Increased Expression of Vasopressin V1a Receptors after Traumatic Brain Injury

JOURNAL OF NEUROTRAUMAVolume 21 Number 8 2004copy Mary Ann Liebert IncPp 1090ndash1102

Increased Expression of Vasopressin V1a Receptors afterTraumatic Brain Injury

JOANNA SZMYDYNGER-CHODOBSKA1 INSUNG CHUNG1 EWA KOZNIEWSKA2BAO TRAN1 J FREDERICK HARRINGTON1 JOHN A DUNCAN1

and ADAM CHODOBSKI1

ABSTRACT

Experimental evidence obtained in various animal models of brain injury indicates that vasopressinpromotes the formation of cerebral edema However the molecular and cellular mechanisms un-derlying this vasopressin action are not fully understood In the present study we analyzed the tem-poral changes in expression of vasopressin V1a receptors after traumatic brain injury (TBI) in ratsIn the intact brain the V1a receptor was expressed in neurons located in all layers of the fron-toparietal cortex The V1a receptor-immunoreactive product was predominantly localized to neu-ronal nuclei and had both a diffused and punctate staining pattern The V1a receptors were also ex-pressed in astrocytes especially in layer 1 of the frontoparietal cortex In these cells two distinctivepatterns of immunopositive staining for V1a receptors were observed a diffused cytosolic stainingof cell bodies and processes and a clearly punctate staining pattern that was predominantly local-ized to the astrocytic cell bodies The real-time reverse-transcriptase polymerase chain reactionanalysis of changes in mRNA for the V1a receptor demonstrated that after TBI there is an early (4 h post-TBI) increase in the number of transcripts in the ipsilateral frontoparietal cortex whencompared to the contralateral hemisphere or the sham-injured rats This increase in the messagewas followed by the up-regulation of expression of the V1a receptors at the protein level This wasmost evident in cortical astrocytes in the areas surrounding the lesion The number of the V1a re-ceptor-immunopositive astrocytes in the traumatized parenchyma gradually increased starting at8 h and peaking at 4ndash6 days after TBI Furthermore a redistribution of V1a receptors from the as-trocytic cell bodies to the astrocytic processes was observed In addition to astrocytes an increasedexpression of V1a receptors was found in the endothelium of both blood microvessels and the large-diameter blood vessels in the frontoparietal cortex ipsilateral to injury This increase in the V1a re-ceptor expression was apparent between 2 and 4 days after TBI As early as 1ndash2 h following the im-pact there was also a striking increase in the number of the V1a receptor-immunopositive beadedaxonal processes with greatly enlarged varicosities that were localized to various areas of the in-jured parenchyma It is suggested that the increased expression of V1a receptors plays an impor-tant role in the vasopressin-mediated formation of edema in the injured brain

Key words astrocytes cerebrovascular endothelium neurons rat V1a receptor vasopressin

1Department of Clinical Neurosciences Brown University School of Medicine Providence Rhode Island2Department of Neurosurgery Medical Research Center of Polish Academy of Sciences Warsaw Poland

INTRODUCTION

CENTRALLY RELEASED VASOPRESSIN (VP) has beendemonstrated to play an important role in the phys-

iological regulation of brain fluid homeostasis (Raichleand Grubb 1978 Rosenberg et al 1986 DePasquale etal 1989 Hertz et al 2000 Niermann et al 2001) How-ever increasing evidence indicates that VP also promotesthe formation of edema in various types of brain injurysuch as cryogenic brain injury cerebral ischemia and intracerebral hemorrhage (Dickinson and Betz 1992Rosenberg et al 1992 Kagawa et al 1996 Bemana andNagao 1999 Shuaib et al 2002) Furthermore centrallyadministered VP has been shown to produce edema inintact animals including VP-deficient Brattleboro rats(Rosenberg et al 1990 Dickinson and Betz 1992)These observations are in line with the increased con-centrations of VP in plasma and cerebrospinal fluid (CSF)found in patients with traumatic brain injury (TBI) ischemic stroke and subarachnoid hemorrhage (Matheret al 1981 Joynt et al 1981 Soslashrensen et al 1985 Bar-reca et al 2001 Huang et al 2003)

Cerebral edema is generally classified into two typescytotoxic and vasogenic (Kimelberg 1995) Cytotoxicedema refers to cellular swelling which usually involvesthe astrocytic cell bodies and processes neuronal den-drites and cerebrovascular endothelium (Liu et al2001) In comparison vasogenic edema is related to aswelling of the brain parenchyma associated with the dis-ruption of the bloodndashbrain barrier (BBB) A leaky bar-rier allows for the interstitial accumulation of blood-borne osmotically active solutes and water which resultsin formation of edema The two types of edema fre-quently coincide and for example in TBI astrocytic andendothelial swelling (Dietrich et al 1994 Vaz et al1997 Castejoacuten et al 1998) is accompanied by the open-ing of the BBB and formation of vasogenic brain edema(Baskaya et al 1997)

VP is a ligand for the three types of receptors V1aV1b and V2 These receptors belong to a large family ofG proteinndashcoupled receptors (Schoumlneberg et al 1998Thibonnier et al 1998) The signal transduction in V1

receptors involves the activation of phospholipases C Dand A2 the production of inositol 145-triphosphate anddiacylglycerol the stimulation of protein kinase C andthe mobilization of intracellular Ca2 (Thibonnier et al1998) In comparison the intracellular signaling in V2 re-ceptors involves the activation of adenylyl cyclase theproduction of adenosine 35-cyclic monophosphate andthe stimulation of protein kinase A (Thibonnier et al1998) While the V1a and V1b receptors are widely dis-tributed within the central nervous system (Ostrowski etal 1994 Szot et al 1994 Hernando et al 2001) the

V2 receptors do not appear to be expressed in any partof the adult brain except the cerebellum (Kato et al1995) These data are consistent with the functional ob-servations made in various models of brain injury inwhich the selective V1 receptor antagonists but not theV2 receptor blockers decreased the permeability of theBBB and reduced edema (Rosenberg et al 1992 Ka-gawa et al 1996 Bemana and Nagao 1999 Shuaib etal 2002)

The aim of the present study was to characterize thechanges in expression of the V1a receptors following TBIOur results demonstrated for the first time that in the in-jured parenchyma there is a considerable increase in ex-pression of V1a receptors in astrocytes neurons and thecerebrovascular endothelium The increased expressionof V1a receptors may play an important role in VP-mediated formation of post-traumatic brain edema

MATERIALS AND METHODS

Reagents and Antibodies

ThermoScript RNase H reverse transcriptase RNaseinhibitor RNaseOut and RNase-free DNase I were ob-tained from Invitrogen (Carlsbad CA) HotStart Taq andFastStart Taq DNA polymerases were purchased fromQiagen (Valencia CA) and Roche Molecular Biochem-icals (Indianapolis IN) respectively SYBR Green I wasfrom Molecular Probes (Eugene OR)

