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Journal of Neurochemistry, 2005, 95, 254–262 doi:10.1111/j.1471-4159.2005.03362.

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 significantly higher intracranial pressure (mean ± SEM 27 ± 2


Brain abscess is associated with local vasogenic edema, vs. 17 ± 2 mmHg; p < 0.001) and brain water content
which leads to increased intracranial pressure and significant (81.0 ± 0.3 vs. 79.3 ± 0.5 % water by weight in the
morbidity. Aquaporin-4 (AQP4) is a water channel expressed abscess-containing hemisphere; p < 0.01) than wild-type
in astroglia at the blood–brain and brain–CSF barriers. To mice. Reactive astrogliosis was found throughout the
investigate the role of AQP4 in brain abscess-associated abscess-containing hemisphere; however, only a subset of
edema, live Staphylococcus aureus (105 colony-forming units) astrocytes in the peri-abscess region of wild-type mice had
was injected into the striatum to create a focal abscess. Wild- increased AQP4 immunoreactivity. Our findings demonstrate
type and AQP4-deficient mice had comparable immune a protective effect of AQP4 on brain swelling in bacterial
responses as measured by brain abscess volume ( 3.7 mm3 abscess, suggesting that AQP4 induction may reduce vaso-
at 3 days), bacterial count and cytokine levels in brain hom- genic edema associated with cerebral infection.
ogenates. Blood–brain barrier permeability was increased Keywords: aquaporin-4, cerebral edema, intracranial pres-
comparably in both groups as assessed by extravasation of sure, vasogenic edema, water channel.
Evans blue dye. However, at 3 days the AQP4 null mice had J. Neurochem. (2005) 95, 254–262.

Brain abscess can occur as the result of local extension of an are cleared from the ECS by bulk flow of fluid into the
extracranial infection, hematogenous spread of infected emboli, cerebral ventricles and subarachnoid space (Reulen et al.
penetrating head trauma or as a complication of neurosurgery 1978; Marmarou et al. 1994). The rates of fluid influx into
(Mathisen and Johnson 1997). Streptococcal species and the brain across the leaky BBB and clearance into the CSF
Staphylococcus aureus are the most common organisms that space determine the magnitude of ECS water accumulation
cause bacterial brain abscesses (Mathisen and Johnson 1997; and thus the severity of the swelling.
Townsend and Scheld 1998). In addition to the direct tissue There is recent evidence that the astroglial water channel
destruction caused by the infectious organism and the resulting aquaporin-4 (AQP4) plays an important role in brain water
immune response, substantial brain edema develops around the balance (Papadopoulos et al. 2002; Manley et al. 2004).
abscess. The mass effect produced by the abscess and peri- AQP4 is highly expressed in perivascular astrocyte foot
abscess brain swelling leads to increased intracranial pressure processes at the BBB and at the ependymal and glial limiting
(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 Received April 20, 2005; revised manuscript received June 2, 2005;
accepted June 3, 2005.
at 10% (Seydoux and Francioli 1992). Address correspondence and reprint requests to Alan S. Verkman,
The edema associated with brain abscess is thought to be MD, PhD, 1246 Health Sciences East Tower, Cardiovascular Research
primarily extracellular, resulting from increased blood–brain Institute, University of California, San Francisco, CA 94143–0521,
barrier (BBB) permeability in response to the elaboration of USA. E-mail: verkman@itsa.ucsf.edu; http://www.ucsf.edu/verklab
cytokines from the abscess (Lo et al. 1994). Extravasation of Abbreviations used: AQP4, aquaporin-4; BBB, blood–brain barrier;
BHI, brain heart infusion; CFU, colony-forming units; DPBS, Dul-
solutes and water across a leaky BBB into the brain becco’s phosphate-buffered saline; ECS, extracellular space; GFAP, glial
extracellular space (ECS) causes a ‘vasogenic’ (leaky-vessel) fibrillary acidic protein; ICP, intracranial pressure; IL, interleukin; TNF,
edema. The traditional view is that excess water and solutes tumor necrosis factor.

