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Short-term Preoperative Dietary Restriction Is
Neuroprotective in a Rat Focal Stroke Model
Ka
¨rt Varendi
1.
, Mikko Airavaara
1.
, Jenni Anttila
1
, Sarah Vose
2
, Anu Planken
1¤
, Mart Saarma
1
,
James R. Mitchell
2
*
"
, Jaan-Olle Andressoo
1
*
"
1Institute of Biotechnology, University of Helsinki, Helsinki, Finland, 2Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston,
Massachusetts, United States of America
Abstract
Stroke is a major complication of cardiovascular surgery, resulting in over 100,000 deaths and over a million postoperative
encephalopathies annually in the US and Europe. While mitigating damage from stroke after it occurs has proven elusive,
opportunities to reduce the incidence and/or severity of stroke prior to surgery in at-risk individuals remain largely
unexplored. We tested the potential of short-term preoperative dietary restriction to provide neuroprotection in rat models
of focal stroke. Rats were preconditioned with either three days of water-only fasting or six days of a protein free diet prior
to induction of transient middle cerebral artery occlusion using two different methods, resulting in either a severe focal
stroke to forebrain and midbrain, or a mild focal stroke localized to cortex only. Infarct volume, functional recovery and
molecular markers of damage and protection were assessed up to two weeks after reperfusion. Preoperative fasting for 3
days reduced infarct volume after severe focal stroke. Neuroprotection was associated with modulation of innate immunity,
including elevation of circulating neutrophil chemoattractant C-X-C motif ligand 1 prior to ischemia and suppression of
striatal pro-inflammatory markers including tumor necrosis factor a, its receptor and downstream effector intercellular
adhesion molecule-1 after reperfusion. Similarly, preoperative dietary protein restriction for 6 days reduced ischemic injury
and improved functional recovery in a milder cortical infarction model. Our results suggest that short-term dietary
restriction regimens may provide simple and translatable approaches to reduce perioperative stroke severity in high-risk
elective vascular surgery.
Citation: Varendi K, Airavaara M, Anttila J, Vose S, Planken A, et al. (2014) Short-term Preoperative Dietary Restriction Is Neuroprotective in a Rat Focal Stroke
Model. PLoS ONE 9(4): e93911. doi:10.1371/journal.pone.0093911
Editor: Cesar V. Borlongan, University of South Florida, United States of America
Received January 31, 2014; Accepted March 7, 2014; Published April 4, 2014
Copyright: ß2014 Varendi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by
the Sigrid Juselius Foundation; the Academy of Finland [grant number 250275, 256398 to MA; 136591, 140983 and 263700 to JOA; 1126735 to MS]; the Doctoral
Program Brain & Mind to KV; JRM was supported by the National Institutes of Health [AG036712 and DK090629]; and SV by Radiation Biology T32CA009078.
Competing Interests: JRM has consulted with a company that makes medical foods for cancer treatment. This study does not deal with cancer, but stroke, and
JRM has no ownership in the aforementioned company (L-Nutra). This does not alter adherence to PLOS ONE policies on sharing data and materials. None of the
other authors declares any competing interests.
* E-mail: jmitchel@hsph.harvard.edu (JRM); Jaan-Olle.Andressoo@helsinki.fi (JOA)
.These authors contributed equally to this work.
"These authors also contributed equally to this work.
¤ Current address: Department of Research and Development, North Estonian Medical Center, Tallinn, Estonia
Introduction
Perioperative stroke occurring during or soon after surgery is a
major cause of morbidity and mortality, with an average incidence
of 2–13% in cardiovascular procedures and 0.08–0.7% in non-
cardiovascular procedures [1,2]. With 7 million cardiovascular
and 21 million non-cardiovascular surgeries performed annually in
the US [3] and similar numbers in Europe (extrapolated from data
from the Netherlands [4]), annual deaths are calculated to be in
excess of 180,000 on these two continents alone. Those left with
the debilitating consequences of perioperative stroke/encephalop-
athy number an order of magnitude higher [5,6]. Treatment of
perioperative stroke accounts for a quarter of the resources spent
annually for stroke treatment in the USA [5].
