Nicotine Exacerbates Brain Edema during In Vitro and In Vivo
Focal Ischemic Conditions
Jennifer R. Paulson, Tianzhi Yang, Pradeep K. Selvaraj, Alexander Mdzinarishvili,
Cornelis J. Van der Schyf, Jochen Klein, Ulrich Bickel, and Thomas J. Abbruscato
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University, Health Sciences Center, Amarillo, Texas
(J.R.P., T.Y., U.B., T.J.A.); Department of Internal Medicine, Texas Tech University Health Sciences Center, Amarillo, Texas
(P.K.S.); Department of Pharmaceutical Sciences, Northeastern Ohio Universities Colleges of Medicine and Pharmacy,
Rootstown, Ohio (A.M., C.J.V.); and University of Frankfurt College of Pharmacy, Frankfurt, Germany (J.K.)
Received June 16, 2009; accepted November 3, 2009
We have previously shown that nicotine, the addictive compo-
nent of tobacco products, alters the blood-brain barrier (BBB)
Na?,K?,2Cl?cotransporter (NKCC) during in vitro hypoxia-
aglycemia exposure. Attenuation of abluminal NKCC suggests
that accumulation of ions in the brain extracellular fluid would
result in an increase of fluid or cytotoxic edema in the brain
during hypoxia-aglycemia or stroke conditions. To further in-
vestigate whether nicotine products have the potential to
worsen stroke outcome by increasing edema formation, two
separate models to mimic stroke conditions were utilized to
decipher the effects of short-term and long-term administra-
tions of nicotine products on brain edema following stroke.
Oxygen glucose deprivation (OGD) was studied in rat hip-
pocampal slices with short-term or long-term exposure to nic-
otine and cigarette smoke constituents. During short-term ex-
posure, the presence of nicotine at a concentration mimicking
heavy smokers increased water content of hippocampal slices
during OGD. Furthermore, long-term 1-week administration
of nicotine increased water content in hippocampal slices
that could be attenuated with nicotine acetylcholine receptor
(nAChR) antagonists, suggesting nicotine increase edema
during OGD via nAChRs. A second model of focal ischemia,
middle cerebral artery occlusion, showed an increase of
infarct size during short-term exposure to nicotine and an
increase of edema during both short-term and long-term
administration of nicotine, compared with saline controls.
These findings support the paradigm that nicotine products
not only increase the incidence of stroke but also have the
potential to worsen stroke outcome by increased edema
Stroke has the third highest mortality rate of 160,000 per
year and is a leading cause of neurological disease resulting
in long-term disability, with 3.5 million survivors in the
United States (Hankey, 1999; Carandang et al., 2006). Cig-
arette smoking and even second-hand smoke have been as-
sociated with increased incidence of stroke in both men and
women (Bonita et al., 1999). Smoking cigarettes clearly have
been shown to be a risk factor for stroke (Bonita et al., 1999;
Ueshima et al., 2004). Twenty-five percent of strokes is as-
sociated with smoking (Hankey, 1999). The risk of stroke is
dose-dependent and increases from 3.57 to 4.65 with 5 to 15
cigarettes per day (Bonita et al., 1999). In addition to en-
hanced risk factors for brain ischemia, there is a growing
body of evidence that nicotine alters BBB permeability char-
acteristics that have a direct influence on stroke outcome and
the pathophysiology of brain ischemia (Abbruscato et al.,
2002, 2004; Hawkins et al., 2002, 2004). More importantly,
stroke outcome has been shown to be worsened by increased
edema observed in a 2-week exposure to nicotine after focal
ischemia in rats (Wang et al., 1997).
The controlled water movement and maintenance of ion
balance in the brain extracellular fluid are imperative for the
reduction of edema and neuronal survival during stroke con-
ditions. Other diseases also have brain water balance as a
central point of damage, including head trauma, brain can-
cer, and certain types of epilepsy (Agre et al., 2004). Edema is
thought to play a central role at all levels of the neurovascu-
lar unit during neuronal damage associated with ischemia.
This work was supported by the National Institutes of Health [Grant
R01-NS046526] (to T.J.A.).
J.R.P. and T.Y. contributed equally to this work.
Article, publication date, and citation information can be found at
ABBREVIATIONS: BBB, blood-brain barrier; N-CSE, nicotine-containing cigarette smoke extract; NF-CSE, nicotine-free cigarette smoke extract;
NKCC, Na,K,2Cl?cotransporter; aCSF, artificial cerebrospinal fluid; MCAO, middle cerebral artery occlusion; OGD, oxygen glucose deprivation;
nAChR, nicotinic acetylcholine receptor; PG/DMSO, propylene glycol/dimethyl sulfoxide; RIA, radioimmunoassay; HPLC, high-performance liquid
chromatography; N/C, nicotine/cotinine; ANOVA, analysis of variance.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET 332:371–379, 2010
Vol. 332, No. 2
Printed in U.S.A.
Cellular swelling can be seen at the endothelial cell, peri-
cytes, and astrocytic foot processes within the first 90 min of
focal occlusion (Liu et al., 2001). Edema formation contrib-
utes to stroke morbidity and mortality, and Na?accumula-
tion in the brain occurs during ischemic assault before BBB
breakdown (Dzialowski et al., 2004).
Two types of edema are associated with stroke, cytotoxic
cellular edema and vasogenic edema (Unterberg et al., 2004).