254  2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262
Aquaporin-4 and brain abscess 255

borders that form the brain–CSF barrier, suggesting a role in grown on Brain Heart Infusion (BHI) agar at 37C. Ten hours before
water movement between brain fluid compartments (Badaut bead preparation, several colonies were suspended in trypticase soy
et al. 2002). Deletion of AQP4 in mice markedly reduces broth (Fisher Scientific, Los Angeles, CA, USA) and incubated at
brain swelling in mouse models of cytotoxic (cell swelling) 37C to mid-log phase as assessed by optical absorbance at 600 nm
(compared with a predetermined standard curve). A volume
edema, including water intoxication and focal cerebral
containing 108 colony-forming units (CFU) was centrifuged at
ischemia (Manley et al. 2000). However, in mouse models
3000 g for 15 min and the pellet was resuspended in 1 mL sterile
of vasogenic edema produced by cortical freeze injury or Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St
brain tumor, AQP4 deletion worsens the severity of the edema Louis, MO, USA). The bacteria were added to a solution of 1.4%
(Papadopoulos et al. 2004), suggesting that under some low-melting-point agarose (BMA, Rockland, ME, USA) at 40C,
conditions AQP4 may facilitate the clearance of excess brain which was then added to rapidly swirling heavy mineral oil (Sigma-
water. Here, we focus on the role of AQP4 in brain abscess- Aldrich) at 37C through a 27-G needle to form the agarose beads.
associated edema, testing the hypothesis that AQP4 is The mixture was cooled quickly to 0C on crushed ice with
protective in reducing brain swelling. Although AQP4 continued stirring for 2 min. Beads were pelleted at 800 g and
deletion in mice has been shown to be protective in meningitis washed four times with DPBS to remove the mineral oil. The final
(Papadopoulos and Verkman 2005), resulting in less increase 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
in ICP and improved survival, the edema associated with
determined by phase-contrast microscopy were used for injection.
meningitis was determined to be primarily cytotoxic, and so
Sterile beads for control injections were prepared by the same
different from that accompanying parenchymal infection. technique but without addition of bacteria. Bacterial viability (and
Understanding the molecular mechanisms of edema forma- sterility of control beads) was determined by culture for 24 h on
tion and clearance is important in developing therapies to BHI agar. Serial dilutions and quantitative culture of bacterial beads
improve clinical outcome in brain abscess, and may be of were also done to verify the injected bacterial load.
even greater value in the treatment of diffuse cerebritis and
consequent brain swelling resulting from bacterial, parasitic Experimental brain abscess
or viral infections for which there is no effective therapy. Mice were anesthetized with 2.5% avertin (2,2,2-tribromoethanol,
To investigate the role of AQP4 in brain abscess-associated 125 mg/kg i.p.; Sigma-Aldrich). The head was secured in a
edema, we created a mouse model of brain abscess involving 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
parenchymal injection of live S. aureus encased in agarose
diameter burr hole was made 1 mm anterior and 2 mm lateral to the
beads, modified from a procedure described previously in rats
bregma using a micromotor drill (Foredom, Bethel, CT, USA). A 30-G
(Flaris and Hickey 1992). Wild-type and AQP4 null mice had needle attached to a gas-tight glass syringe (Hamilton, Reno, NV,
similar severity of cerebral infection as measured by abscess USA) was introduced stereotactically through the burr hole to a depth
size, bacterial count, cytokine levels and BBB permeability. of 3 mm below the surface of the calvarium (Fig. 1a, top left). Five
However, the AQP4 null mice developed significantly greater microliters of the bead suspension (105 CFU) was infused into the
ICP and brain water content than the wild-type mice. AQP4 striatum over 5 min, and 2 min later the needle was slowly removed to
protein expression was found to be greatly increased in peri- prevent reflux. The burr hole was sealed with sterile bone wax and the
abscess brain of wild-type mice, which may represent a skin was closed with sutures. Control wild-type and AQP4 null mice
protective response to limit brain swelling. The involvement received injections of sterile agarose beads. More than 90% of
of AQP4 in the clearance of excess brain water in focal mice survived the injection procedure. After recovery from anesthesia,
mice were kept at room temperature (22C) and provided with
bacterial infection suggests a potential therapeutic option to
standard chow and water ad libitum. Mice were examined daily for
improve clinical outcome in cerebral infections.
clinical signs of illness. Weight and rectal temperature were recorded
daily and global neurological function was assessed.