Polyclonal antibody to the V1a receptor a generous gift of Dr Melvyn S Soloff (University of Texas) was raised in rabbits against the synthetic peptide H2N-CHSMAQKFAKDDSDS-COOH This antibody has pre-viously been characterized (Strakova et al 1997) and inthis study affinity-purified antibody was used at 150 dilution In addition to anti-V1a receptor antibody mono-clonal mouse antibodies were used as follows anti-neuronal nuclei (NeuN) (clone A60) anti-glial fibrillaryacidic protein (GFAP) (clone G-A-5) and anti-rat CD31(clone TLD-3A12) from Chemicon International (Temec-ula CA) Anti-NeuN antibody was used at a concentra-tion of 05 gmL whereas other monoclonal antibodieswere applied at a concentration of 2 gmL Secondaryantibodies were purchased from Molecular Probes Thesewere goat anti-rabbit and anti-mouse IgGs conjugatedwith Alexa 594 (red fluorophore) and Alexa 488 (greenfluorophore) respectively They were used at a concen-tration of 4 gmL Normal goat serum was obtained fromJackson Immunoresearch Labs (West Grove PA) Tis-sue-Tek OCT Compound and Vectashield mountingmedium were from Sakura Finetek (Torrance CA) andVector Labs (Burlingame CA) respectively

EXPRESSION OF V1a RECEPTORS IN THE INJURED BRAIN

Animals and the TBI Model

Adult male Sprague-Dawley rats weighing 280ndash320 gwere purchased from the Charles River Breeding Labs(Wilmington MA) They were kept at 22degC with a 12-hlight cycle and maintained on standard pelleted rat chowand water ad libitum A weight-drop model as previouslydescribed (Chodobski et al 2003) was used to produceTBI In brief animals were anesthetized intraperitoneallywith chloral hydrate (450 mgkg) Rectal temperature wascontinuously monitored and maintained at 37degC Ratswere placed in a stereotaxic frame and before the inci-sion the scalp was infiltrated with 2 lidocaine solutionA 4-mm craniotomy was performed over the right fron-toparietal cortex to expose the dura with the center ofthe opening located 15ndash20 mm posterior to bregma and25 mm lateral to the midline A 25-g weight was droppedon the intact dura from a height of 8 cm The impactorrsquosdiameter was 25 mm and the depth of brain deformationwas set at 25 mm Immediately after TBI the scalp wasclosed with a silk suture and the animals were allowedto recover in their cages In the sham-injured animals thesame surgical procedures were performed but the weightwas not dropped on the dura

Real-Time Reverse-Transcriptase PolymeraseChain Reaction (RT-PCR)

At 4 and 8 h and at 1 2 4 6 and 14 days after TBIrats (4 animals per time point) were reanesthetized withintraperitoneal pentobarbital sodium (50 mgkg) and wereperfused transcardially with 100 ml of ice-cold 09NaCl The samples of the frontoparietal cortex adjacent

to the lesion and those from the contralateral side werecollected Samples of the frontoparietal cortex from thebrains of the sham-injured rats were collected as wellTotal RNA was isolated using the acid guanidinium thio-cyanate-phenol-chloroform extraction method (Chom-czynski and Sacchi 1987) Ethanol-precipitated RNAwas resuspended in H2O and stored at 80degC Before the first-strand cDNA synthesis RNA was treated withRNase-free DNase I for 15 min at room temperature us-ing 1 U of DNase I First-strand cDNAs were synthe-sized using oligo(dT)20 primer (05 g) and 15 U of Ther-moScript RNase H reverse transcriptase Forty units ofRNase inhibitor RNaseOut were also added to the re-verse-transcription reaction For each 20-L reaction 1g of total RNA was used and the reaction was carriedout for 1 h at 50degC

The following primers were used 5-CGACACAG-CAAGGGTGACAAGG-3 (forward primer for the V1a

receptor) 5-AGGAAGCCAGCAACGCCG-3 (reverseprimer for the V1a receptor) 5-ACCCCACCGTGTT-CTTCG-3 (forward primer for cyclophilin A) and 5-CTTGCCATCCAGCCACTC-3 (reverse primer forcyclophilin A) Cyclophilin A was used for the normal-ization of mRNA for the V1a receptor The predicted sizesof the PCR products were 265 and 368 bp for the V1a

receptor and cyclophilin A respectively Single bandscorresponding to these predicted sizes were observed on agarose gels and the identity of these PCR productswas confirmed by Southern blotting as previously de-scribed (Chung et al 2003) Real-time PCR was per-formed using the DNA Engine Opticon System (MJ Research Waltham MA) The 50-l PCR reaction mix-

SZMYDYNGER-CHODOBSKA ET AL

FIG 1 Real-time RT-PCR analysis of the changes in expression of the V1a receptor after TBI The changes in mRNA for theV1a receptor in the ipsilateral frontoparietal cortex adjacent to the impact area (Ipsi Cx) as compared to those in the contralat-eral cortex (Contra Cx) and in the cortex from the sham-injured brains (Sham Cx) were analyzed The number of copies of tran-scripts for the V1a receptor relative to the message for cyclophilin A (Cycl-A) is shown p 005 p 001 for Ipsi Cx vsContra Cx daggerp 005 daggerdaggerp 001 for Ipsi Cx vs Sham Cx

tures contained 02 mM mixed dNTPs 02 M eachprimer 2 mM MgCl2 2 U HotStart Taq DNA polymerase(V1a receptor) or FastStart Taq DNA polymerase (cy-clophilin A) SYBR Green I diluted 1100000 and 120(V1a receptor) or 12000 (cyclophilin A) of the reverse-transcription reaction product The reaction mixtures

were heated to 95degC for 15 min (HotStart Taq) or 4 min(FastStart Taq) and then were subjected to 40 cycles ofdenaturation (94degC 30 sec) annealing (67degC for the V1a

receptor or 59degC for cyclophilin A 30 sec) and exten-sion (72degC 1 min) The final extension was carried outat 72degC for 10 min

