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ORIGINAL ARTICLE
Intermittent fasting attenuates increases in neurogenesis after
ischemia and reperfusion and improves recovery
Silvia Manzanero
1
, Joanna R Erion
2
, Tomislav Santro
1
, Frederik J Steyn
1
, Chen Chen
1
, Thiruma V Arumugam
1,3
and Alexis M Stranahan
2
Intermittent fasting (IF) is neuroprotective across a range of insults, but the question of whether extending the interval between
meals alters neurogenesis after ischemia remains unexplored. We therefore measured cell proliferation, cell death, and
neurogenesis after transient middle cerebral artery occlusion (MCAO) or sham surgery (SHAM) in mice fed ad libitum (AL) or
maintained on IF for 3 months. IF was associated with twofold reductions in circulating levels of the adipocyte cytokine leptin in
intact mice, but also prevented further reductions in leptin after MCAO. IF/MCAO mice also exhibit infarct volumes that were less
than half those of AL/MCAO mice. We observed a 30% increase in basal cell proliferation in the hippocampus and subventricular
zone (SVZ) in IF/SHAM, relative to AL/SHAM mice. However, cell proliferation after MCAO was limited in IF mice, which showed
twofold increases in cell proliferation relative to IF/SHAM, whereas AL/MCAO mice exhibit fivefold increases relative to AL/SHAM.
Attenuation of stroke-induced neurogenesis was correlated with reductions in cell death, with AL/MCAO mice exhibiting twice the
number of dying cells relative to IF/MCAO mice. These observations indicate that IF protects against neurological damage in
ischemic stroke, with circulating leptin as one possible mediator.
Journal of Cerebral Blood Flow & Metabolism (2014) 34, 897–905; doi:10.1038/jcbfm.2014.36; published online 19 February 2014
Keywords: caloric restriction; hippocampus; intermittent fasting; neurogenesis; stroke; subventricular zone
INTRODUCTION
Proliferation and differentiation of new neurons are ongoing
features of the hippocampus and olfactory bulb. Cell division,
migration, differentiation, and functional integration are regulated
by environmental demands in both regions.
1
Rates of progenitor
cell proliferation and neurogenesis also fluctuate in the context of
pathological conditions, such as traumatic brain injury, epilepsy,
and stroke.
2
Transient or permanent interruptions in cerebral
blood flow induce neurogenesis, which may occur in response to
widespread neuronal loss under ischemic conditions.
3
Although
this initially spurred substantial interest in stroke-induced
neurogenesis as a mechanism for brain repair, more recent data
suggest that new neurons formed after middle cerebral artery
occlusion (MCAO) fail to integrate properly and may in fact
contribute to circuit dysfunction in the hippocampus.
4
In this
regard, new neurons born into a pathological molecular
environment may impede, rather than promote, recovery after
stroke due to aberrant functional integration into the dentate
gyrus circuitry.
Rates of adult neurogenesis in the intact brain increase after
energetic challenges, such as exercise
5
and intermittent fasting
(IF).
6
Although the quantitative impact of neurogenesis on the
function of neural circuitry in the intact and injured brain has yet
to be entirely resolved, computational studies suggest that both
the number and functional integration of newly generated
neurons may contribute to plasticity under intact conditions,
and recovery after injury.
7
The IF regimen, which involves
increasing the interval between meals, enhances hippocampal
neurogenesis, but the extent to which this enhancement extends
to the subventricular zone (SVZ) remains unclear. Because newly
generated cells in the ischemic SVZ can migrate into the striatum
and differentiate into neurons,
8
it is important to understand how
SVZ progenitors can potentially be recruited for neuroprotection
after stroke. Moreover, while IF reduces infarct size and evokes a
molecular profile consistent with neuroprotection in mice after
transient MCAO,
9
the question of whether this dietary regimen
alters the magnitude and extent of stroke-induced neurogenesis
has yet to be explored. IF evokes widespread alterations in
metabolism and alters numerous neuroendocrine signals known
to regulate adult neurogenesis, such as the adipocyte cytokine
leptin.
10
Given the consistently demonstrated relationship
between circulating factors and adult neurogenesis, and the
broad nature of physiological responses to IF, we hypothesize that
metabolic intervention exerts opposite effects on neurogenesis
under intact and ischemic conditions. Specifically, we predict that
extending the interval between meals will increase neurogenesis
in the intact brain and reduce the extent of cellular damage in
the ischemic brain, thereby constraining the proliferative response
to stroke.
To address this question, we measured changes in cell death,
cell proliferation, and neurogenesis after IF under basal conditions
and after stroke. Under intact conditions, extending the interval
between meals reduces body weight and circulating leptin levels,
and enhances basal neurogenesis in the hippocampus and SVZ.
After an ischemic insult, IF limits increases in cell proliferation and
neurogenesis, reduces the extent of cell death, and prevents the
ischemia-induced drop in circulating leptin. These observations
suggest that IF enhances basal neurogenesis and improves the
1
School of Biomedical Sciences, The University of Queensland, St Lucia, Queensland, Australia and
2
Department of Physiology, Georgia Regents University, Augusta, Georgia, USA.