Materials and methods Neurological score


Mice were scored for global neurological function using a modified
Mice neurological scale as follows: 5, normal; 4, decreased scavenging
AQP4 null mice were generated by targeted gene disruption (Ma activity, decreased scatter reflex; 3, no spontaneous scavenging, loss
et al. 1997). All experiments were performed on male wild-type and of scatter reflex, ataxia; 2, non-purposeful movements, seizure;
AQP4 null mice matched for age and weight (age 6–8 weeks; 25– 1, loss of righting reflex; 0, dead. Mice were assessed before
30 g) in a CD1 genetic background. Investigators were blinded to bacterial (or control) injection and every 24 h thereafter.
genotype information in all experiments. Protocols were approved
by the University of California San Francisco Committee on Animal Abscess bacterial load
Research. Mice were killed at 3 days after injection and brains were removed
immediately. The injected hemisphere was homogenized in 500 lL
S. aureus agarose beads DPBS using a rotor/stator homogenizer (Tissue-Tearor; Biospec
Live Staphylococcus aureus was purchased from the American Type Products, Bartlesville, OK, USA). Serial 10-fold dilutions of the
Culture Collection (#25923; ATCC, Manassus, VA, USA) and

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256 O. Bloch et al.

(a)

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-
(b) injected mice at 3 days (five AQP4+/+ vs.
(c)
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.

brain homogenate were plated on BHI agar and incubated at 37C eosin (Fisher Scientific). Stained sections were imaged in the bright-
for 24 h. Titers were calculated from bacterial colony counts and are field mode at 2.5· magnification (DM4000B microscope; Leica,
expressed as mean log10 CFU per hemisphere. Bannockburn, IL, USA) equipped with a color charge coupled
device camera (Spot RT-KE; Diagnostic Instruments, Sterling
Cytokine production Heights, MI, USA). The abscess area in each section was measured
Freshly excised mouse brains were divided by hemisphere, and the using image analysis software (ImageJ; NIH, Bethesda, MD, USA)
injected hemisphere was homogenized in 500 lL DPBS containing for computation of total abscess volume.
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 Immunodetection of AQP4
15 min at 4C and the supernatants were used for cytokine analysis. For immunohistochemistry, paraffin sections 10 lm thick were
Murine interleukin (IL)-1b and tumor necrosis factor (TNF)-a deparaffinized in Citrisolv (Fisher Scientific) and rehydrated in
concentrations were measured using ELISA kits (R & D Systems, graded ethanols. After blocking with goat serum, sections were
Minneapolis, MN, USA) according to the manufacturer’s instruc- incubated with a rabbit AQP4 polyclonal antibody (1 : 200; Chemi-
tions. Reported values were expressed as picograms of cytokine per con, Temecula, CA, USA) or rabbit GFAP polyclonal antibody
milligram total protein content determined by a DC protein assay (1 : 500; Chemicon) and washed in phosphate-buffered saline. Bound
(Bio-Rad, Hercules, CA, USA). antibody was detected using the Vectastatin avidin-biotin complex kit
(Vector Laboratories, Burlingame, CA, USA). Slides were developed
Processing of tissues for histology/immunohistochemistry using the substrate 3,3-diaminobenzidene. Some sections were
Mice were perfused by cardiac puncture with 10 mL 10% formalin, incubated with rabbit anti-AQP4 followed by a Cy3-conjugated
after which brains were removed and immersed in 10% formalin for sheep anti-rabbit IgG secondary antibody (1 : 200; Sigma-Aldrich)
24 h. The fixed tissue was processed through graded concentrations for immunofluorescence. For immunoblot analysis, brains of wild-
of ethanol, followed by Citrisolv (Fisher Scientific, Los Angeles, type mice were excised and homogenized in 250 mM sucrose, 10 mM
CA, USA), and embedded in paraffin. Coronal sections 10 lm thick Tris-HCl, 2 lg/mL aprotinin, 2 lg/mL pepstatin A, 2 lg/mL
were cut through the entire abscess for abscess volume determin- leupeptin and 100 lM Pefabloc, pH 7.4. The homogenate was
ation and immunostaining. 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%
Brain abscess volume sodium dodecyl sulfate–polyacrylamide gel (10 lg/lane). Protein
Paraffin tissue sections (10 lm thick) were sampled every 100 lm was then transferred to a polyvinylidene difluoride membrane and
through the entire abscess volume and stained with hematoxylin and incubated with rabbit anti-AQP4 (1 : 1000) followed by horseradish