FIG 2 Immunohistochemical localization of V1a receptors in the intact brain (A) The microphotograph shows the V1a recep-tor-immunopositive staining of neuronal nuclei and astrocytic processes in the frontoparietal cortex Note that some of these as-troglial processes are associated with the pial blood vessels penetrating the brain parenchyma (arrows) (B) The control experi-ment in which the brain section was incubated with the primary antibody that had been preabsorbed overnight with the antigenicpeptide fragment of the V1a receptor (100 gmL) (C) Confocal microscopy image of the choroid plexus Consecutive opticalsections through the choroidal tissue were acquired at 05ndash1-m intervals and were subsequently projected into one image Notethat the V1a receptor-immunoreactive product having a punctate staining pattern is localized to the apical (CSF-facing) plasmamembrane domain of choroidal epithelial cells The nuclei of these cells are also diffusely stained (D) Control experiment inwhich the choroid plexus was incubated with the primary antibody preabsorbed with the antigenic peptide (E) Double stainingof cortical neurons with anti-V1a receptor (red fluorophore) and anti-neuronal nuclei (NeuN) (green fluorophore) antibodies ex-amined with confocal microscopy Note that the neuronal cytoplasm is also stained with anti-NeuN antibody (F) Higher magni-fication confocal microscopy images of cortical neurons A single optical section across neuronal nuclei is shown These imagesdemonstrate that the V1a receptor-immunoreactive product is predominantly localized to neuronal nuclei and has both a diffusedand punctate staining pattern (G) Double staining with anti-V1a receptor (red fluorophore) and anti-glial fibrillary acidic protein(GFAP) (green fluorophore) antibodies examined with confocal microscopy The co-localization of the immunoreactive productsfor the V1a receptor and GFAP confirms the astroglial expression of V1a receptors and their cytosolic distribution Note that somelong astroglial processes have unusually strong cytosolic staining (arrows) (H) High-magnification confocal microscopy imagesof cortical astrocyte with a long heavily stained process Double staining for the V1a receptor and GFAP is shown Bar 100m (AB) 20 m (CD) 50 m (EG) 10 m (FH)

Immunohistochemistry

Separate rats were used for immunohistochemistryTwo to three animals per group were sacrificed after TBIor sham injury at time points described above In addi-tion two early time points ie 1 and 2 h post-TBI wereanalyzed Rats were reanesthetized as described above

and were perfused transcardially with ice-cold 09NaCl followed by ice-cold 4 paraformaldehyde in 005M phosphate-buffered saline (PBS pH 74) Brains wereremoved and postfixed for an additional 4 h in theparaformaldehydePBS solution at 4degC They were thenincubated overnight in 20 sucrose in PBS and embed-ded in Tissue-Tek OCT Compound The coronal brainsections were cut on a cryostat at 10 m

Immunohistochemical procedures were performed atroom temperature except for the incubation with primaryantibodies that was completed at 4degC All incubationswere performed in PBS containing 05 of bovine serumalbumin (BSA) and 02 of Triton X-100 (TX-100) Forwashes PBS containing 01 BSA and 01 TX-100was used To minimize non-specific staining the sectionswere incubated for 30 min with 10 normal goat serumFour percent of normal goat serum was also includedwhen the specimens were incubated with primary or sec-ondary antibodies Following the initial blocking step thesections were incubated overnight with primary antibod-ies Six 10-min washes were then performed and the sec-tions were incubated for 1 h with secondary antibodiesAfter four 10-min washes the sections were mountedwith Vectashield mounting medium The specimens wereviewed with either a conventional fluorescence OlympusBH2-RFCA microscope (Figs 2ABD and 4BD) or aNikon PCM2000 confocal laser-scanning microscope(the rest of the images)

Statistical Analysis

The results of real-time RT-PCR are presented as meannumber of copies of mRNA for the V1a receptor per 100

FIG 3 Schematic illustration of injured areas (AB) Coro-nal brain sections cut at the level of septum 0ndash1 mm posteriorto bregma and at the hippocampal level 25ndash35 mm posteriorto bregma respectively Since the post-TBI changes in the V1a

receptor expression occurred predominantly in the frontopari-etal cortex adjacent to the lesion this brain area was mainly an-alyzed (marked with rectangles) CL LV 3rdV and FH are thearea of cortical lesion lateral and 3rd ventricles and fimbriahippocampi respectively

FIG 4 The up-regulation of the V1a receptor expression in cortical astrocytes 4 days after TBI Double staining for V1a re-ceptors (red fluorophore) and GFAP (green fluorophore) was examined with confocal microscopy (AC) Images of an area ofthe frontoparietal cortex adjacent to the lesion and of the contralateral frontoparietal cortex respectively Note that the changesin the intensity of astrocytic staining following TBI are most evident in the cortical layer 1 however they are also readily no-ticeable in deeper cortical layers in the vicinity of the impact These astrocytes have a diffused cytoplasmic staining pattern andare frequently of a reactive hypertrophic type with increased levels of GFAP expression (BD) The frontoparietal cortex ipsi-lateral and contralateral to injury respectively from the brain section that was incubated with the primary antibody preabsorbedwith the antigenic peptide (for further details see the legend to Fig 2B) (EF) High-magnification images of the frontoparietalcortex (layers 1ndash2) ipsilateral and contralateral to injury respectively Note that after TBI there is a redistribution of the V1a re-ceptor-immunoreactive product with a punctate staining pattern from the astrocytic cell bodies (arrowheads) to the astrocyticprocesses (arrows) Bar 50 m for (AndashD) 10 m (EF)

FIG 5 The up-regulation of the V1a receptor expression in cortical astrocytes associated with parenchymal blood vessels (BVs)Confocal microscopy images were acquired from the coronal brain sections doubly stained for V1a receptors and GFAP (AB)Astrocytic processes closely associated with blood microvessels in the frontoparietal cortex ipsilateral and contralateral to injuryrespectively 4 days post-TBI Note that the astrocytes surrounding the lesion area are reactive and hypertrophied and expresshigh levels of V1a receptors and GFAP compared to the contralateral cortex (CD) The ipsilateral and contralateral frontopari-etal cortices respectively at 14 days after the trauma The images of astrocytes associated with the large-diameter blood vesselsare shown Bar 10 m (AB) 20 m (CD)

copies of cyclophilin A mRNA SEM For statisticalevaluation of data ANOVA was used followed by theNewman-Keuls test for multiple comparisons amongmeans p 005 was considered statistically significant

RESULTS

RT-PCR

The real-time RT-PCR analysis of the changes inmRNA for the V1a receptor in the ipsilateral frontopari-etal cortex adjacent to the impact area demonstrated thatat 4 and 8 h after TBI there was a significant increase inthe number of transcripts compared to the contralateralhemisphere or the sham-injured rats (Fig 1) This in-crease in the message preceded the up-regulation of theV1a receptor expression at the protein level (see below)The elevated levels of mRNA for the V1a receptor werealso found 1 4 and 6 days post-TBI however thesechanges did not attain statistical significance The ex-pression of the V1a receptor in the frontoparietal cortexcontralateral to injury or in the brains of the sham-injuredanimals did not change at any time point following TBI(Fig 1)