Correspondence: Dr AM Stranahan, Georgia Regents University, Physiology Department, 1120 15th Street, room CA3145, Augusta, GA 30912, USA.
E-mail: astranahan@gru.edu
This work was supported by an ARC Future Fellowship (FT100100427) awarded to TVA, and by start-up funds from the Medical College of Georgia (AMS).
3
Current address: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Received 21 November 2013; revised 22 January 2014; accepted 24 January 2014; published online 19 February 2014
Journal of Cerebral Blood Flow & Metabolism (2014) 34, 897 –905
&
2014 ISCBFM All rights reserved 0271-678X/14 $32.00
www.jcbfm.com
defense of a proliferative set point after an ischemic insult, possibly
by maintaining leptin levels, which may emerge as a novel
biomarker for the extent of neurological damage after stroke.
MATERIALS AND METHODS
Animals, Diets, and Bromodeoxyuridine Administration
Male C57Bl6J mice were obtained from the Animal Resources Centre in
Canning Vale, Australia, and group housed on arrival in the University of
Queensland animal facility. At 10 weeks of age, mice were randomly
assigned to either the ad libitum (AL, n¼27) or IF (n¼27) diet conditions.
IF mice were fed for 8 hours out of every 24-hour period, with food
available between 0700 and 1500 hours (lights on at 0600 hours, lights out
at 1800 hours). This feeding schedule was applied for 3 months before
random assignment into either the SHAM (n¼8) or MCAO (n¼19)
condition. Within the MCAO condition, (n¼10) mice from each diet group
were used for 2,3,5-triphenyltetrazolium chloride (TTC) staining, and the
remainder were used for the adult neurogenesis studies. Mortality rates in
the MCAO group were comparable across diet conditions (n¼3 from AL;
n¼2 from IF) and food was freely available to mice in both dietary
conditions during the 1-week recovery period after MCAO. Body weights
were collected on a weekly basis and all procedures followed guidelines
set out by the National Institutes of Health (USA) and were approved by
the Animal Care and Use Committee of the University of Queensland.
The DNA synthetic marker bromodeoxyuridine (BrdU; Sigma-Aldrich,
St Louis, MO) was administered at a dose of 50 mg/kg (intraperitoneally)
every 12 hours for 2 days, beginning 5 days after MCAO or sham surgery.
This BrdU treatment regimen is widely used for pulse labeling of neuronal
progenitor cells
11
and does not induce toxicity, even at doses greater than
that used in the present study.
12
A week after MCAO or sham surgery, mice
used for immunohistochemistry were transcardially perfused with 4%
paraformaldehyde in phosphate buffer under deep Isoflurane anesthesia.
Subsets of MCAO mice from each diet condition were not perfused;
instead, these mice were sacrificed by decapitation under Isoflurane
anesthesia three days after MCAO or sham surgery, and the brains were
immediately processed for TTC staining, as described.
9
In brief, 2.0-mm-
thick brain sections were stained in 2% TTC in phosphate buffer and
photographed. The resultant images were analyzed in a masked fashion
using NIH ImageJ software; infarct areas were determined by subtracting
the area of intact tissue in the ipsilateral hemisphere from the area of the
contralateral hemisphere and summing the infarct area across all slices
from each brain.
Ischemia/Reperfusion and Behavioral Assessment of Neurological
Damage
MCAO was carried out under Isoflurane anesthesia as described.
9
Briefly,
mice were anesthetized 15 minutes before occlusion and maintained
under anesthesia throughout the 1-hour occlusion period. To induce
MCAO, an incision was made along the midline of the neck to expose the
left external carotid and pterigopalatine arteries, which were isolated and
tied off with 6-0 silk thread. The internal carotid artery (ICA) was then
occluded distal to the bifurcation of the ICA and the pterigopalatine artery
using small clip, and the common carotid artery was ligated with 6-0 silk
thread. The external carotid artery (ECA) was then incised and a blunt-tip
6-0 nylon monofilament was inserted into the ECA. The junction below the
ECA and the inserted nylon thread was tightened with 6-0 silk suture to
prevent bleeding during manipulation of the nylon thread, which was
rotated during its advancement into the ICA. The clip at the ICA was then
removed and the nylon thread was advanced to a distance slightly more
than 6 mm from the tip of the nylon thread the ICA-pterygophalatine
artery bifurcation. This distance relative to the ICA-ECA bifurcation is
slightly less than 9 mm. During MCAO, the parietal bone on the occluded
side becomes slightly transparent, and laser Doppler flowmetry revealed
that blood flow in this area decreases to less than 10% of baseline. After a
1-hour occlusion period, the nylon thread and the common carotid artery
ligature are removed and the neck incision is closed with wound clips. In
the sham surgery group, the arteries are visualized but not disturbed.
Functional outcomes were assessed daily throughout the 7 days after
ischemia and reperfusion injury. Behavioral deficits were evaluated using a
5-point score as described
9
(0, no deficit; 1, failure to extend right paw; 2,
circling to the right; 3, falling to the right; and 4, unable to walk
spontaneously), with higher scores reflecting greater sensorimotor
impairment. All behavioral analyses were conducted by an experimenter
who was blind to the dietary condition of the animals.
