systemic hypoxia differentially affects neurogenesis during early mouse brain maturation

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Brain & Development 34 (2012) 261–273

Original article

Systemic hypoxia differentially affects neurogenesis duringearly mouse brain maturation

Christina Schneider a, Gudrun Krischke a, Wolfgang Rascher a, Max Gassmann b,Regina Trollmann a,⇑

a Department of Pediatrics, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germanyb Institute of Veterinary Physiology, Vetsuisse Faculty, Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Switzerland

Received 17 February 2011; received in revised form 7 July 2011; accepted 8 July 2011

Abstract

Background: Cerebral tissue oxygen level modifies crucial processes of neurogenesis, glial and neuronal development during phys-iological and hypoxic conditions. Whether hypoxia-sensitive factors such as doublecortin (DCX) and hypoxia-inducible transcrip-tion factor (HIF)-regulated CXCR4 and SDF-1 modify and activate adaptation to hypoxia in developing brain is not wellunderstood. Present study investigated maturational regulation of oxygen-sensitive developmental genes and proteins in developingmouse brain in relation to the degree of hypoxia. Methods: Physiological expression of HIF-1, CXCR4, SDF-1 and DCX were ana-lyzed in the brain of C57/BL6 mice (P0–P60). In addition, mice (P0, P7) were exposed to normoxia, acute (8% O2, 6 h) or chronichypoxia (10% O2, 7 d) followed by reoxygenation. Gene expression was analyzed by quantitative PCR, proteins were quantified byWestern blot analysis and immunohistochemistry. Results: Cerebral HIF-1a protein, CXCR4 and DCX mRNA levels showed mat-urational stage-related peak levels at P0/P1, whereas SDF-1 mRNA levels were highest at P17. CXCR4 and SDF-1 mRNA levelswere not altered in response to hypoxia. Whereas DCX mRNA levels significantly increased during acute hypoxia, down-regulationof DCX transcripts was found in response to chronic hypoxia compared to controls, and these changes were related to specificallyvulnerable brain regions. Conclusions: Maturational stage-related dynamic changes of HIF-1a, CXCR4, SDF-1 and DCX mayreflect involvement of hypoxia-regulated systems in important developmental regulatory processes of the developing brain. Extend-ing the knowledge of differential effects of hypoxia on neurogenesis and dynamic regulatory networks present data provide a basisfor future research on gestational age-specific neuroprotective options.� 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Keywords: Neurogenesis; Doublecortin; Chemokine receptor 4; Stromal cell derived factor 1; Hypoxia-inducible factor-1a

0387-7604/$ - see front matter � 2011 The Japanese Society of Child Neuro

doi:10.1016/j.braindev.2011.07.006

Abbreviations: CNS, central nervous system; CXCR4, chemokinereceptor 4; DCX, doublecortin; HIF-1a, hypoxia-inducible factor-1a;PBGD, porphobilinogen deaminase; SDF-1, stromal cell derived fac-tor 1⇑ Corresponding author. Address: Department of Pediatrics, Fried-

rich-Alexander University of Erlangen-Nuremberg, Loschgestrasse 15,91054 Erlangen, Germany. Tel.: +49 9131 853 3753; fax: +49 9131 8533389.

E-mail address: [email protected] (R. Trollmann).

1. Introduction

Early cerebral developmental processes including vas-culogenesis [1,2], neuronal migration and differentiation[3,4] as well as synaptogenesis, cytoskeletal maturationand physiological apoptosis [5] are crucially modifiedby tissue oxygenation. Among oxygen-sensitive regula-tory systems, hypoxia-inducible transcription factors(HIF) are characterized as main regulators of cellularadaptation to hypoxia [6,7]. The crucial role of theHIF system during development has been demonstrated

logy. Published by Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.braindev.2011.07.006

mailto:[email protected]

http://dx.doi.org/10.1016/j.braindev.2011.07.006

262 C. Schneider et al. / Brain & Development 34 (2012) 261–273

by HIF knock-out mouse experiments indicating thatHIFs represent a basic prerequisite for physiologicembryonic [1,2] as well as brain development [8,9].

