Strain Differences in Behavioral and Cellular Responses to Perinatal Hypoxia and Relationships to Neural Stem Cell Survival and Self-Renewal
2009; Elsevier BV; Volume: 175; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.090354
ISSN1525-2191
AutoresQi Li, Jaimei Liu, Michael Michaud, Michael L. Schwartz, Joseph A. Madri,
Tópico(s)Anesthesia and Neurotoxicity Research
ResumoPremature infants have chronic hypoxia, resulting in cognitive and motor neurodevelopmental handicaps caused by suboptimal neural stem cell (NSC) repair/recovery in neurogenic zones (including the subventricular and the subgranular zones). Understanding the variable central nervous system repair response is crucial to identifying "at risk" infants and to increasing survival and clinical improvement of affected infants. Using mouse strains found to span the range of responsiveness to chronic hypoxia, we correlated differential NSC survival and self-renewal with differences in behavior. We found that C57BL/6 (C57) pups displayed increased hyperactivity after hypoxic insult; CD-1 NSCs exhibited increased hypoxia-induced factor 1α (HIF-1α) mRNA and protein, increased HIF-1α, and decreased prolyl hydroxylase domain 2 in nuclear fractions, which denotes increased transcription/translation and decreased degradation of HIF-1α. C57 NSCs exhibited blunted stromal-derived factor 1-induced migratory responsiveness, decreased matrix metalloproteinase-9 activity, and increased neuronal differentiation. Adult C57 mice exposed to hypoxia from P3 to P11 exhibited learning impairment and increased anxiety. These findings support the concept that behavioral differences between C57 and CD-1 mice are a consequence of differential responsiveness to hypoxic insult, leading to differences in HIF-1α signaling and resulting in lower NSC proliferative/migratory and higher apoptosis rates in C57 mice. Information gained from these studies will aid in design and effective use of preventive therapies in the very low birth weight infant population. Premature infants have chronic hypoxia, resulting in cognitive and motor neurodevelopmental handicaps caused by suboptimal neural stem cell (NSC) repair/recovery in neurogenic zones (including the subventricular and the subgranular zones). Understanding the variable central nervous system repair response is crucial to identifying "at risk" infants and to increasing survival and clinical improvement of affected infants. Using mouse strains found to span the range of responsiveness to chronic hypoxia, we correlated differential NSC survival and self-renewal with differences in behavior. We found that C57BL/6 (C57) pups displayed increased hyperactivity after hypoxic insult; CD-1 NSCs exhibited increased hypoxia-induced factor 1α (HIF-1α) mRNA and protein, increased HIF-1α, and decreased prolyl hydroxylase domain 2 in nuclear fractions, which denotes increased transcription/translation and decreased degradation of HIF-1α. C57 NSCs exhibited blunted stromal-derived factor 1-induced migratory responsiveness, decreased matrix metalloproteinase-9 activity, and increased neuronal differentiation. Adult C57 mice exposed to hypoxia from P3 to P11 exhibited learning impairment and increased anxiety. These findings support the concept that behavioral differences between C57 and CD-1 mice are a consequence of differential responsiveness to hypoxic insult, leading to differences in HIF-1α signaling and resulting in lower NSC proliferative/migratory and higher apoptosis rates in C57 mice. Information gained from these studies will aid in design and effective use of preventive therapies in the very low birth weight infant population. Preterm birth is known to result in cognitive and motor disabilities and recent evidence suggests that there can be significant recovery over time in some patients.1Wilson-Costello D Friedman H Minich N Fanaroff AA Hack M Improved survival rates with increased neurodevelopmental disability for extremely low birth weight infants in the 1990s.Pediatrics. 