Expression of V1a receptors in the intact brain Thefrontoparietal cortex was mainly analyzed to character-ize the expression of V1a receptors in the normal brainand in the brains of rats subjected to TBI For this pur-pose the coronal brain sections were cut at the level ofseptum 0ndash1 mm posterior to bregma and at the hip-pocampal level 25ndash35 mm posterior to bregma In theintact brain the most prominent immunopositive stain-ing for V1a receptors was associated with neurons locatedin all layers of the frontoparietal cortex (Fig 2A) Neu-ronal localization of these receptors was confirmed byco-staining with anti-NeuN antibody (Fig 2EF) Inter-estingly the V1a receptor-immunoreactive product waspredominantly localized to neuronal nuclei and had botha diffused and punctate staining pattern (Fig 2F) Thisnuclear distribution of V1a receptors was clearly seen in high-magnification confocal microscopy images ofcortical neurons doubly stained with anti-V1a receptorand anti-NeuN antibodies (Fig 2F) The V1a receptor-immunopositive beaded axonal processes were also spo-radically observed in various layers of the frontoparietalcortex (data not shown)

In addition to neurons the V1a receptors were ex-pressed in astrocytes especially in layer 1 of the fronto-parietal cortex (Fig 2A) The V1a receptor-immunoreac-tive product appeared as a diffused cytosolic staining of

astrocytic cell bodies and processes that were frequentlyassociated with parenchymal blood vessels (Fig 2A) Occasionally very strong cytosolic staining of long as-troglial processes was observed (Fig 2GH) The V1a re-ceptor-immunopositive product also had a clearly punc-tate staining pattern and was predominantly localized tothe astrocytic cell bodies [shown in high-magnificationimages of astrocytes in the frontoparietal cortex con-tralateral to injury (Fig 4F)] The astrocytic localizationof V1a receptors was confirmed by a double staining with anti-V1a receptor and anti-GFAP antibodies (Fig2GH) The immunopositive staining of neurons and as-trocytes was completely eliminated when the primary an-tibody had been pre-absorbed with the antigenic peptide(Fig 2B)

Among the non-parenchymal cells the choroid plexusepithelium was found to express high levels of the V1a

receptor (Fig 2C) In this tissue the V1a receptor-immunopositive product seen as a punctate staining waslocalized to the apical (CSF-facing) plasma membranedomain of epithelial cells The nuclei of these cells werealso diffusely stained This staining of the choroid plexuswas abolished when the primary antibody had been pre-absorbed with the antigenic peptide (Fig 2D)

Changes in expression of V1a receptors after TBI Fig-ure 3 illustrates schematically the location of injury thatmostly involved the frontoparietal cortex The changes inexpression of V1a receptors in astrocytes cerebrovascu-lar endothelium and neurons were examined at 1 2 4and 8 h and 1 2 4 6 and 14 days post-TBI Since thepost-TBI changes in the V1a receptor expression occurredpredominantly in the cortical areas adjacent to the lesionthese brain regions were mainly analyzed

Beginning at 8 h post-TBI there was a gradual increasein the expression of V1a receptors in cortical astrocytessurrounding the lesion area (Fig 4AE) The changes inthe intensity of astrocytic staining were most evident inlayer 1 of the ipsilateral frontoparietal cortex howeverthey were also readily noticeable in deeper cortical layersin an area adjacent to the impact (Fig 4A) The numberof astrocytes expressing the V1a receptor peaked at 4ndash6days after TBI but the increased astroglial expression ofthis receptor was maintained to the end of the observationperiod ie 14 days post-TBI These astrocytes had a dif-fused cytoplasmic staining pattern and were frequently ofa reactive hypertrophic type with increased levels ofGFAP expression (Fig 4AE) Furthermore after the in-jury there appeared to be a redistribution of V1a receptorsfrom the astrocytic cell bodies to the astrocytic processes(compare the changes in a punctate astrocytic staining pat-tern in Fig 4EF) In contrast to the traumatized hemi-sphere in the frontoparietal cortex contralateral to injury

FIG 6 The increased expression of the V1a receptor in the cerebrovascular endothelium 2 days post-TBI Double staining forV1a receptors (red fluorophore) and the endothelial marker CD31 (green fluorophore) was examined with confocal microscopy(AB) Blood microvessels (arrowheads) from an area of the frontoparietal cortex adjacent to the lesion and from the contralateralfrontoparietal cortex respectively Note a robust clearly distinguishable punctate staining of endothelial cells in the traumatizedparenchyma which contrasts with a weak endothelial staining in the contralateral cortex Also note a punctate V1a receptor-pos-itive staining of endothelial nuclei (arrows) in the ipsilateral cortex (CD) Large-diameter blood vessels (BVs) from the ipsilat-eral and contralateral frontoparietal cortices respectively Note a distinctive punctate staining of endothelial cells in the injuredparenchyma that is similar to the staining of blood microvessels shown in A Also note both a diffused and punctate nuclearstaining of endothelial cells (arrows) in the ipsilateral cortex Bar 10 m (AB) 20 m (CD)

there were no obvious changes in astrocytic expression of V1a receptors (Fig 4CF) compared to the sham-injured rats (not shown) or the intact animals The V1a re-ceptor-immunoreactive staining in the injured brains wascompletely eliminated when the primary antibody hadbeen pre-absorbed with the antigenic peptide (Fig 4BD)Many astrocytes located in the frontoparietal cortex wereintimately associated with blood microvessels After theinjury in an area adjacent to the lesion these astroglia fre-quently became reactive and hypertrophied and expressedhigh levels of V1a receptors when compared to the con-tralateral hemisphere (Fig 5A vs 5B) Similar changes inthe expression of V1a receptors were observed in corticalastrocytes whose processes were in close contact with thelarge-diameter blood vessels (Fig 5C vs 5D)

During a short period between 2 and 4 days after TBIthe increased expression of V1a receptors was observedin the endothelium of both blood microvessels and thelarge-diameter blood vessels in the frontoparietal cortexipsilateral to injury The distribution of blood vessels ex-pressing high levels of V1a receptor was not uniform witha number of the V1a receptor-positive microvessels rang-ing between 0 and 4 per 001 mm2 The changes in theexpression of the V1a receptor included a robust clearlyseen punctate staining of endothelial cell bodies in thetraumatized parenchyma (Fig 6AC) which contrastedwith a weak endothelial staining noted in the contralat-eral cortex (Fig 6BD) or in the brains of the sham-injured rats (not shown) Moreover both a diffused andpunctate nuclear staining of endothelial cells especially

noticeable in the large-diameter blood vessels was foundin the ipsilateral cortex (Fig 5AC)