Two isoforms, HIF-1 and HIF-2, are well character-ized both consisting of an oxygen-sensitive a- and aconstitutively expressed b-subunit. Under normoxia,the a-subunit is immediately destabilized mediatedhydroxylation of specific prolyl residues by specific pro-lyl hydroxylases (PHD). Subsequently, HIF-a interactswith the von-Hippel-Lindau protein that comprises anubiquitin ligase allowing destruction of HIF-a by theubiquitin–proteasome pathway. Under hypoxia, degra-dation of HIF-a is prevented, and HIF-a protein accu-mulates. Translocated into the nucleus the a-subunitheterodimerizes (a/b), and the dimer acts as transcrip-tion factor for many target genes involved in angiogen-esis, vasculogenesis, erythropoiesis, metabolic processes,neuronal differentiation and migration [7]. Of note,beside modulation of developmental processes andadaptive mechanisms to hypoxia also proapoptoticproperties of HIF have been described such as activa-tion of Bnip3 and p53 under conditions of severehypoxia [10]. Thus, activation and functional role ofthe cerebral HIF system seem to be related to severityof hypoxia, cell type and developmental stage [7,11].

Chemokine receptor CXCR4 and its physiologicligand stromal-derived factor-1 (SDF-1) are HIF-regu-lated factors [4,12] mediating directed migration of neu-ral and oligodendrocyte precursors [4,13] and regulationof axonal pathfinding during early brain development[14,15]. Shown by knock-out mouse experiments, defi-ciency of CXCR4 or SDF-1 leads to disturbed migrationof neurons [16], hippocampal [17,18] and cerebellar [19]cells as well as cortical Cajal-Retzius cells [20]. CXCR4and SDF-1 have been detected in various rodent brainregions from early fetal stage through adulthood includ-ing cerebral cortex, hippocampus, thalamus, cerebellum,brain stem and spinal cord [14,21]. During brain devel-opment, spatial expression patterns of CXCR4 andSDF-1 pass through dynamic changes which arestrongly related to cell type and developmental stage[22–30]. It has been shown that CXCR4 is expressed inearly generated Cajal-Retzius cells of the cortical mar-ginal zone [23] and neurons of germinal and intermedi-ate zone [15,23] as well as basal ganglia during lateembryonic development [23]. Moreover, CXCR4 andSDF-1 are involved in cerebral regenerative stem cellmigration as shown by adult rodent stroke models [24]as well as in hypoxia/ischemia-induced migratory pro-cesses in the postnatal developing rat brain [25]. In addi-tion, migrational activity and chemotactic SDF-1response of N9 microglia cells [26] and HUVECs [12]were enhanced by hypoxia.

Doublecortin (DCX) characterized as marker of neu-rogenesis is detectable in migrating and differentiatingdeveloping neurons and radial gliocytes during early

embryonic stages [27] including early hippocampaldevelopment [28]. During early postnatal development(P0–P7) DCX was found in granule precursor cells ofmouse cerebellar cortex and white matter as well as pre-cursors of late migrating molecular layer interneurons[29,32]. Disrupted microtubule development and anom-alies of cortical structure such as lissencephaly in malesand subcortical heterotopy in females caused by DCXgene mutations reflect the essential role of DCX forphysiological brain development [29,32]. In addition,DCX plays a role in adult neurogenesis of the hippo-campus and subventricular zone [30,31]. Under condi-tions of perinatal cerebral hypoxia/ischemia, activationof neurogenesis estimated from accumulation ofDCX-positive cells has been shown in postnatal (P6)rat brain [33]. Directed migration of neuronal precursorcells into hypoxic areas of the cerebral cortex and stria-tum was associated with increased DCX expression[34,35]. However, the role of DCX in the complex pro-cesses of recovering and remodeling of the developingbrain following hypoxic brain injury remains to beestablished [36].

The aims of the present study were to investigatecerebral regulation of HIF-dependent (CXCR4, SDF-1) and HIF-independent developmental factors (DCX)in relation to the stage of mouse brain maturation(P0–P60), and to the degree of systemic hypoxia duringearly mouse brain development (P0–P7).

2. Material and methods

2.1. Animal experiments

Acute hypoxia: Neonatal (P0; n = 40; P7; n = 40)C57/Bl6 wild type mice were exposed to acute systemichypoxia with FiO2 of 8% O2 for 6 h (Hypoxic Worksta-tion INVIVO2 1000, Biotrace International, UK). Toenable adjustment to the hypoxic environment O2 depri-vation was done gradually by decreasing the FiO2 from21% to 8% in 2% O2 steps during 60 min before keepingthe mice in continuous systemic hypoxia for 6 h.Controls were incubated under room air. After theincubation period, brains (n = 10 per group) were imme-diately dissected avoiding reoxygenation. Anothergroups of pups (n = 10 per group) ran through reoxy-genation for 24 h, 72 h or 7 d under normoxic condi-tions (room air) together with their dams beforedecapitation and brain dissection. Chronic hypoxia:

Pregnant (n = 15) and neonatal (P7; n = 40) C57BL/6mice were kept at continuous hypoxia with FiO2 of10% O2 for 7 d (Hypoxic Workstation INVIVO2 1000,Biotrace International, UK) starting at embryonic day14 (E14, intrauterine hypoxia) or postnatal day 7 (P7),respectively. Adaptation to hypoxic environment wasperformed as described above. Controls were kept underroom air. After 7 d of incubation, developing brains

C. Schneider et al. / Brain & Development 34 (2012) 261–273 263

(n = 6–11 pups per pregnant mouse; P7, n = 10 pergroup) were immediately dissected. During reoxygena-tion, pups (n = 10 per group) were kept under roomair for 24 h, 72 h or 7 d.

