Journal of Neurochemistry, 2005, 95, 254–262
Aquaporin-4 gene deletion in mice increases focal edema
associated with staphylococcal brain abscess
Orin Bloch,* Marios C. Papadopoulos,* Geoffrey T. Manley  and A. S. Verkman*
*Departments of Medicine and Physiology, Cardiovascular Research Institute, and  Department of Neurological Surgery, University
of California, San Francisco, California, USA
Abstract
Brain abscess is associated with local vasogenic edema,
which leads to increased intracranial pressure and significant
morbidity. Aquaporin-4 (AQP4) is a water channel expressed
in astroglia at the blood–brain and brain–CSF barriers. To
investigate the role of AQP4 in brain abscess-associated
edema, live Staphylococcus aureus (105 colony-forming units)
was injected into the striatum to create a focal abscess. Wild-
type and AQP4-deficient mice had comparable immune
responses as measured by brain abscess volume ( 3.7 mm3
at 3 days), bacterial count and cytokine levels in brain hom-
ogenates. Blood–brain barrier permeability was increased
comparably in both groups as assessed by extravasation of
Evans blue dye. However, at 3 days the AQP4 null mice had
Brain abscess can occur as the result of local extension of an
extracranial infection, hematogenous spread of infected emboli,
penetrating head trauma or as a complication of neurosurgery
(Mathisen and Johnson 1997). Streptococcal species and
Staphylococcus aureus are the most common organisms that
cause bacterial brain abscesses (Mathisen and Johnson 1997;
Townsend and Scheld 1998). In addition to the direct tissue
destruction caused by the infectious organism and the resulting
immune response, substantial brain edema develops around the
abscess. The mass effect produced by the abscess and peri-
abscess brain swelling leads to increased intracranial pressure
(ICP), which may result in ischemia, herniation and death. Even
with modern imaging techniques, surgical interventions and
antibiotic therapy, the mortality rate from brain abscess remains
at 10% (Seydoux and Francioli 1992).
The edema associated with brain abscess is thought to be
primarily extracellular, resulting from increased blood–brain
barrier (BBB) permeability in response to the elaboration of
cytokines from the abscess (Lo et al. 1994). Extravasation of
solutes and water across a leaky BBB into the brain
extracellular space (ECS) causes a ‘vasogenic’ (leaky-vessel)
edema. The traditional view is that excess water and solutes
254
2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
doi:10.1111/j.1471-4159.2005.03362.
significantly higher intracranial pressure (mean ± SEM 27 ± 2
vs. 17 ± 2 mmHg; p < 0.001) and brain water content
(81.0 ± 0.3 vs. 79.3 ± 0.5 % water by weight in the
abscess-containing hemisphere; p < 0.01) than wild-type
mice. Reactive astrogliosis was found throughout the
abscess-containing hemisphere; however, only a subset of
astrocytes in the peri-abscess region of wild-type mice had
increased AQP4 immunoreactivity. Our findings demonstrate
a protective effect of AQP4 on brain swelling in bacterial
abscess, suggesting that AQP4 induction may reduce vaso-
genic edema associated with cerebral infection.
Keywords: aquaporin-4, cerebral edema, intracranial pres-
sure, vasogenic edema, water channel.
J. Neurochem. (2005) 95, 254–262.
are cleared from the ECS by bulk flow of fluid into the
cerebral ventricles and subarachnoid space (Reulen et al.
1978; Marmarou et al. 1994). The rates of fluid influx into
the brain across the leaky BBB and clearance into the CSF
space determine the magnitude of ECS water accumulation
and thus the severity of the swelling.
There is recent evidence that the astroglial water channel
aquaporin-4 (AQP4) plays an important role in brain water
balance (Papadopoulos et al. 2002; Manley et al. 2004).
AQP4 is highly expressed in perivascular astrocyte foot
processes at the BBB and at the ependymal and glial limiting
Received April 20, 2005; revised manuscript received June 2, 2005;
accepted June 3, 2005.
Address correspondence and reprint requests to Alan S. Verkman,
MD, PhD, 1246 Health Sciences East Tower, Cardiovascular Research
Institute, University of California, San Francisco, CA 94143–0521,
USA. E-mail: verkman@itsa.ucsf.edu; http://www.ucsf.edu/verklab
Abbreviations used: AQP4, aquaporin-4; BBB, blood–brain barrier;
BHI, brain heart infusion; CFU, colony-forming units; DPBS, Dul-
becco’s phosphate-buffered saline; ECS, extracellular space; GFAP, glial
fibrillary acidic protein; ICP, intracranial pressure; IL, interleukin; TNF,
tumor necrosis factor.

