Baseline Cognitive Function, Recurrent Stroke, and Risk

of Dementia in Patients With Stroke

Pamela M. Rist, ScD; John Chalmers, MD; Hisatomi Arima, MD; Craig Anderson, MD;
Stephen MacMahon, PhD; Mark Woodward, PhD; Tobias Kurth, MD, ScD; Christophe Tzourio, MD, PhD

Background and PurposeTo determine the interrelationships between baseline Mini-Mental State Examination (MMSE)
score and risk of overall dementia, post-recurrent stroke dementia, and dementia without recurrent stroke among patients
with a history of stroke.
MethodsProspective cohort study among participants enrolled in the Perindopril Protection Against Recurrent Stroke
Study (PROGRESS) for whom baseline MMSE score was available. Baseline MMSE score was divided into 4 categories:
30, 2927, 2624, and <24. Participants were followed for incident dementia and recurrent stroke. Logistic regression
models were used to examine the association between MMSE score and dementia.
ResultsOf the 6080 participants included in this analysis, 2493 had an MMSE score of 30, 1768 had a score of 2928,
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1369 had a score of 2624, and 450 had a score of <24. Average follow-up time was 3.8 years. There were 407 cases
of dementia, 106 of which were preceded by a recurrent stroke. The risk of overall dementia increased with decreasing
MMSE score. However, the impact of MMSE score on the risk of dementia without recurrent stroke was much stronger
than the impact of MMSE score on the risk of post-recurrent stroke dementia. For those with MMSE score <24, the risk of
dementia without recurrent stroke was 47.89 (95% confidence interval, 28.5780.26), whereas the risk of post-recurrent
stroke dementia was only 7.17 (95% confidence interval, 3.7013.89). Higher MMSE scores were even less strongly
associated with the risk of post-recurrent stroke dementia.
ConclusionsPatients with stroke with low MMSE scores are at high risk of dementia over time, even in the absence of a
recurrent stroke, and should therefore be followed closely for further cognitive decline.(Stroke. 2013;44:1790-1795.)
Key Words: cerebrovascular disease cognitive functioning dementia epidemiology

S troke and dementia are 2 of the largest morbidity burdens

worldwide.1 Previous research has shown that cognitive
status and stroke occurrence are strongly related to the risk
of future dementia. Individuals who experience a stroke have
double the risk of dementia, including delayed dementia com-
pared to those who do not experience stroke.2 Other studies
have also shown that lower scores on tests of cognitive func-
tion like the Mini-Mental State Examination (MMSE) are also
highly predictive of future risk of dementia.3,4
However, little research has examined whether prestroke
cognitive function is still a strong predictor of the risk of
dementia after a stroke event because most previous stud-
ies have not assessed cognitive function before the stroke
event. A review article on the impact of stroke on the risk of
dementia concluded that prestroke cognitive decline did not
seem to account for the association between stroke and cogni-
tive impairment and did not find evidence for an interaction
between incident stroke and prestroke cognition on the risk of
dementia.5 However, very few studies have specifically tested
this interaction or assessed the impact of baseline cognitive
status on the risk of dementia preceded by or not preceded by
stroke. Learning more about the associations among baseline
cognitive functioning, stroke, and risk of dementia would help
determine whether the risk of dementia is driven primarily by
the stroke event, which would suggest a vascular pathology, or
by the level of cognitive functioning before the stroke event,
which would suggest pathologies independent of the stroke
event.
The Perindopril Protection Against Recurrent Stroke
Study (PROGRESS) was a randomized trial among people
with a history of cerebrovascular disease in which a blood
pressure regimen was tested against placebo for the secondary
prevention of stroke. The study assessed participants cognitive
function at baseline and followed them for recurrent stroke

Received October 18, 2012; accepted April 11, 2013.

