Apoptosis and Neurogenesis after Transient
Hypoxia in the Developing Rat Brain
Jean-Luc Daval* and Paul Vert†
Perinatal brain damage following a hypoxic-ischemic episode has been considered for a long time as
an irreversible phenomenon. However, recent studies have shown that various insults may induce de
novo neurogenesis in the adult rodent brain. The present study tested the hypothesis that acute
hypoxia may trigger neurogenesis in the developing brain. In vitro, the influence of transient hypoxia
was analyzed on the outcome of embryonic rat neurons in culture. In vivo, the temporal profile of
brain damage was monitored at the level of the CA1 layer of the hippocampus after the exposure to
hypoxia of 1-day-old rats. The extent of cell loss and regeneration was evaluated after staining with
DAPI. The characterization of newly generated cells was performed in the subventricular zone at 20
days postexposure by immunohistochemistry. Following hypoxia for 6 hours, neuronal viability in the
culture dishes was reduced by 36% at 96 hours, with a significant number of cell nuclei showing
apoptosis features. In contrast, a 3-hour hypoxia apparently did not damage cultured neurons whose
number increased by 14%. The Bax/Bcl-2 ratio tended to increase after 6-hour hypoxia and to
decrease after 3-hour hypoxia. In vivo, hypoxia induced cell damage in the CA1 subfield of the
hippocampus, where the total number of cells was reduced by 27% at days 6-7 postreoxygenation,
with histopathological hallmarks of apoptosis. This cell deficit was followed by a gradual recovery
observable from day 20, suggesting a repair mechanism. Brain incorporation of BrdU in the
subventricular zone revealed an accumulation of proliferating cells expressing the neuronal marker
NeuroD. The present data demonstrate that a posthypoxic neurogenesis does occur during development and may account for brain protection.
© 2004 Elsevier Inc. All rights reserved.
erinatal brain damage following a hypoxicischemic insult has been considered for a
long time as an irreversible phenomenon responsible for ever-lasting sequelae.1-3 Numerous
studies have provided extensive descriptions of
the pathophysiologic sequences of posthypoxic
neuronal injury leading to substantial anatomical and functional impairments.4-6
However, it is also known that, in developing— or newborn—animals, the brain is more resistant to oxygen deprivation than in adults.7,8 Furthermore, experiments performed in various
species have shown a variety of evidence suggesting
that cerebral plasticity, even not specific to the
neonatal period, is more efficient early in the development.9-11 In this respect, recent studies have
shown that insults like ischemia or seizures may
trigger de novo neurogenesis at specific sites like
the subventricular zone in the adult brain.12,13
In the studies reported herein, we aimed to
elucidate the fate of developing brain neurons
in response to global hypoxia, and we tested the
hypothesis that acute hypoxia may trigger neurogenesis which in turn could participate in
brain repair and functional restoration. For this
P
purpose, two series of experiments were designed: in vitro and in vivo. In vitro, embryonic
rat neurons in culture were exposed to hypoxic
episodes of various duration, and cellular mechanisms which influence neuronal outcome were
analyzed. In vivo, we monitored the temporal
profile of brain damage, especially in the vulnerable CA1 subfield of the hippocampus, following
exposure to hypoxic conditions that approximate birth hypoxia.
Materials and Methods
In Vitro Studies on Cultured Neurons
Primary cultured neurons were obtained from
14-day-old rat embryo forebrains as previously
From the *Laboratoire de Biochimie, INSERM EMI 0014, Faculté de
Médecine de Nancy, France; and †Service de Médecine Néonatale,
Maternité Régionale Universitaire Nancy, France.
Address reprint requests to Jean-Luc Daval, PhD, INSERM EMI
0014, Faculté de Médecine, 9 avenue de la Fôret de Haye, 54500
Vandoeuvre-les-Nancy, France e-mail: Jean-Luc.Daval@nancy.
inserm.fr
© 2004 Elsevier Inc. All rights reserved.