borders that form the brain–CSF barrier, suggesting a role in
water movement between brain fluid compartments (Badaut
et al. 2002). Deletion of AQP4 in mice markedly reduces
brain swelling in mouse models of cytotoxic (cell swelling)
edema, including water intoxication and focal cerebral
ischemia (Manley et al. 2000). However, in mouse models
of vasogenic edema produced by cortical freeze injury or
brain tumor, AQP4 deletion worsens the severity of the edema
(Papadopoulos et al. 2004), suggesting that under some
conditions AQP4 may facilitate the clearance of excess brain
water. Here, we focus on the role of AQP4 in brain abscess-
associated edema, testing the hypothesis that AQP4 is
protective in reducing brain swelling. Although AQP4
deletion in mice has been shown to be protective in meningitis
(Papadopoulos and Verkman 2005), resulting in less increase
in ICP and improved survival, the edema associated with
meningitis was determined to be primarily cytotoxic, and so
different from that accompanying parenchymal infection.
Understanding the molecular mechanisms of edema forma-
tion and clearance is important in developing therapies to
improve clinical outcome in brain abscess, and may be of
even greater value in the treatment of diffuse cerebritis and
consequent brain swelling resulting from bacterial, parasitic
or viral infections for which there is no effective therapy.
To investigate the role of AQP4 in brain abscess-associated
edema, we created a mouse model of brain abscess involving
parenchymal injection of live S. aureus encased in agarose
beads, modified from a procedure described previously in rats
(Flaris and Hickey 1992). Wild-type and AQP4 null mice had
similar severity of cerebral infection as measured by abscess
size, bacterial count, cytokine levels and BBB permeability.
However, the AQP4 null mice developed significantly greater
ICP and brain water content than the wild-type mice. AQP4
protein expression was found to be greatly increased in peri-
abscess brain of wild-type mice, which may represent a
protective response to limit brain swelling. The involvement
of AQP4 in the clearance of excess brain water in focal
bacterial infection suggests a potential therapeutic option to
improve clinical outcome in cerebral infections.
Materials and methods
Mice
AQP4 null mice were generated by targeted gene disruption (Ma
et al. 1997). All experiments were performed on male wild-type and
AQP4 null mice matched for age and weight (age 6–8 weeks; 25–
30 g) in a CD1 genetic background. Investigators were blinded to
genotype information in all experiments. Protocols were approved
by the University of California San Francisco Committee on Animal
Research.
S. aureus agarose beads
Live Staphylococcus aureus was purchased from the American Type
Culture Collection (#25923; ATCC, Manassus, VA, USA) and

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Aquaporin-4 and brain abscess 255
grown on Brain Heart Infusion (BHI) agar at 37C. Ten hours before
bead preparation, several colonies were suspended in trypticase soy
broth (Fisher Scientific, Los Angeles, CA, USA) and incubated at
37C to mid-log phase as assessed by optical absorbance at 600 nm
(compared with a predetermined standard curve). A volume
containing 108 colony-forming units (CFU) was centrifuged at
3000 g for 15 min and the pellet was resuspended in 1 mL sterile
Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St
Louis, MO, USA). The bacteria were added to a solution of 1.4%
low-melting-point agarose (BMA, Rockland, ME, USA) at 40C,
which was then added to rapidly swirling heavy mineral oil (Sigma-
Aldrich) at 37C through a 27-G needle to form the agarose beads.
The mixture was cooled quickly to 0C on crushed ice with
continued stirring for 2 min. Beads were pelleted at 800 g and
washed four times with DPBS to remove the mineral oil. The final
pellet was resuspended in DPBS and allowed to settle for 5 min to
separate beads by size. Beads with diameters of 50–150 lm as
determined by phase-contrast microscopy were used for injection.
Sterile beads for control injections were prepared by the same
technique but without addition of bacteria. Bacterial viability (and
sterility of control beads) was determined by culture for 24 h on
BHI agar. Serial dilutions and quantitative culture of bacterial beads
were also done to verify the injected bacterial load.
Experimental brain abscess
Mice were anesthetized with 2.5% avertin (2,2,2-tribromoethanol,
125 mg/kg i.p.; Sigma-Aldrich). The head was secured in a
stereotactic frame (Benchmark; Neurolab, St Louis, MO, USA) and
a 1-cm midline incision was made in the scalp from eye to ear. A 1-mm
diameter burr hole was made 1 mm anterior and 2 mm lateral to the
bregma using a micromotor drill (Foredom, Bethel, CT, USA). A 30-G
needle attached to a gas-tight glass syringe (Hamilton, Reno, NV,
USA) was introduced stereotactically through the burr hole to a depth
of 3 mm below the surface of the calvarium (Fig. 1a, top left). Five
microliters of the bead suspension (105 CFU) was infused into the
striatum over 5 min, and 2 min later the needle was slowly removed to
prevent reflux. The burr hole was sealed with sterile bone wax and the
skin was closed with sutures. Control wild-type and AQP4 null mice
received injections of sterile agarose beads. More than 90% of
mice survived the injection procedure. After recovery from anesthesia,
mice were kept at room temperature (22C) and provided with
standard chow and water ad libitum. Mice were examined daily for
clinical signs of illness. Weight and rectal temperature were recorded
daily and global neurological function was assessed.