As early as 1ndash2 h following the impact there was a striking increase in the number of the V1a receptor-immunopositive beaded axonal processes with greatlyenlarged varicosities that were localized both to the cor-tical lesion area and to the cortex adjacent to the lesionAt later time points including the longest observation pe-riod (14 days post-TBI) these axonal processes were notonly found ipsilaterally in the frontoparietal cortex adja-cent to the impact but also in the brain parenchyma sur-rounding the lateral ventricle (Fig 7A) and in the fim-bria hippocampi (Fig 7C) of the injured hemisphere Incomparison a few beaded axonal processes with smallvaricosities that were immunoreactive for the V1a recep-tor were noted in the contralateral hemisphere (Fig 7BD)or in the brains of the sham-injured rats (not shown)

DISCUSSION

In the present study a weight-drop model of TBI orig-inally described by Feeney et al (1981) was used This

model produces an injury that is reminiscent of surfacecontusion observed in humans (Povlishock et al 1994)A similar experimental model was employed more re-cently to analyze the neutrophilic invasion following TBI(Clark et al 1996 Carlos et al 1997 Chodobski et al2003) In our hands a reproducible injury with no mor-tality was obtained with this model of TBI

This study demonstrated that after TBI there is a grad-ual and relatively long-lasting (8 h to 14 days) increasein expression of the V1a receptor in cortical astrocytessurrounding the impact area Astrocytic swelling is usu-ally the most prominent feature of cytotoxic brain edema(Kimelberg 1995) and based on our observations theastroglia appear to be important target cells for VP in thetraumatized parenchyma Consistent with this idea VPhas been shown to increase the astrocytic cell volume(Latzkovits et al 1993 Sarfaraz and Fraser 1999) ThisVP action could be blocked by bumetanide an inhibitorof the Na-K-2Cl co-transporter that plays a criticalrole in cell volume regulation (Russell 2000) In a morerecent study (Johnson and OrsquoDonnell 2003) direct evi-dence for stimulatory effect of VP on the Na-K-2Cl

co-transporter activity has been provided VP has alsobeen shown to stimulate the activity of the Na-K-2Cl

FIG 7 The changes in neuronal expression of the V1a receptor 6 days after TBI (AB) Beaded axonal processes (arrows) sur-rounding the lateral ventricle (LV) in the ipsilateral and contralateral hemispheres respectively The coronal brain section wascut at the level of septum Note that the V1a receptor-immunopositive axons in the ipsilateral hemisphere have greatly enlargedvaricosities This contrasts with a few beaded axonal processes with small varicosities seen in the contralateral hemisphere Alsonote an intense nuclear staining of ependymal cells (arrowheads) (CD) Fimbria hippocampi (see Fig 3 for anatomical location)in the ipsilateral and contralateral hemispheres respectively Note the presence of numerous axonal processes with large vari-cosities in the ipsilateral fimbria hippocampi These beaded axonal processes are absent in the contralateral side Bar 50 m

co-transporter in cerebral microvascular endothelium(OrsquoDonnell et al 1995) and aortic endothelial cells (Orsquo-Donnell 1991) in which this VP action was in part me-diated by Ca2 Interestingly the stimulation of proteinkinase C inhibited the Na-K-2Cl co-transporter ac-tivity suggesting that protein kinase C plays a negativeregulatory role in VP-mediated control of the activity ofthis co-transporter While these studies have demon-strated the VPrsquos ability to regulate a cell volume it is important to note that in these experiments only short-lasting effects of VP were analyzed It is therefore un-clear whether VP-dependent increase in intracellular wa-ter content would contribute to prolonged swelling ofastrocytes and vascular endothelium observed in the in-jured brain (Dietrich et al 1994 Vaz et al 1997 Caste-joacuten et al 1998) A bumetanide-mediated reduction ofedema found in rats subjected to a transient (Yan et al2001) or a permanent (OrsquoDonnell et al 2003) occlusionof the middle cerebral artery warrants further investiga-tions to clarify the above matter

In addition to astroglia we showed that in the trau-matized parenchyma the expression of V1a receptors isup regulated in the endothelial cells of blood microves-sels and the large-diameter blood vessels The augmentedexpression of the V1a receptors on the cerebrovascularendothelium was observed between 2 and 4 days afterTBI which coincided with the secondary increase in thepermeability of the BBB and the brain water content pre-viously found in the weight-drop and controlled corticalimpact models of brain injury (Holmin and Mathiesen1995 Baskaya et al 1997) It is thus possible that theincreased expression of the endothelial V1a receptorsplays a role in VP-mediated changes in the permeabilityof the BBB and the formation of edema in the injuredbrain Our immunohistochemical analysis did not allowfor the precise localization of V1a receptors to the lumi-nal vs abluminal plasma membrane of endothelial cellsHowever the previous functional studies (Reith et al1987) implied that these receptors are expressed on theluminal side of blood vessels Consistent with the lumi-nal expression of V1a receptors is the reduction in thepermeability of the BBB and the brain water contentfound after peripheral administration of the V1 receptorantagonist in a rat model of cryogenic brain injury (Be-mana and Nagao 1999)

The possible mechanisms underlying the VP-mediatedincrease in the BBB permeability may involve the for-mation of stress fibers in endothelial cells followed byincreased paracellular permeability (Nathanson et al1992 Gohla et al 1999) However it cannot be excludedthat VP modulates the cerebrovascular permeability in-directly by promoting the astrocytic synthesis andor se-cretion of vascular endothelial growth factor (VEGF) a

potent vascular permeability factor (Bates et al 1999)Indeed the processes of the V1a receptor-positive astro-cytes make a close contact with blood microvessels (Fig5AB) and VP has previously been shown to have theability to up-regulate VEGF synthesis (Tahara et al1999) The astroglial expression of this growth factor israpidly increased following TBI (Chodobski et al 2003)and in the injured brain parenchyma many astrocytes ex-pressing high VEGF levels were found to co-express theV1a receptor (data not shown) The redistribution of V1a

receptors from the astrocytic cell bodies to the astrocyticprocesses observed after TBI (Fig 4EF) may facilitatethe access of these receptors by circulating VP thatcrossed the disrupted BBB This concept is also supportedby the observations of post-traumatic increase of VP syn-thesis in the hypothalamus a major source of circulatingVP (unpublished observations)

In the present study an early (within 1ndash2 h after TBI)increase in neuronal expression of V1a receptors in thetraumatized parenchyma was found The immunohisto-chemical analysis revealed the presence of numerous V1a

receptor-immunopositive beaded axonal processes withvastly enlarged varicosities that were localized to vari-ous areas of the injured hemisphere In this context it isinteresting to note that axotomy has previously been re-ported to increase the VP binding and the message forthe V1a receptor in the cranial and spinal motor nuclei(Tribollet et al 1994 Chritin et al 1999) The physio-logical significance of these findings is unclear but maybe related to the neurotrophic activity of VP that has pre-viously been observed in the cultures of cortical neurons(Chen et al 2000)

