Sulforaphane Enhances Aquaporin-4Expression and Decreases Cerebral EdemaFollowing Traumatic Brain Injury

Jing Zhao, Anthony N. Moore, Guy L. Clifton, and Pramod K. Dash*

The Vivian L. Smith Center for Neurologic Research and Department of Neurobiology and Anatomy,The University of Texas Medical School, Houston, Texas

Brain edema, the infiltration and accumulation of ex-cess fluid causing an increase in brain tissue volume,often leads to a rise in intracranial pressure and is akey contributor to the morbidity and mortality associ-ated with traumatic brain injury (TBI). The cellular andmolecular mechanisms contributing to the development/resolution of TBI-associated brain edema are poorly un-derstood. Aquaporin-4 (AQP4) water channel is ex-pressed at high levels in brain astrocytes, and the bidir-ectional transport of water through these channels iscritical for the maintenance of brain water homeostasis.By using a rodent injury model, we show that TBIdecreased AQP4 level in the injury core and modestly in-creased it in the penumbra region surrounding the core.Postinjury administration of sulforaphane (SUL), an iso-thiocyanate present in abundance in cruciferousvegetables such as broccoli, attenuated AQP4 loss inthe injury core and further increased AQP4 levels in thepenumbra region compared with injured animals receiv-ing vehicle. These increases in AQP4 levels were ac-companied by a significant reduction in brain edema(assessed by percentage water content) at 3 days post-injury. These findings suggest that the reduction ofbrain edema in response to SUL administration couldbe due, in part, to water clearance by AQP4 from theinjured brain.VVC 2005 Wiley-Liss, Inc.

Key words: head trauma; intracranial pressure; Nrf2;phase II enzymes; vasogenic edema

Brain edema, the infiltration and accumulation ofexcess fluid in the brain, which leads to an increase inbrain tissue volume, is a key determinant of the morbid-ity and mortality following traumatic brain injury (TBI;Marmarou, 1994; Graham et al., 1995; Papadopouloset al., 2002). In many human TBI cases, edema developson the second or third day postinjury and either pro-gresses to untreatable elevated intracranial pressure (ICP)or resolves by about the tenth day after injury. Twotypes of edema contribute to the overall increase in braintissue volume: 1) vasogenic, in which water enters thebrain as a result of the blood–brain barrier (BBB) com-

promise and accumulates in the extracellular space, and2) cytotoxic, in which water enters cells causing them toswell (Klatzo,1967). However, the cellular and molecularmechanisms contributing to the development/resolutionof TBI-associated brain edema are not well understood.

Aquaporin channels play an important role in watertransport in many cell types. In the functional form,aquaporin channel is a tetramer of identical 28-kDa sub-units. Each subunit forms a water channel with a poreabout 0.38 nm in diameter (which is only slightly largerthan the diameter of a water molecule) that allows bidir-ectional water transport in response to osmotic gradient(Borgnia et al., 1999; Verkman and Mitra, 2000; Agre,2004). In the aquaporin water channel family, aqua-porin-4 (AQP4) is the predominant subtype in the cen-tral nervous system (CNS) and is highly expressed inbrain astrocytes, notably in the end-feet that surroundbrain capillaries (Nielsen et al., 1997; Rash et al., 1998).Recent studies have demonstrated that osmotic waterflow through AQP4 is a mechanism that underlies cyto-toxic brain edema (Manley et al., 2000, 2004). The ex-pression of AQP4 is induced in the periinfarcted tissuethat is associated with the formation of the brain edemafollowing focal cerebral ischemia (Taniguchi et al., 2000).Mice lacking aqp4 show significantly reduced brainedema and lethality in response to acute water intoxica-tion or stroke (Manley et al., 2000). These and otherfindings have led to the suggestion that water entry intocells through AQP4 may be detrimental under theseconditions (water intoxication and cerebral ischemia) inwhich cytotoxic brain edema is predominant. However,it has also been proposed that AQP4 may function to

Grant sponsor: National Institutes of Health, TIRR/Mission Connect;

Grant number: NS35457; Grant number: NS049160.

