1 Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch,
Robert-Rössle-Str. 10, 13125 Berlin, Germany
2 VolkswagenStiftung Research Group, Dept. of Experimental Neurology,
Charité University Hospital, Humboldt University, Schumannstr. 20/21,
10117 Berlin, Germany
3 Department of Psychiatry, Freie Universität Berlin, Eschenallee 3, 14050
Berlin, Germany
4 Department of Physiology, Graduate School of Medicine, University of Tokyo,
Bunkyo-ku, Tokyo 113-0033, Japan
5 The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 North
Torrey Pines Rd., La Jolla, CA 92037, USA
* Author for correspondence (e-mail: gerd.kempermann{at}mdc-berlin.de)
Accepted 11 October 2002
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SUMMARY |
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Key words: Stem cell, Progenitor cell, Adult neurogenesis, Mouse
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INTRODUCTION |
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Neurogenesis is defined as the series of developmental steps that leads from the division of a neural stem or progenitor cell to a mature, functionally integrated neuron. Therefore, quantification of `neurogenesis' and its regulation is influenced by the time, at which it is measured during this development.
Some studies have only assessed cell proliferation as a measure of
neurogenesis; however, cell proliferation in the subgranular zone (SGZ) also
leads to gliogenesis and angiogenesis
(Palmer et al., 2000) and most
of the new cells die (Biebl et al.,
2000
; Kempermann and Gage,
2002b
; Kempermann et al.,
1997a
). Investigation of several time-points after cell division
is required to obtain a reasonable estimate of neurogenesis. Most studies on
adult hippocampal neurogenesis have followed the new cells for about 4 weeks
after division. This survival time is sufficient for the cells to become
recognizable as neurons or glial cells by immunohistochemistry. Van Praag et
al. (Van Praag et al., 2002
)
showed that the electrophysiological properties of the new granule cells are
indistinguishable from the older cells, but also found evidence that full
maturation might take additional weeks or even months.
In addition, the answer to the question of whether the new neurons are
generated transiently or persistently influences functional interpretations of
adult neurogenesis. There is good, although still correlational evidence that
functional stimuli promote the survival of new hippocampal neurons
(Gould et al., 1999;
Kempermann et al., 1997b
;
Nilsson et al., 1999
). We
theorize that the functional role of adult hippocampal neurogenesis lies in
enabling the hippocampus to cope better with novelty and to adjust the dentate
gyrus to processing new and greater levels of complexity
(Kempermann, 2002b
). Adult
neurogenesis cumulatively and strategically adds to the neuronal network in
the dentate gyrus, in order to optimize its functionality at the smallest
possible size (Kempermann,
2002b
). This hypothesis is supported by the fact that the
genetically determined baseline levels of adult hippocampal neurogenesis
correlate with the acquisition of the water-maze task, but not with the recall
of the newly learned information
(Kempermann and Gage, 2002a
).
Several studies indicate that the total number of granule cells increases over
the first year of life in a rodent (Bayer,
1985
; Boss et al.,
1985
). Consequently, our morphological hypothesis is that adult
hippocampal neurogenesis leads to a fast, stable and lasting integration of
new neurons.
To test this hypothesis, we labeled dividing cells with the thymidine analog bromodeoxyuridine (BrdU) and studied the numbers of BrdU-labeled cells in the dentate gyrus at various time-points after division in an attempt to assess a time-course of cellular survival, migration and differentiation. We analyzed the distribution of phenotypes among the newly generated cells in order to understand better at what ratio transiently expressed neuroectodermal markers give way to markers associated with cellular maturation.
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MATERIALS AND METHODS |
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BrdU injections
For 12 consecutive days, all mice in the first set received one daily
intraperitoneal injection of 10 mg/ml BrdU (5-bromo-2-deoxyuridine; Sigma) in
sterile 0.9% NaCl solution (daily dose: 50 µg/g body weight). From the
first set of mice, 5 animals each were perfused 1 day, 3 days, 7 days, 4
weeks, 3 months, 6 months and 11 months after the last injection of BrdU, as
described below. The transgenic animals in set 2 received two injections of
BrdU (50 µg/g body weight) 6 hours apart and were perfused 2 hours or 24
hours later.