The real-time RT-PCR analysis indicated that afterTBI an increase in the message preceded the up-regulation of expression of the V1a receptor at the proteinlevel The molecular mechanisms underlying this increasein mRNA for the V1a receptor are currently unknown butit is possible that the expression of this receptor is con-trolled by the transcription factors nuclear factorndashB (NF-B) and AP-1 These transcription factors are activatedafter TBI (Yang et al 1994 1995 Hayes et al 1995Nonaka et al 1999) and the promoter region of the V1a

receptor gene has the putative consensus binding sites forNF-B and AP-1 (Murasawa et al 1995) Further workwill be needed to determine whether NF-B and AP-1play a mediatory role in transcriptional regulation of theV1a receptor expression in the injured brain

In addition to the increased expression of V1a recep-tors observed after TBI a new finding of this study is thenuclear localization of these receptors in neurons thecerebrovascular endothelium and choroidal epithelialcells A similar cellular distribution that is to both theplasma membrane and the nucleus has previously been

described for another G protein-coupled receptor an an-giotensin type 1 (AT1) receptor (Lu et al 1998) Thefunctional studies have also suggested that the nuclear re-ceptors for endothelin-1 exist (Bkaily et al 2000) Thenuclear retention appears to depend on the presence of aputative nuclear localization signal (NLS) consensus se-quence (Boulikas 1993) in the AT1 receptor since a pep-tide containing the putative NLS sequence interfered withthe nuclear targeting of this receptor (Lu et al 1998)Unlike the AT1 receptor the V1a receptor does not havethe putative NLS sequence and therefore other thanNLS-mediated molecular interactions must exist to main-tain the nuclear retention of this latter receptor The phys-iological role of nuclear V1a receptors is presently un-clear The biochemical studies have demonstrated thatunlike endothelin-1 VP does not bind to the nuclei of rathepatocytes (Hocher et al 1992) even though the V1a

receptors are localized to the nuclei of these cells (un-published observations) Further work will therefore berequired to clarify the physiological importance of nu-clear distribution of V1a receptors

ACKNOWLEDGMENTS

We would like to thank Dr Melvyn S Soloff (De-partment of Obstetrics and Gynecology University ofTexas Medical Branch) for his generous gift of anti-V1a

receptor antibody We also thank Virginia Hovanesian atthe Core Research Facilities of Rhode Island Hospital forher assistance in acquiring and processing the confocalmicroscopy images This work was supported by grantfrom NIH NS-39921 (to AC) and by research fundsfrom the Neurosurgery Foundation and Lifespan RhodeIsland Hospital

REFERENCES

BARRECA T GANDOLFO C CORSINI G et al (2001)Evaluation of the secretory pattern of plasma arginine vaso-pressin in stroke patients Cerebrovasc Dis 11 113ndash118

BASKAYA MK RAO AM DOGAN A et al (1997) Thebiphasic opening of the blood-brain barrier in the cortex andhippocampus after traumatic brain injury in rats NeurosciLett 226 33ndash36

BATES DO LODWICK D and WILLIAMS B (1999)Vascular endothelial growth factor and microvascular per-meability Microcirculation 6 83ndash96

BEMANA I and NAGAO S (1999) Treatment of brainedema with a nonpeptide arginine vasopressin V1 receptorantagonist OPC-21268 in rats Neurosurgery 44 148ndash154

BKAILY G CHOUFANI S HASSAN G et al (2000)Presence of functional endothelin-1 receptors in nuclearmembranes of human aortic vascular smooth muscle cells JCardiovasc Pharmacol 36 S414ndashS417

BOULIKAS T (1993) Nuclear localization signals (NLS)Crit Rev Eukaryot Gene Expr 3 193ndash227

CARLOS TM CLARK RSB FRANICOLA-HIGGINSD et al (1997) Expression of endothelial adhesion mole-cules and recruitment of neutrophils after traumatic brain in-jury in rats J Leukoc Biol 61 279ndash285

CASTEJOacuteN OJ (1998) Morphological astrocytic changes incomplicated human brain trauma A light and electron mi-croscopic study Brain Inj 12 409ndash427

CHEN Q PATEL R SALES A et al (2000) Vasopressin-induced neurotrophism in cultured neurons of the cerebralcortex dependency on calcium signaling and protein kinaseC activity Neuroscience 101 19ndash26

CHODOBSKI A CHUNG I KOZNIEWSKA E et al(2003) Early neutrophilic expression of vascular endothelialgrowth factor after traumatic brain injury Neuroscience 122853ndash867

CHOMCZYNSKI P and SACCHI N (1987) Single-stepmethod of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162 156ndash159

CHRITIN M ROQUETTE P SCHULZ MF et al (1999)Up-regulation of vasopressin V1a receptor mRNA in rat fa-cial motoneurons following axotomy Mol Brain Res 70210ndash218

CHUNG I BURKART A SZMYDYNGER-CHODOB-SKA J et al (2003) Expression of two membrane fusionproteins synaptosome-associated protein of 25 kDa and vesi-cle-associated membrane protein in choroid plexus epithe-lium Neuroscience 116 349ndash357

CLARK RSB CARLOS TM SCHIDING JK et al(1996) Antibodies against Mac-1 attenuate neutrophil accu-mulation after traumatic brain injury in rats J Neurotrauma13 333ndash341

DEPASQUALE M PATLAK CS and CSERR HF (1989)Brain ion and volume regulation during acute hypernatremiain Brattleboro rats Am J Physiol 256 F1059ndashF1066

DICKINSON LD and BETZ AL (1992) Attenuated de-velopment of ischemic brain edema in vasopressin-deficientrats J Cereb Blood Flow Metab 12 681ndash690

DIETRICH WD ALONSO O and HALLEY M (1994)Early microvascular and neuronal consequences of traumaticbrain injury a light and electron microscopic study in ratsJ Neurotrauma 11 289ndash301

GOHLA A OFFERMANNS S WILKIE TM et al (1999)Differential involvement of G12 and G13 in receptor-mediated stress fiber formation J Biol Chem 274 17901ndash17907

HAYES RL YANG K RAGHUPATHI R et al (1995)Changes in gene expression following traumatic brain injuryin the rat J Neurotrauma 12 779ndash790

HERNANDO F SCHOOTS O LOLAIT SJ et al (2001)Immunohistochemical localization of the vasopressin V1b re-ceptor in the rat brain and pituitary gland anatomical sup-port for its involvement in the central effects of vasopressinEndocrinology 142 1659ndash1668

HERTZ L CHEN Y and SPATZ M (2000) Involvementof non-neuronal brain cells in AVP-mediated regulation ofwater space at the cellular organ and whole-body level JNeurosci Res 62 480ndash490

HOCHER B RUBENS C HENSEN J et al (1992) Intra-cellular distribution of endothelin-1 receptors in rat livercells Biochem Biophys Res Commun 184 498ndash503