Dissected brains were immediately frozen in liquidnitrogen and stored at �80 �C until protein (n = 3 pergroup) and mRNA (n = 5 per group) extraction. Forimmunohistochemical studies tissues were embedded in4% paraformaldehyde (n = 3 per group). Coronal sec-tions (n = 3) at the level of the dorsal hippocampus wereperformed to analyze cerebral cortex and hippocampusreflecting areas of highly selective vulnerability tohypoxia [37,38]. Animal experiments were performedaccording to protocols approved by the KantonalesVeterinaramt Zurich.

2.2. RNA isolation and Real-Time-PCR

Total RNA was extracted from whole brains using Tri-zol isolation method (Invitrogen, Germany) according tothe manufacturer’s instructions. RT-PCR was performedas described previously [37]. Commercial reagents (Taq-Man PCR Reagent Kit, Eurogentec, Germany) and con-ditions were applied according to the manufacturer’sprotocol. The PCR reaction was performed in an ABI7500 RT PCR thermocycler (Applied Biosystems, Ger-many). All reactions were performed in duplicate usingb-actin and porphobilinogen deaminase (PBGD) asendogenous controls. The following primers and Taq-Man probes based on published reports were used:

PBGD

Forward: 50-ACAAGATTCTTGATACTGCACTC-TCTAAG-30;Reverse: 50-CCTTCAGGGAGTGAACAACCA-30;TaqMan probe: 50(FAM)-TCTAGCTCCTTGG-TAAACAGGCTCTTCTCTCCA-(TAMRA)-30.b-ActinForward: 50-ATGCTCCCCGGGCTGTAT-30;Reverse: 50-TCACCCACATAGGAGTCCTTCTG-30;TaqMan probe: 50(FAM)-ATCACACCCTGGT-GCCTAGGGCG-(TAMRA)-30.HIF-1aForward: 50-AGACAGACAAAGCTCATCCAA-GG-30;Reverse: 50-GCGAAGCTATTGTCTTTGGGTT-TAA-30;TaqMan probe: 50(FAM)-CTGCCACTTTGAATC-AAAGAAATACTGTTCCTGAG-(TAMRA)-30.CXCR4

Forward: 50-GCTGGCTGAAAAGGCAGTCTA-30;Reverse: 50-CGTCGGCAAAGATGAAGTCA-30;TaqMan probe: 50(FAM)-TCTGGATCCCAGCC-CTCCTCCTG-(TAMRA)-30.SDF-1c

Forward: 50-GAGCCAACGTCAAGCATCTG-30;Reverse: 50-TCTTCAGCCGTGCAACAATC-30;TaqMan probe: 50(FAM)-AAATCCTCAACACTC-CAAACTGTGCCCTTC-(TAMRA)-30.Doublecortin

Forward: 50-GCGCCGCAGCAAGTCT-30;Reverse: 50-TTGAGAGCTGACTGCTGGAAGTT-30;TaqMan probe: 50(FAM)-CTGACTCAGGTAAC-GACCAAGACGCAAATG-(TAMRA)-30.

2.3. Protein extraction and quantification

Whole brains were homogenized in lysis buffer A(10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mMdithiothreitol, 5 mM sodium fluoride, 1% NP-40 andprotease inhibitors in distilled water). Samples were cen-trifuged (13,000 rpm) at 4 �C for 5 min. The superna-tant, containing cytosolic extracts, was separated andstored at �80 �C. The extant pellet was resuspended inbuffer B (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2,0.5 mM dithiothreitol, 5 mM sodium fluoride, 420 mMNaCl, 25% glycerol and protease inhibitors in distilledwater). After incubation on ice for 20 min samples werecentrifuged (13,000 rpm) for 5 min at 4 �C. Superna-tants, containing nuclear extracts, were kept at�80 �C. Nuclear and cytosolic protein contents wereanalyzed by commercial protein assay (BioRad,Munich, Germany).