2005; 115: 997-1003Crossref PubMed Scopus (562) Google Scholar, 2Tyson JE Saigal S Outcomes for extremely low-birth-weight infants: disappointing news.JAMA. 2005; 294: 371-373Crossref PubMed Scopus (35) Google Scholar, 3Saigal S Stoskopf B Streiner D Boyle M Pinelli J Paneth N Goddeeris J Transition of extremely low-birth-weight infants from adolescence to young adulthood: comparison with normal birth-weight controls.JAMA. 2006; 295: 667-675Crossref PubMed Scopus (197) Google Scholar, 4Hack M Flannery DJ Schluchter M Cartar L Borawski E Klein N Outcomes in young adulthood for very-low-birth-weight infants.N Engl J Med. 2002; 346: 149-157Crossref PubMed Scopus (947) Google Scholar, 5Saigal S Doyle LW An overview of mortality and sequelae of preterm birth from infancy to adulthood.Lancet. 2008; 371: 261-269Abstract Full Text Full Text PDF PubMed Scopus (1975) Google Scholar, 6Schafer RJ Lacadie C Vohr B Kesler SR Katz KH Schneider KC Pugh KR Makuch RW Reiss AL Constable RT Ment LR Alterations in functional connectivity for language in prematurely born adolescents.Brain. 2009; 132: 661-670Crossref PubMed Scopus (118) Google Scholar, 7Ment LR Constable RT Injury and recovery in the developing brain: evidence from functional MRI studies of prematurely-born children.Nat Clin Prac Neurol. 2007; 3: 558-571Crossref PubMed Scopus (33) Google Scholar More than 1% of all live infants in the United States weigh less than 1000 g, and the survival rate for this population ranges from 60 to 85%.8Bassan H Feldman HA Limperopoulos C Benson CB Ringer SA Veracruz E Soul JS Volpe JJ du Plessis AJ Periventricular hemorrhagic infarction: risk factors and neonatal outcome.Pediatr Neurol. 2006; 35: 85-92Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar The individuals that do survive exhibit a high rate of neonatal morbidities and are frequently severely compromised.9Paul DA Leef KH Locke RG Bartoshesky L Walrath J Stefano JL Increasing illness severity in very low birth weight infants over a 9-year period.BMC Pediatr. 2006; 6: 2Crossref PubMed Scopus (22) Google Scholar, 10Cooke RW Preterm mortality and morbidity over 25 years.Arch Dis Child Fetal Neonatal Ed. 2006; 91: F293-F294Crossref PubMed Scopus (31) Google Scholar Many of these very low birth weight infants experience cerebral hypoxemia resulting from apnea and respiratory distress syndrome. Behavioral studies of this cohort have documented that approximately one quarter are functioning in the mentally retarded or borderline ranges at school age, 10% have cerebral palsy, and one half of these neonates need special assistance in school. The effects of hypoxia in the perinatal period include altered neuronal differentiation and synaptogenesis. The loss of neurons, glia, and their progenitor cells are thought to be the consequences of altered neuronal differentiation and synaptogenesis.11Curristin SM Cao A Stewart WB Zhang H Madri JA Morrow JS Ment LR Disrupted synaptic development in the hypoxic newborn brain.Proc Natl Acad Sci USA. 2002; 99: 15729-15734Crossref PubMed Scopus (96) Google Scholar Interestingly, significant improvement in academic functioning over time in this population has been reported.1Wilson-Costello D Friedman H Minich N Fanaroff AA Hack M Improved survival rates with increased neurodevelopmental disability for extremely low birth weight infants in the 1990s.Pediatrics. 2005; 115: 997-1003Crossref PubMed Scopus (562) Google Scholar Although encouraging, this cognitive improvement is variable, and the repair/recovery mechanisms involved are not yet understood. The variable recovery observed in the very low birth weight infant population may be a result of the responsiveness of neurogenic zones (neurovascular niches) in the brain, namely the subventricular zone and the subgranular zone, due to a range of responsiveness to the chronic hypoxic insult of hypoxia- induced factor 1α (HIF-1α) induction and its downstream signaling cascades. Consistent with this notion, investigators, using a murine model mimicking the chronic hypoxia associated with premature birth, have demonstrated twice as many 5-bromo-2′-deoxyuridine-labeled cells expressing neuronal markers in the neocortex in mice recovering from hypoxia compared with normoxic-reared controls.12Fagel DM Ganat Y Silbereis J Ebbitt T Stewart W Zhang H Ment LR Vaccarino FM Cortical neurogenesis enhanced by chronic perinatal hypoxia.Exp Neurol. 