Tissue preparation
The mice were killed with an overdose of ketamine and perfused
transcardially with 4% paraformaldehyde in cold 0.1 M phosphate buffer. The
brains were stored in the fixative overnight and then transferred into 30%
sucrose. One day later, 40 µm coronal sections were cut from a dry
ice-cooled block on a sliding microtome (Leica). The sections were stored at
-20°C in cryoprotectant containing 25% ethylene glycol, 25% glycerin and
0.05 M phosphate buffer.
Antibodies
All antibodies were diluted in TBS containing 0.1% Triton X-100, 0.05%
Tween 20 and 3% donkey serum (TBS-plus).
The primary antibodies used in this study were: monoclonal rat anti-BrdU (Harlan Seralab), 1:500; monoclonal mouse anti-NeuN (Chemicon), 1:100; monoclonal mouse anti-doublecortin (Santa Cruz); monoclonal mouse anti-ß-III-tubulin (Promega) and polyclonal rabbit-anti S100ß (SWant, Bellinzona, Switzerland), 1:2000; and polyclonal rabbit-anti GFP (Abcam), 1:400.
For indirect immunofluorescence the following secondary antibodies were used (all 1:250): donkey anti-rabbit IgG (Jackson) conjugated with CY5 or FITC; donkey anti-mouse IgG (Jackson) conjugated with FITC or CY5; and goat anti-rat IgG (Jackson) conjugated with Rhodamine-X. For immunohistochemistry with the peroxidase technique, biotinylated donkey anti-mouse IgG (Jackson) (1:250) was used as secondary antibody and detected with avidinbiotin-peroxidase complex (ABC, Vectastain Elite, Vector Laboratories) (9 µl/ml).
Pretreatment for BrdU immunohistochemistry
After quenching endogenous tissue peroxidases with 0.6%
H2O2 in TBS for 30 minutes the sections were incubated
in 2 N HCl for 30 min at 37°C and washed in 0.1 M borate buffer (pH 8.5)
for 10 minutes.
Immunohistochemistry
For light microscopic quantification of BrdU-labeled cells a series of
every sixth 40 µm section was used. After BrdU pretreatment (see above) and
washing in TBS, sections were blocked in TBS-plus with 3% horse serum for 1
hour, followed by incubation in primary antibody in TBS-plus overnight at
4°C. After rinses in TBS, the sections were incubated in the secondary
antibody in TBS-plus for 4 hours at room temperature. After another set of
rinses, ABC Elite reagent (Vector Laboratories) was applied for 1 hour. As
substrate for the peroxidase reaction, diaminobenzedine (DAB, Sigma) was
applied for 5 minutes at a concentration of 0.25 mg/ml in TBS with 0.01%
hydrogen peroxide and 0.04% nickel chloride. Sections were thoroughly washed,
mounted, air dried and coverslipped.
Immunofluorescence
For immunofluorescent triple-labeling of BrdU, NeuN and S100ß; BrdU,
ßIII-tubulin and S100ß; and of BrdU, doublecortin (DCX) and NeuN
every twelfth section throughout the dentate gyrus was used. After
pretreatment (see above) and a blocking step with TBS-plus containing 3%
donkey serum, sections were incubated in a mixture of the three antibodies of
each series for 36 hours at 4°C. After washing in TBS and TBS-plus, a
cocktail of secondary antibodies (Rhodamine X to detect BrdU, FITC for NeuN,
DCX, and ß-III-tubulin, and CY5 for S100ß and NeuN) was applied for
4 hours at room temperature. Sections were washed again, mounted and
coverslipped in polyvinyl alcohol with diazabicyclo-octane (DABCO) as an
anti-fading agent.
Fluorescent signals were detected using a confocal laser scanning microscope (Leica TCS SP2). For each series the phenotypes of 50 BrdU-labeled cells per animal were determined. Images were processed with Adobe Photoshop 6.0 (Adobe Systems).