Neurological score
Mice were scored for global neurological function using a modified
neurological scale as follows: 5, normal; 4, decreased scavenging
activity, decreased scatter reflex; 3, no spontaneous scavenging, loss
of scatter reflex, ataxia; 2, non-purposeful movements, seizure;
1, loss of righting reflex; 0, dead. Mice were assessed before
bacterial (or control) injection and every 24 h thereafter.
Abscess bacterial load
Mice were killed at 3 days after injection and brains were removed
immediately. The injected hemisphere was homogenized in 500 lL
DPBS using a rotor/stator homogenizer (Tissue-Tearor; Biospec
Products, Bartlesville, OK, USA). Serial 10-fold dilutions of the
256 O. Bloch et al.
(a)
(b)
(c)
brain homogenate were plated on BHI agar and incubated at 37C
for 24 h. Titers were calculated from bacterial colony counts and are
expressed as mean log10 CFU per hemisphere.
Cytokine production
Freshly excised mouse brains were divided by hemisphere, and the
injected hemisphere was homogenized in 500 lL DPBS containing
2 lg/mL aprotinin, 2 lg/mL pepstatin A, 2 lg/mL leupeptin and
100 lM Pefabloc. Homogenates were centrifuged at 21 000 g for
15 min at 4C and the supernatants were used for cytokine analysis.
Murine interleukin (IL)-1b and tumor necrosis factor (TNF)-a
concentrations were measured using ELISA kits (R & D Systems,
Minneapolis, MN, USA) according to the manufacturer’s instruc-
tions. Reported values were expressed as picograms of cytokine per
milligram total protein content determined by a DC protein assay
(Bio-Rad, Hercules, CA, USA).
Processing of tissues for histology/immunohistochemistry
Mice were perfused by cardiac puncture with 10 mL 10% formalin,
after which brains were removed and immersed in 10% formalin for
24 h. The fixed tissue was processed through graded concentrations
of ethanol, followed by Citrisolv (Fisher Scientific, Los Angeles,
CA, USA), and embedded in paraffin. Coronal sections 10 lm thick
were cut through the entire abscess for abscess volume determin-
ation and immunostaining.
Brain abscess volume
Paraffin tissue sections (10 lm thick) were sampled every 100 lm
through the entire abscess volume and stained with hematoxylin and

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Fig. 1 Mouse model of S. aureus brain
abscess. (a) Diagram showing bead injec-
tion into the striatum (top left). Hematoxylin
and eosin-stained brain section at 3 days
after injection from a wild-type mouse at low
magnification (top right) shows a well cir-
cumscribed abscess (black arrowheads); at
high magnification (bottom left) agarose
beads (white arrowheads) containing col-
onies of S. aureus (black arrowheads) sur-
rounded by a leukocyte infiltrate are seen.
High-magnification image at the abscess
edge (bottom right) shows a well defined
abscess margin surrounded by a thin cap-
sule and normal brain (black arrowheads).
(b) Brain abscess volume in S. aureus-
injected mice at 3 days (five AQP4+/+ vs.
five AQP4–/–; p not significant). (c) Bac-
terial titers from brain homogenates of
S. aureus-injected hemispheres at 3 days
after injection (five AQP4+/+ vs. five
AQP4–/–; p not significant). Values are
mean ± SEM.
eosin (Fisher Scientific). Stained sections were imaged in the bright-
field mode at 2.5· magnification (DM4000B microscope; Leica,
Bannockburn, IL, USA) equipped with a color charge coupled
device camera (Spot RT-KE; Diagnostic Instruments, Sterling
Heights, MI, USA). The abscess area in each section was measured
using image analysis software (ImageJ; NIH, Bethesda, MD, USA)
for computation of total abscess volume.