Cytotoxic edema, experienced at the beginning of an ischemic
incident, is associated with increased extracellular ion accu-
mulation resulting in increased water content in cells, in-
cluding glia and neurons (Unterberg et al., 2004). Vasogenic
edema later amplifies the damage done by cellular edema
and is contributed by blood-brain barrier breakdown (Unter-
berg et al., 2004). Brain water content begins to immediately
accumulate before BBB breakdown and has been monitored
with computed tomography scan in a rat middle cerebral
artery occlusion (MCAO) model (Dzialowski et al., 2004).
Accumulation of water in astrocytes has been suggested to
produce the most damage due to the volume of astrocytes
versus neurons in the brain (20:1 in humans and 10:1 in rats)
(Kimelberg et al., 1995; Unterberg et al., 2004).
Previously, we have shown that nicotine, the addictive
component of tobacco products, is responsible for alteration of
the Na?,K?,2Cl?cotransporter 1 (NKCC1) on the abluminal
(brain facing) surface of the BBB during in vitro hypoxia/
aglycemia conditions used to model stroke. This could result
in the accumulation of ions, including Na?, K?, and Cl?in
the brain extracellular fluid, resulting in increased fluid or
brain edema during hypoxic or stroke conditions with or
without reperfusion (Abbruscato et al., 2004; Paulson et al.,
2006). However, there is no further information on how cig-
arette smoke constituents, not only nicotine, will affect
edema formation in animal models of stroke, especially under
To further investigate the possibility that nicotine prod-
ucts have the potential to worsen stroke outcome by in-
creased edema formation, two separate models to mimic
stroke conditions were utilized to decipher the effects of
short-term and long-term administration of cigarette smoke
constituents or nicotine on brain edema following stroke. The
first model, oxygen glucose deprivation (OGD), was studied
in rat hippocampal slices with short-term exposure to nico-
tine or long-term exposure to cigarette smoke constituents. A
second model of focal ischemia, MCAO, was used to evaluate
infarct size and edema during short-term and long-term ex-
posure to nicotine. Knowledge gained from these studies
could lead to better therapeutic approaches to protect the
central nervous system from neurological damage associated
with nicotine and/or stroke insults.
Materials and Methods
Preparation of Smoke Condensates. Cigarettes (Marlboro fil-
ter cigarettes; Philip Morris Inc., Richmond, VA; and Quest 3; Vector
Tobacco Inc., Research Triangle Park, NC) were obtained through
commercial sources. Marlboro filtered cigarettes contain 1.1 mg of
nicotine per cigarette, and Quest 3 contains no more than 0.05 mg of
nicotine per cigarette and is therefore regarded to be nicotine-free for
these studies. Mainstream smoke was bubbled through 50 ml of
acetone by a slight vacuum drawn repetitiously through a custom
manufactured apparatus. A cigarette smoke extract-acetone solution
was generated, which was concentrated under vacuum. The residue
was subsequently redissolved in 1.626-ml propylene glycol/dimethyl
sulfoxide (PG/DMSO, 1:1). The products of the Marlboro and Quest
cigarette extraction were designated as N-CSE (nicotine-containing
cigarette smoke extract) and NF-CSE (nicotine-free cigarette smoke
extract), respectively. These methods have successfully been used
in vitro to model exposure to cigarette smoke chemicals (Sherratt et
al., 1988; Paulson et al., 2006).
Hippocampal Slice Oxygen Glucose Deprivation. Experi-
mental design was formed by varying a method of hippocampal brain
slicing and subsequent exposure to oxygen glucose-deprived artificial
CSF (MacGregor et al., 2003). Female Sprague Dawley rats were
anesthetized and euthanized through cervical dislocation. The brain
was extracted, and the hippocampus identified by stereotaxic coor-
dinator (Franklin and Paxinos, 2001) was sliced with a McIlwain
Tissue Chopper (Mickle Laboratory Engineering Co. Ltd., Guilford,
Surrey, UK) at 400 ?m. Hippocampal slices were pre-equilibrated in
a solution of ice-cold artificial cerebrospinal fluid (aCSF; 137 mM
NaCl, 2.7 mM KCl, 0.2 mM CaCl2, 1.2 mM MgCl2, 0.2 mM NaH2PO4,
11.9 mM NaHCO3, and 5.6 mM glucose), with the addition of 1 mM
ascorbic acid. Preincubation with ascorbic acid prevents artificial
edematous conditions produced by in vitro conditions (Brahma et al.,
2000). To ensure cellular survival, the time period from brain extrac-
tion to placement in artificial CSF was consistently kept under 3
Hippocampal slices were treated under normoxic or OGD condi-
tions in a PermeGear permeation chamber (AMIE Systems, Riegels-
ville, PA), with the slices placed between thin nylon 200 mesh screen
(Ted Pella Inc., Redding, CA). Artificial CSF flowed through the
chamber at a constant rate of 0.7 ml/min at a temperature of 37°C.
Normoxic conditions were maintained through control aCSF, with
the presence of glucose bubbled with oxygen (137 mM NaCl, 2.7 mM
KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 0.2 mM NaH2PO4, 11.9 mM
NaHCO3, and 5.6 mM glucose). OGD was maintained through aCSF
with the absence of glucose and bubbled with 95% nitrogen and 5%
CO2and kept at a temperature of 37°C.
Short-term and long-term treatments were tested in this study.
Short-term treatment of nicotine/cotinine was administered through
the aCSF at varying concentrations ranging from 1/10, 10/100, 100/
1000, and 1000/10,000 ng/ml nicotine and cotinine, respectively.