2006; 199: 77-91Crossref PubMed Scopus (139) Google Scholar In addition, in both hypoxic-reared infant/juvenile mice, neuroblasts were noted detaching from the forebrain subventricular zone, migrating through the subcortical white matter, and entering the lower cortical layers several days after their last mitotic division.12Fagel DM Ganat Y Silbereis J Ebbitt T Stewart W Zhang H Ment LR Vaccarino FM Cortical neurogenesis enhanced by chronic perinatal hypoxia.Exp Neurol. 2006; 199: 77-91Crossref PubMed Scopus (139) Google Scholar These data suggest that neurogenesis probably plays a role in neuronal recovery after neonatal hypoxic injury. Observations made on adult mice demonstrating cortical, striatal, and hippocampal neurogenesis after a variety of injuries and responses to several treatment modalities are also consistent with this concept.13Warner-Schmidt JL Duman RS Hippocampal neurogenesis: opposing effects of stress and antidepressant treatment.Hippocampus. 2006; 16: 239-249Crossref PubMed Scopus (653) Google Scholar Thus, whereas neurogenesis after a hypoxic insult in the very low birth weight newborn may explain the cognitive improvement noted over time,12Fagel DM Ganat Y Silbereis J Ebbitt T Stewart W Zhang H Ment LR Vaccarino FM Cortical neurogenesis enhanced by chronic perinatal hypoxia.Exp Neurol. 2006; 199: 77-91Crossref PubMed Scopus (139) Google Scholar the variability of the improvement requires a better understanding of the mechanisms involved in modulating neurogenesis occurring in the subventricular zone (SVZ) before the development of treatment modalities geared to providing a greater and more complete recovery. Our studies described here reveal that CD-1 NSC self-renewal ability is appreciably greater than that observed in C57BL/6 (C57) NSCs and that this may explain the differences in behavior observed in these two strains after exposure to hypoxia. Our previous findings documented significant differences in HIF-1α, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), stromal-derived factor 1 (SDF-1), neuropilin-1, and SDF-1 receptor CXCR4 protein expression in brain tissues and NSC lysates of these two strains. The current studies showed that CD-1 NSCs exhibit increased HIF-1α mRNA and protein levels and increased HIF-1α and decreased prolyl hydroxylase domain 2 (PHD2) proteins in their nuclear fractions compared with C57 NSCs. These data are consistent with increased levels of transcription and translation and decreased degradation of HIF-1α in the CD-1 NSCs. We also found that, compared with CD-1 NSCs, C57 NSCs exhibit blunted migratory responsiveness to SDF-1, decreased induction of matrix metalloproteinase (MMP)-9 and increased neuronal differentiation after migratory stimuli. Last, we demonstrated significant differences in SVZ vascular density, NSC proliferation, and selected behaviors in adult C57 mice compared with those in CD-1 adult mice after 8 days of hypoxic insult (10% O2) at postnatal day (P) 3 to P11. This is consistent with our hypothesis that the behavioral data exhibited by the C57 pups is a consequence of their lower NSC proliferative and migratory rates and higher susceptibility to undergo apoptosis in response to a reduction in O2% because of their relatively blunted HIF-1α response compared with that of CD-1 mice. The information gained from these and future studies will aid in the rational design and effective use of novel preventive therapies (directed at specific receptors and signaling pathway components) in the very low weight infant population. All animal studies were performed with approval and in full compliance with the Yale University Animal Care committee (protocol 2008-07366). CD-1 and C57BL/6 (C57) male and female breeders were obtained from Charles River Laboratories (Wilmington, MA) or laboratory stock derived from Charles River Laboratories breeders.14Li Q Michaud M Stewart W Schwartz M Madri JA Modeling the neurovascular niche: murine strain differences mimic the range of responses to chronic hypoxia in the premature newborn.J Neurosci Res. 2008; 86: 1227-1242Crossref PubMed Scopus (24) Google Scholar At P3 (approximates a 23-week gestational age in humans),15Barrett RD Bennet L Davidson J Dean JM George S Emerald BS Gunn AJ Destruction and reconstruction: hypoxia and the developing brain.Birth Defects Res C Embryo Today. 