Immunodetection of AQP4
For immunohistochemistry, paraffin sections 10 lm thick were
deparaffinized in Citrisolv (Fisher Scientific) and rehydrated in
graded ethanols. After blocking with goat serum, sections were
incubated with a rabbit AQP4 polyclonal antibody (1 : 200; Chemi-
con, Temecula, CA, USA) or rabbit GFAP polyclonal antibody
(1 : 500; Chemicon) and washed in phosphate-buffered saline. Bound
antibody was detected using the Vectastatin avidin-biotin complex kit
(Vector Laboratories, Burlingame, CA, USA). Slides were developed
using the substrate 3,3-diaminobenzidene. Some sections were
incubated with rabbit anti-AQP4 followed by a Cy3-conjugated
sheep anti-rabbit IgG secondary antibody (1 : 200; Sigma-Aldrich)
for immunofluorescence. For immunoblot analysis, brains of wild-
type mice were excised and homogenized in 250 mM sucrose, 10 mM
Tris-HCl, 2 lg/mL aprotinin, 2 lg/mL pepstatin A, 2 lg/mL
leupeptin and 100 lM Pefabloc, pH 7.4. The homogenate was
centrifuged at 2700 g for 10 min at 4C to remove debris and the
supernatant containing crude membranes was loaded on to a 4–12%
sodium dodecyl sulfate–polyacrylamide gel (10 lg/lane). Protein
was then transferred to a polyvinylidene difluoride membrane and
incubated with rabbit anti-AQP4 (1 : 1000) followed by horseradish

peroxidase-linked anti-rabbit IgG (1 : 10 000; Amersham Biosciences,
Piscataway, NJ, USA), and visualized using enhanced chemilumi-
nescence (Boehringer Mannheim, Indianapolis, IN, USA).
Measurement of ICP
Anesthetized mice were immobilized in a stereotactic frame and a
midline incision was made over the vertex of the skull. A 1-mm burr
hole was drilled 3 mm posterior and 3 mm lateral to the bregma. A
parenchymal pressure catheter (SPR320; Millar Instruments, Hous-
ton, TX, USA) with a tip diameter of 660 lm connected to a
pressure control unit (TC-510; Millar Instruments) was inserted to a
depth of 2 mm. ICP was recorded at 50 Hz using a TC-100
recording system (Biopac, Santa Barbara, CA, USA). Rectal
temperature was maintained at 37–38C using a heating lamp
during ICP measurements. ICP values were averaged over 2–5 min.
Brain water content
Mice were anesthetized and killed by cervical dislocation. Brains
were removed immediately, and divided into right and left
hemispheres and cerebellum. Each segment was weighed immedi-
ately and then dried in a vacuum oven at 110C for 12 h. Dried
brains were reweighed and brain water content was calculated as
[(wet weight–dry weight)/wet weight] · 100.
BBB permeability
Three days after bacterial injection, 0.1 mL 4% Evans blue dye in
phosphate-buffered saline (Sigma-Aldrich) was injected into a jugular
vein. Thirty minutes after dye injection, mice were perfused
intracardially with 20 mL phosphate-buffered saline and brains were
removed. The brain was divided into right and left hemispheres and
cerebellum. Each brain segment was weighed and immersed in 2 mL
formamide at 55C for 36 h. Extracted dye was measured by optical
absorbance at 620 nm and compared with Evans blue/formamide
standards. Results are reported as nanograms Evans blue per
milligram brain tissue.
Statistical analysis
Data are expressed as mean ± SEM. The significance of differences
between experimental groups was determined by two-tailed Stu-
dent’s t-test assuming a 95% confidence interval (Excel 2000;
Microsoft, Redmond, WA, USA).
Results
Brain abscess model
Injection of S. aureus produced a focal brain abscess with a
clinical course similar to that in previously reported models
(Baldwin and Kielian 2004). Previous studies in mice
indicated maximal peri-abscess edema at 3 days after
injection, which was confirmed in wild-type and AQP4 null
mice (data not shown). Histological examination of brains at
3 days after injection demonstrated a well circumscribed
abscess containing a neutrophil and macrophage infiltrate,
with significant mass effect and midline shift (Fig. 1a, top
right). There was no gross histological difference in abscess
morphology in brains of wild-type and AQP4 null mice.

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Aquaporin-4 and brain abscess 257
Encapsulation of bacteria in agarose beads (Fig. 1a, bottom
left) served to confine the infection within the abscess
volume, preventing a diffuse cerebritis or meningitis.
CSF samples obtained from mice in both experimental
groups at the time of death demonstrated minimal growth
(< 100 S. aureus CFU/mL at 72 h). In addition, there was no
histological evidence of inflammation or leukocyte infiltrates
extending outside of the well demarcated abscess volume
(Fig. 1a, bottom right).
To compare abscess growth in wild-type and AQP4 null
mice, total abscess volume at 3 days after injection was
quantified by summing abscess areas in serial sections through
the entire abscess volume. Abscess volume was similar in
wild-type and AQP4 null mice (Fig. 1b). Bacterial growth was
assessed by quantitative culture of S. aureus in brain homo-
genates. Three days after injection of 105 CFU S. aureus
encased in beads, 107 CFU were recovered from the injected
hemispheres of both wild-type and AQP4 null mice (Fig. 1c).