0146-0005/04/2804-0000$30.00/0
doi:10.1053/j.semperi.2004.08.002
Seminars in Perinatology, Vol 28, No 4 (August), 2004: pp 257-263
257
258
Daval and Vert
described.14 Cultures were grown for 6 days at
37°C in a humidified atmosphere of 95%
air/5% CO2. Hypoxic conditions were then produced by transferring culture dishes to a humidified and thermoregulated incubation chamber
flushed by a gas mixture corresponding to 95%
N2/5% CO2. Hypoxia was achieved for either 3
or 6 hours, and for subsequent analyses, dishes
were then returned for the ensuing 96 hours to
normal atmosphere, whereas matched controls
were constantly maintained under standard normoxic conditions. Reduction in oxygen delivery
to the neurons was scored by measuring O2 content in samples of extracellular medium sheltered from ambient air, by means of a gas analyzer.
Cell morphology was routinely assessed by
phase-contrast microscopic observations. Purity
of neuronal cultures was regularly evaluated by
using monoclonal antibodies against neuronspecific enolase (NSE, a neuronal marker) and
glial fibrillary acidic protein (GFAP, a marker for
glial cells). Cell viability was monitored by
Trypan blue exclusion and by the spectrophotometric method using 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT), according to Hansen and coworkers.15
Morphological hallmarks of apoptosis, necrosis, and mitosis were analyzed by staining fixed
cultured cells with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI, 0.5 ␮g/mL).16
Using this procedure, healthy neurons exhibit
intact round-shaped nuclei with diffuse fluorescence, indicative of homogeneous chromatin.
Necrotic neurons are characterized by highly
refringent smaller nuclei with uniformly dispersed chromatin, while condensation and fragmentation of chromatin lead to shrinked nuclei
in apoptotic neurons.17 Typical morphological
features of mitosis are easily recognizable. Apoptosis was also monitored by studying the immunohistochemical expression of prototypic regulating proteins, such as Bax, Bcl-2, caspase-3, or
p53.14
100% N2, whereas the remaining pups were
taken as controls and exposed for the same time
to 21% O2/79% N2 (a mixture corresponding to
air). The temperature inside the chamber was
adjusted to 36°C to maintain body temperature
in the physiological range. All pups were allowed
to recover for 20 minutes in normoxic conditions, and they were then returned to their
dams. Under such experimental conditions, the
overall mortality was 4% in hypoxic rats, and the
litter size was finally reduced to 10 pups, corresponding to 5 controls and 5 hypoxic rats, for
homogeneity in subsequent investigations. In
some experiments, blood samples were rapidly
withdrawn by decapitation and sheltered from
air for the measurement of pH, pO2, and pCO2.
To evaluate the extent of cell loss resulting
from hypoxia, brain sections of 20 ␮m in thickness were generated at various time intervals at
the level of anterior hippocampus according to
the developing rat brain atlas of Sherwood and
Timiras19; they were fixed and stained with thionin, and morphometric analyses were conducted by means of a microscope coupled to a
computerized image-processing system. Adjacent brain sections were stained with DAPI for
the measurement of cell density by counting cell
nuclei, and for monitoring of apoptosis and necrosis. In addition, the expression level of apoptosis regulating proteins was studied by immunohistochemistry.18
In some experiments, cell proliferation was
evaluated at 20 days postexposure to hypoxia in
the subventricular zone, which is known as a
neurogenic site, including in the adult rodent
brain.20 Rats received daily injections of bromodeoxyuridine (BrdU, 50 mg/kg, IP) for 9
consecutive days before sacrifice. BrdU was subsequently visualized in brain sections by immunohistochemistry. Colabeling experiments were
performed with specific cell markers to identify
the phenotype of newly generated cells.21
In Vivo Studies on Newborn Rats
Effects of In Vitro Hypoxia
To approach the consequences of global brain
hypoxic conditions around the birth period, we
have used a model approximating birth asphyxia
in newborn rats.18 Within 24 hours after birth,
half of the litter was placed for 20 minutes in a
thermostated plexiglass chamber flushed with
When compared with control cultures maintained in normoxia, transient exposure to anaerobic gas mixture routinely reduced by 80% the
partial pressure of oxygen (pO2) in the culture
medium, irrespectively of the duration of the
hypoxic episode.
Results
Neurogenesis after Hypoxia
Following exposure to hypoxia for 6 hours,
neuronal alterations could be first observed by
48 hours after reoxygenation, as previously reported,22,23 and the number of living cells was
finally reduced by 36% at 96 hours (Fig 1). At
this experimental time point, a significant number of cell nuclei stained by the fluorescent dye
DAPI exhibited characteristic apoptosis-related
morphological features, such as condensed
chromatin and apoptotic bodies. The presence
of necrotic cells was also detected, but cell
counts revealed that the percentage of apoptotic
nuclei increased more sharply in response to the
insult (Fig 2, upper panel).
