Journal of Behavioral and Brain Science, 2011, 1, 199-209
doi:10.4236/jbbs.2011.14027 Published Online November 2011 (
Copyright © 2011 SciRes. JBBS
Repeated Physical Training and Envir onmental E nrichment
Induce Neurogenesis and Synaptogenesis Following
Neuronal Injury in an Inducible Mouse Model
Fabiana Morroni, Masashi Kitazawa†*, Denise Drago, David Cheng,
Rodrigo Medeiros, Frank M. LaFerla*
Department of N e uro bi ology and Behavior, Institute for Memory Impairments and Neurological Disorders,
University of California, Irvine, USA
E-mail: *, *
Received July 19, 2011; revised August 20, 2011; accepted September 5, 2011
Neuronal loss as a consequence of brain injury, stroke and neurodegenerative disorders causes functional
impairments ranging from cognitive impairments to physical disabilities. Extensive rehabilitation and train-
ing may lead to neuroprotection and promote functional recovery, although little is known about the molecu-
lar and cellular mechanisms driving this event. To investigate the underlying mechanisms and levels of func-
tional recovery elicited by repeated physical training or environmental enrichment, we generated an induc-
ible mouse model of selective CA1 hippocampal neuronal loss. Following the CA1 neuronal injury, mice
underwent one of the above mentioned conditions for 3 months. Exposure to either of these stimuli promoted
functional cognitive recovery, which was associated with increased neurogenesis in the subgranular zone of
dentate gyrus and enhanced synaptogenesis in the CA1 subfield. Notably, a significant correlation was found
between the functional recovery and increased synaptogenesis among survived CA1 neurons. Collectively,
these results support the utilization of cognitive and physical stimulation as approaches to promote recovery
after neuronal loss and demonstrate the potential of this novel mouse model for the development of therapeu-
tic strategies for various neurological disorders associated with focal neuronal loss.
Keywords: Hippocampus, Water Maze, Environmental Enrichment, Cognition, Head Injury
1. Introduction
Neuronal loss is the signature feature of numerous neu-
rological conditions, including head injury, stroke, and
neurodegenerative disorders such as Alzheimer disease.
Depending on the brain regions impacted by the neuronal
loss, individuals may experience physical disabilities and/or
cognitive impairments. Currently, stroke is the most com-
mon single cause of disability, and Alzheimer disease is
the leading cause of dementia among elderly, positioning
these diseases as major medical concerns in our society
[1,2]. Notably, no effective therapeutic strategies have been
developed for these conditions so far. Experimentally, stem
cell transplant has shown some potential in functional re-
covery in mouse models [3-5]. However, the technical hur-
dle implementing of stem cell-based therapeutic approaches
in humans is still high and other issues including ethical
and safety concerns need to be further evaluated.
Traditionally, rehabilitation has been practically applied
to help recovery of lost or impaired functions caused by
disease or injury. In the case of brain injuries, physical
rehabilitation and social/environmental enrichment have
been proposed to stimulate new neuronal connections and
enhance neuronal plasticity among survived neurons, pro-
moting functional recovery [6-8]. Endogenous neuroge-
nesis stimulated by rehabilitation has been reported to
play a critical role in functional recovery following brain
damage [9-12]. However, the underlying mechanisms as-
sociated with the functional recovery have not yet been
well understood. Therefore, we sought to examine the
potential mechanisms and changes following rehabilita-
tion in mice with defined neuronal injury.
To evaluate the effectiveness of rehabilitation after
neuronal injury, we developed a novel genetic model of
These authors contributed equally to this work.
hippocampal neuronal loss that is mediated by the ex-
pression of diphtheria toxin A (DTA) and tightly regu-
lated by tetracycline-inducible system (Tet-off). Utiliza-
tion of the calmodulin kinase II (CaMKII) promoter
allows the induction of DTA expression primarily in
hippocampal CA1 pyramidal neurons followed by corti-
cal neurons in a time-dependent manner. Of note, our pre-
vious studies have demonstrated that induction of this
CaMKII/TetDTA genetic system for 20 - 25 days re-
sults in extensive neuronal loss specifically in the CA1,
which causes severe cognitive decline in hippocam- pal-
dependent tasks [5]. Using this model, mice underwent
3-months of repeated physical training within the Morris
water maze or 3-months of environmental enrichment,
mimicking a physical or social rehabilitation paradigm,
respectively, following a 21-day induction of neuronal
injury. Data presented indicates that both training proto-
cols markedly increased BrdU-positive neurons in the
subgranular zone (SGZ) of the dentate gyrus. Of great
relevance, we also determined that repeated physical
training and environmental enrichment rescues the neu-
ronal loss-induced cognitive impairment as well as the
synaptic density in the SGZ and CA1 in mice with neu-
ronal injury. Taken together, our data indicates that reha-
bilitation following neuronal injury helps functional recov-
ery through upregulation of neurogenesis in the SGZ and
synaptogenesis within survived neurons.