Numerous pharmacological compounds have been tested for
their ability to provide neuroprotection after stroke, including 5-
HT
1a
agonists, free radical scavengers, immunosuppressants and
agents that block excitotoxicity. Despite efficacy in rodent models,
most have failed in clinical trials [7]. While perioperative stroke
risk assessment prior to surgery is a common practice, general
prophylactic methods are lacking [1,2], underpinning a need for
basic research.
Strategies that provide neuroprotection when initiated before
the ischemic period are known as preconditioning. Ischemic
preconditioning is a phenomenon in which brief periods of
ischemia protect against subsequent, longer insults to various
organs, including heart [8] and brain [9]. In preclinical models of
stroke, ischemic preconditioning prevents subsequent ischemic
injury by suppressing the expression of pro-inflammatory cyto-
kines, chemokines, adhesion molecules and transcription factors
[10]. Other low-dose stressors such as hypoxia [11], endotoxin
[12] or heat shock [13] can also precondition against ischemic
injury. However, the clinical application of such methods has
remained a matter of debate in large part due to the potential
PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e93911
safety concerns, highlighting a need for safer preoperative
prophylactic methods.
Dietary restriction (DR), defined as reduced food intake without
malnutrition, extends lifespan and increases resistance to a variety
of acute stressors in multiple species, including rodents [14]. Long-
term application of DR for 3 months or longer is neuroprotective
in rodent models of stroke [15] and excitotoxicity [16–18].
Mechanistically, upregulation of neurotrophic and growth factors,
such as brain-derived neurotrophic factor (BDNF) induced by
long-term DR [19,20] could be partially responsible for increased
protection, as BDNF has been shown to reduce neuronal injury
after ischemia [21,22]. Long-term DR also offers benefits against
ischemic injury in other organs, such as heart, through a variety of
mechanisms including immunosuppression, elevation of reactive
oxygen and nitrogen species scavenging mechanisms and upregu-
lation of heat shock protein levels [23,24].
Despite its potential as a safe and effective prophylactic method,
the relatively long periods of food restriction employed in
preclinical studies (3 months or longer) are not considered feasible
in a clinical setting [25]. However, recent data indicate that
dietary preconditioning against ischemic injury can be realized in
a clinically relevant time frame in rodent models [26]. For
example, 2 weeks of 30% reduced daily food intake or 3 days of
water-only fasting protect against ischemia reperfusion injury to
kidney or liver [27]. Protein restriction in the absence of calorie
restriction, or restriction of individual essential amino acids such as
tryptophan, can also impart benefits within 6 days [28]. Here, we
describe two different pre-operative manipulations – 3 days of
water-only fasting and 6 days of protein-free DR – with benefits on
focal stroke outcomes in rats.
Methods
Please also see Methods S1 for further details.
Animals
All animal experiments were carried out according to the
National Institute of Health (NIH) guidelines for the care and use
of laboratory animals and approved by the appropriate local or
national ethics board (permit number ESAVI/5459/04.10.03/
2011, issued by ELA
¨INKOELAUTAKUNTA – ELLA Etela¨-
Suomen aluehallintovirasto, Finland). Adult male Sprague-Dawley
(SD) rats weighing 240–300 g were housed under standard
conditions with ad libitum access to food and water unless indicated
otherwise.
Dietary preconditioning regimens
Fasting was performed by removing the complete chow diet
(Harlan Teklad Global 2016 Rodent Diet) for 3d while
maintaining free access to water at all times. Protein-free dietary
restriction was performed by first acclimating all animals to a
complete diet made of refined ingredients (Research Diets
D12450B) consisting of 18% calories from protein (casein), 72%
from carbohydrate (corn starch, maltodextrin, sucrose) and 10%
from fat (soybean oil, lard) for 6d. The control group was then
maintained on the complete diet, and the protein-free group was
given restricted access to an isocaloric diet lacking protein
(Research Diets D08043003, consisting of 90% calories from
carbohydrate and 10% from fat) at 60% of the average daily intake
of the complete diet group for 6d prior to tMCAO and 2d after
reperfusion.