In contrast, a 3-hour episode of hypoxia apparently did not damage cultured neurons, and
even increased significantly (14%, P ⬍ 0.01) the
viability score over control values at 96 hours
postreoxygenation (Fig 1). Accordingly, hypoxia
for 3 hours led to an elevated number of characteristic mitotic nuclei, as depicted by DAPI
labeling (Fig 2, upper panel). It is noteworthy
that hypoxia for 3 hours was not followed by a
significant increase of the final proportion of
glial cells (data not shown), suggesting that increased mitotic activity likely reflects neuronal
proliferation.
As shown in Fig 2 (lower panel), immunohistochemical studies revealed detectable baseline
levels of the prototypic apoptosis-related proteins Bcl-2 and Bax in control cultures. Consistently with morphological observations, the
Figure 1. Evolution of cell viability in cultured neurons exposed to hypoxia for either 6 hours (H6) or 3
hours (H3) as compared with matched controls maintained in normoxic conditions. Data are expressed as
means ⫾ standard deviations (SD) and were obtained
from five separate experiments. Statistically significant differences from controls: **, P ⬍ 0.01 (Dunnett’s test for multiple comparisons).
259
Figure 2. (Upper panel) Proportions of necrosis,
apoptosis, and mitosis in cultured neurons at 96 hours
posthypoxia. Morphological hallmarks were scored in
the different culture sets following nuclear incorporation of DAPI and subsequent cell counts. Data were
obtained from at least five separate experiments and
are expressed as means ⫾ SD. Statistically significant
differences from controls: *, P ⬍ 0.05; **, P ⬍ 0.01
(Dunnett’s test). (Lower panel) Evolution of the Bax/
Bcl-2 protein ratio in cultured neurons subjected to
hypoxia for either 6 hours (H6) or 3 hours (H3) and
their controls. Immunohistochemical studies were
performed at 1 hour after the onset of hypoxia and
then at 48 and 96 hours postreoxygenation as well as
in matched controls. Fluorescence activity was computerized by means of Adobe Photoshop software,
and the protein ratio was calculated at each time
point. Data are means ⫾ SD and were obtained from
three separate experiments. Statistically significant
differences from controls: *, P ⬍ 0.05; **, P ⬍ 0.01
(Dunnett’s test).
Bax/Bcl-2 ratio tended to increase as a function
of time in response to hypoxia for 6 hours. This
phenomenon was linked to a temporally regulated robust expression of Bax, a process indicative of spreading apoptotic cell death. When
the hypoxic stress was imposed for 3 hours, the
protein ratio progressively declined, due to an
increased expression of the antiapoptotic Bcl-2
protein. Although not illustrated, cultured neurons subjected to hypoxia for 3 hours also
strongly expressed various regulatory components of the cell cycle, including PCNA (proliferative cell nuclear antigen) and Rb (retinoblastoma susceptibility gene product).14
260
Daval and Vert
Effects of In Vivo Hypoxia
In vivo exposure of newborn rats to N2 for 20
minutes appeared to mimic birth asphyxia in
human infants, eliciting a pronounced hypoxemia (11.8 ⫾ 4.8 versus 60.7 ⫾ 7.4 mm Hg
(mixed blood), P ⬍ 0.01) associated with hypercapnia (130.5 ⫾ 6.9 versus 35.6 ⫾ 7.3 mm Hg,
P ⬍ 0.01) and subsequent acidosis (6.62 ⫾ 0.03
versus 7.40 ⫾ 0.03, P ⬍ 0.01) which persisted
after reoxygenation. In our model, neonatal
hypoxia induced a long-lasting reduction of
body weight, and only a transient alteration of
brain weight.18
Cell damage was repeatedly observed in various brain regions of hypoxic rats, including
those which are known to be particularly sensitive to oxygen supply, such as the cerebral cortex
and the CA1 subfield of the hippocampus. Fig 3
(upper panel) shows that cell density was progressively reduced after hypoxia in the CA1 hippocampus. The decline was maximal around 6-7
days postreoxygenation, reaching 27% as compared with controls. Moreover, histopathological studies showed the accumulating presence of
neurons with nuclear condensation, clumping,
and fragmentation into spheric entities corresponding to apoptotic bodies. Immunohistological analyses and cell counts revealed that NSEpositive cells were predominantly affected (data
not shown), suggesting that the reduction of cell
density mostly reflects the loss of neurons. Bcl-2
was transiently overexpressed at 3 days postinsult, and its expression gradually declined thereafter, to be reduced by 38% by comparison to
matched controls at 13 days. Bax expression was
similar to controls at 3 days, and then markedly
augmented in response to hypoxia, leading to a
significant elevation of the Bax/Bcl-2 protein
ratio at 13 days postinsult (Fig 3, lower panel).