2. Materials and Methods
2.1. Animals and Induction of Neuronal Injury
Double transgenic CaMKII/Tet-DTA mice or single
transgenic Tet-DTA mice were maintained on a 12-hr li-
ght/dark cycle and freely accessible to food and water as
previously described [5]. Doxycycline (2 mg/g of chow)
containing food was given to all mice all the time. CA1
neuronal injury was induced in 6 - 9 month old CaMKII/-
Tet-DTA mice by withdrawing doxycycline-containing
food and replacing it with regular food for 21 days (re-
ferred to as lesioned mice). Doxycycline was also with-
drawn in Tet-DTA mice for 21 days, but no neuronal loss
was triggered due to a lack of CaMKII-TRE transgene
(referred to as non-lesioned control mice). All procedures
were performed in accordance with the regulations of the
Institutional Animal Care and Use Committee of the Uni-
versity of California, Irvine.
2.2. Rehabilitation Strategies
1) Repeated physical training (Morris water maze, MWM) -
Two weeks after the induction of neuronal injury, one
group of mice underwent a repeated MWM experience as
a physical training group, and another group of mice was
not exposed to MWM at all (no training group, n = 11 - 13
per group). Mice in the training group received physical
training in the water maze once a month for 3 months
(please see below for detailed MWM procedure). During
the 3-month training, both groups also received BrdU (50
mg/kg, ip) injections twice a week.
2) Environmental enrichment - After the induction of
neuronal injury, mice were placed in environmentally en-
riched cages or regular cages for 3 months (n = 7 - 8 per
group). Each environmentally-rich cage (45 × 30 × 30 cm)
consisted of running wheels, various types of plastic or
wooden shelters, tube maze, bells, plastic balls and other
toys. 3 - 4 mice shared one cage to stimulate social inter-
action, and toys were cleaned weekly and rotated to dif-
ferent cages. Regular cages (30 × 15 × 20 cm) contained
the same bedding but did not have any toys. All mice re-
eived BrdU (50 mg/kg, ip) injections every other day in
the last 2 weeks of the experimental period.
2.3. Behavioral Tests
1) Morris water maze (MWM) test—The apparatus used
for the water maze task was a circular aluminum tank
(1.2 m diameter) painted white and filled with water
maintained at 27˚C. The maze was located in a room con-
taining several simple visual, extramaze cues. To reduce
stress, mice were placed on the platform for 10 s prior to
the first training trial. Mice were trained to swim to a 14
cm diameter circular clear Plexiglas platform submerged
1.5 cm beneath the surface of the water and invisible to
the mice while swimming. On each trial, the mouse was
placed into the tank at one of four designated start points
in a pseudorandom order. If a mouse failed to find the plat-
form within 60 s, it was manually guided to the platform
and allowed to remain there for 10 s. After the trial, each
mouse was placed into a holding cage under a warming
lamp for 25 s until the start of the next trial. To ensure that
memory differences were not due to lack of task learning,
mice were given four trials a day for as many days as
were required to reach criterion (<20 s mean escape la-
tency before the first probe trial was run). To control for
overtraining, probe trials were run for each group, both as
soon as they reached group criterion and after all groups
had reached criterion. Retention of the spatial training
was assessed 24 h after the last training trial. Both probe
trials consisted of a 60 s free swim in the pool without
the platform. The parameters measured during the probe
trial included (1) time spent in the platform quadrant, (2)
latency to cross the platform location, and (3) number of
platform location crosses.
2) Novel object recognition test—Each mouse was
first habituated to an empty Plexiglass arena (45 × 25 ×
Copyright © 2011 SciRes. JBBS
20 cm) for 3 consecutive days prior to the actual test. On
the first day of testing, mice were exposed to two identi-
cal objects placed at opposite ends of the arena for 5 min.
In the probe trial 24 h later, mice were presented for 5
min with one of the familiar objects and a novel object of
similar dimensions. Exploration counted if the mouse’s
head was within one inch of the object with its neck ex-
tended and vibrissae moving. The recognition index repre-
sents the percentage of the time that mice spent exploring
the novel object.
3) Contextual fear conditioning test—Each mouse was
placed in the fear conditioning chamber (San Diego In-
struments, San Diego, CA, USA) and allowed to explore
for 2 min before receiving three electric foot-shocks (dura-
tion, 1 s; intensity, 0.2 mA; inter-shock interval, 2 min).
The mouse was returned to the home cage 30 sec after
the last foot-shock. Twenty-four hours later, the mouse
was placed back in the chamber, and freezing behavior
was recorded during a 5 min examination period.
2.4. Immunohistochemistry and
Immunofluorescent Staining
Fixed brain halves were sliced on a vibratome at 50 μM
thickness. Brain sections were mounted onto slides, and
hematoxylin and eosin (H&E) staining was performed as
previously described [5,13].