*Correspondence to: P.K. Dash, Department of Neurobiology and Anat-

omy, The University of Texas Medical School, P.O. Box 20708, Hous-

ton, TX 77225. E-mail: p.dash@uth.tmc.edu

Received 17 June 2005; Revised 2 August 2005; Accepted 10 August

2005

Published online 6 October 2005 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.20649

Journal of Neuroscience Research 82:499–506 (2005)

' 2005 Wiley-Liss, Inc.

clear excess water from the brain, thereby decreasingvasogenic edema and intracranial pressure (Manley et al.,2004). Consistent with this, direct infusion of isotonicfluid into the parenchyma caused a marked increase inbrain water content and ICP in aqp4 –/– mice comparedwith wild-type siblings (Papadopoulos et al., 2004). Inso-far as TBI primarily causes vasogenic edema as a resultof compromised BBB, a decrease or loss of AQP4 levelscould delay the clearance of the excess water out of thebrain. In contrast, strategies to enhance AQP4 expressionfollowing TBI may be beneficial.

Employing a rodent controlled cortical impact(CCI) injury model, we examined the AQP4 level inthe injured brain. Consistent with previous studies (Keet al., 2001; Kiening et al., 2002), we found that TBIdecreased AQP4 level in the contusion core and mod-estly increased it in the penumbra region that surroundsthe core (but see Sun et al., 2003). Postinjury adminis-tration of sulforaphane (SUL), an isothiocyanate foundabundantly in cruciferous vegetables, especially broccoli,attenuated AQP4 loss in the core and further increasedAQP4 protein levels in the penumbra region comparedwith injured animals receiving vehicle. This increase inAQP4 was accompanied by a significant reduction ofbrain edema.

MATERIALS AND METHODS

Materials

SUL was purchased from LKT Laboratories (St. Paul,MN). Antibodies for AQP4 (Abcam, Cambridge, MA), glialfibrillary acidic protein (GFAP; Chemicon, Temecula, CA),and human von Willebrand factor (vWF; Sigma, St. Louis,MO) were purchased for the present study. SuperScript IIreverse transcriptase was purchased from Invitrogen (Carlsbad,CA) and AmpliTaq DNA Polymerase from Applied Biosys-tems (Foster City, CA).

Animals

Male Sprague-Dawley rats (280–320 g) were purchasedfrom Charles River Laboratories (Wilmington, MA). The ani-mals were housed on a 12-hr light/dark cycle with ad libitumaccess to food and water. All experimental procedures involv-ing animals were approved by the Institutional Animal Careand Use Committee and were conducted in accordance withthe recommendations provided in the NIH Guide for theCare and Use of Laboratory Animals.

Controlled Cortical Impact Injury

A controlled cortical impact (CCI) injury model as pre-viously described (Dixon et al., 1991; Hamm et al., 1992;Meaney et al., 1994) was used to cause TBI in the animals.Briefly, rats were initially anesthetized with 5% isoflurane witha 1:1 O2:N2O mixture. Animals were mounted on a stereo-taxic frame and were secured by two ear bars and an incisorbar. Anesthesia was maintained with 2.5% isoflurane with a1:1 O2:N2O mixture. A 6-mm-diameter craniotomy was

made midway between bregma and lambda on the right side,with the medial edge of the craniotomy 1 mm lateral to mid-line. Injury was produced using a pneumatic impactormounted at an angle of 108 from the vertical plane. A singleimpact at a velocity of 6 m/sec with a deformation depth of2.0 mm was delivered. After injury, the incision was closedwith wound clips. Sham animals received all aforementionedsurgical procedures except for the craniotomy and impactinjury. Core body temperature was monitored with a rectalthermometer and maintained at 36.8–37.28C with a heatingpad during the surgery. All rats were allowed to recover fromthe anesthesia completely after the surgery in a warm chamberbefore being sent back to the home cages.

Brain Edema Measurement

Percentage brain water content was determined by usingthe wet–dry method as described previously (McIntosh et al.,1990; Shohami et al., 1993). Animals were killed by decapita-tion, brains were quickly removed, and the cerebella were dis-carded. Ipsi- and contralateral hemispheres were separated,and the wet weight of each hemisphere was measured. Thetissues were then completely dried in a desiccating oven at1008C for 48 hr, and the dry weight of each hemisphere wasrecorded. The percentage water content (%H2O) is calculatedfor each hemisphere as follows: %H2O ¼ [(wet weight – dryweight)/wet weight] 3 100.