From the Division of Preventive Medicine, Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Boston, MA (P.M.R.,
T.K.); Departments of Epidemiology (P.M.R., T.K.) and Social and Behavioral Sciences (P.M.R.), Harvard School of Public Health, Boston, MA; The George
Institute for Global Health, University of Sydney, New South Wales, Australia (J.C., H.A., C.A., S.M., M.W.); and INSERM Unit 708Neuroepidemiology,
Bordeaux, France (T.K., C.T.); and University of Bordeaux, U708, Bordeaux, France (T.K., C.T.).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
111.680728/-/DC1.
Correspondence to Pamela M. Rist, ScD, Division of Preventive Medicine, Brigham and Womens Hospital, 900 Commonwealth Ave, 3rd Floor, Boston,
MA 02215. E-mail prist@mail.harvard.edu
2013 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.111.680728

1790
 Rist et al Cognitive Function, Recurrent Stroke, and Dementia 1791
and incident dementia. Using data from this study, we aimed
to determine the interrelationships between baseline MMSE
score and the risk of overall dementia, post-recurrent stroke
dementia, and dementia without the presence of a recurrent
stroke. We hypothesized that recurrent stroke is a strong risk
factor for the development of dementia and cognitive status
before recurrent stroke may modify the impact of recurrent
stroke on the development of dementia.
Methods
Previous studies have described the design of PROGRESS.6,7 In
brief, PROGRESS was a randomized, double-blind, placebo-con-
trolled trial to determine the effectiveness of a blood pressure
lowering regimen to prevent recurrent stroke and dementia among
6105 participants with previous stroke or transient ischemic attack.
Participants were recruited from 172 collaborating centers in 10
countries from May 1995 to November 1997. To be eligible, partici-
pants needed to have had either a stroke or transient ischemic attack
(but not subarachnoid hemorrhage) within the past 5 years and have
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no clear indication for, nor a contraindication to, treatment with an
angiotensin-converting enzyme inhibitor. In addition, dementia was
an exclusion criterion. After a run-in period, participants who toler-
ated and adhered to perindopril therapy were randomly assigned to
continued active treatment or placebo. Randomization was stratified
by study center, age, sex, systolic blood pressure at entry, inclusion
diagnosis, and the intention to begin combination therapy or single
drug therapy.
Cognitive Decline and Dementia Assessment
At baseline, the 6- and 12-month visits, and annually thereafter, par-
ticipants completed the MMSE.3 One point was awarded for each
successfully completed item (maximum score of 30); no points were
awarded for any missing item. Baseline MMSE score was divided a
priori into 4 categories: 30 (high MMSE score), 2928 (medium-high
MMSE score), 2724 (medium-low MMSE score), and <24 (low
MMSE score). In the event that baseline MMSE score was missing
(n=32), baseline MMSE score was imputed using the MMSE score
from the 6-month visit (n=7). Participants for whom baseline MMSE
score could not be imputed were excluded from the analysis (n=25).
Throughout the follow-up, a 2-phase screening and assessment
process was used to diagnose dementia.8 Participants meeting any
of the following criteria were considered to be screened positive for
dementia: an MMSE score 25 at any follow-up visit, a decline in
the MMSE score of 3 points between any 2 follow-up visits, an
MMSE score missing for 2 scheduled follow-up visits, or a positive
response by the investigator to the question: In your opinion, does this
patient have dementia? All participants who screened positive were
referred to a local specialist experienced in diagnosing dementia. The
specialists were blinded to treatment assignment and to all clinical
data. Participants who screened negative were classified as not hav-
ing dementia.
The local specialist used a checklist based on the criteria for the
diagnosis of dementia as defined in the Diagnostic and Statistical
Manual of Mental Disorder, Fourth Edition.9 The questionnaire in-
cluded systematic questions on the presence of poststroke focal defi-
cits, such as aphasia or motor deficit, and on the presence of more
global problems, such as depressed mood, that could have altered
the diagnosis of dementia. Local specialists were also systematically
asked whether the diagnosis of dementia was reliable. Whenever