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Aquaporin-4 and brain abscess 257

peroxidase-linked anti-rabbit IgG (1 : 10 000; Amersham Biosciences, Encapsulation of bacteria in agarose beads (Fig. 1a, bottom
Piscataway, NJ, USA), and visualized using enhanced chemilumi- left) served to confine the infection within the abscess
nescence (Boehringer Mannheim, Indianapolis, IN, USA). volume, preventing a diffuse cerebritis or meningitis.
CSF samples obtained from mice in both experimental
Measurement of ICP
groups at the time of death demonstrated minimal growth
Anesthetized mice were immobilized in a stereotactic frame and a
(< 100 S. aureus CFU/mL at 72 h). In addition, there was no
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 histological evidence of inflammation or leukocyte infiltrates
parenchymal pressure catheter (SPR320; Millar Instruments, Hous- extending outside of the well demarcated abscess volume
ton, TX, USA) with a tip diameter of 660 lm connected to a (Fig. 1a, bottom right).
pressure control unit (TC-510; Millar Instruments) was inserted to a To compare abscess growth in wild-type and AQP4 null
depth of 2 mm. ICP was recorded at 50 Hz using a TC-100 mice, total abscess volume at 3 days after injection was
recording system (Biopac, Santa Barbara, CA, USA). Rectal quantified by summing abscess areas in serial sections through
temperature was maintained at 37–38C using a heating lamp the entire abscess volume. Abscess volume was similar in
during ICP measurements. ICP values were averaged over 2–5 min. wild-type and AQP4 null mice (Fig. 1b). Bacterial growth was
assessed by quantitative culture of S. aureus in brain homo-
Brain water content
genates. Three days after injection of 105 CFU S. aureus
Mice were anesthetized and killed by cervical dislocation. Brains
encased in beads, 107 CFU were recovered from the injected
were removed immediately, and divided into right and left
hemispheres and cerebellum. Each segment was weighed immedi- hemispheres of both wild-type and AQP4 null mice (Fig. 1c).
ately and then dried in a vacuum oven at 110C for 12 h. Dried Homogenates from the contralateral hemispheres of mice in
brains were reweighed and brain water content was calculated as both groups showed no growth at 48 h of culture (data not
[(wet weight–dry weight)/wet weight] · 100. shown).
The inflammatory response to intraparenchymal bacteria
BBB permeability was quantified by assay of the pro-inflammatory cytokines
Three days after bacterial injection, 0.1 mL 4% Evans blue dye in IL-1b and TNF-a, known to be the primary cytokines
phosphate-buffered saline (Sigma-Aldrich) was injected into a jugular initiating the host immune response to brain abscess (Kielian
vein. Thirty minutes after dye injection, mice were perfused et al. 2004). Cytokine levels were comparable in brains of
intracardially with 20 mL phosphate-buffered saline and brains were
wild-type and AQP4 null mice at 3 days (Fig. 2). Cytokines
removed. The brain was divided into right and left hemispheres and
were undetectable in control brains.
cerebellum. Each brain segment was weighed and immersed in 2 mL
formamide at 55C for 36 h. Extracted dye was measured by optical The S. aureus-injected mice showed mild clinical signs of
absorbance at 620 nm and compared with Evans blue/formamide general illness, including a 15% reduction in bodyweight,
standards. Results are reported as nanograms Evans blue per reduced body temperature and lower neurological score
milligram brain tissue. compared with sterile bead-injected control mice (Fig. 2b).
However, there were no significant differences in these clinical
Statistical analysis indices between the wild-type and AQP4 null groups, with the
Data are expressed as mean ± SEM. The significance of differences exception of a 3% difference in bodyweight loss measured on
between experimental groups was determined by two-tailed Stu- day 3 after injection. Arterial blood gas values and peripheral
dent’s t-test assuming a 95% confidence interval (Excel 2000; white blood cell counts at 3 days after injection were within
Microsoft, Redmond, WA, USA).
normal limits (Table 1), excluding systemic sepsis as an
explanation for the observed signs of clinical illness.
Results
Increased abscess-associated edema in AQP4 null mice
Brain abscess model ICP was measured in the control (injected with sterile beads)
Injection of S. aureus produced a focal brain abscess with a and brain abscess groups using a micropressure transducer
clinical course similar to that in previously reported models introduced into the brain parenchyma. Representative record-
(Baldwin and Kielian 2004). Previous studies in mice ings in Fig. 3(a) (inset) show stably increased ICP in mice
indicated maximal peri-abscess edema at 3 days after from both experimental groups. Mean ICP at 3 days in the
injection, which was confirmed in wild-type and AQP4 null S. aureus-injected AQP4 null mice was significantly greater
mice (data not shown). Histological examination of brains at than that in injected wild-type mice (27 ± 2 vs.
3 days after injection demonstrated a well circumscribed 17 ± 2 mmHg; p < 0.001) (Fig. 3a). This represented an
abscess containing a neutrophil and macrophage infiltrate, 60% greater increase in ICP from baseline in the AQP4 null
with significant mass effect and midline shift (Fig. 1a, top group compared with the wild-type group. ICP values for
right). There was no gross histological difference in abscess control mice were within normal limits and not statistically
morphology in brains of wild-type and AQP4 null mice. different between groups.