Homogenates from the contralateral hemispheres of mice in
both groups showed no growth at 48 h of culture (data not
shown).
The inflammatory response to intraparenchymal bacteria
was quantified by assay of the pro-inflammatory cytokines
IL-1b and TNF-a, known to be the primary cytokines
initiating the host immune response to brain abscess (Kielian
et al. 2004). Cytokine levels were comparable in brains of
wild-type and AQP4 null mice at 3 days (Fig. 2). Cytokines
were undetectable in control brains.
The S. aureus-injected mice showed mild clinical signs of
general illness, including a 15% reduction in bodyweight,
reduced body temperature and lower neurological score
compared with sterile bead-injected control mice (Fig. 2b).
However, there were no significant differences in these clinical
indices between the wild-type and AQP4 null groups, with the
exception of a 3% difference in bodyweight loss measured on
day 3 after injection. Arterial blood gas values and peripheral
white blood cell counts at 3 days after injection were within
normal limits (Table 1), excluding systemic sepsis as an
explanation for the observed signs of clinical illness.
Increased abscess-associated edema in AQP4 null mice
ICP was measured in the control (injected with sterile beads)
and brain abscess groups using a micropressure transducer
introduced into the brain parenchyma. Representative record-
ings in Fig. 3(a) (inset) show stably increased ICP in mice
from both experimental groups. Mean ICP at 3 days in the
S. aureus-injected AQP4 null mice was significantly greater
than that in injected wild-type mice (27 ± 2 vs.
17 ± 2 mmHg; p < 0.001) (Fig. 3a). This represented an
60% greater increase in ICP from baseline in the AQP4 null
group compared with the wild-type group. ICP values for
control mice were within normal limits and not statistically
different between groups.
258 O. Bloch et al.
Table 1 Physiological measurements in wild-type and AQP4 null mice
measured at 3 days after S. aureus injection
Wild type n AQP4 null
Arterial blood gases
pO2 (mmHg) 95 ± 5 5 86 ± 7
pCO2 (mmHg) 47 ± 1 5 46 ± 1
pH 7.37 ± 0.02 5 7.36 ± 0.02
Blood count
Peripheral WBC 8.0 ± 0.7 5 5.0 ± 0.7
(· 103 cells/mm3)
Values are mean ± SEM. pO2, partial pressure of O2; pCO2, partial
pressure of CO2; WBC, white blood cell count.
Because the difference in ICP between wild-type and
AQP4 null mice could not be explained by a difference in
abscess volume or inflammatory response, we investigated
the magnitude of peri-abscess edema. Brain water content, as
measured by wet-to-dry weight ratio, is summarized in
Fig. 3(b) (left). Brain water content was significantly raised
at 3 days after injection in both the S. aureus-injected
(ipsilateral) and contralateral hemispheres of AQP4 null mice
compared with wild-type mice, with most of the increased
water in the ipsilateral hemisphere. There was a small
increase in brain water in the ipsilateral hemisphere
compared with the contralateral hemisphere in sterile bead-
injected mice, suggesting that the injection procedure caused
some edema. The increase in total brain water volume was
calculated from the difference in water content between
S. aureus-injected and control mice. The absolute water
content of each tissue sample was computed as percentage
water content · wet brain weight. This calculation expresses
the increase in brain water in microliters and takes into
account small differences in baseline brain water content
between wild-type and AQP4 null mice. As seen in Fig. 3(b)

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Fig. 2 Inflammation and clinical outcome in
mouse model of brain abscess. (a) Con-
centrations of inflammatory cytokines, IL-1b
(top) and TNF-a (bottom), from homogen-
ates of S. aureus-injected brains (four
AQP4+/+ vs. four AQP4–/–; p not signifi-
cant) and sterile bead-injected controls
(three AQP4+/+ vs. three AQP4–/–) at
3 days. (b) Bodyweight (top), temperature
(middle) and neurological score (bottom) of
mice with brain abscesses (33 AQP4+/+ vs.
34 AQP4–/– from six independent experi-
ments; *p < 0.05 vs. AQP4–/–) and sterile
bead-injected controls (12 AQP4+/+ vs. 12
AQP4–/– from three independent experi-
ments). Values are mean ± SEM.
(right), there was a 120% greater increase in brain water in
the AQP4 null group, corresponding to 14 lL excess fluid.
The Evans blue extravasation assay was done to investi-
n gate whether differences in BBB permeability could account
for the greater ICP and brain water accumulation in the
5 AQP4 null abscess group. At 30 min after intravenous
5 injection of Evans blue dye on day 3 of abscess development,
5 there was significant dye extravasation in the peri-abscess
region (Fig. 3c), confirming a vasogenic mechanism for the
5 abscess-associated edema. There was an approximately
three-fold increase in Evans blue accumulation in the
injected hemisphere compared with sterile bead-injected
mice (Fig. 3d). However, the Evans blue accumulation was
similar in wild-type and AQP4 null groups, suggesting that
the differences in ICP and peri-abscess edema are related to
AQP4-dependent mechanisms of brain water clearance.