possible, the specialist examined the patient. If an interview could
not be conducted, data were sought from all other available sources,
including medical records, interviews with family members, and con-
sultations with other medical professionals. After receiving the infor-
mation from and diagnosis of the local specialist, a 2-person central
Dementia Adjudication Committee confirmed or refuted the diagnosis
and assigned each screen-positive case to 1 of the following 4 catego-
ries: certain dementia, fairly certain (probable) dementia, uncertain
(possible) dementia, or no dementia. No attempt was made to further
classify cases into subtypes of dementia because all participants had
a history of cerebrovascular disease and often had other vascular risk
factors. The main outcome for this analysis was the occurrence of
dementia, either certain dementia or fairly certain dementia.
Stroke Assessment
Recurrent fatal or nonfatal stroke was defined as an acute disturbance
of focal neurological function with symptoms lasting >24 hours (or
resulting in earlier death) thought to be because of either cerebral in-
farction or cerebral hemorrhage.7,10 All suspected strokes and deaths
were first reported by local study investigators and then reviewed by
experts on the central End Point Adjudication Committee. This com-
mittee was provided with a clinical summary of the event and copies
of any available investigation reports (eg, biochemistry, hematology,
radiology, and autopsy findings).11 Strokes were classified as cerebral
hemorrhage, ischemic stroke, or stroke of unknown pathological type.
If dementia occurred before the recurrent stroke event, the person
was classified as dementia without recurrent stroke. If stroke event
occurred before dementia, the person was classified as post-recurrent
stroke dementia.
Statistical Analysis
First, we used logistic regression models to calculate the odds ratio
as a measure for the relative risk (RR) of developing dementia for the
4 categories of the baseline MMSE score using the highest category
of MMSE as the reference category. Next, to examine the impact of
MMSE score on the joint outcome of recurrent stroke and dementia,
we divided incident dementia into post-recurrent stroke dementia and
dementia without recurrent stroke. We then used logistic regression
models to assess the RR of both dementia types compared to no de-
mentia for each of our MMSE categories.
All models were adjusted for variables that we believed could be
potential confounders based on biological mechanisms. These vari-
ables were age (continuous), sex, height (continuous), smoking status
(never smoked regularly, past, current), current alcohol consumption
(currently do not drink more than once/wk, currently drink <8 drinks/
wk, currently drink 8 drinks/wk), educational status (stopped edu-
cation by 14, 16, 19, and >19 years of age), diabetes mellitus status
(yes/no), and systolic blood pressure (continuous). We also adjusted
for randomized treatment assignment.
In secondary analyses, we explored the relationship between base-
line MMSE score and recurrent stroke and risk of dementia strati-
fied by the recurrent stroke subtype (ischemic versus hemorrhagic
stroke). We additionally performed all analyses using only those
randomized to placebo. Finally, we performed separate sensitivity
analyses in which we adjusted for baseline Barthel index in addi-
tion to our potential confounders, excluded patients who screened
positive for dementia at baseline, and excluded patients with MMSE
scores <18 at baseline.
No participant had missing information on age, sex, current alco-
hol consumption, diabetes mellitus status, or baseline systolic blood
pressure. Fewer than 100 people were missing information on height
and smoking and were assigned to the median and past smoker, re-
spectively. More than 100 people had missing information on educa-
tion status so we used the missing indicator method.
All statistical analyses were performed using SAS 9.1.3. All P val-
ues are 2-tailed, and P<0.05 was considered statistically significant.
Results
Of the 6080 participants included in this analysis, 2493 (41.0%)
had an MMSE score of 30, 1768 (29.1%) had an MMSE score
of 2927, 1369 (22.5%) had an MMSE score of 2624, and 450
(7.4%) had an MMSE score of <24. Average follow-up time was
3.8 years. There were 407 cases of dementia, 106 of which were
preceded by a recurrent stroke. A total of 709 strokes occurred
either before dementia onset or the end of the follow-up.
 1792StrokeJuly 2013