Immunohistochemistry

Rats were killed and brains quickly removed and frozenin –808C isopentane. Ten-micrometer-thick coronal sectionswere prepared from frozen tissues using a cryostat. Tissue sec-tions were mounted on 2% gelatin-subbed slides and dried atroom temperature for 1 hr. After being fixed with 100%methanol at –208C for 20 min, sections were permeabilizedand blocked by incubation in phosphate-buffered saline (PBS)containing 5% goat serum and 0.25% Triton X-100 at roomtemperature for 1 hr. Sections were incubated in primary anti-bodies (1.0 lg/ml in PBS containing 2.5% goat serum and0.25% Triton X-100) for 48 hr at 48C, washed in PBS, andthen incubated with Alexa Fluor-conjugated, species-specificsecondary antibodies for 3 hr. Sections were again washed inPBS, coverslipped with Fluoromount G (Fisher Scientific, FairLawn, NJ), and visualized with a Bio-Rad MRC 1024 confo-cal microscope (Bio-Rad, Hercules, CA). Double- and triple-label immunohistochemistry was performed essentially asdescribed above, with the addition of cell type-specific anti-bodies to the primary antibody incubation mixture, followedby detection with appropriate secondary antibodies.

Fluorescence Intensity Quantification

Fluorescence intensity was quantified essentially asdescribed previously (Masliah et al., 1992; Alford et al., 1994;Verschure et al., 1994; Hamann et al., 1995; Brus et al., 2002;Kamuhabwa et al., 2003; Li et al., 2004; Khiabani et al.,2004). Briefly, images of immunofluorescence were capturedwith a Bio-Rad MRC 1024 confocal microscope and Olym-pus BX 50WI camera. The parameters used for image acquisi-

500 Zhao et al.

tion (including laser power, iris size, brightness, offset, etc.)were preset to minimize the background and optimize the sig-nal using a tissue section from an injured animal. Theseparameters were kept constant across all subsequent groups. Astack of pictures was generated for each section by scanningthrough the section at a step thickness of 0.80 lm along thez-axis. MetaMorph 6.1 software was used to determine thefluorescence intensity based on the stack of pictures. Threenonoverlapping regions (844 lm 3 633 lm) in the ipsilateralcortex from each section and two sections from each animalwere used for imaging. The fluorescence intensities of thethree regions were averaged for each section, and this valuefor the two sections were averaged for each animal. The sec-tions containing the contusion core corresponded to 2.0 mmcaudal to bregma level, whereas the sections representing thepenumbra were taken from 1.0 mm caudal to bregma level.

Real-Time PCR

Animals were killed and parietal cortical tissues were quicklydissected out and frozen in dry ice. The frozen tissue was homo-genized in 1.5 ml of TriZol (Invitrogen) per 100 mg tissue, fol-lowed by addition of chloroform (1:5) and incubation on ice for20 min. The homogenate was centrifuged at 14,000g for 30 min,and total RNA was precipitated by isopropanol. One microgramof total RNA was reverse transcribed for 2 hr at 378C in a 20 llmixture containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl,3.0 mMMgCl2, 10mMdithiothreitol (DTT), 2.5 lMrandom hex-amer, 1.0 mM each dNTP, 20 U RNasin, and 200 U SuperscriptII reverse transcriptase. The level of expression of each target genewas quantified by using a Bio-Rad iCycler real-time PCR system.Reactions were carried out in triplicate. Each 30-ll reaction mix-ture consisted of 1.5 ll of the cDNA and 18 mM Tris-HCl, pH8.3, 55 mM KCl, 2.0 mM MgCl2, 0.2 mM of each dNTP,0.5 lM of each primer, 10 nM fluorescein, 1:75,000 dilution ofSybr green I, and 2 U AmpliTaq DNA polymerase. The follow-ing primer pairs were utilized for target mRNA amplification: forAQP4, forward, 50-CCAGCTGTGATTCCAAACGGA-30;reverse, 50-GCACAGCGCCTATGATTGGTC-30; for b-actin, forward, 50-CCCCATTGAACACGGCATT-30; reverse,50-CATCTTTTCACGGTTGGCCTTA-30. The amplificationprotocol consisted of one cycle at 958C for 3 min, followed by40 cycles at 958C for 30 sec, 588C for 30 sec, then 728C for 30sec. The melt-curve protocol, performed at the end of theamplification, consisted of 80 cycles beginning at 558C for 10sec, after which the temperature was increased by 0.58C/cycle.A standard curve for each target gene was generated to deter-mine the linear range and amplification efficiency of each sam-ple. The threshold cycle of each sample was fitted to the standardcurve to calculate the relative copy number of the initial cDNA.The resultant data was analyzed with the iCycler iQ Real-TimeDetection System software (Redell et al., 2003).