Surgical procedures
Two different stroke models were employed. In experiments
testing the effects of fasting, a severe focal stroke involving
forebrain and midbrain was induced by intraluminal occlusion of
the middle cerebral artery (MCA) with a filament for 60 min,
followed by reperfusion as described previously [29]. Stroke
involving forebrain and midbrain is associated with fever [30]. To
determine whether the MCA occlusion surgery produced a lesion,
core body temperature in each animal one hour after reperfusion
was measured. Experiments evaluating the effect of fasting on
severe focal stroke were performed at Charles River Laboratories
(CRL), Kuopio, Finland.
In experiments testing the effects of protein-free DR, a mild
focal stroke involving cortex only was induced by transient direct
occlusion of the right MCA and bilateral CCAs with a 10-0 suture
for 60 min followed by reperfusion as described previously [31].
Cortical-only stroke does not result in fever in rats [32] (Figure S1)
thus the presence of lesion was verified by behavioral tests up to
two weeks post-stroke and/or by TTC staining 48 hours after
stroke. Experiments evaluating the effect of protein-free diet on
‘‘mild’’ cortical stroke were performed at the University of Helsinki
(UH), Finland.
Behavioral procedures
In the mild suture-induced cortical stroke model, neurological
deficits were evaluated using body swing, Bederson’s score [33]
and cylinder tests [31]; and locomotor activity was measured using
an infrared activity monitor (MedAssociates Inc.). All tests were
conducted by an investigator blinded to the treatment groups.
Analysis of infarction volume
For determination of infarct volume 7d after severe filament-
induced stroke, T2-weighted multi-slice (12–14 continuous slices)
MRI images were acquired using a Varian Inova console
interfaced to a 4.7T horizontal magnet (Magnex Scientific Ltd,
Abington, UK). Lesion quantitation was done by manually
delineating total lesion outlines from MRI images based on T2
contrast between lesioned and healthy tissue using MATLAB
software by an observer blinded to the treatment groups.
Infarct volume 2d after mild suture-induced cortical stroke was
assessed with triphenyltetrazolium chloride (TTC) staining by an
observer blinded to the treatment groups as described previously
[31].
Blood measurements
Glucose levels were measured from fresh blood with a
Glucocard II Super device (Akray Factory Inc., Shiga, Japan).
Plasma cytokines were measured on the Rat Demonstration Multi-
Spot plate (Meso Scale Discovery, Gaithersburg, MD) according
to the manufacturer’s instructions. Clinical chemistry analyses
from plasma samples were performed with an automatic analyzer
(Konelab 30i, Thermo Fisher Scientific, Vantaa, Finland) accord-
ing to manufacturer’s instructions.
Real-time quantitative PCR
Striatum was dissected from snap frozen brains before or 24hr
after severe filament-induced stroke for isolation of RNA for
cDNA synthesis. Real-time quantitative PCR (qPCR) was
performed on a LightcyclerH480 real-time PCR system (Roche
Diagnostics) using LightcyclerH480 SYBR Green I Master
complemented with 2.5pmol of primers (Table S1). Reactions
were performed in triplicate and analyzed with LightcyclerH480
Software. Gene expression was normalized to peptidylprolyl
Preoperative Dietary Restriction Is Neuroprotective in Stroke
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isomerase A. Similar methods were employed for gene expression
analysis from cortex and striatum before or 24hr after mild suture-
induced stroke.
Statistics
All values are presented as mean 6standard error of the mean
(SEM). Differences were considered to be statistically significant at
the p,0.05 level. Statistical analyses including Student’s t-test,
Mann-Whitney U-test and one- or two-way ANOVA or Kruskal-
Wallis non-parametric ANOVA followed by appropriate post hoc
analysis were performed with SPSS 15.0 software.
Results
Neuroprotection against severe focal stroke by
preoperative fasting
In hypoxia and global brain ischemia models, short-term water-
only fasting is protective [34–38]. However, while global brain
hypoperfusion accounts for less than 10% of perioperative strokes
in humans [2], the majority (62%) of perioperative strokes are
caused by focal ischemic insults that are mechanistically and
pathologically different from hypoperfusion. Our first objective
was to assess the effect of short-term fasting on severe focal brain
ischemia. Rats were subjected to pre-operative water-only fasting
for 3d as shown in Fig 1A. As expected, fasted rats exhibited a
significant reduction in body weight (Fig 1B). Fasting also
significantly reduced blood glucose levels (Fig 1C) and body
temperature (Fig 1D) prior to induction of focal stroke by
intraluminal occlusion of the middle cerebral artery (MCA) with
a filament for 60 min. One hour after reperfusion, body
temperature was elevated in both groups, consistent with lesion
induction (Fig 1D). Analysis of infarction volumes obtained from
T2-MRI images revealed a significant reduction 7d after tMCAO
in the fasted group relative to the ad libitum-fed group (Fig 1E and
F).