Increased AQP4 expression in brain abscess
Based on evidence that various types of insult to the brain
alter AQP4 expression (Papadopoulos et al. 2001; Saadoun
et al. 2002, 2003; Badaut et al. 2003), often but not always
in a protective manner, we investigated AQP4 protein
expression in abscess-containing brains of wild-type mice.
AQP4 immunohistochemistry in brain sections at 3 days
showed remarkable up-regulation of AQP4 immunoreactivity
surrounding the abscess capsule compared with levels in
regions far from the abscess in the ipsilateral and contra-
lateral hemispheres (Figs 4a and b). The pattern of expres-
sion away from the abscess capsule was similar to AQP4
expression in sterile bead-injected controls (Fig. 4c) and
normal mice (data not shown). The immunoblot shown in
Fig. 4h (inset) confirms the antibody specificity; there was a
single band in samples from wild-type mice that was absent
in AQP4 null mice. Demonstration of AQP4 up-regulation in
protein samples from a whole hemisphere was not possible
because AQP4 up-regulation was restricted to a small area

(a) (b)
(c) (d)
Fig. 3 Increased ICP and brain water in AQP4 null mice in brain
abscess model. (a) ICP values for individual mice (and mean ± SEM)
for sterile bead-injected control mice (six per group; p not significant)
and S. aureus-injected mice (13 AQP4+/+ vs. 12 AQP4–/– from three
independent experiments; **p < 0.001 vs. AQP4+/+) measured at
3 days. Representative ICP tracings (inset) show stably elevated ICP
in AQP4+/+ and AQP4–/– mice over several minutes. (b) Left:
mean ± SEM brain water content at 3 days measured by wet-to-dry
weight ratios in S. aureus-injected mice (six per group, two inde-
pendent experiments) and sterile bead-injected controls (three mice
per group), divided into ipsilateral (injected) and contralateral hemi-
(a) (b)
(e) (f)
(i) (j)
Fig. 4 Reactive astrogliosis and AQP4 expression in brain abscess
model. Histological sections from the S. aureus-injected (a, e, i) and
contralateral (b, f, j) hemispheres of wild-type mice, the sterile bead-
injected hemisphere (c, g, k) of control wild-type mice, and the
S. aureus-injected (d, h, l) hemisphere of AQP4 null mice killed 3 days
after injection. Dotted lines indicate abscess margin. Asterisks indicate
the needle tract in control mice. Sections were stained for AQP4 by

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Aquaporin-4 and brain abscess 259
spheres (*p < 0.01 vs. AQP4+/+). Right: increase in brain water con-
tent, calculated as mean water content difference, for abscess minus
control mice for each genotype. Gray bars indicate contralateral
hemisphere and white bars indicate ipsilateral hemisphere (*p < 0.01
vs. AQP4+/+). (c) Axial view (left) and coronal section (right) of
S. aureus-injected brain of wild-type mouse 30 min after injection of
Evans blue dye. Black arrowheads indicate extravasated dye and
white arrowhead indicates abscess mass. (d) Mean ± SEM Evans
blue extravasation in S. aureus-injected mice (five mice per group) and
sterile bead-injected controls (three mice per group) at 3 days after
injection (p not significant).
(c) (d)
(g) (h)
(k) (l)
immunoperoxidase (a, b, c, d) or immunofluorescence (e, f, g, h), or for
GFAP (i, j, k, l) to demonstrate reactive astrogliosis. The immunoblot
(h, inset) demonstrates the specificity of the AQP4 antibody. The inset
in (i) shows a reactive astrocyte in an S. aureus-injected wild-type
mouse at high magnification stained for GFAP (green, left) and AQP4
(red, right). White arrowhead indicates the astrocyte foot process that
co-localizes with AQP4 expression.
260 O. Bloch et al.
surrounding the abscess. In contrast to the pattern of
increased AQP4 expression, glial fibrillary acidic protein
(GFAP) immunostaining demonstrated glial activation in a
much broader distribution (Fig. 4i). Reactive astrocytes were
observed throughout the entire injected hemisphere of both
wild-type and AQP4 null mice, extending from the abscess
capsule to the brain surface, but rarely into the contralateral
hemisphere. At higher magnification, AQP4 expression was
seen localized to foot processes of reactive astrocytes in the
peri-abscess region (Fig. 4i, inset). Control mice showed
reactive astrocytes surrounding the needle tract (Fig. 4k).