Statistical Analysis

Student’s t-test for unpaired variables was used for com-paring AQP4 mRNA levels, AQP4 immunoreactivities, andbrain edema between vehicle and SUL treatment groups.Results were considered significant at P < 0.05. Data are pre-sented as mean 6 SEM.

RESULTS

Change in AQP4 Immunoreactivity FollowingCCI Injury

Previous reports have demonstrated that brain edema ismaximal between 24 hr and 3 days following CCI of rodents(Baskaya et al., 1996; Markgraf et al., 2001). It has beenhypothesized that decreased AQP4 level may aggravate vaso-genic edema following brain injury (Manley et al., 2004;Papadopoulos et al., 2004) and that strategies to increase itslevels may be beneficial. AQP4 is expressed primarily inastroctyes, especially in end-feet that surround the braincapillaries (Nielsen et al., 1997; Nagelhus et al., 1998). Con-sistent with this, our high-magnification confocal micro-scope pictures show that the immunoreactivity for AQP4(green in Fig. 1A,B) was observed surrounding the braincapillary, identified by the endothelial cell marker vWF (redin Fig. 1A,B) and in close proximity to the astrocyte markerGFAP (blue in Fig. 1B). At low magnification, numerousAQP4-immunopositive vessel-like structures can be seen insham-operated animals (Fig. 1C). CCI injury, by compari-son, causes a marked reduction in AQP4 immunoreactivityin the contusion core at 24 hr postinjury (Fig. 1D). Thesefindings are consistent with previous reports regardingAQP4 mRNA level following TBI using a rodent weight-drop brain injury model (Ke et al., 2001).

SUL Attenuates the Loss of AQP4Immunoreactivity in the Injury Core

To determine the influence of SUL on TBI-associ-ated loss of AQP4 immmunoreactivity in the injury

Fig. 1. Immunohistochemical localization of AQP4. A: A representativeconfocal image of a capillary in the parietal cortex demonstrating thatAQP4 immunoreactivity (green) is found surrounding the capillary, asindicated by endothelial cell marker von Willebrand factor immunoreac-tivity (red). B: Triple-label immunohistochemistry shows that AQP4immunoreactivity localizes to the same area where astrocyte (demon-strated by GFAP, blue) end-feet coat the capillary. Low-magnificationimages showing AQP4 immunoreactivity in the parietal cortex of asham animal (C) and the contusion core of an injured animal (D) at 24hr following injury. Scale bars ¼ 20 lm in A,B, 100 lm in C,D.

Sulforaphane and AQP4 501

core, groups of injured animals were intraperitoneally(i.p.) injected with either SUL (5 mg/kg prepared incorn oil) or an equal volume of vehicle at 6 hr afterinjury. This dose is consistent with a previous studyshowing that an i.p. dose of 0.5 mg/mouse effectivelyincreased the expression of target genes in the retina ofmice (Tanito et al., 2005). The delay period was chosenbased on the observation that, on average, 6 hr elapsesfrom the time of injury to the initiation of pharmacolog-ical treatment for TBI patients (G.L. Clifton, unpub-lished observation). AQP4 immunoreactivity was visual-ized by using confocal microscopy. The representativelow-magnification pictures show that the marked reduc-tion in AQP4 immunoreactivity in the contusion core at24 hr after injury (sham animal, Fig. 2A, vs. vehicle-treated animal, Fig. 2B) was lessened as a result of SULtreatment (Fig. 2C). Double-label immunohistochemistryof representative brain capillaries (indicated by vWFimmunoreactivity in red) shows that AQP4 immunor-eactivity (green) was dramatically decreased along vesselsin the contusion core (Fig. 2E) compared with shamanimals (Fig. 2D). In comparison with the vehicle con-trol, the representative blood vessel from an SUL-treatedanimal shows increased immunoreactivity for AQP4(Fig. 2F). The quantification of AQP4 immunofluores-cence intensity shown in Figure 2G indicates that post-injury SUL administration modestly but significantlyattenuated the loss of AQP4 immunoreactivity caused bythe injury (24 hr injury-vehicle: 13.79% 6 2.22%, 24 hrinjury-SUL: 35.25% 6 3.13%, P < 0.05). Interestingly,SUL appears also to have attenuated the loss of vWFimmunoreactivity (Fig. 2E,F).