Table1 shows covariates by baseline MMSE categories for
our study population. Those with the lowest MMSE scores
were older, shorter in stature, had stopped schooling at a
younger age, more likely to be female, consumed alcohol less
frequently, had higher systolic blood pressure, were more
likely to have diabetes mellitus, and were less likely to be cur-
rent smokers than participants with higher MMSE scores.
The impact of baseline MMSE score on the risk of overall
dementia is presented in Table2. The risk of dementia increased
with decreasing MMSE scores. Those with an MMSE score of
2829 had an RR of dementia of 2.15 (95% confidence interval
[CI], 1.433.24), whereas those with an MMSE score of <24
had an RR of dementia of 26.81 (95% CI, 18.0839.76) com-
pared to those with an MMSE score of 30.
The Figure shows the proportion of post-recurrent stroke
dementia and dementia without recurrent stroke by MMSE cate-
gory. Among those with an MMSE score of 30, 50% of those who
develop dementia had dementia without recurrent stroke. In con-
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The effect estimates for the association between baseline
MMSE score and post-recurrent stroke dementia and between
MMSE score and dementia without recurrent stroke can be
seen in Table2. For both outcomes, we observed an increase in
the risk of dementia with decreasing MMSE scores. However,
the impact of MMSE score on risk of dementia without recur-
rent stroke was much stronger than the impact of MMSE score
on the risk of post-recurrent stroke dementia. For those with
an MMSE score <24, the risk of dementia without stroke was
47.89 (95% CI, 28.5780.26), whereas the risk of post-recur-
rent stroke dementia was only 7.17 (95% CI, 3.7013.89).
Higher MMSE scores were even less strongly associated with
the risk of post-recurrent stroke dementia. The risk of post-
recurrent stroke dementia was only 1.32 (95% CI, 0.702.49)
among those with an MMSE score 2928, whereas the risk of
dementia without stroke was 2.99 (95% CI, 1.735.18).
Results stratified by stroke subtype (ischemic versus
hemorrhagic; results not shown) were similar to those shown

trast, among those with a low MMSE score (<24), nearly all those above. Results among those assigned to placebo were similar
who develop dementia had dementia without recurrent stroke. to those seen for the full cohort (results not shown). Results of

Table 1. Baseline Characteristics of Participants in the PROGRESS Trial by Baseline MMSE Score (n=6080)
MMSE Score
Characteristic 30 2928 2624 <24
Age, mean (SE) 61.4 (9.5) 64.8 (9.5) 66.0 (8.8) 67.6 (8.9)
Female, % 27.8 30.7 32.1 37.6
Height, mean (SE) 167.3 (8.9) 167.2 (8.9) 165.4 (9.4) 164.1 (9.0)
Smoking status, %
Never smoked regularly 44.7 40.0 40.0 42.7
Past smoker 33.8 41.7 39.7 39.8
Current smoker 21.6 18.3 20.4 17.6
Current alcohol consumption, %
<1/wk 60.5 56.6 59.9 66.7
1 to 8 drinks/wk 18.0 20.8 20.0 17.1
8 drinks/wk 21.5 22.6 20.1 16.2
Age at which schooling stopped, y
14 22.3 36.3 42.5 61.5
1516 20.1 25.4 25.1 19.2
1719 26.4 18.4 17.2 13.1
>19 31.3 19.9 15.2 6.3
Diabetes mellitus, % 12.0 11.8 12.5 17.8
Systolic blood pressure, mean (SE) 145.4 (18.8) 147.2 (19.0) 148.5 (18.7) 150.4 (20.4)
Type of qualifying event, %
TIA 22.9 24.4 18.5 18.2
Ischemic stroke 64.6 61.4 64.5 64.7
Hemorrhagic stroke 9.8 8.9 11.8 10.9
Unknown 2.7 5.3 5.2 6.2
Medication, %
Antihypertensive therapy 57.6 57.1 61.5 61.1
Antiplatelet therapy 71.9 73.9 71.6 71.3
Oral anticoagulants 7.1 11.9 8.7 11.8
Lipid-lowering therapy 15.8 13.9 12.4 10.7
PROGRESS indicates Perindopril Protection Against Recurrent Stroke Study; and TIA, transient ischemic attack.
 Rist et al Cognitive Function, Recurrent Stroke, and Dementia 1793