Quantification of Serum Leptin
Circulating levels of leptin were determined using a commercial ELISA kit
(EZML-82K, Mouse Leptin ELISA; Millipore, Temecula, CA) in serum samples
collected from sham and MCAO animals 3 days after surgery. Assay
procedures followed the manufacturer’s specifications.
Immunohistochemistry and Immunofluorescence
Brains were postfixed for 24 hours in 4% paraformaldehyde in phosphate
buffer, then moved into 0.1 M phosphate-buffered saline, before genera-
tion of 40-mm transverse sections using a vibrating tissue slicer (Leica).
Sections were collected as a 1:6 series and stored in cryoprotectant at
20 1C in preparation for immunohistochemical and immunofluorescence
reactions. Peroxidase detection of BrdU and the endogenous proliferation
marker, Ki67, followed previously published methodology,
13
with the
exception that the current study used different primary antibodies for
detection of BrdU (1:200; BD Pharmingen, San Jose, CA) and Ki67 (1:500,
Millipore). Primary antibodies against BrdU and Ki67 were detected with
biotin-conjugated secondary antibodies (Vector Laboratories, Burlingame,
CA), amplified via avidin–biotin–horseradish peroxidase, and visualized
with diaminobenzadine. Nuclei were counterstained with Cresyl Violet
(Sigma-Aldrich).
For TUNEL staining, we used a peroxidase labeling kit from Promega
(Madison, WI, USA), according to the manufacturer’s instructions. For
Fluoro-Jade B histology, slides were pretreated with 0.06% KMnO
4
for
15 minutes, rinsed, and incubated in Fluoro-Jade B working solution
(0.01% Fluoro-Jade B from Molecular Probes (Carlsbad, CA) in ddH
2
O with
0.1% acetic acid) overnight. Fluoro-Jade-positive nuclei were quantified
throughout the rostrocaudal extent of the dentate gyrus, including the
hilar region. For the SVZ, virtually all Fluoro-Jade-positive cells localized to
the striatum, and these cells were counted and normalized to the sampled
area with the aid of StereoInvestigator software (Microbrightfield, Williston,
VT). Immunofluorescent staining of BrdU and phenotypic markers followed
previously published protocols,
14
with minor modifications. Free-floating
tissue sections were denatured in 2 N HCl:Tris-buffered saline (TBS) for
30 minutes, blocked in 5% normal goat serum in TBS containing 0.5%
Triton X 100, then reacted overnight in primary antibody mouse anti-BrdU
(1:250, BD Pharmingen). The BrdU antibody was detected using a
biotinylated secondary and chromated with Streptavidin Alexa 568
(Molecular Probes). Antibodies directed against the immature neuronal
marker doublecortin (DCX; 1:250, Abcam, Cambridge, MA), the mature
neuronal marker NeuN (1:500, Millipore), and the astroglial marker glial
fibrillary acidic protein (GFAP) (1:1,000, Pierce, Rockford, IL) were then
applied overnight. Bound primary antibodies for the cell type markers
were visualized with Alexa 488-conjugated secondary antibodies
(Molecular Probes) directed against the appropriate host, followed by
nuclear counterstaining with DAPI. Images were collected on a Zeiss
LSM 510 Meta confocal microscope (Jena, Germany).
Stereological Quantification of Labeled Cells
For quantification of BrdU-, Ki67-, and TUNEL-immunoreactive nuclei
visualized with peroxidase immunohistochemistry, unbiased stereological
sampling schemes were applied using StereoInvestigator software
(Microbrightfield) as described.
14
All cell counts were conducted blind to
the treatment condition of the animals. For analysis of BrdU-, Ki67-, and
TUNEL-positive cells in the hippocampus, total cell counts were generated
using an XY step size of 150 mm, a disector height of 10 mm, and a 25-mm
sampling frame. For quantification of labeled nuclei in the SVZ, the XY step
size was reduced to 50 mm, with other sampling parameters consistent
with those used in the hippocampus. Numbers of BrdU-, Ki67-, and TUNEL-
positive nuclei in the SVZ were normalized to the sampled volume of the
SVZ and expressed as cell densities.
For analysis of colabeling, 100 BrdU-labeled cells were sampled from
both blades of the dentate gyrus, with sampling anatomically balanced
along the dorsal–ventral and septotemporal axes. Similarly, 100 BrdU-
labeled cells were scanned throughout the extent of the SVZ. Double-
labeled cells were visualized using a 63 objective (numerical
aperture ¼1.4) on a Zeiss LSM 510 Meta confocal microscope, with
images captured under 2 optical zoom at 512 512 pixel density with a
1-mm step size in the zplane. To assess colocalization, cells were analyzed
offline in ImageJ software, and fluorescence signals in the two channels
used for visualization of BrdU and phenotypic markers were required to
exhibit 480% spatial overlap across each captured plane in which the cell
nucleus (visualized with DAPI) was visible. For MCAO animals, colabeling
Intermittent fasting improves stroke recovery
S Manzanero et al
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Journal of Cerebral Blood Flow & Metabolism (2014), 897 – 905 &2014 ISCBFM
was assessed on the contralateral (right) hemisphere; to be consistent, cells
scanned in the hippocampus and SVZ of the sham-operated mice were
also sampled from the right hemisphere. BrdU-labeled cells in the SVZ
were further analyzed based on their proximity to the ventricle, derived
from the Euclidean distance (in mm) from the nucleus to the ventricular
wall. Nuclei within 20 mm of the ventricle were classified as SVZ
progenitors, whereas nuclei 420 mm from the ventricles were character-
ized as putative differentiated cells. All of the potentially differentiated
cells localized exclusively to the striatum.