SUL Increases AQP4 Immunoreactivity in thePenumbra Region

In the penumbra region surrounding the contusioncore, TBI modestly increased AQP4 immunoreactivity(Fig. 3B,E) compared with sham controls (Fig. 3A,D).Postinjury SUL administration further augmented AQP4levels (Fig. 3C,F). Summary results for fluorescenceintensity measurements (Fig. 3G) shows that postinjuryadministration of SUL significantly increased AQP4 lev-els in the penumbra region at both 24 hr and 3 days fol-lowing injury. The increase in AQP4 does not appear tobe due to the enhancement in the number or the activa-tion of astrocytes following injury as indicated by a lackof difference in GFAP immunoreactivity betweenvehicle- and SUL-treated animals (Fig. 3H). This sug-gests that SUL may increase the expression of AQP4.

Systemic Administration of SUL Increases AQP4mRNA Levels in the Brain

To assess whether SUL can induce AQP4 expres-sion, rats were injected i.p. with either 5 mg/kg SULprepared in corn oil or an equal volume of vehicle.Twenty-four hours following SUL or vehicle adminis-tration, animals were killed, and cortical tissues were dis-sected for RNA extraction. AQP4 mRNA levels wereevaluated via real-time PCR. Figure 4A shows the rela-tionship between the starting quantity of total RNA andthe number of PCR cycles required to detect the ampli-fied AQP4 product. The threshold signal was linear fromat least 6.25 ng to 200 ng of the starting RNA, with acorrelation coefficient of 0.994 and a reaction efficiency

Fig. 2. Postinjury sulforaphane administration attenuates the loss ofAQP4 in the injury core. Low-magnification confocal images dem-onstrating AQP4 immunoreactivity in the parietal cortex of a sham(A), an injured animal receiving vehicle (B), and an injured animalreceiving SUL (C) at 24 hr following TBI. High-magnification dou-ble-label immunohistochemistry pictures showing AQP4 (green) andvWF (red) immunoreactivities in a sham (D), an injured animal

receiving vehicle (E), and an injured animal receiving SUL (F) at24 hr following TBI. G: Summary data of fluorescence intensity forAQP4 showing that TBI markedly decreases AQP4 immunoreactiv-ity in the injury core, which is attenuated by SUL. Data are mean 6SEM. Sham n ¼ 4, vehicle n ¼ 4, SUL n ¼ 4. *P < 0.05. Scalebars ¼ 100 lm in A–C, 20 lm in D–F.

502 Zhao et al.

of 84.1%. Representative profiles from a vehicle- and anSUL-treated animal are shown in Figure 4B. The grayhorizontal line indicates the threshold level at whichquantification was performed. The amplified product insamples prepared from the SUL-treated animals crossedthe threshold in earlier cycles compared with samplesfrom vehicle-treated animals, suggesting increased AQP4mRNA. The summary data (from three independentexperiments) in Figure 4C show that SUL administrationsignificantly increases AQP4 mRNA level at the 24-hrtime point (vehicle: 100.00% 6 18.16%, SUL: 289.76%6 16.52%, P < 0.05). The specificity of amplified prod-

ucts was confirmed by melting curve analysis (data notshown) and gel electrophoresis (Fig. 4D). The levels ofb-actin were used as an internal control to evaluate theamount of starting material for each sample. No signifi-cant difference in b-actin mRNA level was observed inany of the samples.