Table 2. Multivariate-Adjusted* Relative Risks of Overall Dementia, Post-recurrent stroke Dementia, and Dementia Without
Recurrent Stroke by Baseline MMSE Score
No
Dementia
(n=5673) All Dementia (n=407) Post-recurrent stroke Dementia (n=106) Dementia Without Recurrent Stroke (n=301)
Crude Incidence Crude Incidence Crude Incidence
Rate (Per 1000 Rate (Per 1000 Rate (Per 1000
Baseline MMSE n No. Cases Person-Years) RR (95% CI) No. Cases Person-Years) RR (95% CI) No. Cases Person-Years) RR (95% CI)
30 2455 38 3.86 1.00 19 1.93 1.00 19 1.93 1.00
2829 1703 65 9.54 2.15 21 3.08 1.32 44 6.46 2.99
(1.433.24) (0.702.49) (1.735.18)
2427 1218 151 28.87 6.59 44 8.41 3.67 107 20.45 9.56
(4.549.55) (2.106.43) (5.7915.80)
<24 297 153 105.08 26.81 22 15.11 7.17 131 89.97 47.89
(18.0839.76) (3.7013.89) (28.5780.26)
CI indicates confidence interval; MMSE, Mini-Mental State Examination; and RR, relative risk.
*Results have been adjusted for age, sex, height, smoking status, current alcohol consumption, educational status, diabetes mellitus, baseline systolic blood pressure,
and randomized treatment assignment.
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our other sensitivity analyses were also similar to those seen
for the full cohort (online-only Data Supplement).
To further explore why baseline MMSE score was not as
strongly associated with the risk of post-recurrent stroke demen-
tia, we examined the associations between baseline MMSE
and the risk of recurrent stroke as well as between recurrent
stroke and the risk of dementia. Baseline MMSE score was not
associated with the risk of recurrent stroke (results not shown).
However, recurrent stroke was significantly associated with the
risk of dementia (RR, 2.93; 95% CI, 2.243.82).
Discussion
In this prospective cohort of patients with stroke, baseline
MMSE score was strongly associated with the risk of devel-
oping dementia. Analyses examining post-recurrent stroke
dementia versus dementia without recurrent stroke suggested
stronger associations between baseline MMSE score and risk
of dementia without recurrent stroke than with post-recurrent
stroke dementia.
Previous studies have shown that both lower MMSE scores
and stroke predict the risk of subsequent dementia.24 However,
data on the interrelationships among prestroke MMSE, stroke,
and the risk of dementia in one study are sparse, mainly
because small numbers of stroke or dementia events prohibits
the evaluation of joint effects. Three studies have examined
the interrelationships among prestroke cognitive functioning,
stroke, and cognitive impairment. Data from the Framingham
Heart Study showed that participants who experienced a
stroke had significantly lower mean MMSE scores prestroke
and poststroke compared to the stroke-free participants.12 The
Baltimore Longitudinal Study of Aging found that those with
mild cognitive impairment who experience a stroke are at
increased risk of developing dementia (odds ratio, 12.4; 95%
CI, 1.599) compared to those with mild cognitive impair-
ment who do not experience a stroke. In addition, those who
were cognitively normal at the time of their stroke did not
have an increased risk of dementia compared to those with-
out stroke.13 Another study using data from the Health and
Retirement Study found that the rate of cognitive decline was
faster among those who later survived a stroke compared to
those who remained stroke-free throughout follow-up. Those
who died after stroke had even faster rates of decline. After the
stroke event, the rate of decline among stroke survivors was
similar to their rate of decline before the stroke event.14
Although these studies were able to examine the relationships
between cognitive functioning and first stroke, research on the
interrelationships among cognitive function, recurrent stroke,
and risk of subsequent dementia is sparse. A few studies that
assessed cognitive functioning in patients with stroke have
shown links between cognitive decline and dementia,15,16 but