However, time course studies showed that the
observed cell deficits were followed by a gradual
recovery, starting from day 20 after reoxygenation (Figs 3 and 4), suggesting the existence of
compensatory repair processes. As illustrated in
Fig 5, further investigations by measuring brain
incorporation of BrdU revealed the accumulation of proliferating cells in the subventricular
zone of hypoxic rats at 20 days postexposure.
Newly generated cells tended to migrate along
the posterior periventricle toward the hippocampus.21 Moreover, the use of specific cell
Figure 3. (Upper panel) Influence of in vivo hypoxia for 20 minutes on subsequent cell density in the
CA1 layer of the rat hippocampus. At various time
intervals, total number of cells per mm2 was measured
using an ocular grid of 1/400 mm2 in control and
hypoxic rats after nuclear staining by DAPI. Data were
obtained from three to five separate experiments and
are expressed as percentages of change from matched
controls (means ⫾ SD). Statistically significant differences from controls: **, P ⬍ 0.01 (Dunnett’s test).
(Lower panel) Evolution of the Bax/Bcl-2 protein
ratio in the CA1 hippocampal cell layer of rats exposed to neonatal hypoxia and their controls. Immunohistochemical studies were performed at 3, 6, and
13 days posthypoxia as well as in control rat pups. The
data represent mean values (⫾ SD) obtained from
three separate experiments. Statistically significant
differences from controls: **, P ⬍ 0.01 (Dunnett’s
test).
markers showed that BrdU-positive cells expressed the neuronal marker NeuroD.
Discussion
The different steps of our studies demonstrate a
similar process of cell division after a hypoxic
episode both in vitro, on embryonic rat neurons,
and in vivo, in the newborn rat.
In vitro, a significant number of cultured cells
died in response to hypoxia for 6 hours, mainly
through an apoptotic process, as previously documented.11,14,24 Apoptosis was also shown to pre-
Neurogenesis after Hypoxia
261
signals for cell cycle activation and cell cycle
arrest.28,29 Accordingly, a treatment by a cell cycle inhibitor has been shown to protect neurons
from hypoxia-induced injury.14,23 Coupled to
our previous data,14,23 our observations suggest
that mild hypoxia is able to trigger neuronal
proliferation by stimulating the expression of
neurogenic and survival-associated proteins, including proliferating cell nuclear antigen
(PCNA) and Bcl-2.
In the newborn rat, the observation of neurogenesis induced by hypoxia in the germinative
subventricular zone confirms previous studies in
adult animals where generation of new neurons
occurs not only as a transient repair mechanism
but appears to be a continuous phenomenon
over lifespan.30,31 The new neurons can migrate
to the CA1 layer of the hippocampus where they
seem to integrate the hippocampal circuitry.32
Neurogenesis has also been reported in the cerebral cortex.33
Figure 4. Evolution of cell density in the CA1 hippocampus of control and hypoxic rats as illustrated by
DAPI nuclear staining at 6 and 20 days postexposure
to hypoxia (⫻40 magnification). Similar profiles were
observed in three separate experiments.