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258 O. Bloch et al.

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.

Table 1 Physiological measurements in wild-type and AQP4 null mice (right), there was a 120% greater increase in brain water in
measured at 3 days after S. aureus injection the AQP4 null group, corresponding to 14 lL excess fluid.
The Evans blue extravasation assay was done to investi-
Wild type n AQP4 null n gate whether differences in BBB permeability could account
Arterial blood gases for the greater ICP and brain water accumulation in the
pO2 (mmHg) 95 ± 5 5 86 ± 7 5 AQP4 null abscess group. At 30 min after intravenous
pCO2 (mmHg) 47 ± 1 5 46 ± 1 5 injection of Evans blue dye on day 3 of abscess development,
pH 7.37 ± 0.02 5 7.36 ± 0.02 5 there was significant dye extravasation in the peri-abscess
Blood count region (Fig. 3c), confirming a vasogenic mechanism for the
Peripheral WBC 8.0 ± 0.7 5 5.0 ± 0.7 5 abscess-associated edema. There was an approximately
(· 103 cells/mm3) three-fold increase in Evans blue accumulation in the
injected hemisphere compared with sterile bead-injected
Values are mean ± SEM. pO2, partial pressure of O2; pCO2, partial
pressure of CO2; WBC, white blood cell count.
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
Because the difference in ICP between wild-type and AQP4-dependent mechanisms of brain water clearance.
AQP4 null mice could not be explained by a difference in
abscess volume or inflammatory response, we investigated Increased AQP4 expression in brain abscess
the magnitude of peri-abscess edema. Brain water content, as Based on evidence that various types of insult to the brain
measured by wet-to-dry weight ratio, is summarized in alter AQP4 expression (Papadopoulos et al. 2001; Saadoun
Fig. 3(b) (left). Brain water content was significantly raised et al. 2002, 2003; Badaut et al. 2003), often but not always
at 3 days after injection in both the S. aureus-injected in a protective manner, we investigated AQP4 protein
(ipsilateral) and contralateral hemispheres of AQP4 null mice expression in abscess-containing brains of wild-type mice.
compared with wild-type mice, with most of the increased AQP4 immunohistochemistry in brain sections at 3 days
water in the ipsilateral hemisphere. There was a small showed remarkable up-regulation of AQP4 immunoreactivity
increase in brain water in the ipsilateral hemisphere surrounding the abscess capsule compared with levels in
compared with the contralateral hemisphere in sterile bead- regions far from the abscess in the ipsilateral and contra-
injected mice, suggesting that the injection procedure caused lateral hemispheres (Figs 4a and b). The pattern of expres-
some edema. The increase in total brain water volume was sion away from the abscess capsule was similar to AQP4
calculated from the difference in water content between expression in sterile bead-injected controls (Fig. 4c) and
S. aureus-injected and control mice. The absolute water normal mice (data not shown). The immunoblot shown in
content of each tissue sample was computed as percentage Fig. 4h (inset) confirms the antibody specificity; there was a
water content · wet brain weight. This calculation expresses single band in samples from wild-type mice that was absent
the increase in brain water in microliters and takes into in AQP4 null mice. Demonstration of AQP4 up-regulation in
account small differences in baseline brain water content protein samples from a whole hemisphere was not possible
between wild-type and AQP4 null mice. As seen in Fig. 3(b) because AQP4 up-regulation was restricted to a small area

