kidney, liver, and lung were removed as rapidly as possible for RNA isolation. Total RNA was isolated with TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. Briefly, the tissue pellets were homogenized in TRIzol Reagent using a syringe. After extraction with chloroform (an additional step of phenol/chloroform extraction is added to the manufacturer's protocol), RNA was precipitated by isopropyl alcohol and subjected to further purification using an RNeasy mini kit (Qiagen, Valencia, CA). The quality and quantity of extracted total RNA samples were examined by loading 5 μg of each sample on a denaturing agarose gel. Microarray expression analysis was performed according to the Affymetrix expression analysis technical manual (Affymetrix GeneChip® Expression Analysis Manual, P/N 700217–700222, Affymetrix, Santa Clara, CA). Double-stranded cDNA is synthesized from total RNA. To synthesize the first cDNA strand, a high pressure liquid chromatography-purified oligo(dT) primer was annealed to the RNA, and extension by reverse transcriptase was performed in the presence of deoxyoligonucleotides. The second strand was synthesized using DNA polymerase I. Double-stranded cDNA was purified using a modified phenol/chloroform extraction procedure (Eppendorf, 5 Prime → 3 Prime, Inc., Boulder, CO), followed by ethanol precipitation. An in vitro transcription was performed to produce biotin-labeled cRNA from the cDNA. cRNA was synthesized using T7 RNA polymerase and biotin-labeled ribonucleotides and purified with affinity columns (Qiagen) followed by ethanol precipitation. No amplification procedure was performed to produce the final cRNA. The amount of product was quantified by spectrophotometric analysis, and the quality of the cRNA was assessed by gel electrophoresis and a test array (Test-2 chip). Once prepared, cRNA was hybridized to Affymetrix U34A rat arrays (Affymetrix), which contains more than 7000 genes and 1000 expressed sequence tags (ESTs are referred to as genes in the following text). The hybridization mixture, containing the cRNA sample, bovine serum albumin, and herring sperm DNA, was incubated at 99°C, transferred to 45 °C, and then injected into the microarray chamber (Affymetrix). Hybridization was performed automatically in a chamber at 45 °C for 16 h while being rotated at 60 rpm. The hybridized probe array was then automatically washed, dried, and scanned two times at an excitation wavelength of 488 nm. For microarray analysis, we used total RNA prepared from 2 animals for each group (6 groups in total) as follows: 4 “hypoxia 3 h groups” with 0, 6, 18, or 24 h of re-oxygenation and 2 “controls groups” (6-day-old rat pups that correspond to the sham-control of the hypoxia 3 h with 0 h of re-oxygenation, and 7-day-old rat pups that correspond to the sham-control of the hypoxia 3 h with 24 h of re-oxygenation) (Fig. 1). Two animals were used for each experimental group; thus 12 arrays were used in total. The Affymetrix rat U34A array contains more than 7000 genes and 1000 ESTs. The Affymetrix Genechip software MAS 4.0 (Affymetrix) was used first to collect and process the original expression data from the 12-rat Affymetrix arrays. Each gene on the array is assessed using 16 probe pairs. Each probe pair consists of an oligomer (25 bases long) that is designed to be perfectly complementary to a particular message (called the perfect match or PM) and a companion oligomer that is identical to the PM probe except for a single base difference in a central position (called the mismatch or MM probe). The mismatch probe serves as a control for hybridization specificity and helps subtract nonspecific hybridization. After hybridization intensity data are captured, the Affymetrix Genechip software automatically calculates intensity values for each probe cell and uses these probe cell intensities to calculate an average intensity for each gene (called average difference), which directly correlates with mRNA abundance. The software also gives each gene a qualitative assessment of “present” or “absent” based on a “voting scheme,” with the number of instances in which the PM signal is significantly larger than the MM signal across the whole probe set. Prior to comparing any two measurements, a scaling procedure is performed so that all signal intensities on an array are multiplied by a factor that makes the mean PM-MM value for each array equal to a preset value of 1500. The scaling corrects for any inter-array differences or small differences in sample concentration, labeling efficiency, or fluorescence detection and makes inter-array more