Discussion
Edema is an important factor contributing to morbidity and
mortality in cerebral infections. Although several studies
have investigated the role of immune activation and host
response in bacterial brain abscess (Kielian and Hickey 2000;
Kielian et al. 2001, 2004; Baldwin and Kielian 2004), this
study is the first to report the magnitude of peri-abscess
edema and increase in ICP in an experimental mouse model.
The time course and disease severity in our model were
similar to those described in previous reports of bacterial
brain abscess in mice (Baldwin and Kielian 2004). We found
similar brain abscess generation and progression in wild-type
and AQP4 null mice. The inflammatory response to intrapa-
renchymal bacteria, as measured by levels of the cytokines
IL-1b and TNF-a, was comparable in wild-type and AQP4
null mice, resulting in morphologically similar abscesses of
comparable size. The clinical severity of disease was also
comparable, with minor decreases in bodyweight and
temperature compared with control mice that were probably
attributable to increased levels of brain cytokines. However,
despite the similarity in abscess characteristics and inflam-
matory response, the AQP4 null mice developed a signifi-
cant, 60% greater increase in ICP compared with wild-type
mice. Furthermore, there was a more than two-fold greater
accumulation of excess brain water in the AQP4 null mice
compared with wild-type mice, the majority of the increase
occurring in the abscess-containing hemisphere. A lesser but
significant increase in brain water in the contralateral
hemisphere was probably related to fluid tracking along
comissural white matter tracts.
Cerebral edema is generally divided into cytotoxic and
vasogenic types, according to the mechanism of edema
formation. Cytotoxic edema results from disruption of
normal osmotic gradients across the plasma membrane,
causing an osmotically induced flux of water into cells and a
primarily intracellular edema (Klatzo 1994; Kimelberg
1995). AQP4 water channels in the foot processes of
perivascular astrocytes facilitate water movement from the
vascular space across the BBB into cells. AQP4 deletion
significantly reduced the accumulation of intracellular fluid
in mouse models of cytotoxic edema (Manley et al. 2000). In

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
contrast, vasogenic edema develops by an aquaporin-inde-
pendent mechanism involving increased BBB permeability
(Klatzo 1994; Kimelberg 1995), resulting in extracellular
accumulation of edema fluid. Increased BBB permeability
can occur as a result of physical disruption or biochemical
signals such as pro-inflammatory cytokines (Chan et al.
1983; Ohnishi et al. 1992). We reported recently that AQP4
null mice have increased brain water accumulation and ICP
compared with wild-type animals in several models of acute
vasogenic edema (Papadopoulos et al. 2004), as well as
increased ECS volume as quantified by a cortical surface
photobleaching method (Papadopoulos et al. 2005). These
findings suggest that AQP4 facilitates the clearance of
extracellular fluid in the brain, although the mechanism is not
fully understood.
Several lines of evidence show that the edema associated
with intracerebral infection is primarily vasogenic, resulting
from cytokine production in response to immune activation.
Magnetic resonance imaging of human brain abscesses
demonstrates enhancement of the wall (i.e. opening of the
BBB) and a vasogenic pattern of edema in peri-abscess brain,
similar to that seen in brain tumors (Falcone and Post 2000).
Diffusion-weighted magnetic resonance imaging demon-
strates restricted diffusion within the abscess cavity and
increased diffusion around the abscess, typical of the ECS
expansion seen with vasogenic edema (Leuthardt et al. 2002;
Sener 2004). In agreement with previous studies of brain
abscess in rodents (Baldwin and Kielian 2004), we also
found a significant increase in BBB permeability using the
Evans blue assay, with a three-fold increase in dye extrava-
sation in the abscess-containing hemisphere compared with
that in the contralateral hemisphere and sterile bead-injected
mice. The magnitude of the dye extravasation was compar-
able to that reported for cortical freeze injury (Papadopoulos
et al. 2004), an established model of acute vasogenic edema.
However, BBB permeability was similar in wild-type and
AQP4 null mice, suggesting that the greater ICP and brain
water in AQP4 deficiency result from impaired clearance of
excess brain water through an AQP4-dependent pathway.
Prolonged differences in brain edema and ICP of wild-type
and AQP4 null mice may also produce secondary effects on
immune function and so differences in abscess resolution at
late stages.
Although AQP4 null mice have reduced ability to clear
ECS fluid in their normal state, as suggested by a slightly
increased brain water content at baseline (Fig. 3b), the
difference in the clearance of excess brain water between
wild-type and AQP4 null mice is probably exaggerated by
the up-regulation of AQP4 in wild-type mice. AQP4 protein
expression in the abscess-containing hemisphere of wild-type
mice was greatly up-regulated in the capsule surrounding the
abscess, probably facilitating fluid reabsorption from the
abscess core and peri-abscess parenchyma. The up-regulation
of AQP4 expression is likely to be an adaptive response to

protect the brain from excess extracellular fluid accumulation
secondary to BBB disruption.