 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 254–262


Aquaporin-4 and brain abscess 259

(a) (b)

(c) (d)

Fig. 3 Increased ICP and brain water in AQP4 null mice in brain spheres (*p < 0.01 vs. AQP4+/+). Right: increase in brain water con-
abscess model. (a) ICP values for individual mice (and mean ± SEM) tent, calculated as mean water content difference, for abscess minus
for sterile bead-injected control mice (six per group; p not significant) control mice for each genotype. Gray bars indicate contralateral
and S. aureus-injected mice (13 AQP4+/+ vs. 12 AQP4–/– from three hemisphere and white bars indicate ipsilateral hemisphere (*p < 0.01
independent experiments; **p < 0.001 vs. AQP4+/+) measured at vs. AQP4+/+). (c) Axial view (left) and coronal section (right) of
3 days. Representative ICP tracings (inset) show stably elevated ICP S. aureus-injected brain of wild-type mouse 30 min after injection of
in AQP4+/+ and AQP4–/– mice over several minutes. (b) Left: Evans blue dye. Black arrowheads indicate extravasated dye and
mean ± SEM brain water content at 3 days measured by wet-to-dry white arrowhead indicates abscess mass. (d) Mean ± SEM Evans
weight ratios in S. aureus-injected mice (six per group, two inde- blue extravasation in S. aureus-injected mice (five mice per group) and
pendent experiments) and sterile bead-injected controls (three mice sterile bead-injected controls (three mice per group) at 3 days after
per group), divided into ipsilateral (injected) and contralateral hemi- injection (p not significant).

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

Fig. 4 Reactive astrogliosis and AQP4 expression in brain abscess immunoperoxidase (a, b, c, d) or immunofluorescence (e, f, g, h), or for
model. Histological sections from the S. aureus-injected (a, e, i) and GFAP (i, j, k, l) to demonstrate reactive astrogliosis. The immunoblot
contralateral (b, f, j) hemispheres of wild-type mice, the sterile bead- (h, inset) demonstrates the specificity of the AQP4 antibody. The inset
injected hemisphere (c, g, k) of control wild-type mice, and the in (i) shows a reactive astrocyte in an S. aureus-injected wild-type
S. aureus-injected (d, h, l) hemisphere of AQP4 null mice killed 3 days mouse at high magnification stained for GFAP (green, left) and AQP4
after injection. Dotted lines indicate abscess margin. Asterisks indicate (red, right). White arrowhead indicates the astrocyte foot process that
the needle tract in control mice. Sections were stained for AQP4 by co-localizes with AQP4 expression.

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260 O. Bloch et al.