SUL Reduces Brain Edema Following TBI

Previous studies have suggested that, under patho-logical conditions causing vasogenic edema, AQP4 mayplay a beneficial role by facilitating the clearance of

Fig. 3. Postinjury sulforaphane administration further enhancesAQP4 immunoreactivity in the penumbra region. Representativeconfocal images of AQP4 immunoreactivity from a sham (A,D), aninjured animal receiving vehicle (B,E), and an injured animal receiv-ing SUL (C,F) at 24 hr after injury. G: Summary data show thatAQP4 fluorescence intensity is augmented by SUL at both 24 hr and

3 days following injury. H: Summary data show that there is no dif-ference in GFAP fluorescence intensity in the penumbra regionbetween injury-vehicle and injury-SUL groups at 24 hr after injury.Data are mean 6 SEM. Sham n ¼ 8, vehicle n ¼ 8, SUL n ¼ 8.*P < 0.05. Scale bars ¼ 100 lm in A–C, 20 lm in D–F.

Sulforaphane and AQP4 503

accumulated water from the brain (Manley et al., 2004;Papadopoulos et al., 2004). To test whether theenhanced AQP4 expression observed following SULtreatment is associated with a reduction in brain edema,groups of injured animals were injected with eithervehicle or 5 mg/kg SUL 6 hr following TBI. Our datashow that TBI caused a significant increase in brainwater content in the ipsilateral hemisphere both at 24 hr(sham: 78.68% 6 0.11%, injured: 80.68% 6 0.30%, P <0.05) and at 3 days after injury (sham: 78.58% 6 0.09%,injured: 80.89% 6 0.14, P < 0.05). Postinjury SULtreatment did not show any effect on the brain edemalevel at 24 hr (injured-vehicle: 80.68% 6 0.30%,injured-SUL: 80.39% 6 0.21%). By 3 days, however, asignificant reduction in brain edema was observed inSUL-treated animals (injured-vehicle: 80.89% 6 0.14%,injured-SUL: 80.47% 6 0.11%, P < 0.05; Fig. 5B).When an additional administration of SUL was given 12hr postinjury, animals receiving the two-administration(6 and 12 hr) paradigm still showed a significant decreasein brain edema only at 3 days (injured-vehicle: 80.32%

6 0.11%, injured-SUL: 79.68% 6 0.11%, P < 0.05) butnot 24 hr following injury (Fig. 5C).

DISCUSSION

TBI increases BBB permeability, leading to theentry and accumulation of circulating fluid into the brain(Betz et al., 1989; Klatzo, 1994). AQP4 has been shownto be important for regulating water homeostasis and hasbeen proposed to play a role in the clearance of waterfollowing vasogenic edema (Manley et al., 2004). Thepresent study revealed three findings: 1) TBI decreasedAQP4 level in the core of the injury and modestlyincreased this protein in the penumbra region; 2) postin-jury administration of SUL attenuated AQP4 loss in theinjury core and further increased AQP4 level in thepenumbra region; and 3) postinjury administration ofSUL reduced brain edema at 3 days following TBI.Taken together, these findings suggest that enhancedexpression of AQP4 by SUL facilitates water clearancefrom the injured brain, thereby lessening edema.

Two caveats limit the interpretation of our find-ings. First, the present study is correlative in nature anddoes not demonstrate a direct link between TBI-associ-ated brain edema and AQP4 expression. Second, inaddition to increasing the expression of AQP4, SUL hasbeen shown to enhance the mRNA levels for severalgenes encoding for antioxidant and detoxifying enzymesthat may offer cellular protection. Thus, the ability ofSUL to reduce cerebral edema following TBI couldresult from protection of brain vasculature, in additionto enhancing the expression of AQP4. Consistent withthis, we observed that postinjury SUL administrationincreased the immunoreactivity for vWF (Fig. 2F) andrat endothelial cell antigen-1 (RECA-1; J. Zhao, unpub-

Fig. 4. Systemic administration of sulforaphane increases AQP4mRNA in the brain. A: Standard curve showing the relationshipbetween the starting quantity of total RNA and the number of PCRcycles that are required to detect the amplified AQP4 product.B: Representative real-time PCR profiles for AQP4 amplificationusing RNA prepared from a vehicle- and an SUL-treated animal.The dashed line indicates the threshold value used for quantification.C: Summary data showing that SUL increases AQP4 mRNA levelmeasured at 24 hr postadministration. D: The specificity of theamplified product was confirmed by gel electrophoresis showing asingle amplified product for AQP4. b-Actin mRNA levels were usedas an internal control to evaluate the amount of starting material foreach sample. No significant difference in b-actin mRNA level wasobserved in any of the samples. Data are mean 6 SEM. *P < 0.05(n ¼ 3 for each group).