For BrdU double labeling, sections were first treated
with 2 N HCl for 30 min at 37˚C, then neutralized by 0.1
M borate buffer (pH 8.5). Prior to overnight incubation
with primary antibody in Tris-buffered saline (TBS) con-
taining 3% serum and 2% BSA at 4˚C, sections were
permeabilized with 0.1% Triton X-100 in TBS and blocked
in solution containing 2% BSA. After the incubation with
primary antibodies, sections were washed and incubated
with the appropriate secondary antibody for 1 hr at ambi-
ent temperature. Primary antibodies used in this study were
anti-BrdU antibody (1:500; Acculate Chemical, West- bury,
NY), anti-Iba1 antibody (1:500; Wako, Richmond, VA),
anti-GFAP antibody (1:1000; Dako, Glostrup, Denmark),
anti-NG2 antibody (1:500; Chemicon, Temecula, CA), anti-
NeuN antibody (1:000; Millipore, Billerica, MA), anti-
PSD-95 antibody (1:500; Millipore), and anti-synapto-
physin antibody (1:500; Sigma, St. Louis, MO). Secon-
dary antibodies were anti-mouse or anti-rabbit conjugated
with Alexafluor 488 or Alexafluor 555 (1:200; Invitrogen,
Carlsbad, CA). All fluorescent images were captured us-
ing a Bio-Rad 2000 confocal microscopy (Bio-Rad Labo-
ratories, Hercules, CA). Fluorescent intensity was quanti-
fied by averaging 3 - 5 random fields at 60× objective or
higher in each section. Grayscale images were inverted
and optical density was quantified using the Image J soft-
ware. Pixel intensity from two sections per animal were
averaged and compared.
2.5. Statistical Analysis
All data were analyzed using one-way ANOVA with post-
test (Dunnett or Bonferroni post-test) when comparing
three or more groups, or using unpaired t-test when com-
paring two groups. p < 0.05 or lower was considered to be
statistically significant.
3. Results
3.1. Repeated Physical Training Improves
Cognitive Function after Neuronal Injury
Twenty-one-day induction of DTA by withdrawing do-
xycycline significantly damaged neurons in CA1 hippo-
campus and dentate gyrus (DG) of CaMII/Tet-DTA
(lesioned) mice (Figures 1(a) and (b)). In CA1 region,
the fluorescent in- tensity of NeuN labeling reduced by
44% in lesioned mice (intensity 26.3 ± 0.8) compared to
non-lesioned mice (intensity 47.1 ± 1.1), and the somatic
layer of CA1 was clearly thinner due to neuronal loss
(Figure 1 and Supplemental Figure 1). Similarly, in the
molecular layer of DG, the intensity of NeuN labeling
reduced by 30% in lesioned mice (intensity 52.8 ± 2.2)
Figure 1. Twenty-one days of induction results in a focal
CA1 neuronal lesioning in CaMKII/Tet-DTA transgenic
mice. (a) Hematoxylin and eosin (H & E) staining clearly
indicates thinning in CA1 pyramidal cell layer in lesioned
mice, while other areas in hippocampus are not damaged.
(b) Magnified images in CA1 pyramidal neuronal loss fol-
lowing the induction.
Copyright © 2011 SciRes. JBBS
Copyright © 2011 SciRes. JBBS
compared to non-lesioned mice (intensity 75.5 ± 1.9).
However, the thickness of the molecular layer of DG was
not as clear as that of CA1, suggesting that the neuronal
loss in DG was not as robust as that in CA1 at 21-day
induction (Supplemental Figure 1). The pattern and de-
gree of neuronal injury were controlled and well-defined,
consistent with previously reported observations [5]. All
induced mice resulted in a similar degree of lesions,
minimizing potential variability due to the severity of
neuronal injury. After the completion of three repeated
MWM training over the three-month period, we first ex-
amined cognitive function. Lesioned mice with repeated
MWM training were indistinguishable from non-lesioned
control mice with or without repeated training in the es-
cape latency and number of platform crosses in MWM,
whereas lesioned mice without repeated training showed
significant impairments on both parameters (Figures 2(a) and
(b)). The improvement of cognitive function of lesioned mice
by the MWM test was not simply due to the repeated ex-
posures to the MWM task as we found no difference in
cognitive outcomes between no trained and repeatedly
trained non-lesioned mice. To further rule out this possi-
bility, we examined cognition using other tests that these
mice had never been exposed to. Similarly, place-based
object recognition as well as contextual fear conditioning,
both hippocampus-dependent memory tasks, were mark-
edly rescued by repeated training in lesioned mice (Figures
2(c) and (d)). On the other hand, context-based object rec-
ognition, which evaluates hippocampus-independent and
corticaldependent memory, was not different in all four
groups, and the recognition index was fairly consistent
with our previous observation (Supplemental Figure 2).
(a) (b)
(c) (d)
Fi gu re 2 . A repeated physical training hel ps to recover the CA1 lesion-induced cognitive impairment. After 3 months of physi cal
training, all mice were tested w ith a battery of behavi oral te sts. (a) Escape latency and (b) number of platform crosses for a 24-hr
probe trial of MWM show that a repeate d physical trai ning promotes a functi onal recovery in lesioned mice. (c ) Place rec ognition
test and (d) contextual fear conditioning test also detect a marked recovery of hippocampus- dependent cognitive function in le-
sioned mice. Each graph represents mean ± S.E.M. for n = 11 - 13, and *p < 0.05.