Long-term 1-week and 3-week administration of vehicle control,
nicotine, N-CSE, or NF-CSE was given to rats using Alzet 2ML4
osmotic pumps (Alzet, Cupertino, CA) at a concentration to mimic
heavy smokers (4.5 mg/kg/day) in accordance with previous studies
(Murrin et al., 1987; Wang et al., 1997). Nicotine and cotinine plasma
levels were verified by RIA and HPLC to confirm comparable plasma
levels within groups. In addition, hippocampal slices subjected to
OGD with N/C and nAChR antagonists (25, 125, and 250 ?M
mecamylamine or 10 nM ?-bungarotoxin) were tested to evaluate the
edematous effects involved with nAChRs.
Edema formation was evaluated by determining the percentage of
water content of each slice. Hippocampal slices were initially
weighed on a Cahn microbalance immediately following the treat-
ment incubation on preweighed aluminum foil and considered “wet
weight.” After a 24-h period of desiccation at 95°C, the slices were
again weighed and recorded as “dry weight.” Water content was then
calculated as [(wet weight ? dry weight)/wet weight] ? 100.
In Vivo MCAO Model. The methods used here were a slight
modification of previously published procedures (Mdzinarishvili et
al., 2005). CD-1 female mice were pretreated with nicotine, N-CSE,
or NF-CSE in short-term dosages of 1, 3, or 6 h or long-term dosages
of 1 or 3 weeks (nicotine in 0.9% NaCl; N-CSE and NF-CSE in
PG/DMSO). Short-term injections of nicotine or CSEs were equiva-
lent to 4.5 mg/kg/day calculated from the known nicotine yield. For
short-term nicotine dosages of 1, 3, and 6 h, intraperitoneal injec-
tions of 187.5, 562.5, and 1125 ?g/kg nicotine, respectively, were
given. The doses were calculated as done for 1 h, 4.5 mg/kg divided
by 24 h ? 187.5 ?g/kg and so on for 3- and 6-h doses. This dose was
selected because it did not result in gross signs of toxicity; the
Paulson et al.
resulting nicotine and cotinine plasma concentrations reached were
similar to those found in heavy smokers (80–100 ng/ml) (Murrin et
al., 1987; Lockman et al., 2005). Data from our previous studies
(Lockman et al., 2005a,b) demonstrate that this dosage of nicotine
does not alter regional BBB integrity or regional cerebral perfusion
flow. Long-term administration of nicotine was delivered through
subcutaneously placed Alzet 2001 mini-osmotic pumps at a concen-
tration to mimic heavy smokers (4.5 mg/kg/day) in accordance with
previous studies (Murrin et al., 1987; Wang et al., 1997). Nicotine
and cotinine plasma levels were verified by RIA and HPLC to con-
firm comparable plasma levels within groups.
Animals were anesthetized with 4% isoflurane and maintained
with 1% isoflurane in 30% O2/70% N2O using a face mask and a
SurgiVet vaporizer (Smiths Medical North America, Waukesha, WI).
Surgery was performed using a Zeiss OPMI pico surgical microscope
(Carl Zeiss GmbH, Jena, Germany). Local cortical blood flow in the
left middle cerebral artery territory was monitored with laser-Dop-
pler flowmetry and observed online in real time on computer using a
software program (Moor Instruments, Ltd., Axminster, Devon, UK).
After occlusion of the common carotid artery by a microclip, the left
external carotid artery was ligated, coagulated, and cut distally to
the cranial thyroid artery. A small incision in the external carotid
was made, and a 20-mm monofilament nylon suture (5-0; Harvard
Apparatus Inc., Holliston, MA), which had been rounded at the tip by
heat (final diameter: 0.2–0.3 mm), was inserted into the external
carotid and gently advanced through the internal carotid artery until
its tip occluded the origin of the MCA. Correct placement of the
suture was indicated by a sudden drop of the local cortical blood flow
in the left MCA territory to 10 to 15% baseline as seen by laser-
Doppler flowmetry. After successful occlusion, the monofilament was
secured in place with ligature, and the incision was closed by micro-
MCAO was sustained for a period of 24 h, after which the animals
were deeply anesthetized with isoflurane and euthanized by decap-
itation. It has been reported that permanent MCAO (24 h) creates
predictable neuronal injury that is caused by both cytotoxic and
vasogenic edema (Kumar et al., 2006; Mdzinarishvili et al., 2007).
The brains were quickly removed and sectioned coronally into slices
of 1 mm thickness using McIlwain Tissue Chopper. Slices were
incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride in
phosphate-buffered saline at 37°C for 15 min and fixed by immersion
in 4% buffered formaldehyde solution. 2,3,5-Triphenyltetrazolium
chloride stains viable brain tissue dark red based on intact mitochon-
drial function, whereas infarcted tissue areas remain unstained
(white) (Gorgulu et al., 2000). Images were acquired by digital cam-
era (Nikon, Melville, NY), and areas of both hemispheres and the
infarcted regions were quantified for each slice using image analysis
software (Image J 1.30 and Scion Image version Beta 4.0.2; National
Institutes of Health, Bethesda, MD). We elected to measure the
areas of damage and brain swelling by area increase in the ipsilat-
eral (ischemic) hemisphere and compare these with the contralateral
(nonischemic) hemisphere (Elliot and Jasper, 1949; Sydserff et al.,
1996). This allowed direct comparison of areas of damage with brain
swelling and allowed us to use each animal as its own control in
addition to interexperimental comparisons (Sydserff et al., 1996).