Statistical Analysis
Body weights and functional outcomes after MCAO were compared across
AL and IF mice using repeated measures analysis of variance (ANOVA).
Leptin levels and the percentages of cells expressing markers of a given
phenotype were compared across diet and surgical conditions using a
22 ANOVA. Infarct volumes were compared between AL and IF mice
using a bidirectional t-test. Comparison of the numbers and densities of
BrdU- or Ki67-labeled nuclei in the hippocampus and SVZ used separate
22 ANOVA designs to evaluate the effects of AL or IF on cell numbers
after MCAO or sham surgery in the ipsilateral or contralateral sides. Data
from quantitative measures of cell death were analyzed using the same
statistical design applied to measures of cell birth. Relationships between
markers of cell proliferation and markers of cell death were assessed using
Pearson’s correlation. All statistical analyses were carried out in SPSS
(version 18.0) or GraphPad Prism (version 4.0) with significance at Po0.05.
Because these analyses require normally distributed data sets, comparisons
between two groups used t-tests to compare variances, and comparisons
across more than two groups used ANOVA to compare variances. None of
the tests revealed significant differences, and as such were considered
normally distributed.
RESULTS
IF Reduces Body Weight and Lowers Circulating Factors Associated
with Adipose Tissue
Most previous studies examining the physiological and neurolo-
gical consequences of IF used alternating 24-hour cycles of fasting
and refeeding.
15
To determine whether a 16:8 hours schedule of
fasting and refeeding might exert comparable physiological
effects, we monitored body weight and food intake, and
measured serum levels of leptin, a reliable correlate of adiposity.
Male C57Bl6J mice maintained on IF under the 16:8 hours
schedule gained weight more slowly than mice with ad libitum
food access (Figure 1A; F
1,11
¼6.49, P¼0.001). Reductions in body
weight were accompanied by reduced levels of the adipocyte
cytokine leptin in serum from IF mice (Figure 1B; for the effect of IF
in sham-operated mice, F
1,13
¼7.73, P¼0.001). Unlike the 24-hour
fasting and refeeding cycle, in which 48-hour food intake is
comparable between IF and AL mice,
15
the 16:8 hours schedule
was accompanied by reduced food intake (t
9
¼7.12, P¼0.009). IF
mice on the 16:8 hours fasting and refeeding cycle ate
3.35±0.09 g/24 h, whereas AL mice ate 4.32±0.08 g/24 h (values
represent mean±s.e.m.). This pattern of reduced food intake
indicates that the 16:8 schedule resembles caloric restriction, with
associated declines in body weight and circulating markers of
adiposity.
IF Reduces Sensorimotor Impairment and Infarct Size After
Ischemia and Reperfusion
The functional consequences of ischemia and reperfusion injury
were evaluated over a 1-week period, and throughout that period,
mice maintained on the IF diet consistently demonstrated less
impairment relative to mice fed ad libitum (Figure 2A; F
1,13
¼2.73,
P¼0.02). Protection against stroke-induced sensorimotor deficits
was accompanied by complete prevention of the drastic reduction
in circulating leptin observed after stroke in AL mice (Figure 1B).
Leptin levels fell to less than 15%t of sham-operated animals at 3
days post MCAO in AL mice, but were maintained at this same
time point in IF mice (for the interaction between diet and stroke,
F
1,13
¼4.5, P¼0.01). We measured leptin levels 3 days after MCAO
because this is the time point when previous studies
9
detected
differences in molecular markers of neuroprotection between IF/
MCAO and AL/MCAO mice. In this regard, changes occurring
72 hours post-stroke may determine the transition between the
acute phase, characterized by widespread cellular damage, and
the post-stroke recovery phase, involving recruitment of cell
growth and plasticity pathways.
IF mice exhibit smaller infarcts 3 days after ischemia
and reperfusion injury, relative to mice on the ad libitum diet
Figure 1. Intermittent fasting (IF) reduces body weight and prevents
stroke-induced reductions in circulating leptin levels. (A) Weight
gain is reduced in mice exposed to IF on a 16:8 hours fasting and
refeeding schedule. Asterisk (*) indicates statistical significance
at Po0.05 after repeated measures analysis of variance (ANOVA).
(B) Stroke dramatically reduces circulating leptin levels in mice on
the ad libitum diet, but not in mice maintained on IF. Asterisk (*, **)
indicates statistical significance at Po0.05 (*) or Po0.01 (**) follow-
ing 2 2 ANOVA. Leptin levels were assessed 3 days after middle
cerebral artery occlusion (MCAO) or sham operation and error bars
represent the s.e.m. AL, ad libitum.