dominantly account in vivo for CA1 neuronal
loss in the newborn rat brain. It is now widely
accepted that this kind of cell death is a key
event in delayed neuronal injury consecutive to
severe hypoxia, especially in the developing
brain, which retains a part of the physiological
cell death program involved in normal development.25-27 Beyond characteristic morphological
hallmarks of dying cells, apoptotic death is reflected in our experimental models by temporal
changes in selective proteins, such as Bax and
Bcl-2. Indeed, apoptosis can be distinguished
from necrosis in that cell death is directly dependent on the regulation of specific genes, and it is
the balance in expression of pro- (eg, bax) and
antiapoptotic (eg, bcl-2) genes that determines
the fate of neurons exposed to hypoxia.14,27 In
the case of nonlethal hypoxia (3 hours in our
culture model), overexpression of defense proteins, which include Bcl-2, promotes cell survival. Nevertheless, a strong relationship between apoptosis and the cell cycle has been well
documented. Apoptosis has been described as
an abortive re-entry into the cell cycle, a process
that would be due to abnormal or conflicting
Figure 5. Characterization of cell proliferation in
the rat subventricular zone at 20 days postexposure to
hypoxia. Rats received daily injections of BrdU (50
mg/kg, IP) for 9 consecutive days before sacrifice.
BrdU was subsequently visualized in brain sections by
immunohistochemistry. Colabeling experiments with
specific cell markers showed that BrdU-positive cells
expressed a neuronal marker, ie, NeuroD (LV, lateral
ventricle). Similar observations were made in six separate experiments.
262
Daval and Vert
The neurogenetic process was observed after
an apoptotic phase, suggesting a relationship
between these two processes. But, in a more
recent study, Pourié and coworkers34 showed
that, in the newborn rat, the process of neurogenesis can be triggered after a 5-minute hypoxia which does not induce apoptosis. In this
study, the neuronal phenotype of newly generated cells was shown to be preceded by a transient glial phenotype.
The mechanisms involved in the postnatal
neurogenesis are not clearly understood. It has
been suggested that neurotrophic factors like
IGF-1, EGF, FGF-2, NGF, or erythropoietin play
a significant role, but we still lack the precise
triggering factor.12 Among factors involved, adrenal steroids have been shown to regulate neurogenesis in the adult dentate gyrus via N-methylD-aspartate (NMDA) receptors35; this may
explain the detrimental effect of pre- or postnatal stress on learning deficits, along with an inhibition of hippocampal neurogenesis both in
rats and in monkeys.36,37 Also, the fate of the
newly generated neurons is not yet fully elucidated. Studies have shown that, after a period of
neurogenesis, the new neurons do persist in the
hippocampus of adult rodents,33 and Pourié and
coworkers34 have observed that the new neurons
express the synaptic protein synapsin 1, suggesting that they may be functional.
There are some concerns about the migration
of new neurons in several structures, considering that they might not integrate adequately the
architectural structures and be the cause of aberrant networks somewhat like in the cortical
heterotopia observed in the fetal alcoholism syndrome.38
Posthypoxic neurogenesis might account for
a process of cerebral protection involving mechanisms totally different from those involved either in preconditioning tolerance39 or in the
preventive effect of hypothermia on brain damage. However, it should be noted that some
experiments on preconditioning-induced tolerance showed a posthypoxic neuronal proliferation in vitro.40 The neurogenesis observed in our
experiments might also be viewed as a mechanism of cerebral plasticity, like in the hypertrophy of controlateral brain regions after hemispherectomy in the rat.41
Gathering our results with similar data obtained in other models by several authors,12,13,42
they challenge the fundamental dogma thought
for decades, and stating that the central nervous
system could never be the site of neuronal division after the fetal period, or at least beyond the
early postnatal period.
To what extent these promising experimental
results apply to the human newborn brain remains questionable. But, at least it has been
shown that postnatal neurogenesis is a persisting
phenomenon in humans.43,44 Until demonstrated as damageable, the observation of a posthypoxic neurogenesis may be accepted as good
news, supporting the concept of the beneficial
effects of early psychomotor intervention in infants at risk for the sequelae of an acute brain
hypoxia-ischemia. Indeed, it has been documented that a stimulated environment has positive consequences on neurobehavioral performances45-47 and that learning affects the cell
survival in the adult rat dentate gyrus.48
Acknowledgments
We would like to thank Carine Bossenmeyer-Pourié,
Grégory Pourié, Valérie Lièvre, and Stéphanie
Grojean for having performed a large part of the
reported experiments.
References
1. Rivkin MJ, Volpe JJ: Asphyxia and brain injury, in Spitzer
AR (ed): Intensive Care of the Fetus and Neonate. St.