surrounding the abscess. In contrast to the pattern of contrast, vasogenic edema develops by an aquaporin-inde-
increased AQP4 expression, glial fibrillary acidic protein pendent mechanism involving increased BBB permeability
(GFAP) immunostaining demonstrated glial activation in a (Klatzo 1994; Kimelberg 1995), resulting in extracellular
much broader distribution (Fig. 4i). Reactive astrocytes were accumulation of edema fluid. Increased BBB permeability
observed throughout the entire injected hemisphere of both can occur as a result of physical disruption or biochemical
wild-type and AQP4 null mice, extending from the abscess signals such as pro-inflammatory cytokines (Chan et al.
capsule to the brain surface, but rarely into the contralateral 1983; Ohnishi et al. 1992). We reported recently that AQP4
hemisphere. At higher magnification, AQP4 expression was null mice have increased brain water accumulation and ICP
seen localized to foot processes of reactive astrocytes in the compared with wild-type animals in several models of acute
peri-abscess region (Fig. 4i, inset). Control mice showed vasogenic edema (Papadopoulos et al. 2004), as well as
reactive astrocytes surrounding the needle tract (Fig. 4k). increased ECS volume as quantified by a cortical surface
photobleaching method (Papadopoulos et al. 2005). These
findings suggest that AQP4 facilitates the clearance of
Discussion
extracellular fluid in the brain, although the mechanism is not
Edema is an important factor contributing to morbidity and fully understood.
mortality in cerebral infections. Although several studies Several lines of evidence show that the edema associated
have investigated the role of immune activation and host with intracerebral infection is primarily vasogenic, resulting
response in bacterial brain abscess (Kielian and Hickey 2000; from cytokine production in response to immune activation.
Kielian et al. 2001, 2004; Baldwin and Kielian 2004), this Magnetic resonance imaging of human brain abscesses
study is the first to report the magnitude of peri-abscess demonstrates enhancement of the wall (i.e. opening of the
edema and increase in ICP in an experimental mouse model. BBB) and a vasogenic pattern of edema in peri-abscess brain,
The time course and disease severity in our model were similar to that seen in brain tumors (Falcone and Post 2000).
similar to those described in previous reports of bacterial Diffusion-weighted magnetic resonance imaging demon-
brain abscess in mice (Baldwin and Kielian 2004). We found strates restricted diffusion within the abscess cavity and
similar brain abscess generation and progression in wild-type increased diffusion around the abscess, typical of the ECS
and AQP4 null mice. The inflammatory response to intrapa- expansion seen with vasogenic edema (Leuthardt et al. 2002;
renchymal bacteria, as measured by levels of the cytokines Sener 2004). In agreement with previous studies of brain
IL-1b and TNF-a, was comparable in wild-type and AQP4 abscess in rodents (Baldwin and Kielian 2004), we also
null mice, resulting in morphologically similar abscesses of found a significant increase in BBB permeability using the
comparable size. The clinical severity of disease was also Evans blue assay, with a three-fold increase in dye extrava-
comparable, with minor decreases in bodyweight and sation in the abscess-containing hemisphere compared with
temperature compared with control mice that were probably that in the contralateral hemisphere and sterile bead-injected
attributable to increased levels of brain cytokines. However, mice. The magnitude of the dye extravasation was compar-
despite the similarity in abscess characteristics and inflam- able to that reported for cortical freeze injury (Papadopoulos
matory response, the AQP4 null mice developed a signifi- et al. 2004), an established model of acute vasogenic edema.
cant, 60% greater increase in ICP compared with wild-type However, BBB permeability was similar in wild-type and
mice. Furthermore, there was a more than two-fold greater AQP4 null mice, suggesting that the greater ICP and brain
accumulation of excess brain water in the AQP4 null mice water in AQP4 deficiency result from impaired clearance of
compared with wild-type mice, the majority of the increase excess brain water through an AQP4-dependent pathway.
occurring in the abscess-containing hemisphere. A lesser but Prolonged differences in brain edema and ICP of wild-type
significant increase in brain water in the contralateral and AQP4 null mice may also produce secondary effects on
hemisphere was probably related to fluid tracking along immune function and so differences in abscess resolution at
comissural white matter tracts. late stages.
Cerebral edema is generally divided into cytotoxic and Although AQP4 null mice have reduced ability to clear
vasogenic types, according to the mechanism of edema ECS fluid in their normal state, as suggested by a slightly
formation. Cytotoxic edema results from disruption of increased brain water content at baseline (Fig. 3b), the
normal osmotic gradients across the plasma membrane, difference in the clearance of excess brain water between
causing an osmotically induced flux of water into cells and a wild-type and AQP4 null mice is probably exaggerated by
primarily intracellular edema (Klatzo 1994; Kimelberg the up-regulation of AQP4 in wild-type mice. AQP4 protein
1995). AQP4 water channels in the foot processes of expression in the abscess-containing hemisphere of wild-type
perivascular astrocytes facilitate water movement from the mice was greatly up-regulated in the capsule surrounding the
vascular space across the BBB into cells. AQP4 deletion abscess, probably facilitating fluid reabsorption from the
significantly reduced the accumulation of intracellular fluid abscess core and peri-abscess parenchyma. The up-regulation
in mouse models of cytotoxic edema (Manley et al. 2000). In of AQP4 expression is likely to be an adaptive response to