Figure 2. Intermittent fasting (IF) improves functional outcomes
and reduces infarct size in male mice. (A) Three months of IF atten-
uates sensorimotor deficits after middle cerebral artery occlusion
(MCAO). (B) Laser Doppler flowmetry shows no differences between
groups in the extent to which MCAO compromises blood flow.
(C) IF reduces infarct size in male C57Bl6J mice. (D) Staining for
triphenyltetrazolium chloride (TTC) in 2-mm-thick brain sections
taken after reperfusion reveals smaller infarcts in mice on the IF
diet. Asterisk (*) indicates statistical significance at Po0.05 follow-
ing t-test (C) or repeated measures analysis of variance (A,B).
AL, ad libitum.
Intermittent fasting improves stroke recovery
S Manzanero et al
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&2014 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2014), 897 – 905
(Figures 2C and 2D; t
13
¼5.18, P¼0.002). Improved outcomes
could not be explained by differences in the extent to which
MCAO compromised blood flow during the stroke, as laser
Doppler flowmetry measures conducted in a subset of AL and IF
mice during MCAO revealed comparable reductions in blood flow
during and immediately after ischemia and reperfusion (Figure 2B;
F
1,13
¼2.08, P¼0.18). Although we cannot rule out vasogenic
contributions to improved functional recovery, it is not likely that
the smaller infarcts observed in IF mice were attributable to
differences in flow during and after MCAO.
IF Enhances Basal and Suppresses Stroke-Induced Cell Proliferation
in the Hippocampus
To assess the proliferative response to MCAO, we administered
the thymidine analog BrdU (50 mg/kg, intraperitoneally) for 2
days, with injections every 12 hours, starting 5 days after MCAO or
sham surgery. This time course was based on early reports
identifying the week after ischemia and reperfusion as a temporal
window for increases in proliferative activity in the hippocampal
dentate gyrus and SVZ.
11
Previous work using 24-hour feeding
and fasting schedules revealed that this schedule increases BrdU-
labeled cell number in the hippocampus.
6
We also detected
increases in the number of BrdU-labeled cells in the dentate gyrus
of sham-operated mice on the 16:8 hours fasting and refeeding
schedule (Figure 3A and 3B; F
1,18
¼25.68, P¼0.001). However,
stroke-induced increases in the number of BrdU-labeled cells were
substantially attenuated in both the ipsilateral and contralateral
hemispheres of mice on the 16:8 hours fasting and refeeding
schedule (F
1,19
¼16.04, P¼0.009).
Administration of multiple pulse injections of BrdU labels a
mixed population of proliferating cells and their progeny. More-
over, BrdU is an exogenous marker, and its incorporation could be
influenced by differences in availability and/or uptake. To measure
cell proliferation without the confound of availability of an
exogenous marker, we performed immunohistochemical detec-
tion of Ki67, an endogenous proliferative marker expressed
throughout the cell cycle. Using this marker, we again detected
increases in the number of labeled cells in the dentate gyrus
of sham-operated mice on the IF diet (Figures 3C and 3D;
Figure 3. Intermittent fasting (IF) enhances cell proliferation and survival in the dentate gyrus of intact mice, and attenuates pathological
increases after stroke. (A) To facilitate anatomical visualization of both the hippocampus and subventricular zone, transverse sections were
used for peroxidase labeling. (B) Graph shows stereological estimates of bromodeoxyuridine (BrdU)-labeled cell numbers in the indicated
hemispheres for each treatment group. Asterisk (*) indicates statistical significance following 2 2 analysis of variance. (C) Stereological
estimates of Ki67-labeled cell number in each treatment condition revealed that IF promotes cell proliferation under intact conditions, and
suppresses pathological increases in mice that received middle cerebral artery occlusion (MCAO). (D) Low-power micrographs of hippocampal
BrdU labeling on transverse sections of the mouse hippocampus. The boxed areas are shown in the insets, which depict clusters of
BrdU-labeled cells in each condition. Arrows indicate labeled cells. Scale bar, 100 mm. (E) Low-power micrographs showing sections of mouse
hippocampus stained with antibodies against the endogenous marker of cell proliferation Ki67. Boxed area for each micrograph is shown in
the inset, which depicts clusters of Ki67-labeled cells in each treatment group. Arrows indicate labeled cells. Scale bar, 100 mm. AL, ad libitum.
Intermittent fasting improves stroke recovery
S Manzanero et al
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Journal of Cerebral Blood Flow & Metabolism (2014), 897 – 905 &2014 ISCBFM
Figure 4. Intermittent fasting (IF) enhances cell proliferation and survival in the intact subventricular zone (SVZ), and protects against
pathological increases with stroke. (A) The density of bromodeoxyuridine (BrdU)-labeled cells in the SVZ is significantly increased with
IF under basal conditions. After stroke, pathological increases in BrdU-labeled cell numbers are suppressed by IF. (B) Micrographs depict
BrdU-labeled cells in the SVZ of mice in each condition. (C) The density of Ki67-labeled cells increases in intact mice on the IF diet, and
pathological elevations in cell proliferation after stroke are suppressed. (D) Images of Ki67-labeled cells in the SVZ of mice in each condition.