Suppression of pro-inflammatory response to severe
focal stroke by fasting
Long-term DR is believed to protect against ischemia reperfu-
sion injury at least in part through suppression of inflammatory
responses [23]. In the context of neuroprotection, long-term DR is
additionally associated with upregulation of neurotrophic factors
and growth factors such as BDNF [16,17,19,20], and increased
expression of proteins involved in cytoprotection [39]. To gain
insight into the molecular mechanisms of protection by fasting
against focal stroke, we analyzed striatal gene expression
immediately before (baseline) and 24hr after tMCAO using qPCR
(Fig 2A). Unlike long-term DR, we found no significant differences
in mRNA expression levels of growth factor-related, cytoprotective
or pro-inflammatory markers at baseline as a result of 3d of fasting
relative to ad libitum (AL) fed controls (Fig 2B, Table S1). 24hr after
reperfusion, expression of mRNAs encoding for the pro-inflam-
matory cytokine tumor necrosis factor alpha (TNFa), its receptor
TNFRSF1A and its downstream target, the intercellular adhesion
molecule 1 (ICAM1) were significantly upregulated in the lesioned
hemisphere compared to the control hemisphere in the ad libitum
group, but not in the fasted group (Fig 2C), suggesting attenuation
of the inflammatory response to stroke in fasted rats. Surprisingly,
mRNAs encoding for several neurotrophic and growth factors
including glial cell line-derived neurotrophic factor (GDNF),
neurturin (NRTN), mesencephalic astrocyte-derived neurotrophic
factor (MANF), transforming growth factor beta 1 (TGFb1),
fibroblast growth factor 2 (FGF2) and GDNF family receptor
alpha 1 (GFRa1) were significantly upregulated in the lesioned
striatum after stroke in the ad libitum rats but not in the fasted rats
(Fig 2C). A similar trend was observed in BDNF mRNA
expression, but this effect did not reach statistical significance.
Interestingly, no significant differences were observed in the
expression of cellular stress response genes (with the exception of
HMOX1, Fig 2C) as a function of diet or stroke. The expression of
all genes analyzed relative to the reference gene PPIA and
normalized to expression in the ad libitum group at baseline is
provided in Table S1; no significant changes were observed in any
of these genes in sham-operated animals 24hr after operation in
either dietary group.
Since DR can modulate innate immune activation, we
measured the concentration of plasma chemokines/cytokines
(CXCL1, IL-1b, IL-4, IL-5, TNFa, IFNcand IL-13) prior to
and 4hrs after tMCAO. Of these, only the pro-inflammatory
neutrophil chemoattractant CXCL1 (C-X-C motif ligand 1) was
significantly differentially regulated by fasting at baseline, with
higher levels in the fasted group than in the ad libitum fed group
(Fig 2D). Four hours after tMCAO, there was a trend in each of
the seven cytokines tested toward being reduced in the pre-fasted
group, but none reached statistical significance (Table 1).
Neuroprotection against mild focal cortical stroke by
protein-free dietary restriction
Because water-only fasting may in some clinical settings be
difficult to tolerate, we next asked whether a milder short-term
food restriction regimen could also precondition against stroke. A
protein-free DR preconditioning regimen was chosen based on its
efficacy in protecting kidney and liver from ischemia reperfusion
injury [28].
The intraluminal filament-induced severe stroke model used in
the fasting experiments above is one of the most widely used focal
stroke models in rats. However, a potential limitation of this
method is the severity of the resulting infarction, since stroke lesion
of comparable size extending from forebrain to midbrain in
humans is most often fatal. We thus continued to probe the
potential benefits of protein-free DR using a stroke model that
causes a milder lesion restricted to cortex [40], which is a common
type of embolic stroke in humans. Our objective was also to assess
infarct size at its maximum, i.e. 48 h after stroke and evaluate
functional outcome at 2, 7 and 14 days post-stroke.