100.0%
90.0%
80.0%
70.0%
Percentage

60.0%
Post-recurrent stroke dementia
50.0%
Dementia without stroke Figure. Proportion of types of dementia
40.0%
according to baseline Mini-Mental State
30.0% Examination (MMSE).
20.0%
10.0%
0.0%
MMSE 30 MMSE 28-29 MSME 24-27 MMSE <24
MMSE category
 1794StrokeJuly 2013
many could not specifically examine the effect of recurrent
stroke on the association between cognitive functioning and
dementia. One study directly tested the interaction between
baseline cognitive status and incident stroke using participants
in the Rotterdam Study who were free of dementia or a history
of stroke at baseline and found no interactive effect of incident
stroke and measures of prestroke cognitive function on the
risk of dementia.17
The present study showed that among those who have
already experienced a stroke or transient ischemic attack, base-
line MMSE score is a strong predictor of the risk of dementia
without recurrent stroke. Results from our study suggest that
patients with stroke with low MMSE score are already on a
trajectory of cognitive decline related to preexisting neuro-
degenerative lesions or other undetermined factors triggered
by the initial stroke event. The stroke event may increase the
risk of dementia either because of the direct impact of vas-
cular disease on other neuropathological changes associated
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with Alzheimer disease or by synergistic effects of Alzheimer
neuropathology and vascular neuropathology.18 This has
important clinical implications because it demonstrates that
for those with low MMSE score, a recurrent stroke is not nec-
essary to develop dementia. This result could seem counter-
intuitive because in patients with stroke, one would expect a
second stroke to result in a greater risk of cognitive decline
and vascular dementia. A review article found that the rate of
dementia was at least twice as high after recurrent stroke as it
was after first stroke and the rate of dementia after recurrent
stroke may depend on the number of recurrent strokes.19
An important finding from the main analyses of PROGRESS
is that a blood pressurelowering regimen decreased the risk
of recurrent stroke and the risk of post-recurrent stroke demen-
tia.20 In the present study, the associations between baseline
MMSE score and post-recurrent stroke dementia were weaker
than those seen for the associations between baseline MMSE
score and dementia without recurrent stroke. Although base-
line MMSE score is highly predictive of dementia in the
absence of stroke, the impact of recurrent stroke on the risk
of dementia outweighs the impact of baseline MMSE score.
Therefore, patients, especially those with high MMSE score
who have the lowest risk of dementia without recurrent stroke,
should be delivered strong stroke prevention messages. Given
the associations seen between blood pressurelowering drugs
and a reduced risk of recurrent stroke, patients with high
MMSE score may want to consider a blood pressurelower-
ing regimen to avoid another stroke and the associated risk of
dementia.
One of the main strengths of this study was that unlike
previous studies, we had information on MMSE score before
recurrent stroke and new diagnoses of dementia after recur-
rent stroke. Other strengths include the large number of out-
come events, which allowed us to assess the interrelationships
among baseline MMSE score, recurrent stroke, and risk of
dementia. By screening all participants at baseline for demen-
tia, the cohort of individuals in this study were free of preex-
isting dementia.
Despite the strengths of this study, some weaknesses should
be noted. Although this study did have a large number of out-
come events, many of our CIs for our effect estimates are
wide. In addition, we did not have information on MMSE
score before first stroke, which prevented us from determining
whether first stroke may be the triggering factor for cogni-
tive decline or whether the patients had already experienced
decline before stroke. We took several steps to obtain a consis-
tent diagnosis of dementia across the various centers, includ-
ing using standardized forms and having a central Dementia
Adjudication Committee to minimize heterogeneity. Although
it is possible that people with undiagnosed or mild cognitive
impairment at baseline may have been included in our study,
dementia was an exclusion criterion for entry into the trial, and
we used the original trial cohort for these analyses. Although
4 different tools were used to screen for dementia, they were
mainly based on MMSE score, which is not sensitive to vas-
cular cognitive impairment. Therefore, it is possible that some
cases of dementia may have been missed.
To summarize, these results carry important messages for
patients with stroke and their physicians that vary accord-
ing to the baseline MMSE score of the patient with stroke.
Patients with stroke with low MMSE scores are at high risk of
dementia over time, even in the absence of a recurrent stroke,
and should therefore be followed closely for further cognitive
decline.
Sources of Funding
The Perindopril Protection Against Recurrent Stroke Study was
funded by grants from Servier (Paris, France), the Health Research
Council of New Zealand (Auckland, New Zealand), and the National
Health and Medical Research Council of Australia (Canberra,
Australia). The study was designed, conducted, analyzed, and inter-
preted by the investigators independent of all sponsors. Dr Rist was
funded by a training grant from the National Institute of Aging (AG-
00158) and by the Rose Traveling Fellowship Program in Chronic
Disease Epidemiology and Biostatistics at the Harvard School of
Public Health.
Disclosures
Drs Chalmers and MacMahon have received lecture fees and research
grants from Servier. Dr Arima holds a Future Fellowship from the
Australian Research Council. Dr Anderson holds a Senior Principal
Research Fellowship from the National Health and Medical Research
Council. Dr Woodward has received lecture fees from Servier. Dr
Kurth has received funds from Allergan and the American Academy
of Neurology for educational lectures. Dr Tzourio has received fees
from the ABBOTT Company for participating in scientific commit-
tees. The other author has no conflict to report.
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 Baseline Cognitive Function, Recurrent Stroke, and Risk of Dementia in Patients With
Stroke
Pamela M. Rist, John Chalmers, Hisatomi Arima, Craig Anderson, Stephen MacMahon, Mark
Woodward, Tobias Kurth and Christophe Tzourio
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Stroke. 2013;44:1790-1795; originally published online May 16, 2013;