2007; 81: 163-176Crossref PubMed Scopus (71) Google Scholar, 16Brazel CY Rosti RT Boyce S Rothstein RP Levison SW Perinatal hypoxia/ischemia damages and depletes progenitors from the mouse subventricular zone.Dev Neurosci. 2004; 26: 266-274Crossref PubMed Scopus (53) Google Scholar cohorts of mothers and pups were subjected to hypoxic (10% O2) treatment; control mice remained under normoxic conditions as described.12Fagel DM Ganat Y Silbereis J Ebbitt T Stewart W Zhang H Ment LR Vaccarino FM Cortical neurogenesis enhanced by chronic perinatal hypoxia.Exp Neurol. 2006; 199: 77-91Crossref PubMed Scopus (139) Google Scholar Pups used for tissue analyses of SVZ NSC survival proliferation14Li Q Michaud M Stewart W Schwartz M Madri JA Modeling the neurovascular niche: murine strain differences mimic the range of responses to chronic hypoxia in the premature newborn.J Neurosci Res. 2008; 86: 1227-1242Crossref PubMed Scopus (24) Google Scholar were sacrificed at P11 (approximates a full-term gestation in humans).15Barrett RD Bennet L Davidson J Dean JM George S Emerald BS Gunn AJ Destruction and reconstruction: hypoxia and the developing brain.Birth Defects Res C Embryo Today. 2007; 81: 163-176Crossref PubMed Scopus (71) Google Scholar, 16Brazel CY Rosti RT Boyce S Rothstein RP Levison SW Perinatal hypoxia/ischemia damages and depletes progenitors from the mouse subventricular zone.Dev Neurosci. 2004; 26: 266-274Crossref PubMed Scopus (53) Google Scholar Mice to be used for behavioral analysis were exposed in the same manner between P3 and P11 and were then kept under normoxic conditions from P12 until the completion of testing. All behavioral testing was performed exclusively on males. We recognize that gender effects on behavior in rodents have been documented17Jonasson Z Meta-analysis of sex differences in rodent models of learning and memory: a review of behavioral and biological data.Neurosci Biobehav Rev. 2005; 28: 811-825Crossref PubMed Scopus (396) Google Scholar and is a potential limitation of our interpretations of this study. The righting and whisker-orienting reflexes and forelimb placing were assessed on P12. The righting reflex was tested by placing the animal on its back and assessing the time and ability to turn over and right to all four feet. The whisker-orienting reflex was assessed by brushing the whiskers with a small brush. Normal mice stop moving the whiskers when touched and will orient the head to the side touched. Finally, forelimb placing was examined by holding the animal by the body and bringing the hanging forelimbs in contact with the tabletop and observing the extension of the forelimbs to the surface of the tabletop. Spontaneous open-field behavior was evaluated in mice between P18 and P19. Nineteen normoxic (9 CD-1 and 10 C57) and 23 hypoxic (11 CD-1 and 12 C57) mice were tested by placing them in a Plexiglas enclosed open field (25 × 25 × 40 cm) equipped with infrared photo beams coupled to a computer running TruScan software (Coulbourn Instruments, Whitehall, PA) to automatically record movements within the field. Activity was monitored during a single 28-minute session and measures of total distance moved (cm/4 minutes), the average velocity of movements (cm/1 minute), the amount of time without movement (rest time; seconds/4 minutes), and center time were recorded. Data were binned into three 4-minute intervals and were analyzed using a multiple analysis of variance test with repeated measures. At 4 months of age mice were assessed using the free-swim task. This test assesses behavioral laterality and cerebral asymmetries and is particularly sensitive to the organization and integrity of the corpus callosum linking the two hemispheres.18Filgueiras CC Manhães AC Effects of callosal agenesis on rotational side preference of BALB/cCF mice in the free swimming test.Behav Brain Res. 2004; 155: 13-25Crossref PubMed Scopus (15) Google Scholar, 19Manhães AC Abreu-Villaca Y Schmidt SL Filgueiras CC Neonatal transection of the corpus callosum affects rotational side preference in adult Swiss mice.Neurosci Lett. 2007; 415: 159-163Crossref PubMed Scopus (9) Google Scholar, 20Schalomon PM Wahlsten D Wheel running behavior is impaired by both surgical section and genetic absence of the mouse corpus callosum.Brain Res Bull. 2002; 57: 27-33Crossref