Rats were acclimated to a complete diet made of refined
ingredients for 6d. They were then divided into two groups,
balanced for body weight and food intake during the acclimation
period (Fig 3A). One group remained on the complete diet for 6d
prior to stroke with average ad libitum daily food intake of 18.9 g/d.
The second group was fed a protein-free diet for 6 d prior to stroke
at the reduced amount of 11.4 g/d (,40% calorie restriction).
Although the initial aim was to normalize food intake among
animals in the short-term protein-free DR group in expectation of
food aversion to an incomplete (protein-free) diet, 36% of rats left
some protein-free food uneaten, indicative of slightly greater
aversion than predicted.
After preconditioning, a mild focal cortical stroke was induced
by transient direct occlusion of the right MCA and bilateral CCAs
with a suture for 60 min. One hour after reperfusion, body
temperatures were not significantly elevated, consistent with
milder lesion induction [41] (Fig S1). Two days after stroke
induction, animals were sacrificed and infarction volumes were
measured by TTC staining of brain sections. Rats in the protein-
free DR group showed a 39% reduced infarction volume relative
to the ad libitum fed group on the complete diet (Fig 3B, C).
Similarly, the maximal infarction area was significantly reduced in
the protein-free DR group compared to the complete diet group
Preoperative Dietary Restriction Is Neuroprotective in Stroke
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(Fig 3D). Prior to sacrifice 2d after stroke induction, rats in the
protein-free diet group had smaller behavioral deficits as shown by
biased body swing activity (Fig 3E) and Bederson’s score (Fig 3F).
Although plasma levels of total protein, albumin and urea were
reduced in the protein-free DR group at the time of sacrifice
consistent with the lack of protein in their diet, a number of other
blood parameters including glucose, triglycerides, creatinine and
prothrombin were not significantly different between diet groups
(Table S2).
Improved functional recovery from mild focal cortical
stroke upon short-term protein-free DR
We next assessed whether short-term protein-free DR could
promote functional recovery up to 2 weeks after stroke. The
experimental diets were applied as described above (Fig 4A).
Short-term protein-free DR significantly reduced body weight
(Fig 4B). Horizontal locomotor activity on d2 after stroke was
significantly improved in the protein-free DR group, indicating
faster recovery (Fig 4C). A similar tendency was observed with
vertical activity on d2 but did not reach statistical significance
(Fig 4D). Body swing activity was significantly reduced in the
protein-free DR group on d14 after the stroke compared to the
complete diet group (Fig 4E). Similarly, Bederson’s score was
significantly reduced on d14 (Fig 4F), with a similar trend on d2
and d7 (p = 0.0586 and p = 0.102, respectively, Mann-Whitney U-
test). Rats in the protein-free DR group also showed improved
performance in the cylinder test on d14 compared to the complete
diet group (Fig 4G).
Finally, in order to shed light on underlying mechanism in
comparison to the fasting paradigm, we performed qPCR analysis
of gene expression changes in both cortex and striatum 24 hours
post-stroke. As expected using this stroke model, gene expression
changes in cortex were greater than in striatum. Nonetheless the
patterns were similar between these two brain regions, including
increased expression of neurotrophic factors BDNF and GDNF,
stress response genes HMOX1 and GRP78, and inflammatory
markers including ICAM1 and TNFRSF1A (Figure S2). However,
with the exception of BDNF in the striatum, changes in expression
of these genes in response to stroke were similar between diet
groups, suggesting that protection afforded by fasting and protein-
free DR could work via different mechanisms or time scale.
Figure 1. Preoperative 3-day water-only fasting is neuroprotective against stroke. (A) Experimental timeline indicating periods of ad
libitum feeding and fasting relative to the onset of tMCAO on day 0. AL, ad libitum fed (n = 11); FA, fasted (n = 14). (B) Average body weights prior to
and after tMCAO; F
8, 23
= 17.69, ***p,0.0001, 2-way ANOVA. (C) Blood glucose levels on the indicated days prior to and after tMCAO; F
2, 23
= 14.09,
***p,0.0001, 2-way ANOVA. (D) Body temperature before tMCAO and 1 hour after reperfusion; ***/
###
p,0.001, Student’s t-test. (E) Infarction
volumes at d7 after tMCAO; *p = 0.0215, Student’s t-test. (F) Representative MRI images of the lesioned brain sections with green lines surrounding
the lesion.