doi: 10.1161/STROKEAHA.111.680728
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 ONLINE SUPPLEMENT

Supplemental Methods

Antibodies
Rabbit polyclonal anti-Akt, anti-p-Akt (Ser473), anti-MKK7, anti-p-MKK7 (Ser171, Thr275),
anti-p-MLK3 (Thr277, Ser281), monoclonal anti-JNK3 (55A8) and monoclonal anti--actin
(13E5) antibodies were purchased from Cell Signaling Biotechnology (Boston, MA). Rabbit
polyclonal anti-GluK2, anti-MLK3, and mouse monoclonal anti-p-JNKs (Thr 183, Tyr 185, G-7)
antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse
monoclonal anti-PSD-95 (clone 7E3-1B8) antibody was obtained from Sigma (Saint Louis,
MO).

Drug Administration
A selective PI3K inhibitor LY294002 (BioMol, Plymouth Meeting, PA), a specific Akt inhibitor
Akt inhibitor IV (Calbiochem) and a potent MEK1 inhibitor PD98059 (Cell Signaling
Biotechnology) were used to detect the role of PI3K-Akt and MEK1 signaling in the
postconditioning-mediated neuroprotection respectively. LY294002 (100 nmol) or PD98059 (20
nmol) in 10l DMSO was infused into the rat cerebral ventricle (from the bregma: posterior, 0.8
mm; lateral, 1.5 mm; depth, 3.5 mm) through a stepper-motorized microsyringe (Stoelting,
Wood Dale, IL) 20 minutes before ischemia. Akt inhibitor IV (100 nmol) in 10l DMSO was
infused into the rat cerebral ventricle 2 hours after ischemia.

Histological Assessment
Rats were perfusion-fixed with 4% paraformaldehyde under anesthesia after 5 days of
reperfusion. Brains were removed and further fixed with the same fixation solution at 4C
overnight. Post-fixed brains were embedded by paraffin and then coronal sections (6m thick)
were prepared using a microtome. The paraffin embedded brain sections were deparaffinized
with xylene and rehydrated in a gradient of ethanol, followed by washing with distilled water.
The sections were stained with cresyl violet for the assessment of neuronal survival in the
hippocampus. The number of surviving hippocampal CA1 neurons per 1 mm length was
counted as the neuronal density.