PubMed Scopus (30) Google Scholar Mice were tested for 5 minutes in each of three test sessions spaced approximately 48 hours apart. In each session the mouse was placed in the center of a tank of water with a depth of 33 cm and measuring 33 cm in diameter. The swimming activity of the mouse was videotaped and the amount of movement in the clockwise and counterclockwise directions, changes in the direction of swimming and the consistency of preferred swimming direction across sessions was analyzed in 30-degree increments. Each swimming activity of 30 degrees was scored as a turn in a given direction (clockwise or counterclockwise). Mice were assessed using the water maze between 3 and 5 months of age. This test provides an assessment of learning and memory abilities related to hippocampal and cortical memory systems.21Morris RGM Garrud P Rawlins JNP O'Keef J Place navigation impaired in rats with hippocampal lesions.Nature. 1982; 297: 681-683Crossref PubMed Scopus (5110) Google Scholar Mice were given four trials per day for 8 consecutive days to find and swim to a transparent Plexiglas platform hidden just below the surface of the water within one quadrant of a circular opaque plastic tub (1.5 m in diameter). A video tracking system (Coulbourn Instruments) monitored the path and time to reach the platform for each trial. To assess the strength of initial learning, a 60-second probe trial was given on the 9th day during which the hidden platform was removed. The total path length, % time spent in the platform (training) quadrant, and the probe preference score (PPS) were calculated from the video records. Probe preference reflects the time spent in the training quadrant (T) relative to time in the other three quadrants (opposite, right, and left) on the probe trial. The PPS is equal to [(T − O) + (T − R) + (T − L)]/3. NSCs were isolated from the brains of P1 C57 and CD-1 pups as described and cultured in NSC medium (Dulbecco's modified Eagle's medium/F12 supplemented with N-2 [Gibco, Carlsbad, CA], 20 ng/ml of epidermal growth factor, 10 ng/ml basic fibroblast growth factor, 1%l-glutamine, 1% penicillin/streptomycin, and Fungizone) and incubated in 5% CO2 at 37°C.14Li Q Michaud M Stewart W Schwartz M Madri JA Modeling the neurovascular niche: murine strain differences mimic the range of responses to chronic hypoxia in the premature newborn.J Neurosci Res. 2008; 86: 1227-1242Crossref PubMed Scopus (24) Google Scholar Primary cultures were incubated for 15 to 20 days, at which time neurospheres form. The NSCs were plated at 1 × 104 or 1 × 105 cells/ml and plated into six-well cluster plates and cultured under either normoxic or hypoxic (5%O2) conditions for 6 days. Although our in vivo model of murine chronic sublethal hypoxia mimics the human counterpart, moving to in vitro models, although valuable and useful for elucidating mechanisms, should be approached with the knowledge that any in vitro culture conditions will elicit changes in cell-cell and cell-matrix interactions and changes in metabolism that can influence any data accrued. Further, standard tissue culture conditions are usually set at 20% O2, a hyperoxic environment. Our approach to this concern has been to assess whether the endothelial cells and NSCs that we use are capable of resetting their normoxic set point when grown in a range of O2 percentages and exhibit induction of HIF-1α in response to lowering O2% values over a range of O2 percentages as has been recently demonstrated.22Khanna S Roy S Maurer M Ratan R Sen CK Oxygen-sensitive reset of hypoxia-inducible factor transactivation response: prolyl hydroxylases tune the biological normoxic set point.Free Radic Biol Med. 2006; 40: 2147-2154Crossref PubMed Scopus (52) Google Scholar Indeed, we have observed that our NSCs are capable of this and exhibit induction of HIF-1α when O2% is changed from 20 to 10%, from 10 to 5%, and from 20 to 5% as illustrated in Figure 1, A–G. When O2% was changed from 5 to 1% considerable apoptosis was observed (not shown). In light of these studies illustrating similar HIF-1α inductions (and BDNF [not shown]) as well as graded effects on proliferation and apoptosis when O2% was changed from 20 to 10%, 20 to 5%, and from 10 to 5% and good correlation between our