doi:10.1371/journal.pone.0093911.g001
Preoperative Dietary Restriction Is Neuroprotective in Stroke
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Figure 2. Molecular mechanisms of fasting-induced neuroprotection. (A) Experimental timeline indicating dietary treatments and
experimental endpoints relative to tMCAO on day 0. (B) Relative expression of the indicated genes in the striatum of ad libitum fed (AL, n = 6) and
fasted (FA, n = 5) rats at baseline, measured by qPCR and expressed relative to the AL group. (C) Relative expression of selected genes in AL (n= 5)
and FA (n = 6) rats 24 hours after tMCAO in the unlesioned left (L) and lesioned right (R) striata, measured by qPCR and expressed relative to the
unlesioned AL group; *p,0.05, **p,0.01, ***p,0.001, 1-way ANOVA. (D) Serum CXCL1 levels in AL (n = 12) and FA (n = 12) rats at baseline;
**p = 0.0021, Student’s t-test.
doi:10.1371/journal.pone.0093911.g002
Preoperative Dietary Restriction Is Neuroprotective in Stroke
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Discussion
In hypoxia and global brain ischemia models, short-term water-
only fasting is protective [34–38]. However, global brain
hypoperfusion accounts for less than 10% of perioperative strokes
in humans [2]. The majority (62%) of perioperative strokes are
caused by focal ischemic insults that are mechanistically and
pathologically different from hypoperfusion. Here we report that
three days of preoperative water-only fasting reduced infarct
volume compared to ad libitum fed rats in a focal stroke model
involving forebrain and midbrain. In contrast to long-term DR
and ischemic preconditioning, fasting did not increase baseline
mRNA expression levels of cellular stress resistance genes such as
the molecular chaperones HSP70 and GRP78 [39], or HMOX1
[42]. Nor did it significantly affect the expression of growth and
neurotrophic factors including BDNF and FGF2 [39] or their
downstream targets in the brain (Fig 2B, Table S1). Rather,
increases in neurotrophic and growth factors and their receptors
24 hours after reperfusion in ad libitum fed animals correlated with
increased infarct size. This is consistent with the notion that
neurotrophic and growth factor upregulation is a relatively late
event in ischemic brain damage, occurring downstream of innate
immune system activation [43]. Our results are also in line with
studies showing that intracranial applications of NTFs are in large
part neuroprotective only when injected before the stroke, but are
neither neuroprotective nor able to facilitate recovery when
applied after the stroke [44]. Taken together, our results suggest
that the mechanism of protection by long-term DR and short-term
fasting in brain may differ, as has been suggested in other organs
including the kidney [27].
In the severe stroke model involving forebrain and midbrain,
protection afforded by fasting correlated with an altered inflam-
matory response. Ischemic injury to thalamic areas causes fever
[45], which along with activation of microglia facilitates the
expression of pro-inflammatory cytokines such as TNFa[46,47].
Binding of TNFato its receptor TNFRSF1A on endothelial cells
induces ICAM1 expression [48], increasing blood-brain barrier
permeability to infiltrating leukocytes and exacerbating tissue
damage [49]. In line with the above, we observed a dramatic
upregulation of TNFain the infarcted right hemisphere of ad
libitum fed animals 24 hours after reperfusion, whereas the
expression of TNFain the lesioned right hemisphere of fasted
rats was not significantly affected. TNFRSF1A and ICAM1
expression in the striatum were also both significantly increased
after tMCAO in ad libitum fed but not fasted rats. Since rats
overexpressing TNFaare more susceptible to ischemic injury and
mitochondrial dysfunction upon tMCAO [50], while TNFa
neutralization is protective [51], our results suggest that suppres-
sion of TNFaexpression upon focal ischemia reperfusion injury
may be an important component of fasting-induced protection
from focal stroke.
We also observed a significant increase in the levels of
neutrophil chemoattractant CXCL1 in the plasma of rats
following a 3d fast. Increased CXCL1 in the blood could reduce
local inflammation by reducing the steepness of the chemokine
gradient driving neutrophil chemotaxis from the vasculature to the
site of brain injury, as well as by affecting adhesion molecule
expression on neutrophils themselves [52]. Interestingly, in
humans, short-term dietary preconditioning elevates serum levels
of IL-8, the human paralogue of CXCL1 [53], warranting further
study.