Immunoprecipitation
The hippocampal CA1 regions were isolated after the indicated times of reperfusion and rapidly
frozen in liquid nitrogen. Samples were homogenized in ice-old homogenization buffer. 1 The
homogenates were centrifuged at 800g'/4C for 10 minutes and the supernatants were collected.
Sample proteins were incubated overnight at 4C with appropriate antibodies diluted in
immunoprecipitation buffer. 1 After the addition of protein A/G, the mixture was incubated at
4C for an additional 2 hours. The bound proteins were collected from Protein A/G by boiling
for 5 minutes in Laemmli sample buffer.

1
Immunoblot
Protein samples were separated by SDS-PAGE and then electrotransferred onto a nitrocellulose
membrane. After blocking, the membranes were probed with primary antibodies overnight at
4C. Detection was carried out by appropriate alkaline phosphatase-conjugated IgG (Sigma) and
developed with NBT/BCIP assay kit (Promega).

Statistical Analysis
The results are expressed as meansstandard deviation (SD). For each type of experiment, data
were obtained from at least three independent measurements. Statistical analysis of the results
was carried out using one-way analysis of variance (ANOVA) followed by the least significant
difference test or Newman-Keuls test. Differences were considered significant at P<0.05.

Supplemental Figure

Figure S1. LY294002 (LY) or Akt inhibitor IV (AI) has no effect on the neuronal survival
in the rat hippocampal CA1 subregion. Nissl staining on neuronal survival after 5 days
following drug administration. (a-c) Low-power views of hippocampus sectors. Scale bars =
500 m. (d-f) High-power views of hippocampal CA1 pyramidal cell layer of (a-c). Scale bars =
50 m. Neuronal density was counted as numbers of surviving pyramidal neurons per 1mm
length. Data are mean SD (n=5).

Supplemental Discussion

The role of MEK1-ERK1/2 pathway remains controversial, since both activation and inhibition
of ERK1/2 are reported to mediate neuronal survival in conditions associated with cerebral
ischemia. 2,3 Although different changes in ERK1/2 phosphorylation after postconditioning have
been reported, 4,5 Pignataro and colleagues found that ERK1/2 may be unrelated to the
protective effect of postconditioning after focal ischemia. 4 In this work, our result showed that
inhibiting MEK1-ERK1/2 signal by PD98059 didnt affect protective effect of postconditioning
after global ischemia, which is consistent with the study by Pignataro and colleagues.

2
Considering dual effects of ERK1/2 in ischemic brain damage and controversial data for
ERK1/2 phosphorylation in postconditioning, more studies are needed to clarify the role of
MEK1-ERK1/2 in the postconditioning neuroprotection.

Supplemental References

1. Du CP, Gao J, Tai JM, Liu Y, Qi J, Wang W, et al. Increased tyrosine phosphorylation of
PSD-95 by Src family kinases after brain ischemia. Biochem J. 2009;417:277-285.
2. Zhuang S, Schnellmann RG. A death-promoting role for extracellular signal-regulated
kinase. J Pharmacol Exp Ther. 2006;319:991-997.
3. Franceschini D, Giusti P, Skaper SD. MEK inhibition exacerbates ischemic calcium
imbalance and neuronal cell death in rat cortical cultures. Eur J Pharmacol.
2006;553:18-27.
4. Pignataro G, Meller R, Inoue K, Ordonez AN, Ashley MD, Xiong Z, et al. In vivo and in
vitro characterization of a novel neuroprotective strategy for stroke: ischemic
postconditioning. J Cereb Blood Flow Metab. 2008;28:232-241.
5. Gao X, Zhang H, Takahashi T, Hsieh J, Liao J, Steinberg GK, et al. The Akt signaling
pathway contributes to postconditioning's protection against stroke; the protection is
associated with the MAPK and PKC pathways. J Neurochem. 2008;105:943-955.