Five animals were used for each experiment, and three measure-
ments were made on each slice to calculate the size of the lesion and
to correct for overestimation due to the effects of brain swelling. The
calculations are: a ? area of infarct (mm2), b ? area of the infarcted
(ipsilateral) hemisphere slice (mm2), c ? area of the noninfarcted
(contralateral) hemisphere slice (mm2), d ? brain swelling (mm2) ?
b ? c (Park and Kang, 2000). The area (Al) of the lesion (mm2),
corrected for swelling, was derived from the equation Al? a ? d. The
swelling area was designated Aeand quantified by determining the
ratio between the areas of the infarcted and noninfarcted hemi-
sphere slices, thus: Ae? b ? c. Infarct and edema ratios of hemi-
spheric areas were expressed as the mean ? S.E.M. and compared
using Student’s t test. Values of P ? 0.05 were considered statisti-
cally significant. All of the animal studies were conducted in accor-
dance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals (NIH Publications 80-23) revised 1996
and approved by the Animal Care and Use Committee at Texas Tech
University Health Sciences Center.
Locomotor Activity Measurements. Six groups of female CD-1
mice (including naive mice control) were included in this study: 1)
mice with 1-h exposure of nicotine 4.5 mg/kg/day; 2) mice with
MCAO for 6 h; 3) mice with MCAO for 6 h plus 1 h before exposure
to nicotine 4.5 mg/kg/day; 4) mice with exposure to nicotine 4.5
mg/kg/day for 3 weeks; and 5) mice with MCAO for 6 h plus 3 weeks
before exposure to nicotine 4.5 mg/kg/day. Long-term administration
of nicotine (4.5 mg/kg/day for 3 weeks) was delivered through sub-
cutaneously placed Alzet 2001 mini-osmotic pumps at a concentra-
tion to mimic heavy smokers (4.5 mg/kg/day) in accordance with
previous studies (Murrin et al., 1987; Wang et al., 1997). Nicotine (45
ng/ml) and cotinine (262.2 ng/ml) plasma levels were verified by RIA
and HPLC to confirm comparable plasma levels within groups.
After the treatments, behavioral data were collected using Versa-
Max animal monitors (Accuscan Instruments Inc., Columbus, OH),
which has been used to monitor stroke outcome using several loco-
motor parameters and behavioral deficits that are predictive of
stroke injury (Vendrame et al., 2004). The chamber cage was 42 ?
42 ? 30 cm, made of clear Plexiglas glass, and covered with a
Plexiglas lid with holes for ventilation. Infrared monitoring sensors
were located every 2.5 cm along the perimeter (16 infrared beams
along each side) and 2.5 cm above the floor. Two additional sets of 16
sensors were located 8.0 cm above the floor on opposite sides. Data
were collected and analyzed by a VersaMax analyzer (Accuscan
Instruments Inc.), which in turn sent information to a computer that
ran the VersaMax and Versadat programs. These programs tabu-
lated and processed a number of variables related to locomotor
behavior. For the present experiments, four different locomotor pa-
rameters were measured every 4 min for 20 min, which has been
shown previously to reflect behavioral changes associated with neu-
rological damage induced by MCAO (Vendrame et al., 2004). Before
the experiments, mice were acclimatized to the behavioral proce-
dure. All testings occurred between 12:00 PM and 5:00 PM. Behav-
ioral tests were performed in all six groups of animals.
Statistical Analysis. All data are expressed as the mean ? S.D.,
and values were compared by ANOVA. When the differences in the
means were significant, post hoc pairwise comparisons were con-
ducted using Newman-Keuls multiple comparison (Prism, version
3.03; GraphPad Software, Inc., San Diego, CA). Differences in
p values less than 0.05 were considered statistically significant.
Effects of Nicotine on Blood Gases and Temperature.
Studies were carried out to determine whether the nicotine
administration altered physiologic parameters (tempera-
ture and blood gas) during MCAO. Arterial blood samples
were drawn (100 ?l/sample) under anesthesia from groups
of mice that were exposed to 24 h of MCAO and or 3 h of
nicotine dosage (0.56525 mg/kg). Blood samples were
drawn 1 h after induction of MCAO as described above.
Blood gases and serum electrolytes were analyzed on a
RapidLab 348 blood gas analyzer (Bayer Diagnostics, Tar-
rytown, NY). We found nicotine exposure did not change
pO2, pCO2, or pH parameters when compared with control.
It is noteworthy that we also found that nicotine exposure
before 24-h MCAO did not change these parameters when
compared with the 24-h MCAO alone. Higher pO2values
are explained by the slightly higher oxygen content of the
gas mixture (30% compared with room air) used for MCAO
Nicotine Worsens Brain Edema Associated with Stroke
Hippocampal Slices Subjected to Short-Term Nico-
tine Exposure. Each cigarette smoking results in absorp-
tion of 1 to 1.5 mg of nicotine by the smoker (Hukkanen et al.,
2005). Nicotine is metabolized in the liver resulting in six
metabolites of which cotinine is the primary metabolite av-
eraging 72% (Benowitz and Jacob, 1994). Plasma levels of
nicotine in smokers range from 10 to 50 ng/ml, and cotinine
levels predominantly range from 250 to 300 ng/ml yet can be
variable up to 900 ng/ml (Hukkanen et al., 2005). We used
levels of nicotine/cotinine, respectively, as 1/10, 10/100, 100/
1000, and 1000/10,000 ng/ml for the dosing paradigm in
During normoxic conditions, nicotine exposure at the high-
est dose of 1000 ng/ml nicotine and 10,000 ng/ml cotinine
showed an increase in water content compared with control
(p ? 0.05) (Fig. 1). OGD exposed slices showed greater sen-
sitivity to nicotine/cotinine exposure with significant in-
crease in water content at doses of nicotine/cotinine: 10/100
(p ? 0.01), 100/1000 (p ? 0.01), and 1000/10,000 (p ? 0.01)
Hippocampal Slices Subjected to Long-Term Ciga-
rette Smoke Constituent Exposure. Our long-term nico-
tine exposure through an Alzet minipump delivery system for
1 week (4.5 mg/kg/day) revealed that, during normoxic con-
ditions, N-CSE significantly increased water content of hip-
pocampal slices compared with the vehicle control PG/DMSO
and NF-CSE (p ? 0.05) (Fig. 2A). There is no significant
difference in water content of hippocampal slices between
vehicle control (PG/DMSO) and saline control (data not
shown). It is noteworthy that nicotine and N-CSE at 4.5
mg/kg/day did not show a significant difference in water
content. During OGD conditions, a 1-week exposure to nico-
tine 4.5 mg/kg/day increased water content compared with
control (p ? 0.01) and NF-CSE (p ? 0.05) (Fig. 2A). Normoxic
and OGD exposure following 3-week minipump delivery of all
cigarette smoke constituents resulted in no significant alter-
ation of hippocampal slice water content (Fig. 2B).