2.4. Western blot analysis

Nuclear proteins (30 lg) were run on a 7.5% SDS–polyacrylamide gel and transferred to a nitrocellulosemembrane, blocked in 5% nonfat milk-TBS for 90 minat room temperature. Afterward membranes were incu-bated overnight at 4 �C with the polyclonal rabbit anti-HIF-1a antibody (Novus Biologicals, Littleton, CO;1:1000). After washing in TBS containing 0.05% Tweenblots were incubated with a horseradish peroxidase-con-jugated secondary antibody (Cell Signaling, Danvers,USA. 1:2000) for 1 h at room temperature, followedby chemiluminescent detection. Three samples per groupwere analyzed. b-Actin was used as a loading control.Densiometric quantification was done using QuantityOne Software (BioRad, Munich, Germany).

2.5. Immunohistochemistry

For immunohistochemical analysis coronal sections(3 lm thick) of paraformaldehyde-embedded mousebrains at the level of the dorsal hippocampus were per-formed (three brains per group). After heat-induced epi-tope retrieval, washing (TBS/0.05% Tween 20) andblocking sections were incubated with the polyclonal


rabbit anti-mouse doublecortin (DCX) antibody(Abcam, Cambridge, UK; 1:100) or the polyclonal rab-bit anti-mouse chemokine receptor 4 (CXCR4) antibody(Imgenex, USA; 1:50) overnight at 4 �C. After washingsections were incubated for 2 h with Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Eugene,USA; 1:100). To differentiate neuronal cells, sectionswere co-stained with DCX and NeuN (neuronal marker)using a monoclonal mouse anti-NeuN antibody (Chem-icon Int., Hampshire, UK; 1:50). Incubation was per-formed at room temperature for 2 h followed by AlexaFluor 594-conjugated secondary antibody (Invitrogen,Eugene, USA; 1:100) at room temperature for 1 h andco-staining with DAPI (Boehringer Ingelheim, Ger-many, 1:3000) at room temperature for 10 min. For allincubations a humidified chamber was used. Negativecontrols were performed by omitting the primary anti-body. All stainings were done in triplicate and were ana-lyzed by fluorescence microscopy (Zeiss, Gottingen,Germany) by an observer who was blinded to the studygroups. DCX- and NeuN-positive cells were counted inthree visual fields of the parietal cortex, hippocampalCA3 region and dentate gyrus (DG) at 40-foldmagnification.

2.6. Statistical analysis

Data are expressed as mean ± SEM. Statistical signif-icance was determined by t-test and One-way ANOVA(p-value <0.05).

P0 P1 P3 P7 P8 P10P14P15P17P21P600.0

0.5

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HIF

-1α

/PB

GD

mR

NA

120 kD

43 kD

P0 P1 P3 P7 P8 P10 P14 P15 P17 P60

A

B

Fig. 1. Quantification of HIF-1a mRNA (A) and protein levels (B, Western bto maturational stage (P0–P60). *p < 0.05, vs P60 (adult mouse brain).

3. Results

To analyze maturational stage-related and hypoxia-induced regulation of markers of neurogenesis, we inves-tigated cerebral expression of the HIF-regulated targetgenes CXCR4 and SDF-1, and the HIF-independentneuronal marker DCX in developing and adult mousebrain (P0–P60) during normoxia as well as in responseto perinatal acute (8% O2, 6 h) and chronic (10% O2,7 d) systemic hypoxia.

3.1. Developmental changes of cerebral HIF-1a, CXCR4,

SDF-1 and DCX

As expected, HIF-1a mRNA levels did not signifi-cantly change during the course of maturation(Fig. 1A). However, strong HIF-1a protein accumula-tion analyzed by Western blot was evident during thefirst postnatal week in contrast to low concentrationsat adult stage (Fig. 1B and C). Under physiological con-ditions (normoxia) cerebral CXCR4 mRNA levels werehighest during the first postnatal week with peak levelsat P0 (Fig. 2A). Afterward concentrations decreased tolow levels which were similar to those of adult brains.These temporal changes were confirmed by immunohis-tochemical analysis of parietal cortex (Fig. 2C). In con-trast, SDF-1 mRNA levels increased during earlydevelopmental stages reaching peak levels at P14–P17.Thereafter, SDF-1 mRNA levels significantly decreasedreaching lowest levels at P60 (Fig. 2B).