Quantification
Sampling of BrdU-positive cells was done exhaustively throughout the GCL in
its rostrocaudal extension. Cells were categorized according to their
localization in the dentate gyrus of both sides (see
Fig. 1B for details; numbers
for set 2 animals represent the left hemisphere only). As BrdU-labeled cells
are comparatively rare, the stereological procedure, described elsewhere
(Kempermann et al., 1997a;
Williams and Rakic, 1988
), was
modified to exclude the uppermost focal plane only. The resulting number of
BrdU-positive cells was then multiplied by 6 (because every sixth section had
been used) to give an estimate of the total number of BrdU-positive cells.
Statistical analyses
All statistical analyses were performed with Statview 4.5.1 for Macintosh.
Factorial ANOVA was performed for all comparisons of morphological data
followed by Fisher post hoc test, where appropriate.
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RESULTS |
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Only limited differences in the regional distribution of new cells in
the dentate gyrus over time
BrdU-positive cells were categorized as to whether they were found in the
left or right hippocampus and whether they were located in the dorsal or the
ventral blade of the dentate gyrus (Fig.
1B). Table 2 shows
that, with regard to the number of BrdU-labeled cells 1 day after the last
injection of BrdU, there was no difference between the left and right brain,
but significantly more BrdU-labeled cells were found in the dorsal than the
ventral blade of the dentate gyrus. If analyzed at each individual time-point
these difference were only significant at some of the time-points (3 months,
P=0.0156; 6 months, P=0.0433) and had low P values
at two additional time-points (1 day, P=0.0890; 4 weeks,
P=0.0622), suggesting high variability and limited immediate
relevance.
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Migration of BrdU-labeled cells ends early after division
Migration of BrdU-labeled cells was assessed by categorizing the cells
according to their position within the GCL. We compared the numbers of labeled
cells in (1) the SGZ plus the inner third of the GCL, (2) the mid third of the
GCL and (3) the outer third of the GCL
(Fig. 1C). The distribution of
BrdU-labeled cells in the SGZ and the GCL shows a discrete shift from the SGZ
towards the mid third of the GCL during the first 4 weeks. Significant
differences between the percentage of cells in the SGZ/inner third of the GCL
and the mid and outer third were found with decreasing frequency at 1, 3 and 7
days after BrdU injection (Fig.
3, Table 3). This
finding implies that after the first few weeks the surviving cells do not
substantially change their position in the GCL.
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Not all cells expressing immature neuronal markers become fully
mature neurons, but the final number of new neurons is determined early
Young immature neurons express markers such as ß-III-tubulin
(Geisert and Frankfurter,
1989; Menezes and Luskin,
1994
) or doublecortin (DCX)
(Cooper-Kuhn and Kuhn, 2002
;
des Portes et al., 1998
;
Gleeson et al., 1998
). As Figs
2,
4 illustrate, BrdU-labeled
cells can show a co-localization with these markers soon after the last
injection of BrdU. These numbers reach 46.5±5.6% and 2083±286 in
absolute numbers (ß-III-tubulin) and 47.5±6.9% and 1629±672
(DCX), but decline thereafter (all numbers are means±s.e.m.). At 3 or 6
months after BrdU injection, only occasional cells co-labeled for BrdU and
either of these markers could be found. As these double-labeled cells were so
rare, they were not explicitly quantified and no data points representing them
were included in Fig. 2.
|
NeuN was used as a marker for mature neurons
(Mullen et al., 1992).
Interestingly, BrdU/NeuN labeled cells could already be found at 1 day after
the last injection of BrdU. Subsequently, the relative number of new neurons
(BrdU+/NeuN+) remained stable over the period investigated (Figs
2,
4). In other words, the total
number of BrdU/NeuN double-positive cells did not decrease either with the
total number of BrdU-labeled cells or with those showing a colocalization with
immature neuronal markers. By contrast, BrdU-labeled astrocytes
(BrdU+/S100ß+) decreased and were almost absent at the 11 months time
point.