HOLMIN S and MATHIESEN T (1995) Biphasic edemadevelopment after experimental brain contusion in rat Neu-rosci Lett 194 97ndash100

HUANG WD YANG YM and WU SD (2003) Changesof arginine vasopressin in elderly patients with acute trau-matic cerebral injury Chin J Traumatol 6 139ndash141

JOHNSON DM and OrsquoDONNELL ME (2003) Estrogeneffects on the Na-K-Cl cotransporter of astrocytes and blood-brain barrier endothelial cells Soc Neurosci Abst (avail-able online at httpsfnscholaronecomitin2001)

JOYNT RJ FEIBEL JH and SLADEK CM (1981) An-tidiuretic hormone levels in stroke patients Ann Neurol 9182ndash184

KAGAWA M NAGAO S and BEMANA I (1996) Argi-nine vasopressin receptor antagonists for treatment of vaso-genic brain edema an experimental study J Neurotrauma13 273ndash279

KATO Y IGARASHI N HIRASAWA A et al (1995)Distribution and developmental changes in vasopressin V2

receptor mRNA in rat brain Differentiation 59 163ndash169

KIMELBERG HK (1995) Current concepts of brain edemaReview of laboratory investigations J Neurosurg 83 1051ndash1059

LATZKOVITS L CSERR HF PARK JT et al (1993)Effects of arginine vasopressin and atriopeptin on glial cellvolume measured as 3-MG space Am J Physiol 264C603ndashC608

LIU K-F LI F TATLISUMAK T et al (2001) Regionalvariations in the apparent diffusion coefficient and the intra-cellular distribution of water in rat brain during acute focalischemia Stroke 32 1897ndash1905

LU D YANG H SHAW G et al (1998) Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) re-ceptor in brain neurons Endocrinology 139 365ndash375

MATHER HM ANG V and JENKINS JS (1981) Vaso-pressin in plasma and CSF of patients with subarachnoidhaemorrhage J Neurol Neurosurg Psychiatry 44 216ndash219

MURASAWA S MATSUBARA H KIJIMA K et al(1995) Structure of the rat V1a vasopressin receptor geneand characterization of its promoter region and completecDNA sequence of the 3-end J Biol Chem 270 20042ndash20050

NATHANSON MH GAUTAM A NG OC et al (1992)Hormonal regulation of paracellular permeability in isolatedrat hepatocyte couplets Am J Physiol 262 G1079ndashG1086

NIERMANN H AMIRY-MOGHADDAM M HOLTHOFFK et al (2001) A novel role of vasopressin in the brainmodulation of activity-dependent water flux in the neocor-tex J Neurosci 21 3045ndash3051

NONAKA M CHEN X-H PIERCE JES et al (1999)Prolonged activation of NF-B following traumatic brain in-jury in rats J Neurotrauma 16 1023ndash1034

OrsquoDONNELL ME (1991) Endothelial cell sodium-potas-sium-chloride cotransport Evidence of regulation by Ca2

and protein kinase C J Biol Chem 266 11559ndash11566

OrsquoDONNELL ME MARTINEZ A and SUN D (1995)Cerebral microvascular endothelial cell Na-K-Cl cotransportregulation by astrocyte-conditioned medium Am J Physiol268 C747ndashC754

OrsquoDONNELL ME TRAN L LAM TI et al (2003)Bumetanide inhibition of the bloodndashbrain barrier Na-K-Clcotransporter reduces edema formation in the rat middle cere-bral artery occlusion model of stroke FASEB J 17 A76

OSTROWSKI NL LOLAIT SJ and YOUNG WS III(1994) Cellular localization of vasopressin V1a receptormessenger ribonucleic acid in adult male rat brain pinealand brain vasculature Endocrinology 135 1511ndash1528

POVLISHOCK JT HAYES RL MICHEL ME et al(1994) Workshop on animal models of traumatic brain in-jury J Neurotrauma 11 723ndash732

RAICHLE ME and GRUBB RL JR (1978) Regulation ofbrain water permeability by centrally-released vasopressinBrain Res 143 191ndash194

REITH J ERMISCH A DIEMER NH et al (1987) Sat-urable retention of vasopressin by hippocampus vessels invivo associated with inhibition of bloodndashbrain transfer oflarge neutral amino acids J Neurochem 49 1471ndash1479

ROSENBERG GA KYNER WT FENSTERMACHERJD et al (1986) Effect of vasopressin on ependymal andcapillary permeability to tritiated water in cat Am J Phys-iol 251 F485ndashF489

ROSENBERG GA ESTRADA E and KYNER WT(1990) Vasopressin-induced brain edema is mediated by theV1 receptor Adv Neurol 52 149ndash154

ROSENBERG GA SCREMIN O ESTRADA E et al(1992) Arginine vasopressin V1-antagonist and atrial natri-uretic peptide reduce hemorrhagic brain edema in rats Stroke23 1767ndash1773

RUSSELL JM (2000) Sodium-potassium-chloride cotrans-port Physiol Rev 80 211ndash276

SARFARAZ D and FRASER CL (1999) Effects of argi-nine vasopressin on cell volume regulation in brain astrocytein culture Am J Physiol 276 E596ndashE601

SCHOumlNEBERG T KOSTENIS E LIU J et al (1998) Mo-lecular aspects of vasopressin receptor function Adv ExpMed Biol 449 347ndash358

SHUAIB A WANG CX YANG T et al (2002) Effectsof nonpeptide V1 vasopressin receptor antagonist SR-49059on infarction volume and recovery of function in a focal em-bolic stroke model Stroke 33 3033ndash3037

SOslashRENSEN PS GJERRIS A and HAMMER M (1985)Cerebrospinal fluid vasopressin in neurological and psychi-atric disorders J Neurol Neurosurg Psychiatry 48 50ndash57

STRAKOVA Z KUMAR A WATSON AJ et al (1997)A new linear V1A vasopressin antagonist and its use in char-acterizing receptorG protein interactions Mol Pharmacol51 217ndash224

SZOT P BALE TL and DORSA DM (1994) Distribu-tion of messenger RNA for the vasopressin V1a receptor inthe CNS of male and female rats Mol Brain Res 24 1ndash10

TAHARA A SAITO M TSUKADA J et al (1999) Va-sopressin increases vascular endothelial growth factor secre-tion from human vascular smooth muscle cells Eur J Phar-macol 368 89ndash94

THIBONNIER M BERTI-MATTERA LN DULIN N etal (1998) Signal transduction pathways of the human V1-vascular V2-renal V3-pituitary vasopressin and oxytocin re-ceptors Prog Brain Res 119 147ndash161