3.2. Repeated Physical Training Increases
Neurogenesis in Dentate Gyrus
We next examined molecular changes in the brain after the
repeated MWM training to explain observed restora- tion
of cognition. The overall neurogenesis in subgranular
zone/dentate gyrus (SGZ/DG) was significantly increased
in mice with physical training regardless of neuronal in-
jury as detected by BrdU and NeuN double labeling
(Figures 3(a) and (b)), suggesting the repeated MWM
training itself stimulated neurogenesis over the period of
3 months, consistent with previous findings [11,12]. No-
tably, it appeared that neuronal injury itself also stimu-
lated neuron-genesis in SGZ as lesioned mice without
physical trainingexhibited a marked increase of neuro-
genesis compared to non-lesioned control mice without
physical training (Figures 3(a) and (b)). The prolifera-
tion of other cell types; microglia and astrocytes, was
also quantitatively analyzed by counting BrdU/Iba1 (mi-
croglia) and BrdU/ GFAP (astrocytes) positive cells in
SGZ/DG and CA1 hippocampus. Although neuronal in-
jury significantly increased proliferation of microglia, the
repeated MWM training itself did not alter proliferation
of both cell types (Figures 3(c) and (d)).
3.3. Repeated Physical Training Stimulates
SynaptoGenesis in Survived Neurons
We further examined whether repeated MWM training
modulated synaptic plasticity in remaining neurons in the
(a) (b)
(c) (d)
Figure 3. A repeated phy sical training increases neuroge nesis in SGZ. (a) Representative low-magnification images of double
immunofluorescent staining of NeuN (green) and BrdU (red) in SGZ. (b) Quantitative analysis of NeuN/BrdU positive new-
born neurons in SGZ and representative high-magnification images of NeuN/BrdU double labeled neurons in SGZ. (c, d)
Quantitative analyses of Iba1/BrdU (c) and GFAP/BrdU (d) and representative high-magnification images of double labeled
cells. Each graph represents mean ± S.E.M. for n = 11 - 13, and *p < 0.05.
Copyright © 2011 SciRes. JBBS
hippocampus, and subsequently contributed to functional
recovery. In this regard, we measured densities of synap-
tophysin, a pre-synaptic protein, and PSD-95, a post-sy-
naptic protein, in DG and CA1 hippocampus. Notably,
synaptophysin in CA1 hippocampus was restored in mice
with repeated MWM training, and the levels were almost
equivalent to those in non-lesioned control mice (Figure
4(a)). Similarly, PSD-95 levels in CA1 were also signify-
cantly increased following repeated MWM training in
lesioned mice (Figure 4(b)). Interestingly, neither synap-
tophysin (Figure 4(c)) nor PSD-95 (data not shown) levels
in DG were significantly different among four groups.
3.4. Environmental Enrichment Restores
Cognition and Stimulates Neurogenesis and
Synaptogenesis in Mice with Neur onal Injury
Repeated physical training represented by monthly MWM
is a relatively stressful procedure for mice. Acute or chron-
ic stress has been reported to suppress learning and mem-
ory function [14-16]. To rule out any negative impacts of
stress that may interfere with the beneficial function of reha-
bilitation, we next evaluated whether less stressful reha-
bilitation similarly promoted functional recovery and neu-
ronal changes in mice with neuronal injury. In this regard,
Figure 4. Synaptogenesis in CA1 hippocampus is upregulated by a repeated physical training in lesioned mice. (a) The fluo-
rescent intensity of synaptophy sin in CA1 hippocampal region was quantified. (b) The fluoresc ent intensity of PSD-95 in api-
cal dendrites of CA1 pyra midal neurons was quantified. (c) The fluoresce nt intensity of synaptophysin in DG was not differ-
ent among the four groups. Each graph represents mean ± S.E.M. for n = 11 - 13, and *p < 0.05.
Copyright © 2011 SciRes. JBBS
lesioned mice were housed in environmentally enriched
cages. This approach mimicked rehabilitation associated
with social and environmental interactions [17]. Three
months of environmental enrichment partially improved
hippocampus-associated cognitive function as determined
by place recognition test. Lesioned mice with environ-
mental enrichment explored novel object significantly
more than chance level (50%) while lesioned mice singly
housed in a regular cage did not distinguish novel object
from familiar object (Figure 5(a)). This functional re-
covery was not due to a recovery of CA1 neurons as both
groups showed similar levels of CA1 neuronal thinning
or loss (Figure 5(b)). Rather, it correlated well with the in-
creased synaptogenesis in survived CA1 neurons (Fig-
ures 5(c) and (d)). Neurogenesis was also markedly in-
creased in mice under the environmental enrichment as
previously described in various studies (data not shown)
[12,18,19]. Taken together, both repeated physical train-
ing and environmental enrichment stimulated neurogenesis
(a) (b)
(c) (d)
Figure 5. Environmental enrichment promotes functional
recovery and synaptogenesis in lesioned mice. (a) Place re-
cognition test was performed after the 3-month environ-
mental enrichment in mice with neuronal injury. Each graph
represents mean ± S.E.M. (n = 7 - 8), and *p < 0.05 from the
chance (50%) le vel. (b) Immunofluorescent stai ning of NeuN
in CA1 hippocampus region. (c) Synaptophysin immuno-
fluorescent staining in CA1 region and (d) PSD-95 im-
munofluorescent staining in CA1 region. Fluorescent inten-
sity was quantified by averaging 3 - 5 random fields at 63x
in each section and expressed as mean ± S.E.M. in the
graph. *p < 0.05 or **p < 0.01 compared to non-enriched
group. Scale bar = 25 m.
in SGZ and synaptic plasticity among survived CA1
neurons and promoted functional recovery after neuronal
4. Discussion
Functional impairments of the brain are serious medical
conditions that adversely affect a patient’s life in many ways.