HIF-1α

β-actin

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lot analysis; C, densitometric analysis; n = 3) in mouse brain in relation

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***

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CXCR4 DAPI

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CXCR4 DAPI

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Fig. 2. Age-related CXCR4 (A) and SDF-1 (B) mRNA levels (normalized to PBGD) in normoxic mouse brain (P0–P60, n = 3). (B) Representativephotomicrographs of immunohistochemical staining of CXCR4 (green) in developing mouse parietal cortex in relation to maturational stage. Nucleiare counterstained with DAPI (blue). *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of this article.)


Developmental changes of cerebral DCX mRNAexpression were most prominent during the first post-natal week with peak levels at P0 (Fig. 3A). Withincreasing age DCX mRNA levels significantlydecreased to nearly adult levels at P10 (Fig. 3A).Age-related decrease was confirmed by immunohisto-chemical analysis showing a higher number of DCX-positive cells in the parietal cortex at P0 comparedto P14 and P21, and thereafter a scattered expressionin the adult cerebral cortex (P60) (Fig. 3B). As sum-marized in Fig. 4, expression levels for HIF-1a,CXCR4, SDF-1 and DCX in the normoxic mouse

brain (P0–P60) most prominently changed during thefirst postnatal week.

3.2. Effects of hypoxia on cerebral CXCR4 and SDF-1

expression

Gene expression levels of CXCR4 and SDF-1remained unchanged in response to acute and chronicsystemic hypoxia at both maturational stages P0 andP7 as well as upon reoxygenation periods analyzed(Fig. 5). Noteworthy, there was a trend of increasingcerebral CXCR4 expression during acute hypoxia (8%

P0 P1 P3 P7 P8 P10 P14 P15 P17 P21 P600

1

2

3

**

**

mean±SEM

DC

X/P

BG

D m

RN

A

DCX DAPI

P0

DCX DAPI

P14

DCX DAPI

P60 P21

DCX DAPI

A

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Fig. 3. Cerebral doublecortin (DCX) mRNA levels normalized to PBGD in relation to maturational stage (A, P0–P60, n = 3). (B)Immunohistochemical staining of DCX (green) in parietal cortex of developing mouse brain in relation to maturational stage. *p < 0.05,**p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


O2, 6 h) at P0. In contrast, decreased levels of CXCR4mRNA were present upon reoxygenation period of 7 din comparison to controls (Fig. 5). Similarly, down-reg-ulation of cerebral CXCR4 mRNA levels was evident inresponse to chronic hypoxia (P0) and reoxygenationperiod of 7 d in comparison to normoxia (Fig. 5).

3.3. Effects of hypoxia on cerebral DCX expression

Differential and age-dependent regulation of cerebralDCX was observed in response to acute and chronic

hypoxia as well as different reoxygenation periods(24 h, 72 h, 7 d).

3.4. Acute hypoxia

Upon exposure to acute systemic hypoxia at P0 cere-bral DCX mRNA levels significantly increased com-pared to controls (Fig. 6A, p < 0.01) but not duringreoxygenation periods. In contrast, at P7 a slightdecrease of DCX mRNA levels was obvious in responseto acute hypoxia and reoxygenation for 24 h in compar-

P0 P1 P3 P7 P8 P10P14P15P17 P30 P600

1

2

3

4 HIF-1α proteinCXCR4 mRNASDF-1 mRNADCX mRNA

Age (postnatal day)

Expr

essi

on le

vels

(mea

n)

Maximum period of neuronal migration, differentiation and

organisation

Fig. 4. Summary of age-related dynamics of cerebral expression ofhypoxia-inducible factor-1a protein (HIF-1a), mRNA levels ofchemokine receptor 4 (CXCR4), stromal-derived factor-1 (SDF-1)and doublecortin (DCX) in relation to maturational stage.


ison to controls. Later on, a significant decrease of DCXmRNA concentrations was present upon 7 d of reoxy-genation (Fig. 6B, p < 0.05). This was confirmed byimmunohistochemical analysis. A decreased number ofimmature neurons (DCX-positive) was detectable inhypoxic cerebral cortex (P7) upon 7 d of reoxygenation(Table 1, and Fig. 6C and D). However, indicating spa-tial differences, in hypoxic dentate gyrus the mean num-ber of DCX-positive cells was unchanged (Table 1, andFig. 6E and F), and in contrast, the mean number ofDCX-positive neurons was increased in the hypoxic hip-pocampal CA3 region compared to controls (Table 1,and Fig. 6G and H).