Fig. 5. Postinjury administration of sulforaphane decreases brainedema. Brain water content in the ipsilateral hemisphere after a 6-hrpostinjury administration of vehicle or SUL measured at 24 hr (A) or3 days (B) following injury (vehicle n ¼ 8, SUL n ¼ 8). C: Brainwater content in the ipsilateral hemisphere after 6 hr and 12 hr post-injury administration of vehicle or SUL measured at 3 days followingTBI (vehicle n ¼ 4, SUL n ¼ 4). Data are mean 6 SEM. *P <0.05.

504 Zhao et al.

lished observation), suggesting enhanced BBB integrity.Therefore, the ability of SUL to decrease brain edemafollowing TBI may be due to a combination of mecha-nisms that includes decreased BBB permeability,enhanced cell survival, and/or increased AQP4 expres-sion.

Our observation that AQP4 protein levels declineddramatically in the injury core prompted us to screen forcompounds that could be used to induce the expressionof the aqp4 gene. SUL, a naturally occurring compoundin cruciferous vegetables such as broccoli, has beenreported to be a potent inducer of genes containing theantioxidant-responsive element (ARE). SUL increasesthe expression of these genes by activating the transcrip-tion factor NF-E2-related factor-2 (Nrf2; McWalteret al., 2004; Lee and Johnson, 2004). In response toSUL exposure, Nrf2 translocates into the nucleus, whereit binds, with high affinity, to the ARE in the promoterregions of target genes. Because the promoter region ofthe aqp4 gene contains putative AREs (Umenishi andVerkman, 1998), we tested the ability of SUL to increaseaqp4 mRNA. Our measurement of AQP4 mRNA levelsfrom real-time PCR indicates that SUL can induce theexpression of this gene in the brain. Consistent with this,we observed that SUL enhanced AQP4 levels in thepenumbra region in the absence of an increase in GFAPimmunoreactivity compared with injured animals receiv-ing vehicle. This suggests that the increased amount ofAQP4 immunoreactivity in this area is likely due, at leastin part, to enhanced expression of the AQP4 gene. How-ever, the role of Nrf2 in SUL-mediated AQP4 gene ex-pression remains to be addressed.

SUL administration increased AQP4 levels at both24 hr and 3 days post-TBI. However, the decrease inbrain water content was observed only at the 3-day timepoint. Previously, it has been demonstrated that CCIcauses a significant elevation in brain water content asearly as 1 hr after injury, which reaches maximal valuesat about 6 hr (Markgraf et al., 2001). This increasedwater content is maintained at the peak level for up to 4days following the insult. Although SUL significantlyincreased AQP4 levels at 24 hr postinjury, it is possiblethat the lack of an effect of SUL on brain water contentat this time point could be due to insufficient time forAQP4 to clear a significant amount of water. In additionto its role in brain water homeostasis, AQP4 has beenshown to be required for optimal buffering of potassium(Amiry-Moghaddam et al., 2003). Mice lacking aqp4 areprone to epileptic seizures (Binder et al., 2004), which isthought to result from impaired clearance of extracellularpotassium. Elevations in extracellular potassium causeneuronal depolarization and glutamate release. As gluta-mate release is thought to be an important sequela ofCNS injury (Faden et al., 1989; Katayama et al., 1990;Miller et al., 1990; Hayes et al., 1992; Hicks et al.,1995; Lyeth et al., 2001; Nesic et al., 2002), SUL-medi-ated increase in AQP4 levels may also serve to decreaseextracellular potassium and glutamate release, furtheringthe protective effect of SUL.

In conclusion, the data in this study show thatAQP4 level is decreased in the injury core and modestlyincreased in the penumbra region following TBI. Postin-jury administration of SUL attenuates the loss of AQP4level in the injury core and increases AQP4 levels in thepenumbra region in comparison with injured animalsreceiving vehicle. In association with these changes inAQP4, brain edema is reduced by SUL at 3 days follow-ing injury. These findings suggest that AQP4 is involvedin the clearance of excess water from the injured brain,thereby reducing edema.

ACKNOWLEDGMENTS

The authors thank Dr. Rong Yu for his scientificinput and Melanie Moody for her technical assistance.

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