Louis, MO, Mosby, 1994, pp 685-695
2. Nyakas C, Buwalda B, Luiten PGM: Hypoxia and brain
development. Prog Neurobiol 49:1-51, 1996
3. Maneru C, Junque C, Botet F, et al: Neuropsychological
long-term sequelae of perinatal asphyxia. Brain Inj 15:
1029-1039, 2001
4. Vannucci R: Experimental biology of cerebral hypoxiaischemia: Relation to perinatal brain damage. Pediatr
Res 27:317-326, 1990
5. Haddad GG, Jiang C: O2 deprivation in the central
nervous system: On mechanisms of neuronal response,
differential sensitivity and injury. Prog Neurobiol 40:277318, 1993
6. Mishra OP, Delivoria-Papadopoulos M: Cellular mechanisms of hypoxic injury in the developing brain. Brain
Res Bull 48:233-238, 1999
7. Stattford A, Wheatherhall JAC: The survival of young rats
in nitrogen. J Physiol 153:457-472, 1960
8. Haddad GG, Donelly DF: O2 deprivation induces a major depolarization in brainstem neurons in the adult but
not in the neonate. J Physiol Lond 429:411-428, 1990
9. Bower AJ: Plasticity in the adult and neonatal central
nervous system. Br J Neurosurg 4:253-264, 1990
10. Black JE: How a child builds its brain: Some lessons from
animal studies of neural plasticity. Prev Med 27:168-171,
1998
Neurogenesis after Hypoxia
11. Gubellini P, Ben-Ari Y, Gaiarsa JL: Activity-and age-dependent GABAergic synaptic plasticity in the developing
rat hippocampus. Eur J Neurosci 14:1937-1946, 2001
12. Kokaia Z, Lindvall O: Neurogenesis after ischaemic
brain insults. Curr Opin Neurobiol 13:127-132, 2003
13. Parent JM: Injury-induced neurogenesis in the adult
mammalian brain. Neuroscientist 9:261-272, 2003
14. Bossenmeyer-Pourié C, Lièvre V, Grojean S, et al: Sequential expression patterns of apoptosis- and cell cyclerelated proteins in neuronal response to severe or mild
transient hypoxia. Neuroscience 114:869-882, 2002
15. Hansen MB, Nielsen SE, Berg K: Re-examination and
further development of a precise and rapid dye method
for measuring cell growth/cell kill. J Immunol Meth
119:203-210, 1989
16. Wolvetang EJ, Johnson KL, Krauer K, et al: Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS
Lett 339:40-44, 1994
17. Park DS, Morris EJ, Greene LA, et al: G1/S cell cycle
blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis. J Neurosci 17:1256-1270, 1997
18. Grojean S, Schroeder H, Pourié G, et al: Histopathological alterations and functional brain deficits after transient hypoxia in the newborn rat pup: A long term
follow-up. Neurobiol Dis 14:265-278, 2003
19. Sherwood NM, Timiras PS: A Stereotaxic Atlas of the
Developing Rat Brain, Berkeley, CA, University of California Press, 1970
20. Alvarez-Buylla A, Garcia-Verdugo JM: Neurogenesis in
adult subventricular zone. J Neurosci 22:629-634, 2002
21. Daval JL, Pourié G, Grojean S, et al: Neonatal hypoxia
triggers transient apoptosis followed by neurogenesis in
the rat CA1 hippocampus. Pediatr Res 55:561-567, 2004
22. Bossenmeyer C, Chihab R, Muller S, et al: Hypoxia/
reoxygenation induces apoptosis through biphasic induction of protein synthesis in central neurons. Brain
Res 787:107-116, 1998
23. Bossenmeyer-Pourié C, Chihab R, Schroeder H, et al:
Transient hypoxia may lead to neuronal proliferation in
the developing mammalian brain: From apoptosis to cell
cycle completion. Neuroscience 91:221-231, 1999
24. Chihab R, Ferry C, Koziel V, et al: Sequential activation
of activator protein-1-related transcription factors and
JNK protein kinases may contribute to apoptotic death
induced by transient hypoxia in developing brain neurons. Mol Brain Res 63:105-120, 1998
25. Beilharz EJ, Williams CE, Dragunow M, et al: Mechanisms of delayed cell death following hypoxic-ischemic
injury in the immature rat: Evidence for apoptosis during selective neuronal loss. Mol Brain Res 29:1-14, 1995
26. Sidhu RS, Tuor UI, Del Bigio MR: Nuclear condensation
and fragmentation following cerebral hypoxia-ischemia
occurs more frequently in immature than older rats.