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Aquaporin-4 and brain abscess 261

protect the brain from excess extracellular fluid accumulation complicated by vasogenic edema, but their use in cerebral
secondary to BBB disruption. infections is complicated by the potential for immunosup-
Increased AQP4 expression has been reported secondary pression and worsening of the infection. Our data suggest
to many neurological pathologies, such as ischemia, hemor- that pharmacological induction of AQP4 expression may
rhage, tumor and trauma (Papadopoulos et al. 2001; Saadoun reduce brain swelling and ICP in cerebral infection. As
et al. 2002, 2003; Badaut et al. 2003). In previous reports, AQP4-inducing compounds would act to increase edema
AQP4 up-regulation was found primarily in reactive glia clearance, whereas corticosteroids act by reducing edema
throughout edematous tissue, suggesting that AQP4 expres- formation, AQP4 up-regulators and steroids may act syner-
sion was secondarily up-regulated by activation of astrocytes. gistically to reduce edema in brain abscess. Although focal
In cerebral infection, glial cell activation is an integral part of brain abscess is treated primarily with potent antibiotics and
the immune response. Direct binding of bacterial antigens to surgical drainage, edema-targeted therapies may be of
toll-like receptors on astrocytes and microglia stimulates the particular benefit in cerebral infections that do not have
release of pro-inflammatory cytokines that promote BBB effective primary treatments. Viral encephalitides cause
opening and leukocyte chemotaxis into the infected brain generalized opening of the BBB resulting in massive global
(Kielian and Hickey 2000; Dong and Benveniste 2001; Esen brain edema (Mathur et al. 1992). Although these viruses
et al. 2004; Kielian et al. 2005). Astrocytes are also activated cause some direct leukocyte-mediated neuronal damage,
by cytokines released from the adaptive immune response, much of their associated morbidity and mortality is a result of
inducing an antigen presentation function (Dong and Ben- raised ICP secondary to vasogenic edema. Patients with such
veniste 2001; Carpentier et al. 2005). Therefore, we antici- encephalitides or other infectious cerebritides might benefit
pated substantial glial cell activation from bacterial antigens from AQP4-modulating drugs to reduce the magnitude of
and inflammatory cytokines in the abscess-containing hemi- vasogenic edema until their immune systems clear the
sphere of S. aureus-injected mice, with a concurrent increase infection.
in AQP4 expression. GFAP staining confirmed diffuse
reactive gliosis throughout the injected hemisphere of wild-
Acknowledgements
type and AQP4 null mice. However, increased AQP4
expression was limited to a small area surrounding the The authors thank Liman Qian for mouse breeding and genotype
abscess capsule, suggesting that astrocyte activation alone is analysis. This work was supported by grants DK35124, EY13574,
not sufficient to induce AQP4 up-regulation. Esen et al. EB00415, HL59198, HL73856 and DK72517 from the National
(2004) demonstrated in primary astrocyte cultures that the Institutes of Health, a Research Development Program grant from
the Cystic Fibrosis Foundation to ASV, a Howard Hughes Medical
addition of bacterial antigens, including heat-inactivated
Institute Student Fellowship to OB. During his tenure in
S. aureus, S. aureus-derived peptidoglycan and E. coli-
Dr. Verkman’s laboratory, MCP was on employee of St. George’s,
derived lipopolysaccharide, was sufficient to activate astro- University of London and was funded by a Wellcome Trust
cytes and induce inflammatory cytokine production through Clinician Scientist Fellowship.
binding to toll-like receptor-2. In a similar experiment,
following the addition of bacterial antigens to primary
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