reproducible. In the case of a pairwise comparison of two array results, the patterns of change of the whole probe set (with consistent voting) is used to make a qualitative call (called difference call) of “Increase,” “Decrease,” “Marginally increase,” “Marginally decrease,” or “No change.” The fold change is derived by the ratio of average differences from one experimental array compared with a control array. In order to eliminate possible expression changes related to the development (difference of RNA expression between 6- and 7-day-old rat pups), all groups were compared with both controls at 6 and 7 days old. To obtain differentially expressed genes for each condition, Affymetrix Genechip software was used to compare 2 experimental arrays to the 4 control arrays (P6 and P7). For example, 6 h (array 1) of re-oxygenation was compared with controls P6 (arrays 1 and 2) and controls P7 (arrays 1 and 2), respectively, and then 6 h (array 2) was compared with controls P6 (arrays 1 and 2) and controls P7 (arrays 1 and 2). As a result, there were 8 comparisons for each condition. Absolute calls (present, marginal, and absent) and the average difference (RNA abundance) for each gene on each chip were then imported into Genespring software (Silicon Genetics, Redwood City, CA) for further analysis. By combining the fold change and the present calls derived from the 8 comparisons, we obtained a list for each condition. Three criteria were used for the lists as follows. 1) The fold change for each of the 8 comparisons was at least 1.5-fold. 2) There were present calls in all the hypoxic groups for increasing gene expression and present calls in both controls for decreasing gene expression. 3) Raw data are greater than 100 in hypoxic samples for up-regulated genes and in control samples for down-regulated genes. To avoid developmental gene expression changes, genes that were increased or decreased more than 1.5-fold between 6- and 7-day-old controls were eliminated from the list of hypoxic changes. To obtain a general view of brain genomic response to hypoxia, genes regulated at different time points following hypoxia were selected by the Significance Analysis of Microarrays (SAM) software (27Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9802) Google Scholar). Among those genes, 285 genes with relatively high expression in most samples (raw data at of least 100 in 7 of 12 samples) were subjected to hierarchical cluster analysis with Genespring software. A standard correlation coefficient of 0.95 was used as the measure for significant statistical similarity. The branching behavior of the tree was controlled using a separation ration setting of 0.5 and a minimum distance setting of 0.001. To validate the microarray data, TaqMan quantitative one-step reverse transcriptase-polymerase chain reaction (Q-RT-PCR) was used to quantitate mRNA levels for selected genes. Two primers and one probe (TaqMan probe) (Applied Biosystems, Foster City, CA) were designed for each gene using PerkinElmer Life Sciences PrimerExpress software (Applied Biosystems). Q-RT-PCR was performed for whole brain hemisphere RNA from at least 3 animals for each of the groups (including the same RNA samples from 2 animals employed for microarrays analysis) and from heart, kidney, liver, and lung RNA from 3 animals submitted or not to 3 h of hypoxia with or without re-oxygenation. Primer and probe sequences are listed for forward primers “F,” reverse primers “R,” and TaqMan probes “T” in Table I. TaqMan probes were labeled with VIC on the 5′-nucleotide and TAMRA (6-carboxytetramethylrhodamine) on the 3′-nucleotide. Assays were run in triplicate on the PerkinElmer Life Sciences ABI 5700 instrument under default conditions (RT, 48 °C for 30 min; AmpliTaqGold activation, 95 °C for 10 min; PCR, 40 cycles of 95 °C, 15 s and 60 °C, 1 min). The Q-RT-PCR protocol was done according to the manufacturer's protocol using the TaqMan Gold RT-PCR kit (Applied Biosystems) with 50 ng of RNA for each sample. The abundance of each gene was determined relative to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) standard transcript using TaqMan GAPDH RNA control reagents kits (Applied Biosystems). The abundance of each gene was also determined relative to the standard transcript 18 S using TaqMan ribosomal control reagents kits (Applied Biosystems). The data obtained with 18S are not shown because the results were identical to those obtained with GAPDH (data not shown). To verify the presence and the predicted size of amplified fragments, PCR products were separated by electrophoresis and