Types and severity of impairments are highly dependent
on the areas and extent of the damage in the brain. Various
diseases and conditions cause brain damage and functional
impairments. For example, one-time stroke, hypoxia or
traumatic brain injuries result in focal damage and neu-
ronal loss in the brain. These are often non-progressive,
and the damaged area is limited. On the other hand, Alz-
heimer disease (AD), Lewy body dementia or frontotem-
poral dementia are progressive neurodegenerative disea-
ses, and the damaged areas spread over time together with
progressive cognitive and functional impairments. Nota-
bly, the incidence of brain injuries and neurodegenerative
disorders are increasing every year, and effective treat-
ments and medical support are in immediate demand.
Rehabilitation is one of the most important post-opera-
tive care strategies for patients with brain injuries. In order
to better understand the therapeutic properties of reha-
bilitation, we examined the some of the mechanisms as-
sociated with the restoration of brain function induced by
physical and social/environmental stimulation following
neuronal injury in mice. Importantly, our mouse model de-
velops a very defined and temporal pattern of neuronal
loss following the induction of DTA by withdrawing do-
xycycline from the diet. Significant pyramidal neuronal
loss in CA1 region is first detected at 15 - 20 days of in-
duction, followed by a neuronal loss in DG and cortecies
at 25 - 30 days of induction. We have chosen the 21-day
induction, which gave us a defined neuronal loss in CA1
and DG with minimum loss of neurons in cortecies. Sub-
sequently, this induction paradigm impaired predominantly
hippocampal-based, but not cortical-based, cognitive func-
tion [5]. Notably, it has been well documented that CA1
pyramidal neurons are selectively damaged during global
cerebral ischemia [20-22]. Therefore, we utilized this model
as a focal neuronal injury model and examined whether
repeated physical and social/environmental stimulations
rescued clinical phenotypes. We applied repeated Morris
water maze as physical training-based stimulation and
group-housed environmental enrichment as a social and
environmental stimulation for neuronal injury mouse model.
A repeated water maze training paradigm has been shown
to ameliorate post-seizure-induced cognitive impairments
in rats [23]. Both paradigms markedly restored injury-
associated cognitive function in our mouse model. Such
improvement in the cognitive performance was clearly
Copyright © 2011 SciRes. JBBS
associated with increased synaptogenesis among survived
neurons in CA1 as well as neurogenesis in SGZ. Similar
findings have been reported in different rodent models of
brain injuries [6,9-12].
Neurogenesis in SGZ has been reported to play a criti-
cal role in memory formation and long-term memory con-
solidation [24,25]. In various rodent stroke models, in-
creased neurogenesis in SGZ is associated with functional
recovery following skilled physical activity [11,22]. Our
study showed that the neurogenesis was significantly up-
regulated not only by repeated physical training but also
by CA1 neuronal injury. Interestingly, the increased neu-
rogenesis in SGZ was not sufficient to facilitate functional
recovery in our neuronal lesioned mouse model. There-
fore, it is speculated that neurogenesis together with in-
creased synaptogenesis among survived neurons in CA1
ameliorate the lesion-induced cognitive impairments. This
idea is supported by our recent findings on neural stem
cell transplant in a mouse model of AD [26]. We have de-
monstrated that brain-derived neurotrophic factor (BDNF)
secreted from transplanted neuronal stem cells promotes
synaptogenesis in hippocampal neurons and rescues AD-
associated cognitive impairments without attenuating AD
neuropathologies [26]. The relationship between synap-
togenesis and BDNF is further supported by multiple
studies demonstrating that treatment with neurotrophic fac-
tors including BDNF, glial cell-derived neurotrophic factor
(GDNF) or insulin-like growth factor-1 (IGF-1) show
neuroprotective effects against brain injuries in rodent
models [27-29]. Environmental enrichment or exercise has
been shown to upregulate BDNF and restore cognitive
function in rats with chronic hypoperfusion or ischemia
and blockade of BDNF production significantly negated
its beneficial effects in rats with ischemia [30-32] Inter-
estingly, BDNF also enhances neurogenesis after the stroke
[33]. Promoting synaptogenesis in post-stroke or brain inju-
ries may be a potential therapeutic strategy. In stroke and
ischemia models, increased synaptogenesis by pharma-
cological agents or skilled training facilitates functional
recovery [6,34,35]. In this context, the repeated physical
and social/environmental stimulations in our paradigm
could promote the release of neurotrophic factors, which
in turn contribute to the induction of synaptogenesis and
functional recovery. However, additional studies are neces-
sary to confirm this hypothesis.