Although the 12-day injection period increases the number of labeled cells that could be studied, it also reduced the temporal resolution at the early time-points after BrdU injection. The time-point `1 day' after the last injection of BrdU actually reflects a range of 1 to 12 days after BrdU injection. To study an early time point after BrdU injection with higher precision, a second set of mice that allows the immediate recognition of cells with progenitor cell properties was used.
Nestin is an intermediate filament that is expressed in neural stem or
progenitor cells and thus can to some degree serve as a progenitor cell marker
(Lendahl et al., 1990;
Reynolds et al., 1992
). In
transgenic mice expressing green fluorescent protein (GFP) under the nestin
promoter, nestin-expressing cells and thus presumable stem or progenitor cells
can be visualized efficiently (Sawamoto et
al., 2001
; Yamaguchi et al.,
2000
). These mice received only two injections of BrdU and were
perfused 2 and 24 hours later. As detection of GFP is strongly reduced by the
treatment with HCl, which is necessary for BrdU immunohistochemistry, we used
an antibody against GFP to identify nestin-expressing cells. We found that
with this method, 24 hours after BrdU injection, 55% of the BrdU-positive
cells were nestin-GFP positive (Fig.
4). Relying on direct GFP emission or using antibodies against
nestin led to many fewer double- or triple-labeled cells (not shown).
Importantly, at 24 hours after BrdU injection none of the BrdU-positive cells
were NeuN positive (Fig.
4).
Two hours after two injections of BrdU, 7.2±1.7% of the BrdU-labeled nestin-GFP expressing cells were also DCX positive (Fig. 4), further suggesting that cell fate decisions towards neuronal development are made extremely soon after division. At 24 hours after BrdU injection, the number of BrdU/DCX/nestin-GFP triple-labeled cells had increased to 24.8±6.4%, indicating that DCX expression increases over time. By contrast, it is difficult to assess at what time-point nestin itself is cleared from the maturing cells, because it is not known how long the reporter gene product remains detectable after the reporter gene promoter is turned off. However, as illustrated in Fig. 4, the immunohistochemical signal for nestin-GFP expression was consistently lower in BrdU-positive cells expressing DCX than in nestin-GFP positive cells that were positive for BrdU only. This finding might be indicative of either the clearance of nestin itself or at least of nestin-GFP from the cells that are in the process of acquiring a neuronal phenotype.
We also investigated whether DCX-immunoreactive cells would show double labeling for nestin-GFP. We found that of 250 DCX-positive cells counted separately in five animals 28.4±2.9% displayed nestin-GFP co-staining whereas only 1.2±1.2% were positive for NeuN. Importantly, NeuN never co-localized with nestin-GFP.
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DISCUSSION |
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Our previous research has demonstrated that functional stimuli, such as
exposure to a challenging complex environment, increase the number of new
neurons by means of a survival-promoting effect
(Kempermann et al., 1997b;
Kempermann et al., 1998
).
Presumably this effect would occur very soon after division. We hypothesize
that a surplus of immature neuronal cells provides a pool of neurogenic
potential from which appropriate functional stimuli, such as environmental
complexity or learning stimuli, can recruit more new neurons, resulting in the
net increase in neurogenesis seen in these other studies
(Gould et al., 1999
;
Kempermann et al., 1997b
;
Kempermann et al., 1998
). This
sensitive `neurogenic window' might range from a stage at which cells still
express nestin only and are undergoing division to a later stage where
immature neuronal markers have been expressed.
Our present knowledge about the exact sequence of neuronal development in adult neurogenesis remains limited. Several steps, such as cell proliferation, survival, expression of neuronal markers, neuritogenesis, synaptogenesis, etc., can be conceptually identified. However, no longitudinal examination is possible in an in vivo study. All examinations are snapshots in a continuum of largely unknown kinetics. Still, our present data do suggest that the fate choice decision for new neurons is made earlier than previously assumed and that this decision has lasting consequences.
BrdU is a proliferation marker that, in contrast to techniques based on the immunohistochemical identification of cell cycle-related proteins, captures the time point when the compound is circulating in the blood stream, not conditions at the time-point when the animal is killed. This `birth-dating' property allows us to identify new postmitotic cells that had divided at the known time of BrdU injection. Because NeuN is confirmed as a truly postmitotic marker here (no BrdU+/NeuN+ cells were found 24 hours after 2 injections of BrdU), we can conclude that for those cells that are recruited to become long-lasting neurons the step to a postmitotic state is made soon after division.