previously published in vivo and in vitro studies,14Li Q Michaud M Stewart W Schwartz M Madri JA Modeling the neurovascular niche: murine strain differences mimic the range of responses to chronic hypoxia in the premature newborn.J Neurosci Res. 2008; 86: 1227-1242Crossref PubMed Scopus (24) Google Scholar we elected to use a 20 to 5% reduction in O2% for our in vitro studies. In addition, when C57 and CD-1 NSC apoptosis (assessed using cleaved caspase 3) and proliferation (assessed using proliferating cell nuclear antigen [PCNA]) were investigated at various O2 levels we observed that C57 NSCs appeared to be more sensitive to reductions in O2% compared with CD-1 NSCs, exhibiting increased cleaved caspase 3 and decreased PCNA expression levels as O2% was reduced from 20 to 10% and from 10 to 5%. Immortalized mouse C57 wild-type brain endothelial cells (BECs) were obtained from Dr. Britta Engelhardt (The Theodor Kocher Institute, Bern, Switzerland) and cultured with BEC medium (Dulbecco's modified Eagle's medium and 10% fetal bovine serum, 1%l-glutamine, 1% nonessential amino acid, 1% sodium pyruvate, 1% HEPES, and 10−5 M β-mercaptoethanol) and incubated in 8% CO2 at 37°C as described previously.23Graesser D Solowiej A Bruckner M Osterweil E Juedes A Davis S Ruddle NH Engelhardt B Madri JA Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice.J Clin Invest. 2002; 109: 383-392Crossref PubMed Scopus (273) Google Scholar, 24Ford MC Bertram JP Hynes SR Michaud M Li Q Young M Segal SS Madri JA Lavik EB A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo.Proc Natl Acad Sci USA. 2006; 103: 2512-2517Crossref PubMed Scopus (171) Google Scholar, 25Li Q Ford MC Lavik EB Madri JA Modeling the neurovascular niche: vEGF- and BDNF-mediated cross-talk between neural stem cells and endothelial cells: an in vitro study.J Neurosci Res. 2006; 84: 1656-1668Crossref PubMed Scopus (163) Google Scholar Total RNA of CD-1 and C57 NSCs was isolated with TRIzol (Invitrogen, Carlsbad, CA). A real-time quantitative RT-PCR was performed using the iCycler iQ system (Bio-Rad Laboratories, Hercules, CA). cDNA was prepared, starting from 1 μg of total RNA using the iScrypt cDNA Synthesis Kit according to the manufacturer's instructions. PCR reactions for HIF-1α were performed with the following primer set: sense primer 5′-TGTGAACCCATTCCTCATCCGTCA-3′ and antisense primer 5′-TCCGGCTCATAACCCATCAACTCA-3′. Primers for the internal control, glyceraldehyde-3-phosphate dehydrogenase were included in each reaction: sense primer 5′-TCCAGTATGATTCCACCCATGGCA-3′ and antisense primer 5′-ACGTACTCAGTGTCAGCATCACCA-3′. PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories) in a final volume of 25 μl, starting with a 3-minute template denaturation step at 95°C followed by 40 cycles of 95°C for 30 seconds and 55°C for 30 seconds. NSCs were homogenized in lysis buffer composed of 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Boehringer Mannheim GmbH, Mannheim, Germany). Equal amounts of total protein (20 μg) were run on 10 or 12% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies according to the manufacturer's instructions. Antibodies used included rabbit anti-HIF-1α (1:200), anti-PHD2 (Novus Biologicals, Inc., Littleton, CO), anti-phospho mammalian target of rapamycin (mTOR), anti-mTOR, anti-phospho AKT, anti-AKT, anti-phospho P70, anti-P70, anti-phospho eIF4E, anti-eIF4E, anti-phospho p4E-BP1, and anti-p4E-BP1 at a dilution of 1:1000 (Cell Signaling Technology, Danvers, MA); anti-cleaved caspase 3; anti-PCNA; (Cell Signaling Technology, Danvers, MA) and anti-β-actin (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bound antibodies were detected by using horseradish peroxidase-conjugated anti-IgG (Cell Signaling Technology) and a chemiluminescence detection system as described previously.25Li Q Ford MC Lavik EB Madri JA Modeling the neurovascular niche: vEGF- and BDNF-mediated cross-talk between neural stem cells and endothelial cells: an in vitro study.J Neurosci Res. 2006; 84: 1656-1668Crossref PubMed Scopus (163) Google Scholar Quantitation was performed on scanned densitometric images (Agfa Arcus II Scanner with Adobe Photoshop CS, Adobe Systems, Beaverton, OR) using Quantity One software (Bio-Rad Laboratories). Western blot data are