Scale bars, 50 mm(B,D). Arrows indicate labeled cells. For all graphs, asterisk (*) indicates significance at Po0.05 following 2 2 analysis of
variance. AL, ad libitum.
Figure 5. Bromodeoxyuridine (BrdU)-labeled cells in the hippocampal dentate gyrus express neuronal and astroglial markers.
(A) Immunodetection of BrdU and NeuN in the hippocampal dentate gyrus for each condition. (B) Coexpression of BrdU and the immature
neuronal marker doublecortin (DCX) in the dentate gyrus of the hippocampus. (C) BrdU-labeled cells also express the astroglial marker glial
fibrillary acidic protein (GFAP) in the hippocampal dentate gyrus. (D) Similar proportions of BrdU-labeled cells express neuronal or astroglial
markers across groups. For the micrographs from middle cerebral artery occlusion (MCAO) mice in panels A–C, the contralateral hippocampus
is shown. AL, ad libitum; IF, intermittent fasting.
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S Manzanero et al
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&2014 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2014), 897 – 905
F
1,18
¼11.53, P¼0.002), indicating enhancement of cell prolifera-
tion in these mice. Similarly, elevations in Ki67-labeled cell
numbers observed after MCAO in AL mice were blunted in mice
on the IF feeding schedule (F
1,19
¼22.56, P¼0.001). This suggests
that the neurogenic response to stroke is commensurate with the
extent of neurological damage, as the IF mice, which exhibit
smaller infarcts, also show attenuation of the proliferative
response to MCAO.
IF Promotes Cell Proliferation in the Intact SVZ and Attenuates
Increases after MCAO
The SVZ also responds to stroke with increases in cell proliferation
and neurogenesis, but the dietary regulation of proliferation
among SVZ stem and progenitor cells has never been assessed,
either in the intact or in the injured brain. We measured the
density of BrdU-labeled cells in the SVZ of mice maintained on AL
or IF feeding schedules, and observed increased numbers of BrdU-
positive cells in sham-operated mice after 3 months of the IF
feeding schedule (Figures 4A and 4B; F
1,18
¼15.48, P¼0.002).
Although IF increased BrdU-labeled cell numbers in shams, the
opposite effect was observed after MCAO, with smaller increases
in BrdU-labeled cell numbers in mice on the IF schedule
(F
1,16
¼9.54, P¼0.007). In this regard, the SVZ responds to IF in
a manner that is similar to the hippocampus, with enhancement
of BrdU-labeled cell numbers in the intact SVZ, and reductions in
stroke-induced BrdU incorporation.
We also examined the number of nuclei expressing the
endogenous marker Ki67 in the SVZ of MCAO or sham-operated
mice exposed to IF or ad libitum feeding. Based on the number of
Ki67-labeled nuclei in sham-operated mice, the IF feeding
schedule elicits increases in the number of immunoreactive
nuclei, suggestive of an expanded pool of proliferating progeni-
tors (Figures 4C and 4D; F
1,16
¼17.65, P¼0.008). By contrast, the
increase in Ki67-labeled cell numbers observed after MCAO was
smaller in mice on the IF feeding schedule (F
1,15
¼9.15, P¼0.007).
Taken together, these observations suggest that the proliferative
response to MCAO is dampened by IF feeding, which also
promotes cell proliferation and survival among neurogenic
regions in the intact brain.
Differentiation of Neurons and Glia is Unaffected by Diet or
Ischemia and Reperfusion
Newly generated cells in the subgranular and SVZs differentiate
into functional neurons and astrocytes, but the proportion of cells
expressing neuronal or astroglial markers is unchanged by MCAO
or dietary manipulation (Figures 5 and 6). After four injections of
BrdU (50 mg/kg), administered every 12 hours starting 5 days after
MCAO or sham surgery, comparable numbers of BrdU-labeled
cells in the subgranular zone expressed the mature neuronal
marker NeuN across all treatment groups (Figures 5A and 5D;
percent double-labeled; mean±s.e.m., SHAM/AL ¼77.43±4.93,
SHAM/IF ¼78.98±5.18, MCAO/AL ¼77.89±3.37, MCAO/IF ¼
82.37±3.64). Coexpression of BrdU and the immature neuronal
marker doublecortin in the dentate gyrus was also unaffected by
diet or MCAO (Figures 5B and 5D; percent double-labeled;
mean±s.e.m., SHAM/AL ¼10.54±1.24, SHAM/IF ¼11.07±1.23,
MCAO/AL ¼12.31±1.62, MCAO/IF ¼9.08±0.86). A smaller pro-
portion of BrdU-labeled cells expressed GFAP, a marker of
astrocytes. Again, the proportion of cells that coexpress BrdU
and GFAP in the dentate gyrus was unaffected by stroke or dietary
intervention (Figures 5C and 5D; percent double-labeled; mean±
s.e.m., SHAM/AL ¼5.43±0.59, SHAM/IF ¼6.71±0.51, MCAO/
AL ¼6.73±0.50, MCAO/IF ¼6.93±0.65).