Several physiological parameters can also affect the outcome of
experimental stroke. These include body temperature, blood
glucose, blood pressure and blood gas levels. Hypothermia has
been shown to effectively reduce ischemic injury in experimental
models of brain injury [54], and body temperature was slightly but
significantly reduced in fasted animals. However, therapeutic
Table 1. Plasma cytokine levels (pg/mL) 4 hours after reperfusion in rats fed ad libitum (AL) or fasted for 3 days (FA) prior to
tMCAO; IFN, interferon; IL, interleukin, CXCL1, C-X-C motif ligand 1; TNF, tumor necrosis factor.
4hr plasma cytokines IFNaIL1bCXCL1 TNFaIL4 IL5 IL13
AL (n = 6) 11.763.8 49.369.7 987462069 25.466.3 7.261.7 132.7626.1 0.360.3
FA (n = 6) 5.062.1 55.3614.3 720562388 12.967.0 5.361.5 87.2623.3 0.360.2
p-value 0.16 0.74 0.42 0.22 0.42 0.22 0.94
doi:10.1371/journal.pone.0093911.t001
Figure 3. Protein-free DR is neuroprotective against stroke. (A)
Experimental timeline indicating periods of ad libitum access to a
complete diet (AL, n = 15) or restricted access to a protein-free diet (PF,
n = 14) relative to the onset of tMCAO on day 0. (B) TTC-stained brain
sections showing infarct size (white area). (C) Total infarction volume on
d2 after tMCAO; *p = 0.0396, Student’s t-test. (D) Average maximal
infarction area from the slice with the largest infarction area per animal;
*p = 0.0320, Student’s t-test. (E) Biased body swing activity in 20 trials;
**p = 0.0016, Mann-Whitney U-test. (F) Behavioral performance assessed
by modified Bederson’s score; *p = 0.0396, Mann-Whitney U-test.
doi:10.1371/journal.pone.0093911.g003
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hypothermia in rodents and humans is protective only if body
temperature is reduced by several degrees [55]. Because fasting
reduced body temperature only by a half of a degree, fasting-
associated neuroprotection is not likely to be a result of
hypothermia. Hyperglycemia and hypoglycemia can both be
deleterious after stroke [56,57]. Although baseline blood glucose
levels in fasted rats were reduced by 30% relative to the ad libitum
fed group, this correlated with protection rather than susceptibility
to ischemic brain damage. Finally, fasting in rats has been
reported not to alter blood pressure or blood gas levels [37], thus
making the above parameters unlikely to contribute significantly to
protection.
Although water-only fasting is simple and effective in preclinical
models of stroke, it may not be well-tolerated in some clinical
settings. Thus, the nutritional basis of protection – whether an
overall reduction in calories or the removal of specific nutrients – is
important not only for understanding the underlying mechanism
but also for evaluating the translational potential of the
intervention. In fruit flies, the benefits of long-term DR on
longevity can be abrogated by the addition of essential amino acids
[58]. Long-term protein or individual amino acid restriction can
also slow aging in rodents and precondition against acute stressors,
including acetaminophen (paracetamol) toxicity [59] and ischemic
injury to kidney and liver [28]. In studies of amino acid deficiency-
mediated protection against renal and hepatic ischemia reperfu-
sion injury, activation of the GCN2-dependent amino acid
starvation response is required for protection in part through
modulation of the systemic inflammatory response to injury and/
or by activation of organ-autonomous stress resistance pathways
[28]. Taken together, these data suggest broad evolutionary
conservation of beneficial adaptive responses to protein/amino
acid restriction.
Consistent with these reported benefits of protein/essential
amino acid restriction, we found protection with a short-term
protein-free DR regimen in a rat model of mild focal stroke,
including reduced infarction volume and improved functional
recovery. Interestingly, however, preliminary analyses of gene
expression changes 24hr after reperfusion to probe candidate
mechanisms of protection did not reveal overlap between the
protein-free DR and fasting paradigms of neuroprotection. This
could be due to different kinetics of gene expression in the severe
vs. mild stroke models, or could indicate true differences in
underlying mechanisms of protection by fasting and protein-free
DR regimens. We focused our gene expression analysis on the
24hr time point in an attempt to uncover primary mechanisms of
neuroprotection, as later time points could be confounded by
differences in initial lesion size. Nonetheless, inflammatory
responses after ischemic brain injury are also time dependent,
beginning immediately after injury and lasting for months [60].