The first time-point investigated here, 1 day after the last injection of
BrdU, can be considered as a gross estimate of `proliferative activity' in the
SGZ. However, this attribution has several limitations
(Hayes and Nowakowski, 2002;
Nowakowski et al., 1989
). With
regard to the size of the proliferating cell population, the cumulative
application of BrdU leads to an overestimate. One reason for this lies in the
kinetics of BrdU incorporation in relation to cell cycle parameters. Another
reason is that spreading out BrdU injections over several days results in a
reduction in temporal resolution. Numbers at `day 1' reflect not only cell
proliferation but also to some degree cellular survival. They are also
influenced by effects such as dilution of the label due to continued
divisions. With the exception of the data from the nestin-GFP transgenic
animals that received only two pulses of BrdU within 6 hours and were killed 2
hours and 24 hours later (Set 2; Fig.
1A), all BrdU counts in the present study refer to cells that have
exited the cell cycle within the period of 12 days during which BrdU was
injected.
The detection of NeuN in BrdU-positive cells at early time-points after
BrdU injection does not imply that mature neurons would divide, because if the
neuronal phenotype is determined at 24 hours after BrdU injection, no
BrdU/NeuN double-labeled cells were found. This observation is in accordance
with data from a study by Palmer et al.
(Palmer et al., 2000).
The important issue as to whether BrdU incorporation also reflects DNA
repair (at the time when BrdU is available in the blood stream) has been
discussed in greater detail elsewhere
(Cooper-Kuhn and Kuhn, 2002;
Palmer et al., 2000
). Briefly,
the two main arguments against the possibility that numbers of BrdU-labeled
cells such as in our study are heavily confounded by DNA repair are: (1)
irradiation that induces DNA strand breaks does not lead to an increased
uptake of BrdU in vivo (Parent et al.,
1999
) and in vitro (Palmer et
al., 2000
); and (2) adult neurogenesis can also be detected using
a retroviral labeling, which would not detect DNA repair
(Van Praag et al., 2002
). Our
present study adds another circumstantial argument, because the data suggest a
progression through different markers of neuronal development among the
BrdU-labeled cells that is inconsistent with the detection of DNA repair in
already matured cells.
Many of the newly generated cells die within the first few days after
division (Biebl et al., 2000).
Also, the increase in adult neurogenesis induced by exposure to an enriched
environment found its counterpart in a reduced level of apoptosis
(Kempermann et al., 1997b
;
Young et al., 1999
). These
findings fit with data from embryology, wherein great numbers of neurons are
produced in surplus and those that are not actively selected for function are
eliminated by apoptosis (Blaschke et al.,
1996
). Most, if not all selective processes that are involved in
integrating the new cells into the hippocampal circuits or in eliminating
cells that are not used, apparently take place within the first weeks after
division. We hypothesize that during adult hippocampal neurogenesis, like
during embryogenesis, new but immature neurons are generated in surplus and
are then selected into functional circuits. This hypothesis is supported by
the fact that larger numbers of DCX- and ß-III-tubulin-expressing cells
are generated than are those showing NeuN as a more mature marker. Once the
consolidation process is over, during which time immature markers remain
expressed, the cells become persistently part of the GCL. The net effect is
the stable integration of a comparatively low number of neurons. This might
imply that adult neurogenesis contributes to a lasting alteration of the
hippocampal network, although this addition is small. Our data indicate that
investigating `neurogenesis' at 4 weeks after the application of the
proliferation marker allows a valid estimate of long-term effects. However,
our data also show that it is not sufficient to use immature neuronal markers
such as ß-III-tubulin or DCX as indicators of net neurogenesis.