3.5. Chronic hypoxia

On the other hand, exposure to chronic hypoxia ledto significantly decreased cerebral DCX mRNA levelsat P0 (Fig. 7A, p < 0.01). Whereas DCX mRNA levelswere slightly lowered upon reoxygenation compared tocontrols (Fig. 7A), immunohistochemical analysis indi-cated spatial differences of DCX expression duringreoxygenation. As demonstrated in Fig. 7, mean numberof DCX-positive cells increased in the cerebral cortexand dentate gyrus (Table 2 and Fig. 7C–F), however,decreased in the CA3 region compared to normoxic con-trols (Table 2, and Fig. 7G and H). At P7, chronichypoxia resulted in markedly decreased DCX mRNAlevels during hypoxia and upon reoxygenation of 72 h(Fig. 7B).

4. Discussion

During late-gestational and early postnatal stage ofbrain development migration and differentiation ofimmature neurons as well as growth of cortical and syn-aptic connections represent crucial processes of cerebralmaturation highly vulnerable to hypoxic injury [39].

Accordingly, to differentiate effects of systemic hypoxiaon neurogenesis and neuronal migration, it is importantto consider the heterogeneity of regulation and timing ofdevelopmental events in the developing brain [5]. Focus-sing on early developmental processes and dynamicregenerative mechanisms of the neonatal mammalianbrain the main findings of the present study were (i) thatchanges of expression levels for HIF-1a, CXCR4, SDF-1and DCX under physiological conditions were mostprominent during the first postnatal week correspondingto the period of murine maximum brain growth spurt(P4–P7, [40]), cell migration, organization of neuronalcells (E11–P7; [41] as well as myelination (P5–P21, [42])and synaptogenesis (first postnatal weeks, [43]), and (ii)that acute and chronic systemic hypoxia differentiallyregulate neurogenesis (DCX) and migratory (CXCR4)factors in selectively vulnerable brain regions. Extendinginsights into age-dependent mechanisms of perinatalneurogenesis and neuronal migration, present data implya basis for future research on gestational age-specificneuroprotective options.

4.1. Age-related cerebral expression of HIF-1a and

developmental factors relevant for neurogenesis and

migration

Present data indicate high cerebral activity of the HIFsystem during the first postnatal week compared toadult brains shown by strong accumulation of HIF-1aprotein and transcriptional activity of the HIF targetgene CXCR4 with peak levels at P0–P7. In former stud-ies [37,44] postnatal cerebral HIF-1a protein levels weremainly determined in relation to hypoxic and ischemicinsults whereas investigations focussing on physiologicaltemporal and spatial expression during early brain mat-uration are limited [45]. Present results on age-relatedprominent HIF-1a accumulation in mouse brain duringthe first postnatal week further confirm significance ofthe HIF system for early developmental processesincluding cerebral vasculogenesis, neurogenesis, migra-tion and synaptogenesis [1,5,8].

In this context we examined physiological age-relatedexpression of chemokine receptor 4 (CXCR4) and itsphysiological ligand SDF-1 representing HIF targetgenes [4,12,26] which are involved in the regulation ofdirected neuronal migration [15,16], migration of neuro-nal precursor cells and oligodendrocytes [4,13], axonaldifferentiation [14,15] during development as well as adultneurogenesis [14,21]. Cerebral CXCR4 mRNA expres-sion peaked at P0 and rapidly decreased to adult levelsduring the first postnatal week. In parallel, SDF-1, thespecific CXCR4 ligand, gradually increased at the mRNAlevel during the first weeks reaching peak levels at P15–P17. This might indicate growing interaction between oli-godendrocytic and astrocytic SDF-1 and CXCR4 proteinwhich is expressed either in the intracellular compartment

Acutehypoxia

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Fig. 5. Gene expression of CXCR4 and SDF-1 (normalized to b-actin) in developing mouse brain in response to systemic acute (8% O2, 6 h) andchronic hypoxia (10% O2, 7 d) and different reoxygenation periods (24 h, 72 h, 7 d) in relation to age.


or at the surface of neuronal precursors and developingneurons [24], and could modify underlying mechanismsof directed migration during mouse brain development

and organization of cortical structures [13,21] as well asinjury-induced directed migration of neuronal precursorcells [24]. The antidromic patterns of age-related CXCR4

0h 24h 72h 7d0.0

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Reoxygenation

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C D

E F

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Fig. 6. Doublecortin (DCX) mRNA levels (normalized to b-actin) in developing mouse brain upon exposure to acute hypoxia (6 h 8% O2) at P0 (A)and P7 (B) followed by reoxygenation (24 h, 72 h, 7 d). (C–H) Immunohistochemical co-staining with DCX (green) and NeuN (red) in postnatalmouse brain exposed to acute hypoxia at P7 and reoxygenation of 7 d. Representative photomicrographs of normoxic (C) and hypoxic (D) parietalcortex, normoxic (E) and hypoxic dentate gyrus (F), and normoxic (G) and hypoxic (H) hippocampal CA3 region. Nuclei are counterstained withDAPI (blue). **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


Table 1Quantification (mean ± SEM) of DCX- and NeuN-positive cells (normalized to DAPI-positive cells) in developing parietal cortex and hippocampalregion (CA3; dentate gyrus, DG) of neonatal mice exposed to acute hypoxia (8% O2, 6 h) and reoxygenation in relation to age compared to controls(n = 3 per group).