The current results provide functional and molecular
evidence indicating that physical and social/environmental
stimulations after neuronal injury are ways to effectively
facilitate recovery without any invasive methods. Such
approaches may have great potential as therapeutic alter-
natives for neurological disorders associated with neu-
ronal loss.
5. Acknowledgements
This study was supported by grants from the National Insti-
tutes of Health (NIH): NIH/NIA R01AG20335 (F.M.L.),
NIH Program Project Grant, AG00538 (F.M.L.), NIH/-
NIAMS K99AR054695 (M.K.).
6. References
[1] J. M. Garbusinski, M. A. van der Sande, E. J. Bartholome,
M. Dramaix, A. Gaye, R. Coleman, O. A. Nyan, R. W.
Walker, K. P. McAdam and G.E. Walraven, “Stroke
Presentation and Outcome in Developing Countries: A
Prospective Study in the Gambia,” Stroke, Vol. 36, No. 7,
2005, pp. 1388-1393.
[2] H. W. Querfurth and F. M. LaFerla, “Alzheimer’s Dis-
ease,” The New England Journal of Medicine, Vol. 362,
No. 4, 2010, pp. 329-344. doi:10.1056/NEJMra0909142
[3] M. M. Daadi, A. S. Davis, A. Arac, Z. Li, A. L. Maag, R.
Bhatnagar, K. Jiang, G. Sun, J. C. Wu and G. K.
Steinberg, “Human Neural Stem Cell Grafts Modify Micro-
glial Response and Enhance Axonal Sprouting in Neonatal
Hypoxic-Ischemic Brain Injury,” Stroke, Vol. 41, No. 3,
2010, pp. 516-523.
[4] P. Stroemer, S. Patel, A. Hope, C. Oliveira, K. Pollock
and J. Sinden, “The Neural Stem Cell Line CTX0E03
Promotes Behavioral Recovery and Endogenous Neuro-
genesis after Experimental Stroke in a Dose-Dependent
Fashion,” Neurorehabilitation & Neural Repair, Vol. 23,
No. 9, 2009, pp. 895-909.
[5] T. R. Yamasaki, M. Blurton-Jones, D. A. Morrissette, M.
Kitazawa, S. Oddo and F. M. LaFerla, “Neural Stem
Cells Improve Memory in an Inducible Mouse Model of
Neuronal Loss,” Journal of Neuroscience, Vol. 27, No. 44,
2007, pp. 11925-11933.
[6] A. M. Auriat, S. Wowk and F. Colbourne, “Rehabilitation
after Intracerebral Hemorrhage in Rats Improves Recov-
ery with Enhanced Dendritic Complexity but No Effect
on Cell Proliferation,” Behavioural Brain Research, Vol.
214, No. 1, 2010, pp. 42-47.
[7] R. P. Allred, M. A. Maldonado, J. E. Hsu and T. A. Jones,
“Training the “Less-Affected” Forelimb after Unilateral
Cortical Infarcts Interferes with Functional Recovery of
the Impaired Forelimb In Rats,” Restorative Neurology
and Neuroscience, Vol. 23, No. 5-6, 2005, pp. 297-302.
[8] J. Biernaskie, A. Szymanska, V. Windle and D. Corbett,
“Bi-Hemispheric Contribution to Functional Motor Re-
covery of the Affected Forelimb Following Focal Ischemic
Brain Injury in Rats,” European Journal of Neuroscience,
Vol. 21, No. 4, 2005, pp. 989-999.
[9] S. H. Im, J. H. Yu, E. S. Park, J. E. Lee, H. O. Kim, K. I.
Copyright © 2011 SciRes. JBBS
Park, G. W. Kim, C. I. Park and S. R. Cho, “Induction of
Striatal Neurogenesis Enhances Functional Recovery in
an Adult Animal Model of Neonatal Hypoxic-Ischemic
Brain Injury,” Neuroscience, Vol. 169, No. 1, 2010, pp.
259-268. doi:10.1016/j.neuroscience.2010.04.038
[10] W. L. Li, S. P. Yu, M. E. Ogle, X. S. Ding and L. Wei,
“Enhanced Neurogenesis and Cell Migration Following
Focal Ischemia and Peripheral Stimulation in Mice,” Devel-
opmental Neurobiology, Vol. 68, No. 13, 2008, pp. 1474-
1486. doi:10.1002/dneu.20674
[11] C. Zhao, J. Wang, S. Zhao and Y. Nie, “Constraint-In-
Duced Movement Therapy Enhanced Neurogenesis and
Behavioral Recovery after Stroke in Adult Rats,” The
Tohoku Journal of Experimental Medicine, Vol. 218, No.
4, 2009, pp. 301-308. doi:10.1620/tjem.218.301
[12] F. Wurm, S. Keiner, A. Kunze, O. W. Witte and C. Re-
decker, “Effects of Skilled Forelimb Training on Hippo-
campal Neurogenesis and Spatial Learning after Focal
Cortical Infarcts in the Adult Rat Brain,” Stroke, Vol. 38,
No. 10, 2007, pp. 2833-2840.