Hippocampal Slices Subjected to OGD with N/C and
nAChR Antagonists. In OGD-exposed hippocampal brain
slices, N/C exposure at 100/1000 ng/ml significantly in-
creased water content (p ? 0.01) compared with control OGD
conditions (Fig. 3). In addition, nAChR antagonists abolished
the findings of N/C exposure by significantly decreasing wa-
ter content in hippocampal slices, even in the presence of
nicotine/cotinine. The increase of water content with N/C
(100/1000) exposure is reduced with various concentrations
of nAChR antagonists (i.e., 25–250 ?M mecamylamine
present in the aCSF): 25 ?M mecamylamine (p ? 0.01), 125
?M mecamylamine (p ? 0.001), and 250 ?M mecamylamine
(p ? 0.001) (Fig. 3). In addition, the presence of 10 ?M
?-bungarotoxin in the presence of N/C (100/1000) inhibits the
water gain induced by N/C (p ? 0.05) but remained higher
than 125 ?M mecamylamine exposure (p ? 0.05) (Fig. 3). All
nAChR antagonist exposures alone did not significantly dif-
fer in water gain compared with OGD control (data not
Fig. 1. Water content of hippocampal slices subjected to normoxia or oxygen glucose deprivation with short-term nicotine/cotinine exposure. Effect of
short-term 1-h exposure to aCSF control, nicotine (1 ng/ml)/cotinine (10 ng/ml) (1/10 NC); nicotine (10 ng/ml)/cotinine (100 ng/ml) (10/100 NC); nicotine
(100 ng/ml)/cotinine (1000 ng/ml) (100/1000 NC); and nicotine (1000 ng/ml)/cotinine (10,000 ng/ml) (1000/10,000 NC) on water content of brain
hippocampal slices following normoxic or oxygen glucose deprivation conditions. Data represent mean ? S.E.M. of 9 to 17 independent determinations.
? denotes significance of p ? 0.05; ?? denotes significance of p ? 0.01, and ??? denotes significance of p ? 0.001 using one-way ANOVA with
Newman-Keuls post hoc analysis.
Blood gas values and temperature
Studies were carried out to determine whether the nicotine administration altered physiologic parameters (temperature and blood gas) during MCAO. Arterial blood samples
were drawn (100 ?l/sample) under anesthesia from groups of mice that were exposed to 24 h of MCAO and or 3 h of nicotine (0.56525 mg/kg). Blood samples were drawn
1 h after induction of MCAO as described above. Blood gases and serum electrolytes were analyzed on a RapidLab 348 blood gas analyzer (Bayer Diagnostics). Higher pO2
values are explained by the slightly higher oxygen content of the gas mixture (30% compared to room air) that is used for MCAO.
Control 3-h Nicotine24-h MCAO 24-h MCAO ? 3-h Nicotine
86.3 ? 4.3
32.1 ? 3.8
7.41 ? 0.18
85.4 ? 3.8
32.7 ? 3.9
7.43 ? 0.25
92.1 ? 2.9
29.8 ? 2.6
7.39 ? 2.0
95.6 ? 3.1
28.3 ? 2.1
7.41 ? 0.8
Paulson et al.
shown), suggesting that these antagonists are acting through
a mechanism involving the nAChR.
Short-Term 1-h Exposure to Cigarette Smoke Con-
stituents in MCAO Model. Infarct ratio increases with a
1-h dosage of 187.5 ?g/kg nicotine (4.5 mg/kg/day, 4.5 mg/24
h ? 187.5 ?g/kg/1 h) (p ? 0.001) and N-CSE (p ? 0.05) at
concentrations that mimic plasma levels of a heavy smoker
(Fig. 4). Exposure to NF-CSE did not significantly differen-
tiate infarct ratio from vehicle control. Edema ratio also
increased for 1-h exposure to nicotine (p ? 0.05) and N-CSE
(p ? 0.05) compared with control (Fig. 4). Again, NF-CSE did
not differ from control in edema ratio same as with infarct
Short-Term 1-, 3-, and 6-h Exposure to Nicotine in
MCAO Model. All short-term intraperitoneal injections of
nicotine equivalent to a total dosage of 4.5 mg/kg/day [1 h (4.5
mg/24 h ? 187.5 ?g/kg) (p ? 0.01), 3 h (4.5 mg/8 h ? 562.5
?g/kg) (p ? 0.05), and 6 h (4.5 mg/4 h ? 1125 ?g/kg) (p ?