Acute hypoxia (P7) + 7 d reoxygenation Parietal cortex Dentate gyrus (DG) CA3

Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxian = 3 n = 3 n = 3 n = 3 n = 3 n = 3

DCX+/total cell count (DAPI) 0.26 ± 0.06 0.19 ± 0.02 0.28 ± 0.03 0.28 ± 0.05 0.15 ± 0.05 0.22 ± 0.01NeuN+/total cell count (DAPI) 0.43 ± 0.08 0.40 ± 0.04 0.23 ± 0.08 0.24 ± 0.03 0.46 ± 0.16 0.45 ± 0.09


mRNA and SDF-1 mRNA levels as well as interaction atthe protein level need to be clarified.

Indeed, studies on expression patterns of SDF-1 andCXCR4 during embryonic and postnatal brain develop-ment are limited [21,46,47]. Tissir et al. [46] who exam-ined CXCR4 and SDF-1 mRNA expression inembryonal and neonatal mice (E9.5–P21) by in situhybridization observed a prominent CXCR4 expressionin ventricular zones of stem cell proliferation and differ-entiating neuronal cells at embryonic stages. In line withour observations, down-regulation of cerebral CXCR4expression during postnatal period was evident [46].Berger et al. [21] characterized postnatal patterns of dis-tribution of SDF-1 and CXCR4 nucleic acids by in situhybridization during the first postnatal weeks (P0–P30)in CD1 wild-type or CD57 and CD1 mixed-backgroundnestin-GFP transgenic mice showing SDF-1 expressionin meninges, Cajal-Retzius cells and maturing dentategranule neurons, as well as CXCR4 expression withinmolecular layer, migratory stream and hilus indicatingregional and age specific regulation. In agreement withothers [21,46] we demonstrated maturational andregion-specific expression patterns of SDF-1 andCXCR4 pointing out differential expression of thesemigratory factors at the mRNA and protein level duringearly development. Further studies on regulatory mech-anisms are necessary. Interestingly, Miller et al. [47]showed a transiently activated, SDF-1-mediated chemo-taxis in perivascular areas in HI injury zones of develop-ing mouse brain (P7) suggesting SDF-1 as potentialtarget for neuroprotective strategies.

Among HIF-independent determinants of neuronalmaturation, DCX expressed in differentiating andmigrating neuroblasts and radial glia cells, is function-ally involved in the regulation of microtubular cytoskel-eton of migrating neurons [29,48]. Here, we show thatcerebral DCX mRNA expression is developmentallyregulated with highest levels at P0 with subsequent rap-idly decreasing concentrations comparable to low adultlevels. These observations are in line with former studieson temporal DCX expression in rodent brains duringembryonic and early postnatal development [29,30] aswell as studies on age-related changes of DCX proteinexpression in postnatal mouse brain (subventricularzone) analyzed at the age between 1 and 24 months

[49]. Functionally, during early period of brain develop-ment, DCX has been shown to play an essential role inneuronal migration and axonal wiring [29,50]. In theadult rodent brain, DCX expression is restricted to spe-cific regions of persistent neurogenesis like hippocam-pus, dentate gyrus and subventricular zone/bulbusolfactorius axis [51] and is assumed to be involved inlearning processes, memory and pattern separation[49]. Further in vivo studies on the age-dependent func-tional role of DCX during early neurogenesis andmigration are necessary which might implicate new ther-apeutic approaches.