[13] M. Kitazawa, K. N. Green, A. Caccamo and F. M.
LaFerla, “Genetically Augmenting Abeta42 Levels in
Skeletal Muscle Exacerbates Inclusion Body Myositis-
Like Pathology and Motor Deficits in Transgenic Mice,”
American Journal of Pathology, Vol. 168, No. 6, 2006,
pp. 1986-1997. doi:10.2353/ajpath.2006.051232
[14] S. J. Lupien, S. Gaudreau, B. M. Tchiteya, F. Maheu, S.
Sharma, N. P. Nair, R. L. Hauger, B. S. McEwen and M.
J. Meaney, “Stress-Induced Declarative Memory Im-
pairment in Healthy Elderly Subjects: Relationship to
Cortisol Reactivity,” Journal of Clinical Endocrinology
& Metabolism, Vol. 82, No. 7, 1997, pp. 2070-2075.
[15] K. Mizoguchi, M. Yuzurihara, A. Ishige, H. Sasaki, D. H.
Chui and T. Tabira, “Chronic Stress Induces Impairment
of Spatial Working Memory Because of Prefrontal Dopa-
Minergic Dysfunction,” Journal of Neuroscience, Vol. 20,
No. 4, 2000, pp. 1568-1574.
[16] C. Sandi, J. C. Woodson, V. F. Haynes, C. R. Park, K.
Touyarot, M. A. Lopez-Fernandez, C. Venero and D. M.
Diamond, “Acute Stress-Induced Impairment of Spatial
Memory Is Associated with Decreased Expression of
Neural Cell Adhesion Molecule in the Hippocampus and
Prefrontal Cortex,” Biological Psychiatry, Vol. 57, No. 8,
2005, pp. 856-864. doi:10.1016/j.biopsych.2004.12.034
[17] F. D. Rose, E. A. Attree, B. M. Brooks and D. A. Johnson,
“Virtual Environments in Brain Damage Rehabilitation:
A Rationale from Basic Neuroscience,” Studies in Health
Technology and Informatics, Vol. 58, 1998, pp. 233-242.
[18] N. Madronal, C. Lopez-Aracil, A. Rangel, J. A. del Rio, J.
M. Delgado-Garcia and A. Gruart, “Effects of Enriched
Physical and Social Environments on Motor Performance,
Associative Learning, and Hippocampal Neurogenesis in
Mice,” PLoS One, Vol. 5, No. 6, 2010, p. e11130.
[19] O. Lazarov, J. Robinson, Y. P. Tang, I. S. Hairston, Z.
Korade-Mirnics, V. M. Lee, L. B. Hersh, R. M. Sapolsky,
K. Mirnics and S. S. Sisodia, “Environmental Enrichment
Reduces Abeta Levels and Amyloid Deposition in Trans-
Genic Mice,” Cell, Vol. 120, No. 5, 2005, pp. 701-713.
[20] O. Bendel, T. Bueters, M. von Euler, S. Ove Ogren, J.
Sandin and G. von Euler, “Reappearance of Hippocampal
CA1 Neurons after Ischemia Is Associated with Recovery
of Learning and Memory,” Journal of Cerebral Blood
Flow & Metabolism, Vol. 25, No. 12, 2005, pp. 1586-
1595. doi:10.1038/sj.jcbfm.9600153
[21] T. Kirino, “Delayed Neuronal Death in the Gerbil Hippo-
Campus Following Ischemia,” Brain Research, Vol. 239,
No. 1, 1982, pp. 57-69.
[22] H. Nakatomi, T. Kuriu, S. Okabe, S. Yamamoto, O. Ha-
tano, N. Kawahara, A. Tamura, T. Kirino and M. Naka-
fuku, “Regeneration of Hippocampal Pyramidal Neurons
after Ischemic Brain Injury by Recruitment of Endoge-
nous Neural Progenitors,” Cell, Vol. 110, No. 4, 2002, pp.
429-441. doi:10.1016/S0092-8674(02)00862-0
[23] S. J. Wong-Goodrich, M. J. Glenn, T. J. Mellott, Y. B.
Liu, J. K. Blusztajn and C. L. Williams, “Water Maze
Experience and Prenatal Choline Supplementation Dif-
ferentially Promote Long-Term Hippocampal Recovery
from Seizures in Adulthood,” Hippocampus, Vol. 21, No.
6, 2011, pp, 584-608.
[24] T. Kitamura, Y. Saitoh, N. Takashima, A. Murayama, Y.
Niibori, H. Ageta, M. Sekiguchi, H. Sugiyama and K.
Inokuchi, “Adult Neurogenesis Modulates the Hippocam-
pus-Dependent Period of Associative Fear Memory,” Cell,
Vol. 139, No. 4, 2009, pp. 814-827.
[25] W. Deng, M.D. Saxe, I.S. Gallina and F.H. Gage, “Adult-
Born Hippocampal Dentate Granule Cells Undergoing
Maturation Modulate Learning and Memory in the Brain,”
Journal of Neuroscience, Vol. 29, No. 43, 2009, pp.
13532-13542. doi:10.1523/JNEUROSCI.3362-09.2009
[26] M. Blurton-Jones, M. Kitazawa, H. Martinez-Coria, N. A.