TRIBOLLET E ARSENIJEVIC Y MARGUERAT A etal (1994) Axotomy induces the expression of vasopressinreceptors in cranial and spinal motor nuclei in the adult ratProc Natl Acad Sci USA 91 9636ndash9640

VAZ R SARMENTO A BORGES N et al (1997) Ultra-structural study of brain microvessels in patients with trau-matic cerebral contusions Acta Neurochir (Wien) 139 215ndash220

YAN Y DEMPSEY RJ and SUN D (2001) Na-K-Cl

cotransporter in rat focal cerebral ischemia J Cereb BloodFlow Metab 21 711ndash721

YANG K MU XS XUE JJ et al (1994) Increased ex-pression of c-fos mRNA and AP-1 transcription factors aftercortical impact injury in rats Brain Res 664 141ndash147

YANG K MU XS and HAYES RL (1995) Increased cor-tical nuclear factor-B (NF-B) DNA binding activity aftertraumatic brain injury in rats Neurosci Lett 197 101ndash104

Address reprint requests to Adam Chodobski PhD

Department of Clinical NeurosciencesBrown University School of Medicine

Aldrich Building Room 405593 Eddy Street

Providence RI 02903

E-mail adam_chodobskibrownedu

INTRODUCTION

CENTRALLY RELEASED VASOPRESSIN (VP) has beendemonstrated to play an important role in the phys-

iological regulation of brain fluid homeostasis (Raichleand Grubb 1978 Rosenberg et al 1986 DePasquale etal 1989 Hertz et al 2000 Niermann et al 2001) How-ever increasing evidence indicates that VP also promotesthe formation of edema in various types of brain injurysuch as cryogenic brain injury cerebral ischemia and intracerebral hemorrhage (Dickinson and Betz 1992Rosenberg et al 1992 Kagawa et al 1996 Bemana andNagao 1999 Shuaib et al 2002) Furthermore centrallyadministered VP has been shown to produce edema inintact animals including VP-deficient Brattleboro rats(Rosenberg et al 1990 Dickinson and Betz 1992)These observations are in line with the increased con-centrations of VP in plasma and cerebrospinal fluid (CSF)found in patients with traumatic brain injury (TBI) ischemic stroke and subarachnoid hemorrhage (Matheret al 1981 Joynt et al 1981 Soslashrensen et al 1985 Bar-reca et al 2001 Huang et al 2003)

Cerebral edema is generally classified into two typescytotoxic and vasogenic (Kimelberg 1995) Cytotoxicedema refers to cellular swelling which usually involvesthe astrocytic cell bodies and processes neuronal den-drites and cerebrovascular endothelium (Liu et al2001) In comparison vasogenic edema is related to aswelling of the brain parenchyma associated with the dis-ruption of the bloodndashbrain barrier (BBB) A leaky bar-rier allows for the interstitial accumulation of blood-borne osmotically active solutes and water which resultsin formation of edema The two types of edema fre-quently coincide and for example in TBI astrocytic andendothelial swelling (Dietrich et al 1994 Vaz et al1997 Castejoacuten et al 1998) is accompanied by the open-ing of the BBB and formation of vasogenic brain edema(Baskaya et al 1997)

VP is a ligand for the three types of receptors V1aV1b and V2 These receptors belong to a large family ofG proteinndashcoupled receptors (Schoumlneberg et al 1998Thibonnier et al 1998) The signal transduction in V1

receptors involves the activation of phospholipases C Dand A2 the production of inositol 145-triphosphate anddiacylglycerol the stimulation of protein kinase C andthe mobilization of intracellular Ca2 (Thibonnier et al1998) In comparison the intracellular signaling in V2 re-ceptors involves the activation of adenylyl cyclase theproduction of adenosine 35-cyclic monophosphate andthe stimulation of protein kinase A (Thibonnier et al1998) While the V1a and V1b receptors are widely dis-tributed within the central nervous system (Ostrowski etal 1994 Szot et al 1994 Hernando et al 2001) the

V2 receptors do not appear to be expressed in any partof the adult brain except the cerebellum (Kato et al1995) These data are consistent with the functional ob-servations made in various models of brain injury inwhich the selective V1 receptor antagonists but not theV2 receptor blockers decreased the permeability of theBBB and reduced edema (Rosenberg et al 1992 Ka-gawa et al 1996 Bemana and Nagao 1999 Shuaib etal 2002)

The aim of the present study was to characterize thechanges in expression of the V1a receptors following TBIOur results demonstrated for the first time that in the in-jured parenchyma there is a considerable increase in ex-pression of V1a receptors in astrocytes neurons and thecerebrovascular endothelium The increased expressionof V1a receptors may play an important role in VP-mediated formation of post-traumatic brain edema

MATERIALS AND METHODS

Reagents and Antibodies

ThermoScript RNase H reverse transcriptase RNaseinhibitor RNaseOut and RNase-free DNase I were ob-tained from Invitrogen (Carlsbad CA) HotStart Taq andFastStart Taq DNA polymerases were purchased fromQiagen (Valencia CA) and Roche Molecular Biochem-icals (Indianapolis IN) respectively SYBR Green I wasfrom Molecular Probes (Eugene OR)

Polyclonal antibody to the V1a receptor a generous gift of Dr Melvyn S Soloff (University of Texas) was raised in rabbits against the synthetic peptide H2N-CHSMAQKFAKDDSDS-COOH This antibody has pre-viously been characterized (Strakova et al 1997) and inthis study affinity-purified antibody was used at 150 dilution In addition to anti-V1a receptor antibody mono-clonal mouse antibodies were used as follows anti-neuronal nuclei (NeuN) (clone A60) anti-glial fibrillaryacidic protein (GFAP) (clone G-A-5) and anti-rat CD31(clone TLD-3A12) from Chemicon International (Temec-ula CA) Anti-NeuN antibody was used at a concentra-tion of 05 gmL whereas other monoclonal antibodieswere applied at a concentration of 2 gmL Secondaryantibodies were purchased from Molecular Probes Thesewere goat anti-rabbit and anti-mouse IgGs conjugatedwith Alexa 594 (red fluorophore) and Alexa 488 (greenfluorophore) respectively They were used at a concen-tration of 4 gmL Normal goat serum was obtained fromJackson Immunoresearch Labs (West Grove PA) Tis-sue-Tek OCT Compound and Vectashield mountingmedium were from Sakura Finetek (Torrance CA) andVector Labs (Burlingame CA) respectively

RESULTS

RT-PCR

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

RESULTS

RT-PCR

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

RESULTS

RT-PCR

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

RESULTS

RT-PCR

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

RESULTS

RT-PCR

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

RESULTS

RT-PCR

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

ACKNOWLEDGMENTS

REFERENCES

Providence RI 02903

Increased Expression of Vasopressin V1a Receptors after Traumatic Brain Injury

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