Glial Cell Aquaporin-4 Overexpression in Transgenic Mice Accelerates Cytotoxic Brain Swelling

Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by ∼2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 ± 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 ± 2 mmHg at 10 min in control mice and 9.8 ± 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant. Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by ∼2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 ± 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 ± 2 mmHg at 10 min in control mice and 9.8 ± 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant. Aquaporin-4 (AQP4) 2The abbreviations used are: AQP4, aquaporin-4; GFAP, glial fibrillary acid protein; ICP, intracranial pressure. 2The abbreviations used are: AQP4, aquaporin-4; GFAP, glial fibrillary acid protein; ICP, intracranial pressure. is a water-selective membrane transport protein expressed in glial cells in brain, particularly at the borders between brain parenchyma and the major fluid compartments in brain (1Rash J.E. Yasumura T. Hudson C.S. Agre P. Nielsen S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11981-11986Crossref PubMed Scopus (498) Google Scholar, 2Furman C.S. Gorelick-Feldman D.A. Davidson K.G. Yasumura T. Neely J.D. Agre P. Rash J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13609-13614Crossref PubMed Scopus (249) Google Scholar). Strong AQP4 expression is found in astroglial cell foot processes at the blood-brain barrier, in glia lining the subarachnoid cerebrospinal fluid space, and in ependyma and subependymal glia lining the ventricular cerebrospinal fluid space. AQP4 expression in glial cells is up-regulated in various brain pathologies, including trauma (3Ghabriel M.N. Thomas A. Vink R. Acta Neurochir. Suppl. 2006; 96: 402-406Crossref PubMed Scopus (40) Google Scholar, 4Neal C.J. Lee E.Y. Gyorgy A. Ecklund J.M. Agoston D.V. Ling G.S. J. Neurotrauma. 2007; 24: 1609-1617Crossref PubMed Scopus (30) Google Scholar), tumor (5Saadoun S. Papadopoulos M.C. Davies D.C. Krishna S. Bell B.A. J. Neurol. Neurosurg. Psychiatry. 2002; 72: 262-265Crossref PubMed Scopus (322) Google Scholar, 6Warth A. Simon P. Capper D. Goeppert B. Tabatabai G. Herzog H. Dietz K. Stubenvoll F. Ajaaj R. Becker R. Weller M. Meyermann R. Wolburg H. Mittelbronn M. J. Neurosci. Res. 2007; 85: 1336-1346Crossref PubMed Scopus (101) Google Scholar), subarachnoid hemorrhage (7Badaut J. Brunet J.F. Grollimund L. Hamou M.F. Magistretti P.J. Villemure J.G. Regli L. Acta Neurochir. Suppl. 2003; 86: 495-498PubMed Google Scholar), ischemia (8Badaut J. FASEB J. 2006; 20: 2423-2424Crossref PubMed Scopus (3) Google Scholar), and inflammation (9Papadopoulos M.C. Verkman A.S. J. Biol. Chem. 2005; 280: 13906-13912Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 10Bloch O. Papadopoulos M.C. Manley G.T. Verkman A.S. J. Neurochem. 2005; 95: 254-262Crossref PubMed Scopus (159) Google Scholar). Whether increased AQP4 expression is functionally significant for water movement between brain and cerebrospinal fluid or blood is not known, as is whether AQP4 is rate-limiting for water movement across the blood-brain barrier. Because water movement from blood to brain parenchyma involves water transport across a tight endothelial cell layer, which does not express AQPs, followed by transport across AQP4-expressing glial cell foot processes surrounding brain microvessels, water movement into the brain may be limited by the endothelial barrier. Evidence from AQP4 knock-out mice indicates slowed water movement both into and out of the brain in AQP4 deficiency, resulting in opposite consequences for cytotoxic versus vasogenic brain edema (reviewed in Ref. 11Verkman A.S. Binder D.K. Bloch O. Auguste K. Papadopoulos M.C. Biochim. Biophys. Acta. 2006; 1758: 1085-1093Crossref PubMed Scopus (252) Google Scholar). AQP4 knock-out mice have reduced brain swelling and improved survival when compared with control, wild-type mice following water intoxication and reduced hemispheric swelling after focal cerebral ischemia (12Manley G.T. Fujimura M. Ma T. Noshita N. Filiz F. Bollen A.W. Chan P. Verkman A.S. Nat. Med. 2000; 6: 159-163Crossref PubMed Scopus (1286) Google Scholar). AQP4-null mice also have greatly improved survival in a mouse model of bacterial meningitis (9Papadopoulos M.C. Verkman A.S. J. Biol. Chem. 2005; 280: 13906-13912Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). According to the Klatzo classification of brain edema, these are primarily models of cytotoxic (cell swelling) edema in which excess water moves from the vasculature into the brain parenchyma through an intact blood-brain barrier. AQP4 also facilitates the elimination of excess brain water. When the blood-brain barrier becomes disrupted as in brain tumor or abscess, water moves from the vasculature into the brain extracellular space in an AQP4-independent manner to form vasogenic edema. Excess water is eliminated primarily through the glia limiting membranes into the cerebrospinal fluid. Greater brain water accumulation and intracranial pressure were found in AQP4-null versus wild-type mice with brain tumor, brain abscess, focal cortical-freeze injury, and after infusion of normal saline directly into brain extracellular space (10Bloch O. Papadopoulos M.C. Manley G.T. Verkman A.S. J. Neurochem. 