4.2. Effects of hypoxia on postnatal neurogenesis andneuronal migration

As known from adult rodent stroke models, HIF-reg-ulated chemokine receptor CXCR4 [26] modifies post-ischemic regenerative processes by activation of directedmigration of neuronal stem cells in vivo [24] andhypoxia-induced microglia migration in vitro [26]. Byour in vivo experimental setting representing anestablished model of perinatal systemic hypoxia [37],no significant hypoxia-induced alterations of CXCR4and SDF-1 mRNA levels in developing mouse brain(P0–P7) have been observed. This is in line within vitro observations on neural progenitor cells derivedfrom neonatal C57BL/6 mouse brain (P1) showing noappreciable transcriptional activation of CXCR4 andSDF-1 expression due to chronic hypoxia (10% O2,6 d) [52]. Likewise, Li et al. [52] demonstratedunchanged CXCR4 mRNA levels in brain lysates of11-day-old C57BL/6 mice exposed to chronic hypoxia(9.5% O2 for 8 d). Considering prominent cerebralCXCR4 expression during early postnatal stage ofmouse brain development as shown by present dataand others [21,46], our hypothesis is that transcriptionalactivity of CXCR4 is maximally up-regulated duringthis early stage of brain maturation as known fromother developmental factors such as VEGF in brainsof human embryos and neonates [53]. Thus, further acti-vation in response to hypoxia might be a function ofpost-transcriptional modification and regulation.

Interestingly, as assessed by cerebral DCX mRNAand protein expression, our data extend present

0h 24h 72h 7d 0.0

0.5

1.0

1.5

2.0normoxiahypoxia

mean±SEMChronic hypoxia at P0

**

Reoxygenation

DC

X/ β

-act

in m

RN

A

0h 24h 72h 7d0.00

0.05

0.10

0.15

0.20normoxiahypoxia

mean±SEMChronic hypoxia at P7

Reoxygenation

DC

X/ β

-act

in m

RN

A

III

C D

E F

G H

A B

Fig. 7. Doublecortin (DCX) mRNA levels (normalized to b-actin) in developing mouse brains upon exposure to chronic hypoxia (10% O2, 7 d) anddifferent reoxygenation periods (24 h, 72 h, 7 d) in relation to age (A, P0; B, P14). (C–H) Immunohistochemical co-staining with DCX (green) andNeuN (red) in postnatal mouse brain exposed to chronic hypoxia (P0) and reoxygenation of 72 h. Representative photomicrographs of normoxic (C)and hypoxic (D) parietal cortex, normoxic (E) and hypoxic dentate gyrus (F), and normoxic (G) and hypoxic (H) hippocampal CA3 region. Nucleiare counterstained with DAPI (blue). **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)


Table 2Quantification (mean ± SEM) of DCX- and NeuN-positive cells (normalized to DAPI-positive cells) in developing parietal cortex and hippocampalregion (CA3; dentate gyrus, DG) of neonatal mice exposed to chronic systemic hypoxia (10% O2, 7 d) and reoxygenation in relation to age comparedto controls (n = 3 per group).

Chronic hypoxia (E14–E20) + 72 h reoxygenation Parietal cortex Dentate gyrus (DG) CA3

Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxian = 4 n = 3 n = 4 n = 3 n = 4 n = 3

DCX+/total cell count (DAPI) 0.25 ± 0.07 0.30 ± 0.11 0.18 ± 0.05 0.27 ± 0.16 0.22 ± 0.07 0.19 ± 0.06NeuN+/total cell count (DAPI) 0.20 ± 0.06 0.28 ± 0.02 0.11 ± 0.03 0.18 ± 0.07 0.14 ± 0.03 0.25 ± 0.05


knowledge on the significance of systemic hypoxia onpostnatal neurogenesis. The transient increase of DCXmRNA levels shown in neonatal mouse brain (P0) uponacute systemic hypoxia might reflect an adaptiveresponse to limit hypoxia-induced damage [33].Decreased DCX expression upon exposure to acutehypoxia and reoxygenation at P7, but not P0, suggeststhat vulnerability to hypoxia depends on maturationalstage [54]. Moreover, modification of DCX regulationby severity of systemic hypoxia is suggested fromobserved decrease of DCX expression under chronichypoxia indicating significant effects of long-lasting sys-temic hypoxia on neurogenesis in developing mousebrain [55]. Furthermore, regional-specific differences ofDCX regulation in relation to the duration of hypoxiawere obvious during early stage of brain maturation.Whereas cortical neurogenesis increased in response tochronic hypoxia, a decreased number of DCX-positivecells was detected in response to acute hypoxia. Similarobservations were found in the hippocampal CA3 regionpointing out selective vulnerability to hypoxia duringearly development [56].

In conclusion, present in vivo observations providefirst postnatal weeks as crucial regulatory period ofHIF-1a, DCX, CXCR4 and SDF-1 expression in devel-oping mouse brain. Moreover, our data point out differ-ential expression patterns of neurogenesis factors inrelation to severity of systemic hypoxia and matura-tional stage.

Acknowledgements

This work was supported by the German ResearchFoundation (Deutsche Forschungsgemeinschaft, DFG)and ELAN-Fonds of the University of Erlangen.

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systemic hypoxia differentially affects neurogenesis during early mouse brain maturation

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