Increased AQP4 expression has been reported secondary
to many neurological pathologies, such as ischemia, hemor-
rhage, tumor and trauma (Papadopoulos et al. 2001; Saadoun
et al. 2002, 2003; Badaut et al. 2003). In previous reports,
AQP4 up-regulation was found primarily in reactive glia
throughout edematous tissue, suggesting that AQP4 expres-
sion was secondarily up-regulated by activation of astrocytes.
In cerebral infection, glial cell activation is an integral part of
the immune response. Direct binding of bacterial antigens to
toll-like receptors on astrocytes and microglia stimulates the
release of pro-inflammatory cytokines that promote BBB
opening and leukocyte chemotaxis into the infected brain
(Kielian and Hickey 2000; Dong and Benveniste 2001; Esen
et al. 2004; Kielian et al. 2005). Astrocytes are also activated
by cytokines released from the adaptive immune response,
inducing an antigen presentation function (Dong and Ben-
veniste 2001; Carpentier et al. 2005). Therefore, we antici-
pated substantial glial cell activation from bacterial antigens
and inflammatory cytokines in the abscess-containing hemi-
sphere of S. aureus-injected mice, with a concurrent increase
in AQP4 expression. GFAP staining confirmed diffuse
reactive gliosis throughout the injected hemisphere of wild-
type and AQP4 null mice. However, increased AQP4
expression was limited to a small area surrounding the
abscess capsule, suggesting that astrocyte activation alone is
not sufficient to induce AQP4 up-regulation. Esen et al.
(2004) demonstrated in primary astrocyte cultures that the
addition of bacterial antigens, including heat-inactivated
S. aureus, S. aureus-derived peptidoglycan and E. coli-
derived lipopolysaccharide, was sufficient to activate astro-
cytes and induce inflammatory cytokine production through
binding to toll-like receptor-2. In a similar experiment,
following the addition of bacterial antigens to primary
astrocytes in culture, we were unable to find a significant
increase in AQP4 expression by immunoblot analysis (data
not shown), further supporting our observation that astrocyte
activation alone is not adequate to up-regulate AQP4. The
peri-capsular up-regulation of AQP4 expression in this study
was observed in the same region as the increase in BBB
permeability seen by Evans blue dye extravasation (Fig. 3c).
We postulate that reactive astrocytes adjacent to a disrupted
BBB are preferentially induced to up-regulate AQP4 by an as
yet undetermined mechanism. Our in vivo results suggest that
modulation of the AQP4 response may be a novel approach
to enhancing the innate protective up-regulation of AQP4
and accelerating the clearance of excess brain water. This
warrants further investigation.
At present, the principal treatment for vasogenic edema is
corticosteroid therapy, which reduces the production of
inflammatory cytokines and limits the increase in BBB
permeability (Fishman 1982; Kaal and Vecht 2004). Steroids
are commonly used in the treatment of brain tumors

2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Aquaporin-4 and brain abscess 261
complicated by vasogenic edema, but their use in cerebral
infections is complicated by the potential for immunosup-
pression and worsening of the infection. Our data suggest
that pharmacological induction of AQP4 expression may
reduce brain swelling and ICP in cerebral infection. As
AQP4-inducing compounds would act to increase edema
clearance, whereas corticosteroids act by reducing edema
formation, AQP4 up-regulators and steroids may act syner-
gistically to reduce edema in brain abscess. Although focal
brain abscess is treated primarily with potent antibiotics and
surgical drainage, edema-targeted therapies may be of
particular benefit in cerebral infections that do not have
effective primary treatments. Viral encephalitides cause
generalized opening of the BBB resulting in massive global
brain edema (Mathur et al. 1992). Although these viruses
cause some direct leukocyte-mediated neuronal damage,
much of their associated morbidity and mortality is a result of
raised ICP secondary to vasogenic edema. Patients with such
encephalitides or other infectious cerebritides might benefit
from AQP4-modulating drugs to reduce the magnitude of
vasogenic edema until their immune systems clear the
infection.
Acknowledgements
The authors thank Liman Qian for mouse breeding and genotype
analysis. This work was supported by grants DK35124, EY13574,
EB00415, HL59198, HL73856 and DK72517 from the National
Institutes of Health, a Research Development Program grant from
the Cystic Fibrosis Foundation to ASV, a Howard Hughes Medical
Institute Student Fellowship to OB. During his tenure in
Dr. Verkman’s laboratory, MCP was on employee of St. George’s,
University of London and was funded by a Wellcome Trust
Clinician Scientist Fellowship.
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