0.001)] dosage before MCAO resulted in a significant in-
crease in edema. Furthermore, infarct ratio was significantly
increased for all three short-term nicotine 4.5 mg/kg/day
injections: 1 h (p ? 0.001) (Fig. 5A), 3 h (p ? 0.05) (Fig. 5B),
and 6 h (p ? 0.001) (Fig. 5C).
Long-Term Exposure to Nicotine in MCAO Model.
Long-term administration of nicotine was delivered through
subcutaneously placed Alzet 2001 mini-osmotic pumps at a
concentration to mimic heavy smokers (4.5 mg/kg/day) in
accordance with previous studies (Murrin et al., 1987; Wang
et al., 1997). Nicotine and cotinine plasma levels were veri-
fied by RIA and HPLC to confirm comparable plasma levels
within groups. Long-term exposure to 4.5 mg/kg/day nicotine
did not alter infarct ratio for 1-day, 1-week, or 3-week
minipump administration (Fig. 6), whereas edema ratio was
significantly altered compared with control: 1 day (p ?
0.001), 1 week (p ? 0.001), and 3 week (p ? 0.01) (Fig. 6).
Locomotor Activity Measurements. A series of locomo-
tor activity parameters were monitored in this set of experi-
ments, including vertical activity, total distance, movement
Fig. 2. Water content of hippocampal slices subjected to normoxia or oxygen glucose deprivation with long-term 1- and 3-week cigarette smoke
constituent exposure. Effect of 1-week exposure to vehicle control, nicotine (4.5 mg/kg), N-CSE, and NF-CSE on water content of brain hippocampal
slices following normoxic or oxygen glucose deprivation conditions. Data represent mean ? S.E.M. of 9 to 20 independent determinations. ? denotes
significance of p ? 0.05, and ?? denotes significance of p ? 0.01 using one-way ANOVA with Newman-Keuls post hoc analysis.
Fig. 3. Water content of hippocampal slices subjected to nicotine/cotinine
and nicotinic acetylcholine receptor antagonists during oxygen glucose
deprivation. Effects of short-term exposure of 100 ng/ml nicotine and
1000 ng/ml cotinine with or without nAChR antagonists on water content
of hippocampal slices subjected to OGD. Control, N/C, or N/C along with
25, 125, 250 ?M mecamylamine, or 10 nM ?-bungarotoxin in aCSF. Data
represent the mean ? S.E.M. of 9 to 20 independent determinations. ?
denotes significance of p ? 0.05, and ?? denotes significance of p ? 0.01
using one-way ANOVA with Newman-Keuls post hoc analysis.
Nicotine Worsens Brain Edema Associated with Stroke
time, and stereotypy time. The results showed that for pa-
rameters, such as vertical activity, total distance, movement
time, and stereotypy time, there was no significant difference
between control group and nicotine (1 h)-treated group (Table 2).
Vehicle control had no effect on these locomotor parameters
(data not shown). However, these locomotor parameters were
significantly decreased with animals subjected to MCAO,
compared with the control (p ? 0.05). Combining treatment
of nicotine (1 h) worsened the stroke outcome by decreasing
these locomotor activity parameters compared with the
MCAO group (Table 2). For long-term nicotine use (3 weeks),
nicotine itself did not affect these selected locomotor activity
parameters compared with the control. Animals subjected to
both nicotine (3 weeks) and MCAO significantly decreased
the locomotor parameters compared with the control (Table
2) (p ? 0.05). However, a statistically significant difference
Fig. 4. Effect of 1-h exposure to cigarette smoke constituents on infarct and edema ratios following MCAO. Effect of parenteral injection 1 h before
24-h MCAO of vehicle control, nicotine (4.5 mg/kg/day), N-CSE, or NF-CSE on infarct ratio (infarct area/brain slice area) and edema ratio
(ipsilateral/contralateral hemisphere slice area). Data represent the mean ? S.E.M. of five independent determinations containing five to six slices
each. ? denotes significance of p ? 0.05, and ??? denotes significance of p ? 0.001 using one-way ANOVA with Newman-Keuls post hoc analysis.
Fig. 5. Effect of 1-, 3-, and 6-h intraperitoneal injection of nicotine on edema and infarct ratios following MCAO. Effect of parenteral injection 1, 3,
and 6 h of nicotine (4.5 mg/kg/day) on infarct ratio (infarct area/brain slice area) and edema ratio (ipsilateral/contralateral hemisphere slice area)
following 24-h MCAO. Data represent the mean ? S.E.M. of five independent determinations containing five to six slices each. ? denotes significance
of p ? 0.05, ?? denotes significance of p ? 0.01, and ??? denotes significance of p ? 0.001 using one-way ANOVA with Newman-Keuls post hoc analysis.
Paulson et al.
could not be detected between MCAO group and MCAO/
nicotine 3-week group (p ? 0.05). It is noteworthy that there
was no significant difference in those parameters between
nicotine (1-h exposure)/MCAO group and nicotine (3-week
exposure)/MCAO group (Table 2).
Studies have shown that nicotine has a detrimental effect
at the cerebral microcirculation involving tissue plasminogen
activator, plasminogen activator inhibitor 1, nitric-oxide syn-
thase, leukocyte migration, and BBB dysfunction of tight
junctional proteins (Hawkins et al., 2002). In our studies
using in situ and in vivo models, we have demonstrated that
nicotine can increase cytotoxic and vasogenic edema after
stroke conditions and therefore worsen the stroke outcome.
Hippocampal slice OGD is a model that represents cellular
edema associated with the accumulation of water content
and has previously been utilized to measure the neuropro-
tective effects of bilobalide (Mdzinarishvili et al., 2007).