visualized in 3% agarose gels with ethidium bromide.Table IList of primers used for real time RT-PCRGene nameACaAC, GenBank™accession number.PrimersPosition/sizebpAdrenomedullinD15069F, 5′-CAGGACAAGCAGAGCACGTC-3′36182T, 5′-CAAGCCAGCACTCAGAGCACAGCC-3′391R, 5′-TCTGGCGGTAGCGTTTGAC-3′442CAR-1U76714F, 5′-ACCCGCTTCCATAAGGCTTTA-3′52142T, 5′-CAGCTTTGCTGTTCTTTGCCTTAGTTGTCC-3′139R, 5′-CAGCCTTGTGCCGAAAGAC-3′193Casein kinase-1αU77582F, 5′-CGGAAGATCGGATCTGGTTC-3′6199T, 5′-TCTAGCGATCAACATCACCAATGGCG-3′96R, 5′-CCTGGCCTTCTGGGATTCTAG-3′159CELFM65149F, 5′-CACGACCCCTGCCATGTATG-3′142118T, 5′-CTTCAGCGCCTACATTGATTCCATGGC-3′181R, 5′-GAAGAGGTCGGCGAAGATCTC-3′259EPOD10763F, 5′-GAATTGATGTCGCCTCCAGA-3′44991T, 5′-CCAAGCCGCTCCACTCCGAACA-3′475R, 5′-TGGAGTAGACCCGGAAGAGCT-3′539EST 188825AA799328F, 5′-ATTGTCCCAGGTTGTGTCCAA-3′481102T, 5′-TCCTCCTAGCACTCTCCATTGTGAATCGTATC-3′504R, 5′-GGACTTGAGTTGTGGAGAGCAA-3′582GLUT-1S68135F, 5′-GGTGTGCAGCAGCCTGTGTA-3′111278T, 5′-CCATCGGCTCGGGTATCGTCAACAC-3′1137R, 5′-GACGAACAGCGACACCACAGT-3′118912-LipoxygenaseL06040F, 5′-GATGGGTGTCTACCGCATCC-3′1998T, 5′-CTCCAAGTACGCGGGCTCCAACAAC-3′55R, 5′-CCTCTCCATGCTGTCCAACC-3′116Metallothionein-1M11794F, 5′-CAAATGCACCTCCTGCAAGA-3′142120T, 5′-CTGCTCCAAATGTGCCCAGGGCT-3′190R, 5′-TCACTTCAGGCACAGCACGT-3′261MKP-1S81478F, 5′-GCAGTGCTTACCATGCTTCC-3′66578T, 5′-ATATGCTCGACGCCTTGGGTATCACTGC-3′692R, 5′-AATTGGCCGAGACGTTGATC-3′742Prolyl 4-hydroxylase αX78949F, 5′-CTGTTCTGCCGCTACCATGA-3′991133T, 5′-CAAGCCTCGCATCATTCGTTTCCATG-3′1068R, 5′-CGATCTCAATCTCGGCATCTG-3′1123t-PAM23697F, 5′-CGTTGCCTGACCAGGGAATA-3′81120T, 5′-CTACAGAGCGACCTGCAGAGATGAACAGACTC-3′130R, 5′-TGGGACGTAGCCATGACTGA-3′200VEGFM32167F, 5′-GCAATGATGAAGCCCTGGAG-3′26178T, 5′-CACGTCGGAGAGCAACGTCACTATGC-3′289R, 5′-GGTGAGGTTTGATCCGCATG-3′338a AC, GenBank™accession number. Open table in a new tab Total and nuclear protein extracts were prepared from whole brain hemisphere tissue lysates after 3 h of hypoxia or 3 h of hypoxia followed by 24 h of re-oxygenation and controls (Fig. 1) as described previously (16Bernaudin M. Nedelec A.S. Divoux D. MacKenzie E.T. Petit E. Schumann-Bard P. J. Cereb. Blood Flow Metab. 2002; 22: 393-403Crossref PubMed Scopus (329) Google Scholar). Proteins (150 μg for total extracts and 75 μg for nuclear extracts) were heated at 100 °C for 3 min in the 2× loading buffer (EC-886, National Diagnostics, Atlanta, GA) and separated on 8% SDS-PAGE gels for 1 h at 150 V. The electrophoresis running buffer contained 25 mm Tris base, 192 mm glycine, and 0.1% SDS. Proteins were transferred onto a nitrocellulose membrane (Optitran BA-S 85, Schleicher & Schuell) in blotting buffer (25 mm Tris base, 192 mmglycine, and 20% methanol (v/v)) for 2 h at 300 mA. Membranes were stained with Ponceau S to verify equal protein loading and transfer. Membranes are blocked with 5% nonfat milk (Bio-Rad) in TBST (10 mm Tris-HCl, pH 8.0, 200 mm NaCl, 0.05% Tween 20) for 2 h at room temperature and then incubated overnight at 4 °C with one of the following antibodies diluted in the blocking solution: mouse monoclonal antibodies for HIF-1α (2 μg/ml, OSA-601; StressGen Biotechnologies, Victoria, British Columbia, Canada), HIF-1β (1 μg/ml, NB 100–124; Novas Biologicals, Littleton, CO), and rabbit polyclonal antibody for HIF-2α (2.2 μg/ml, NB 100–122; Novas Biologicals Inc.). After washing with TBST (4 times, 15 min each), the blots were incubated 1 h at room temperature with peroxidase-coupled goat anti-rabbit IgG (1:15,000; Santa Cruz Biotechnologies, Santa Cruz, CA) or peroxidase-coupled goat anti-mouse IgG (1:5,000; Santa Cruz Biotechnologies) diluted in blocking solution and then washed again in TBST. The blots were developed using the chemiluminescence reagents (RPN 2106, Amersham Biosciences). The same blot was stripped (30 min at 50 °C in 62.5 mm Tris base, 2% SDS, 100 mm β-mercaptoethanol) and re-stained with a monoclonal β-actin antibody (5441, Sigma) as an internal control. HIF-1 is a heterodimer made of two protein subunits, HIF-1α and HIF-1β (28Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5105) Google Scholar). Whereas HIF-1β is constitutively expressed, HIF-1α expression is tightly regulated by cellular oxygen concentration (29Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Thus HIF-1α determines HIF-1 DNA binding activity and transcriptional activity during hypoxia. As 3 h of hypoxia 8% O2 is known to induce expression of HIF-1α and its target genes in neonatal brain (13Bergeron M. Gidday J.M., Yu, A.Y. Semenza G.L. Ferriero D.M. Sharp F.R. Ann. Neurol. 2000; 48: 285-296Crossref PubMed Scopus (397) Go
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