Consistent with previous reports,
11
far fewer BrdU-labeled cells
in the SVZ were colabeled with the mature neuronal marker NeuN,
relative to the dentate gyrus, and virtually all of the NeuN/BrdU
Figure 6. New cells in the subventricular zone (SVZ) express neuronal and astroglial markers, with no change in cell fate acquisition
after intermittent fasting. (A) Bromodeoxyuridine (BrdU)-labeled cells in the SVZ express the mature neuronal marker NeuN. (B) Newly
generated cells also coexpress BrdU and the immature neuronal marker doublecortin (DCX). (C) BrdU-labeled cells in the SVZ also express glial
fibrillary acidic protein (GFAP), a marker of astrocytes. (D) Comparable proportions of cells express DCX, NeuN, and GFAP across conditions. For
the low-magnification micrographs from middle cerebral artery occlusion (MCAO) mice in panels A–C, the ipsilateral hemisphere is indicated
by the arrows and the contralateral hemisphere is shown in the insets. AL, ad libitum; IF, intermittent fasting.
Intermittent fasting improves stroke recovery
S Manzanero et al
902
Journal of Cerebral Blood Flow & Metabolism (2014), 897 – 905 &2014 ISCBFM
double-positive cells localized to the ischemic striatum distal
to the ventricular wall. The proportion of cells double-labeled
with BrdU and NeuN in the striatum was comparable between
sham-operated and MCAO mice in the different dietary
conditions (Figures 6A and 6D; percent double-labeled; mean±
s.e.m., SHAM/AL ¼12.35±1.56, SHAM/IF ¼11.67±1.16, MCAO/
Figure 7. Intermittent fasting (IF) reduces cell death in neurogenic regions, limiting the proliferative response to stroke. (A) Fluoro-Jade B
staining and DAPI counterstaining in the ipsilateral hemisphere of the indicated regions (Hipp, hippocampus; Stri, striatum) after middle
cerebral artery occlusion (MCAO). Graphs depict Fluoro-Jade-labeled cell counts from the indicated regions. For all micrographs, the dorsal
surface is oriented at absolute north and lateral surface is oriented at west. (B) TUNEL staining in the indicated regions from an ipsilateral
section from the brains of mice on the ad libitum (AL) or IF diets. Graphs depict TUNEL-positive cell counts from the indicated regions. (C) Left
graph depicts a positive correlation between the number of Fluoro-Jade B-positive cells in the hippocampus, and the number of
bromodeoxyuridine (BrdU)-labeled cells in the hippocampus after MCAO. Right graph indicates that increased density of Fluoro-Jade
B-positive cells in the ipsilateral striatum is associated with greater numbers of BrdU-labeled cells in the ipsilateral subventricular zone (SVZ).
(D) Left graph shows that the number of TUNEL-positive cells in the dentate gyrus is positively correlated with the number of Ki67-labeled
cells. Right graph demonstrates that TUNEL-positive cell density is positively correlated with BrdU-labeled cell density in the ipsilateral SVZ.
For all graphs, error bars represent the s.e.m. and asterisk (*) indicates statistical significance at Po0.05 following (A,B)22 analysis of
variance or Pearson’s correlation (C,D).
Intermittent fasting improves stroke recovery
S Manzanero et al
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&2014 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2014), 897 – 905
AL ¼10.76±1.37, MCAO/IF ¼14.48±1.56). Although few BrdU-
labeled cells expressed NeuN in the SVZ proper, the vast majority
of BrdU-labeled cells in this region expressed the immature
neuronal marker doublecortin, with no change in the rate
of colabeling across groups (Figures 6B and 6D; percent
double-labeled; mean±s.e.m., SHAM/AL ¼62.36±6.18, SHAM/
IF ¼61.46±2.76, MCAO/AL ¼67.02±5.32, MCAO/IF ¼66.01±
5.32). Coexpression of BrdU and GFAP was also unaffected by
diet or MCAO in the SVZ (Figures 6C and 6D; percent double-
labeled; mean±s.e.m., SHAM/AL ¼4.85±0.41, SHAM/IF ¼4.38±
0.56, MCAO/AL ¼6.17±0.68, MCAO/IF ¼5.43±0.72).
Reduced Cell Loss in Mice on IF and Correlated Rates of Cell Death
and Proliferation
To investigate the relationship between neuron loss and neuro-
genesis in greater detail, we performed Fluoro-Jade B histology
and TUNEL staining in the same set of mice used for measurement
of BrdU- and Ki67-labeled cell numbers. Consistent with their
smaller infarct sizes, mice on the IF diet had fewer cells that stained
positive for Fluoro-Jade B (Figure 7A; for the dentate gyrus,
F
1,16
¼11.10, P¼0.001, for the SVZ, F
1,16
¼4.86, P¼0.04) or TUNEL
(Figure 7B; for the dentate gyrus, F
1,16
¼5.90, P¼0.02, for the SVZ,
F
1,16
¼4.93, P¼0.04). Negligible labeling for Fluoro-Jade B or
TUNEL was detected in mice after sham surgery (data not shown).