Therefore, an in situ 1-h incubation of hippocampal slices in
either normoxic or OGD media with or without the presence
of nicotine was used to investigate cytotoxic cellular edema,
and in vivo 24-h murine MCAO pretreated with nicotine or
vehicle control was used to investigate both cellular and
vasogenic edema. With short-term exposure of nicotine/coti-
nine at concentrations ranging from 10/100 to 1000/10,000
ng/ml N/C, there is a significant increase of water content
during OGD conditions compared with OGD control (Fig. 1).
This shows that the concentration of 10/100 ng/ml N/C, sim-
ilar to plasma levels found in smokers, can increase cellular
edema during stroke conditions, which is consistent with
previous studies (Hukkanen et al., 2005). During normoxic
conditions, only the highest concentration of 1000/10,000
ng/ml N/C, 100-fold higher than concentrations applicable to
heavy smokers, showed a significant effect of increasing wa-
ter content compared with control (Fig. 1). Exposure to nic-
otine and its metabolite cotinine in this model at concentra-
tions similar to cigarette smokers seems to be detrimental
only during OGD and does not produce edematous conditions
during normoxia. This could be explained by the fact that this
hippocampal brain slice model mimics cellular edema only
during OGD conditions (Mdzinarishvili et al., 2007).
Effective administration in the 1- and 3-week long-term
dosage of nicotine through osmotic minipumps was verified
via an RIA kit (Cozart Bioscience UK, Abingdon, Oxford-
shire, UK) for detection of cotinine in plasma and detected as
a positive or negative reading (Table 3). Further HPLC anal-
ysis of plasma from plasma of rats administered 4.5 mg/kg/
day nicotine for 1 day, 1 week, and 2 weeks through Alzet
minipumps detected levels of nicotine and the major metab-
olite cotinine at levels similar to a heavy smoker (Lockman et
al., 2005b) (Table 4). All values correlate well with values
reported in the literature (Benowitz and Jacob, 1994; Huk-
kanen et al., 2005). In addition, brain uptake of nicotine and
cotinine in rats delivered through nicotine-loaded Alzet
minipumps at 4.5 mg/kg/day for 28 days demonstrated a
unidirectional influx across the BBB for nicotine at a rate of
Fig. 6. Effect of long-term exposure to nicotine on infarct and edema ratios following MCAO. Effect of long-term administration of vehicle control,
nicotine (4.5 mg/kg) 1 day, nicotine (4.5 mg/kg) 1 week, and nicotine (4.5 mg/kg) 3 weeks on infarct ratio (infarct area/brain slice area) and edema ratio
(ipsilateral/contralateral hemisphere slice area) following 24-h MCAO. Data represent the mean ? S.E.M. of five independent determinations
containing five to six slices each. ?? denotes significance of p ? 0.01, and ??? denotes significance of p ? 0.001 using one-way ANOVA with
Newman-Keuls post hoc analysis.
Effects of stroke and nicotine treatment on the mouse locomotor activity
Data represent the mean ? S.D. of three to four independent determinations.
Parameters Control Nicotine 1 hMCAO
MCAO ? Nicotine 3 Weeks
Total distance (cm)
Movement time (s)
Stereotypy time (s)
4859 ? 872
3558 ? 934
278 ? 79
178 ? 24
4098 ? 1018
2993 ? 817
217 ? 69
181 ? 38
640 ? 209*
397 ? 247*
40 ? 16*
25 ? 3*
140 ? 52#
244 ? 82#
22 ? 10#
16 ? 6#
4414 ? 1214
4337 ? 627
309 ? 32
167 ? 98
578 ? 262*
216 ? 225*
34 ? 16*
24 ? 16*
* P ? 0.05 compared to the control group; # P ? 0.05 compared to the MCAO group.
A plasma cotinine presence in 1- and 3-week osmotic minipump
administration via RIA cotinine analysis
Sample1 Week 3 Weeks
N.D., not determined.
Nicotine Worsens Brain Edema Associated with Stroke
114 to 143 ?g/g/day and cotinine at a rate of 43 to 61 ?g/g/day
(Lockman et al., 2005a).
Water content representing cytotoxic edema increased af-
ter a 1-week exposure to nicotine when hippocampal slices
were exposed to OGD compared with control and NF-CSE
exposures. It is interesting to note that NF-CSE exposure
resulted in significantly less water gain compared with ad-
ministration of nicotine alone, suggesting that the remaining
components of cigarette smoke are not responsible for in-
creased cytotoxic edema during a 1-week administration
(Fig. 2A). Furthermore, when hippocampal slices were ex-
posed to normoxic conditions, only a 1-week N-CSE exposure
resulted in an increase of water content (Fig. 2A). This sug-
gests that nicotine is the main component of cigarette smoke,
which is detrimental both during normal conditions and also
during OGD conditions exacerbating water gain in the hip-
The edematous effects of 100 ng/ml/1000 ng/ml N/C during
OGD were returned back to control levels with nAChR an-
tagonists mecamylamine (25, 125, and 250 ?M) and ?-bun-
garotoxin (10 nM) (Fig. 3). Mecamylamine inhibits the ability
of nicotine to induce the influx of calcium through the open
position at the ion channel site of the nAChR (Zevin et al.,
2000). The ion channel properties of nAChR of inward Ca?
and Na?movements could facilitate edema formation when
the agonist nicotine is present, and this action is abolished in
the presence of nAChR antagonist mecamylamine and a
more selective ?7-antagonist, ?-bungarotoxin (Colquhoun
and Patrick, 1997). Our studies suggest that nicotine in-
creases edema of the hippocampus during OGD via nAChR
as proven by the ability of mecamylamine and ?-bungaro-
toxin to reduce water content.