Neurosci Lett 223:129-132, 1997
27. Banasiak KJ, Xia Y, Haddad GG: Mechanisms underlying
hypoxia-induced neuronal apoptosis. Prog Neurobiol.
62:215-249, 2000
28. Meikrantz W, Schlegel R: Apoptosis and the cell cycle.
J Cell Biol 58:160-174, 1995
29. Timsit S, Rivera S, Ouaghi P, et al: Increased cyclin D1 in
vulnerable neurons in the hippocampus after ischaemia
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
263
and epilepsy: A modulator of in vivo programmed cell
death? Eur J Neurosci 11:263-278, 1999
Hastings NB, Tanapat P, Gould E: Neurogenesis in the
adult mammalian brain. Clin Neurosci Res 1:175-182, 2001
Lie DC, Song H, Colamarino SA, et al: Neurogenesis in
the adult brain: new strategies for central nervous system
diseases. Annu Rev Pharmacol Toxicol 44:399-421, 2004
Nakatomi H, Kuriu T, Okabe S, et al: Regeneration of
hippocampal pyramidal neurons after ischemic brain
injury by recruitment of endogenous neural progenitors. Cell 110:429-441, 2002
Magavi SS, Leavitt BR, Macklis JD: Induction of neurogenesis in the neocortex of adult mice. Nature 405:951955, 2000
Pourié G, Blaise S, Lièvre V, et al: Non-lesioning neonatal hypoxia can induce long-term neurogenesis in the rat
brain. Pediatr Res 55:25A, 2004
Gould E, Tanapat P, Cameron HA: Adrenal steroı̈ds
suppress granule cell death in the developing dentate
gyrus through an NMDA receptor-dependent mechanism. Brain Res Dev Brain Res 103:91-93, 1997
Lemaire V, Koehl M, Le Moal M, et al: Prenatal stress
produces learning deficits associated with an inhibition
of neurogenesis in the hippocampus. Proc Natl Acad Sci
USA 97:1132-1137, 2000
Gould E, Tanapat P, McEvens BS, et al: Proliferation of
granule cell precursors in the dentate gyrus of adult
monkeys is dimished by stress. Proc Natl Acad Sci USA
95:3168-3171, 1998
Miller MW: Migration of cortical neurons is altered by
gestational exposure to ethanol. Alcohol Clin Exp Res
17:304-314, 1993
Gidday JM, Fitzgibbons JC, Shah AR, et al: Neuroprotection from ischemic brain injury by hypoxic preconditioning in the neonatal rat. Neurosci Lett 168:221-224, 1994
Bossenmeyer-Pourié C, Koziel V, Daval JL: Effects of
hypothermia on hypoxia-induced apoptosis in cultured
neurons from developing rat forebrain: Comparison
with preconditioning. Pediatr Res 47:385-391, 2000
Huttenlocher PR, Raichelson RM: Effects of neonatal
hemispherectomy on location and number of corticospinal neurons in the rat. Dev Brain Res 47:59-69, 1989
Liu J, Solway K, Messing RO, et al: Increased neurogenesis in the dentate gyrus after transient global ischemia
in gerbils. J Neurosci 18:7768-7778, 1998
Del Bigio MR: Proliferative status of cells in adult human
dentate gyrus. Microsc Res Tech 45:353-358, 1999
Eriksson PS, Perfilieva E, Björk-Eriksson T, et al: Neurogenesis in the adult human hippocampus. Nat Med
4:1013-1017, 1998
Kempermann G, Kuhn HG, Gage FH: More hippocampal neurons in adult mice living in an enriched environment. Nature 386:493-495, 1997
Rema V, Ebner FF: Effect of enriched environment rearing on impairments in cortical excitability and plasticity
after prenatal alcohol exposure. J Neurosci 19:1099311006, 1999
Risedal A, Mattsson B, Dahlqvist P, et al: Environmental
influences on functional outcome after a cortical infarct
in the rat. Brain Res Bull 58:315-321, 2002
Ambrogini P, Cuppini R, Cuppini C, et al: Spatial learning affects immature granule cell survival in adult rat
dentate gyrus. Neurosci Lett 286:21-24, 2000