Castello, F. J. Muller, J. F. Loring, T. R. Yamasaki, W.
W. Poon, K. N. Green and F. M. LaFerla, “Neural Stem
Cells Improve Cognition via BDNF in a Transgenic Model
of Alzheimer Disease,” Proceedings of the National Aca-
demy of Sciences of the United States of America, Vol. 106,
No. 32, 2009, pp. 13594-13599.
[27] C. R. Almli, T. J. Levy, B. H. Han, A. R. Shah, J. M.
Gidday and D. M. Holtzman, “BDNF Protects against
Spatial Memory Deficits Following Neonatal Hypoxia-
Ischemia,” Experimental Neurology, Vol. 166, No. 1,
2000, pp. 99-114. doi:10.1006/exnr.2000.7492
[28] S. Katsuragi, T. Ikeda, I. Date, T. Shingo, T. Yasuhara, K.
Mishima, N. Aoo, K. Harada, N. Egashira, K. Iwasaki, M.
Fujiwara and T. Ikenoue, “Implantation of Encapsulated
Glial Cell Line-Derived Neurotrophic Factor-Secreting
Cells Prevents Long-Lasting Learning Impairment Fol-
lowing Neonatal Hypoxic-Ischemic Brain Insult in Rats,”
American Journal of Obstetrics & Gynecology, Vol. 192,
No. 4, 2005, pp. 1028-1037.
Copyright © 2011 SciRes. JBBS
Copyright © 2011 SciRes. JBBS
[29] S. Lin, L.W. Fan, Y. Pang, P. G. Rhodes, H. J. Mitchell
and Z. Cai, “IGF-1 Protects Oligodendrocyte Progenitor
Cells and Improves Neurological Functions Following
Cerebral Hypoxia-Ischemia in the Neonatal Rat,” Brain
Research, Vol. 1063, No. 1, 2005, pp. 15-26.
[30] M. Ploughman, S. Granter-Button, G. Chernenko, B. A.
Tucker, K. M. Mearow and D. Corbett, “Endurance Ex-
ercise Regimens Induce Differential Effects on Brain-
Derived Neurotrophic Factor, Synapsin-I and Insulin-
Like Growth Factor I after Focal Ischemia,” Neurosci-
ence, Vol. 136, No. 4, 2005, pp. 991-1001.
[31] H. Sun, J. Zhang, L. Zhang, H. Liu, H. Zhu and Y. Yang,
“Environmental Enrichment Influences BDNF and NR1
Levels in the Hippocampus and Restores Cognitive Im-
pairment in Chronic Cerebral Hypoperfused Rats,” Cur-
rent Neurovascular Research, Vol. 7, No. 4, 2010, pp.
[32] M. Ploughman, V. Windle, C. L. MacLellan, N. White, J.
J. Dore and D. Corbett, “Brain-Derived Neurotrophic Fac-
tor Contributes to Recovery of Skilled Reaching after
Focal Ischemia in Rats,” Stroke, Vol. 40, No. 4, 2009, pp.
1490-1495. doi:10.1161/STROKEAHA.108.531806
[33] W. R. Schabitz, T. Steigleder, C. M. Cooper-Kuhn, S.
Schwab, C. Sommer, A. Schneider and H. G. Kuhn, “In-
travenous Brain-Derived Neurotrophic Factor Enhances
Poststroke Sensorimotor Recovery and Stimulates Neuro-
genesis,” Stroke, Vol. 38, No. 7, 2007, pp. 2165-2172.
[34] M. K. Sun, J. Hongpaisan, T. J. Nelson and D. L. Alkon,
“Poststroke Neuronal Rescue and Synaptogenesis Medi-
ated in Vivo by Protein Kinase C in Adult Brains,” Pro-
ceedings of the National Academy of Sciences of the United
States of America, Vol. 105, No. 36, 2008, pp. 13620-
13625. doi:10.1073/pnas.0805952105
[35] L. Zhang, R. L. Zhang, Y. Wang, C. Zhang, Z. G. Zhang,
H. Meng and M. Chopp, “Functional Recovery in Aged
and Young Rats after Embolic Stroke: Treatment with a
Phosphodiesterase Type 5 Inhibitor,” Stroke, Vol. 36, No.
4, 2005, pp. 847-852.
Supplemental Figures
Supplemental Figure 1. Twenty-one days of induction c a use s ne ur onal loss in CA1 hippocampus and dentate gyrus in CaM-
KIIα/Tet-DTA transgenic mice. NeuN immunofluorescent staining shows significant reductions of the intensity in CA1 and
dentate gyrus of lesioned mice. Graphs represent changes in the NeuN intensity between the two groups (**p < 0.01). No sig-
nificant changes are observed between no training and trained groups.
Supplemental Figure 2. Cortical-dependent cognitive function is minimally affected in lesioned mice. Cortical-dependent
memory tasks, (a) context recognition and (a) object recognition tests, are also evaluated after the repeated physical training
and found no difference among the 4 groups, indicating that neuronal damages are minimal in cortex areas (n = 11 - 13).
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