All cellular components of the neurovascular unit have
nAChR subunit expression, including neurons, astrocytes,
and endothelial cells (Abbruscato et al., 2002; Xiu et al., 2005;
Brody et al., 2006). Long-term smoking has been associated
with an alteration of nAChR expression in the brain with ?4
subunit expression increased in neurons and dendritic pro-
cesses (Teaktong et al., 2004) and decreased in ?7 subunit
expression in astrocytes and hippocampal regions (Teaktong
et al., 2004). We previously have demonstrated that nicotine
decreases NKCC activity in bovine brain microvessel cells,
which also express ?-3, ?-5, ?-7, ?-2, and ?-3 nAChR subunit
proteins (Abbruscato et al., 2004). The decrease of ?7 subunit
expression in astrocytes of the hippocampus and a possible
modulation of NKCC activity and movement of ions may be
responsible for the lack of edema observed in the 3-week
long-term administration of nicotine in the hippocampal
OGD model. Therefore, we investigated these results using a
second model of focal ischemia, MCAO, to evaluate infarct
size and both forms of vasogenic and cellular edema during
short- and long-term exposure to nicotine.
MCAO (24 h) creates predictable neuronal injury that is
caused by both cytotoxic and vasogenic edema due to the
ability of this model to replicate damage to the neurovascular
unit (Mdzinarishvili et al., 2007). Cytotoxic edema has been
observed within 12 h of occlusion, and water content and
ionic disturbances have been observed to increase for 72 h
following rat brain MCAO (Gotoh et al., 1985). Vasogenic
edema has been observed to peak at 6 h following photochem-
ical MCAO and measured through contrast-enhanced T1-
weighted imaging (Chen et al., 2007). This combination of
edema gives a more predictable model of damage from a
chronic focal stroke. Short-term 1-h exposure to nicotine and
N-CSE resulted in a significant increase in infarct ratio and
edema (p ? 0.001 and p ? 0.05), respectively, which predicts
the size of unrecoverable brain cell loss in the ischemic core
(Fig. 4). It is noteworthy that exposure to NF-CSE did not
result in a change of infarct or edema ratio compared with
control, which is similar to the hippocampal 1-week OGD
experiments suggesting that nicotine alone is responsible for
the infarct volume changes. We then investigated the effects
of nicotine alone on infarct and edema ratio with short-term
administration (1, 3, and 6 h) before 24-h MCAO (Fig. 5). All
short-term intraperitoneal injections of nicotine equivalent
to 4.5 mg/kg/day before MCAO resulted in a significant in-
crease of edema at various time points, such as 1 h (p ? 0.01),
3 h (p ? 0.05), and 6 h (p ? 0.001), and also a significant
increase in infarct ratio, such as 1 h (p ? 0.001) (Fig. 5A), 3 h
(p ? 0.05) (Fig. 5B), and 6 h (p ? 0.001) (Fig. 5C). We did not
observe any increase in infarct area 24 h after MCAO with
long-term dosages of nicotine (4.5 mg/kg/day s.c.: 1 day, 1
week, and 3 weeks). Yet, edema ratio significantly increases
with nicotine exposure for long-term (1 day, 1 week, and 3
weeks) exposures (Fig. 6).
To further explore the nicotine effect of the functional
outcome of the stroke, locomotor studies were designed. This
study shows that activity parameter data were decreased
significantly after MCAO surgery compared with those of
control. Parameters from nicotine plus MCAO treatment
group were statistically lower compared with the MCAO
group, which supports our conclusion that nicotine worsens
stroke outcome (Table 2). It is noteworthy that we did not
observe any differences in locomotor activity parameters be-
tween MCAO group and MCAO ? nicotine 3-week group
(Table 2). It is apparent that some level of tolerance develops
to the worsened infarct size effects of nicotine with long-term
exposure. This may be due to nAChR receptor desensitization
in the hippocampus slice. Desensitization of ?4?2nAChR, the
predominant receptor in the brain, has recently been eluci-
dated in cigarette smokers through positron emission tomog-
raphy scanning (Brody et al., 2006). Plasma levels at 4% of
the level of typical smokers resulted in 50% occupancy of ?4?2
nAChR, and tobacco-dependent smokers with plasma levels
ranging from 10 to 50 ng/ml nicotine maintained 96 to 98%
receptor occupancy/desensitization throughout the day. Fu-
ture studies will investigate nAChR expressions in brain
regions susceptible to stroke damage.
In conclusion, this study shows that the nicotine produces
an increase of both cytotoxic and vasogenic edema as seen in
the hippocampal slice OGD and MCAO models, which simu-
late brain ischemia. Increased edema in the ischemic hemi-
sphere may negatively affect penumbral region recovery and
eventually lead to increased neuronal damage that may oth-
erwise have been recoverable or responsive to neuron-protec-
tive therapy. Prevention of increased edematous conditions
Nicotine and cotinine plasma concentrations via HPLC analysis
1 Day1 Week 2 Weeks
Paulson et al.
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nicotine products in stroke prone individuals. Furthermore,
clinicians should be aware of the propensity for edema in
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Address correspondence to: Thomas J. Abbruscato, School of Pharmacy,
Texas Tech University Health Sciences Center, 1300 S. Coulter, Amarillo, TX
79106. E-mail: email@example.com
Nicotine Worsens Brain Edema Associated with Stroke