expressed as histograms of averages of relative levels (in arbitrary units) of at least three independent determinations for each protein examined. Neurospheres were fixed with 4% paraformaldehyde in PBS, pH 7.2, for 10 minutes. Immunofluorescence staining was performed with the following antibodies: antibodies against mouse nestin (1:200, BD Pharmingen, San Diego, CA), glial fibrillary acidic protein (GFAP) (1:200, Sigma-Aldrich, St. Louis, MO), and β-tubulin III (1:200, Sigma-Aldrich). Secondary antibodies were goat anti-rabbit 594 and goat anti-mouse Alexa Fluor 488 (1:200, Molecular Probes, Eugene, OR). Coverslips were mounted with Vectashield (with 4,6-diamidino-2-phenylindole), and specimens were imaged using confocal microscopy. Confocal images were obtained using a laser-scanning confocal microscope (FluoView, Olympus, Tokyo, Japan) integrated with a microscope (IX70-S1F2, Olympus) with LCPlanFl ×40 NA 0.60 objectives (Olympus) and were acquired using FluoView software (Olympus). We used the three rapid procedures described by Durbec et al26Durbec P Franceschini I Lazarini F Dubois-Dalcq M In Vitro Migration Assays of Neural Stem Cells.in: Weiner LP Humana Press, Totowa, NJ2008: 213Google Scholar to test the effect of specific factors on the NSC migration process. Chemotaxis assays were performed with BD Biocoat growth factor reduced Matrigel invasion chambers (8-μm-pore Transwells, BD Sciences, San Jose, CA). C57 and CD-1 neurospheres were dissociated with 0.1% trypsin and then 2.5 × 104 cells were placed in the wells of the upper compartments of 8-μm-pore Transwell chambers, with the lower compartment containing NSC medium without or with SDF-1 for 24 hours. For the coculture assays, 1 × 105 BECs in 500 μl of BEC medium were added to the lower chambers for 4 hours. The wells were washed with PBS twice and then 2.5 × 104 CD-1 or C57 NSCs were added to the upper chamber with 500 μl of NSC medium including 1 μg/ml anti SDF-1. After 24 hours, the cells on the upper side of the membrane were removed with a cotton swab. The cells on the lower surface of the membrane were air-dried, fixed, and stained with 4,6-diamidino-2-phenylindole. Six random selected ×10 magnification microscopic fields of each membrane were counted and three inserts were counted per condition with the average value was taken as the final result. CD31 brain-derived microvascular endothelial cells (2 × 104) were seeded in 24-well plates and cultured with BEC medium overnight. On the second day the BEC medium was removed, and the BECs were washed once with 1× PBS buffer. Individual C57 and CD-1 neurospheres of approximately the same size (80 to 100 μm in diameter) were selected, gently plated in the center of each well, and covered with 500 μl of NSC medium with or without exogenous SDF-1 (10 ng/ml) or anti SDF-1 (1 μg/ml). The plates were incubated at 37°C in a 5% CO2-humidified incubator. Images were captured at 48 hours with an Olympus IX71 fluorescence microscope using an Optronics Microfire C camera. Distances covered by cells migrating out of the neurosphere were measured by using NIH ImageJ. Three independent experiments were performed, for which each data point is a pool of 12 to 14 neurospheres. Brains from 10-day-old CD-1 and C57 mouse pups were harvested and Vibratome-sectioned. Frontal sections containing the SVZ were placed in Petri dishes; SVZ fragments were then dissected out, cut into small pieces (∼0.2 mm), and embedded in Matrigel in four-well cluster dishes. Four explants/dish were arranged in the Matrigel, and 500 μl of medium was added to cover the three-dimensional gel. To test the effect of SDF-1 on the migration of NSCs in both CD-1 and C57 isolated SVZs, exogenous SDF-1 (10 ng/ml) or anti SDF-1 (1 μg/ml) was added to the culture medium separately. Forty-eight hours after culture, the neuronal precursors were observed to migrate radially out of the explants in chain-like organizations, forming a complex three-dimensional network. Migration quantification was obtained by a direct measurement of cell migration around the explants from the border of the explant to the migration front under microscopy using an Olympus IX71 fluorescence microscope with an Optronics Microfi
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