Thus, studies using unbiased gene expression analysis at multiple
time-points and in different brain areas using gene arrays in
combination with physiological, histological and behavioral
parameters may help to dissect mechanisms involved in diet-
induced neuroprotection and improved recovery after stroke in the
Figure 4. Protein-free DR promotes functional recovery after stroke. (A) Experimental timeline indicating periods of ad libitum access to
a complete diet (AL, n = 14) or restricted access to a protein-free diet (PF, n =14) relative to the onset of tMCAO on day 0 and subsequent
behavioral testing on days 2, 7 and 14. (B) Average body weights on the indicated days relative to tMCAO on day 0; F
1,26
= 96.20, ***p,0.0001, 2-way
ANOVA. (C-G) Behavioral tests on the indicated days after tMCAO: (C) horizontal activity; F
1,26
= 4.994, *p = 0.034, 2-way ANOVA; (D) vertical activity;
F
1,26
= 4.150, p = 0.052, 2-way ANOVA; (E) biased body swing activity in 20 trials; *p = 0.0211, Mann-Whitney U-test; (F) modified Bederson’s score;
**p = 0.0018, Mann-Whitney U-test; (G) cylinder test measured on d14 after tMCAO; *p = 0.0492, Mann-Whitney U-test.
doi:10.1371/journal.pone.0093911.g004
Preoperative Dietary Restriction Is Neuroprotective in Stroke
PLOS ONE | www.plosone.org 7 April 2014 | Volume 9 | Issue 4 | e93911
future. How short-term dietary interventions such as fasting or
short-term protein-free DR affect ischemic stress resistance in
humans is currently unknown. However, there is substantial
empirical and observational evidence that medically supervised
fasting with periods of 7–21 days is efficacious in the treatment of
rheumatic diseases, chronic pain syndromes, hypertension, and
metabolic syndrome [61,62]. Because metabolic responses to
dietary restriction observed in experimental organisms are shared
by humans [63], an expectation that benefits will translate to focal
brain ischemia in humans is warranted [26]. The human
functional equivalents of the 3d fasting or 6d protein-free DR
regimens in rodents tested here are not known, but represent an
important next question in translation to the clinic. In conclusion,
due to its simplicity, cost-effectiveness and presumed low risk,
short-term dietary preconditioning may carry an immediate
potential for clinical application in perioperative risk management.
Supporting Information
Figure S1 Rectal body temperature in rat cortical
stroke model in ad libitum complete (AL, n = 4) and
protein free (PF, n =4) diet groups before and 1 h after
reperfusion.
(TIF)
Figure S2 Expression of selected genes 24 hours after
tMCAO in the unlesioned left (L) and lesioned right (R)
cortices and striata of rats in ad libitum (AL, n = 8) and
protein-free (PF, n = 7) diet groups, measured by qPCR
and expressed relative to the unlesioned AL group.
Asterisks indicate difference between the lesioned and unlesioned
hemispheres in the same diet group. No significant differences
were observed between diet groups; *p,0.05; **p,0.01;
***p,0.001, 1-way ANOVA.
(TIF)
Methods S1 Supplementary Methods.
(DOCX)
Table S1
(PDF)
Table S2 Analysis of the indicated component from
blood plasma from rats fed a complete diet ad libitum
(AL) or a protein free (PF) diet for 6 days prior to and 2
days after induction of cortical stroke.
(DOCX)
Acknowledgments
We thank Eero Castre´n and Brandon Harvey for critical reading of the
manuscript. We are grateful for Virpi Perko for help with the protein free
diet experiments.
Author Contributions
Conceived and designed the experiments: MA MS JRM JOA. Performed
the experiments: KV MA JA SV AP. Analyzed the data: KV MA JA SV
JRM JOA. Contributed reagents/materials/analysis tools: MA JRM MS
JOA. Wrote the paper: MA JRM JOA.
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