Nestin-GFP-expressing cells begin to express DCX very early after division and the number of cells that express DCX increases thereafter. The switch from the immature marker DCX to the more mature marker NeuN appears to be swift and, at least in a certain percentage of cells, must occur early. However, the large number of BrdU/DCX-labeled cells that can be found during the first few weeks after division suggests that the time-window during which such a switch can occur is actually much longer. A more detailed future analysis will address this issue.
To date the question of whether or how new neurons in the adult hippocampus
migrate has not been extensively studied. It can easily be demonstrated,
however, that some migration occurs
(Kempermann and Gage, 2002b;
Kuhn et al., 1996
). In the
olfactory system, newly generated cells migrate over a long distance (several
millimeters) through the rostral migratory stream to the olfactory bulb
(Lois et al., 1996
;
Luskin, 1993
). We show that
new neurons in the hippocampus reach their final position very early. The
maximum distance the new cells migrate lies within a range of 50 to 100 µm.
However, many of them do not seem to migrate at all and remain in the SGZ.
For embryonic development a classical concept of an outside-in gradient
exists with the oldest cells at the molecular layer and the younger cells near
the hilus (Altman and Bayer,
1990; Crespo et al.,
1986
). A study by Martin et al., used chimeras to study the clonal
composition of the dentate gyrus and found that the later-born cells clearly
preferred the inner core of the GCL
(Martin et al., 2002
). Our
data relate to this finding in that we demonstrate that, in the adult brain,
the majority of new cells remains confined to the inner third of the GCL.
During adult granule cell development a sequential activation of transcription
factors can be observed, from Mash1 in putative progenitor cells to NeuroD in
young granule cells (Pleasure et al.,
2000
). In this study, stronger NeuroD expression could be found in
the inner half of the GCL, further strengthening the view that later born
granule cells preferentially gather in this region. This study is also
relevant to the current study because it raises the issue of whether the
developmental steps we have seen with regarding to the sequential expression
of proteins (nestin to doublecortin to NeuN) could be related to the
corresponding expression of transcription factors. Therefore, it will be
important to characterize the developmental steps in adult hippocampal
neurogenesis with regard to the underlying molecular events.
From the data obtained by Martin et al.
(Martin et al., 2002) it can
be concluded that different progenitor cell populations form the outer and the
inner shell of the GCL. Presumably, the progenitor cells from which adult
hippocampal neurogenesis originates constitute yet another population of
precursor cells that, as one might expect, relate to the later progenitors of
the inner core rather than to the earlier progenitors of the outer shell.
According to Altman and Bayer (Altman and
Bayer, 1990
) the stem or progenitor cells of the SGZ form a
tertiary germinative center (after the primary matrix at the ventricle wall
and the secondary matrix that forms the outer shell of the GCL).
This organization does not lead to a restricted distribution of new granule
cells, because in our study a considerable number of new neurons could be
found throughout the entire thickness of the adult GCL. Wojtowicz and
co-workers described different electrophysiological properties (as well as
changed morphological features) in cells at different distances from the SGZ
(Wang et al., 2000). Our data
suggest that new neurons migrate very early to a location within the dentate
gyrus and stay there. We propose therefore that the position within the GCL is
meaningful for the function of the new neurons, and further that the
functional differences within the GCL are not a function of cellular age (with
increasing age equaling a greater distance from the SGZ) but of location per
se. Accordingly, new neurons find their functionally appropriate place,
depending on a concrete functional demand.
Our hypothesis is that new neurons are strategically inserted into the
neuronal network of the dentate gyrus, thus enabling the dentate gyrus to
process novel and more complex information
(Kempermann, 2002b). From this
perspective, a fast and lasting integration of the new cells makes sense. But
even a functional integration of new neurons within a few days will most
likely come too late for processing the information that triggered the
neurogenic response. Confrontation with a challenging new situation rather
serves as a trigger to adapt the network of the processor to challenges to
come. In this sense, both young and old animals benefit from the long lasting
integration of new neurons to the dentate gyrus that we have found in the
current study. However, it is equally conceivable that old animals that
already have had more experience (and thus more opportunities to optimize
their hippocampal network for the tasks encountered) can live with reduced
levels of adult hippocampal neurogenesis.
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ACKNOWLEDGMENTS |
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