Importantly, the hypothesis that increases in cell proliferation after
MCAO represent a response to cellular damage and loss was
upheld in our analysis of correlations between markers of cell
death and birth (Figures 7C and 7D; for the correlation between
BrdU-labeled cell number and Fluoro-Jade-positive cell numbers in
the hippocampus, Pearson’s r¼0.92, P¼0.001; for the correlation
between BrdU-labeled cell densities in the ipsilateral SVZ and
Fluoro-Jade-positive cells in the ipsilateral striatum, Pearson’s
r¼0.94, P¼0.001; for the correlation between hippocampal
Ki67-labeled cell number and hippocampal TUNEL-positive cell
number, Pearson’s r¼0.97, P¼0.001; for the correlation between
ipsilateral BrdU-labeled cell numbers and TUNEL-positive cell
densities in the SVZ, Pearson’s r¼0.75, P¼0.02). This pattern
suggests that the neuroprotective consequences of IF lead to
reduced injury, thereby preventing pathological increases in cell
proliferation and neurogenesis after MCAO.
DISCUSSION
We have observed that extending the interval between meals
reduces basal leptin levels and protects against stroke-induced
reductions in circulating leptin concentrations. This endocrine
pattern was accompanied by an enhancement of basal cell
proliferation and attenuation of stroke-induced cell proliferation in
the hippocampal subgranular zone and in the SVZ, two regions of
prominent ongoing neurogenesis in the adult brain. Dampening
of the proliferative response to stroke may be attributable to the
smaller infarcts and reduced cell death detected in mice on the IF
regimen. Although alterations in the number of new cells
generated across these regions occurred after stroke and dietary
intervention, the proportion of cells expressing neuronal markers
was unchanged, in support of changes in neurogenesis because
alterations in cell proliferation rather than differentiation. Taken
together, these observations suggest that IF enhances plasticity in
the intact brain and is neuroprotective after ischemia and
reperfusion injury.
We used a 16:8 fasting–feeding regimen in this study to
determine whether extending the interval between meals is
neuroprotective in a mouse model of focal cerebral ischemia.
Because infusion of exogenous leptin is neuroprotective in
ischemia and may improve outcome by activating the PI3K/Akt
pathway,
16,17
IF-induced alterations in leptin levels or sensitivity
could be a contributing factor to the decreased infarct sizes
observed in IF animals. IF reduced leptin levels in control animals
and prevented decreases in leptin 3 days after ischemia and
reperfusion, suggestive of improved defense of a metabolic set
point. Given previous work demonstrating that exogenous leptin
reduces infarct size,
16,17
and that leptin receptor deficient mice
exhibit greater impairment and more widespread cell death after
ischemia,
18
changes in leptin levels or leptin sensitivity represent a
likely mechanism for neuroprotection in IF mice. Because both AL
and IF mice had food freely available during the post-stroke
recovery period, we cannot rule out the possibility that IF mice
simply consumed more food than AL mice during the period
immediately after MCAO induction. However, whether due to
intake or other metabolic factors, the pattern of neuroprotection
observed after MCAO in IF mice is likely to be associated with their
maintenance of leptin levels within the range of intact animals.
The observation that circulating leptin levels are substantially
reduced by stroke in AL mice, but not in IF mice, is consistent with
the idea that improved defense of a metabolic set point confers
neuroprotection after ischemia.
Stroke increases cell proliferation in both the SVZ and the
hippocampus,
11,19
but the extent to which new cells promote or
suppress functional recovery is not yet known. The pathological
microenvironment encountered by new neurons generated as a
result of stroke could impair their synaptic integration, leading to
functional deficits across multiple behavioral domains. We
observed that IF reduces both cell death and cell birth after
stroke, and improves sensorimotor outcomes. Future studies will
be necessary to determine whether IF also protects against
functional decrements in other domains, such as learning and
memory. Labeling for BrdU and Ki67 revealed that IF increased
basal cell proliferation in sham-operated mice; based on earlier
studies, this effect likely depends on diet-induced increases in
levels of brain-derived neurotrophic factor.
6,20
As previously
reported,
11
ischemia/reperfusion injury enhanced cell prolifera-
tion, particularly in the ipsilateral hemisphere, but IF animals in the
current experiment exhibit smaller increases in cell proliferation
after ischemia when compared with AL mice. It is not fully known
if less severe infarcts account for a lower degree of neurogenesis,
but the concept of proliferation after injury as a mechanism for
population control is an intriguing possibility that is supported by
the correlations that we observed between markers of cell death
and birth. In this regard, IF appears to precondition neurons,
blunting cell death after ischemic insult and limiting pathological
increases in cell proliferation as a result of reduced damage. This
pattern underscores the importance of energy metabolism and
diet as determinants of cellular and functional outcomes after
stroke.
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGMENTS
The authors are grateful to Alexander Widiapradja and Yi-Lin Cheng for technical
assistance, and to Drs Adviye Ergul and William Hill for comments on the manuscript.
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