2005; 95: 254-262Crossref PubMed Scopus (159) Google Scholar, 13Papadopoulos M.C. Manley G.T. Krishna S. Verkman A.S. FASEB J. 2004; 18: 1291-1293Crossref PubMed Scopus (626) Google Scholar), indicating that vasogenic edema fluid is eliminated by an AQP4-dependent route. Also, in a kaolin injection model of obstructive hydrocephalus, AQP4-null mice develop more marked hydrocephalus than wild-type mice (14Bloch O. Auguste K.I. Manley G.T. Verkman A.S. J. Cereb. Blood Flow Metab. 2006; 26: 1527-1537Crossref PubMed Scopus (160) Google Scholar), probably due to reduced water clearance in AQP4-null mice through the ependymal and blood-brain barriers. These studies support the view that AQP4 is a bidirectional water channel that facilitates water transport into and out of the brain. The studies using AQP4 knock-out mice, however, do not address whether increased AQP4 expression, as occurs in various types of brain injuries and pathologies, could have functional consequences. If endothelial cells in brain microvessels are rate-limiting for water transport under normal conditions, then further increases in AQP4 expression in glial cell foot processes would be without effect. To address this question, we generated glial cell-targeted, AQP4-overexpressing mice using a glial fibrillary acid protein (GFAP) promotor strategy and investigated whether AQP4 overexpression would accelerate cytotoxic brain edema by increasing water transport across the blood-brain barrier. GFAP is expressed in most glial cells, including those lining the blood-brain barrier. The data show accelerated brain water accumulation following acute water intoxication, indicating that under normal conditions, glial cell AQP4 is rate-limiting for water movement into the brain, and thus, that altered AQP4 expression can be functionally significant. Transgenic Mice—A 1.54-kb fragment of mouse AQP4 cDNA (121–1661, GenBank™ accession number NM_009700), containing the full coding region (including the ATG translational start site and the TAG stop codon) and the 3′-untranslated and poly-A sequences, was PCR-amplified from mouse brain cDNA and subcloned into pGEM3Z (Promega). A cytomegalovirus enhancer sequence (∼0.3 kb, subcloned from pcDNA3, Invitrogen) and a PCR-amplified ∼2.2-kb GFAP promoter sequence (4–2177, GenBank accession number M67446) were inserted upstream of the AQP4 cDNA sequence (see Fig. 1A). A ∼4-kb EcoRI/HindIII fragment containing the cytomegalovirus enhancer, GFAP promoter, AQP4 cDNA coding region, and 3′-untranslated and poly-A sequences was isolated, purified, and injected into pronuclei of C57 BL/6JxCBA F1 fertilized oocytes. Fertilized oocytes were transferred into pseudopregnant C57 BL/6JxCBA F1 mice. F0 generation mice carrying the GFAP-AQP4 transgene construct were identified by genomic PCR analysis of tail DNA using sense primer (5′-TGGTCTGGCTCCAGGTACCAC-3′) from the GFAP promoter and antisense primer (5′-AGCAATGCTGAGTCCAAAGC-3′) from the AQP4 cDNA coding region. PCR-positive mice were bred with wild-type CD1 to establish heterozygotic GFAP-AQP4 transgenic strains. Mouse lines with the strongest AQP4 expression were identified by immunoblot analysis of brain homogenates in F1 generation offspring. AQP4 knock-out mice in a CD1 genetic background, generated by targeted gene disruption (15Ma T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Clin. Investig. 1997; 100: 957-962Crossref PubMed Scopus (395) Google Scholar), were used as well. Protocols were approved by the University of California, San Francisco, Committee on Animal Research. Histology and Immunofluorescence—Tissues for histological examination was immersion-fixed in neutral buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin according to standard protocols. For immunofluorescence, brain tissue was fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 24 h and then equilibrated with 30% sucrose in phosphate-buffered saline overnight at 4 °C. Frozen (6-μm) sections were blocked with 3% nonfat milk and then incubated overnight at 4 °C with rabbit anti-AQP4 polyclonal antibody (1:500 dilution, Santa Cruz Biotechnology) and mouse anti-GFAP antibody (1:1000 dilution, Chemicon), washed with phosphate-buffered saline, and incubated with Cy3-labeled anti-rabbit IgG secondary antibody (Sigma) and fluorescein isothiocyanate-labeled anti-mouse secondary antibody (Invitrogen). Sections were also 4′,6-diamidino-2-phenylindole-stained and mounted with Vectashield mounting medium (Vector Laboratories). Immunoblot Analysis—Brains were homogenized by 20 strokes of a glass Dounce homogenizer in 250 mm sucrose, 10 mm Tris-HCl, pH 7.4, 0.2 mm EDTA, 20 μg/ml phenylmethylsulfonyl fluoride. Nuclei were removed by centrifugation at 500 × g for 10 min at 4 °C. Protein concentration was determined in the supernatant using the DC protein assay kit (Bio-Rad). Proteins (5 μg of protein/lane) were resolved by SDS-PAGE, transferred to Hybond-P membranes (GE Healthcare), blocked with 5% nonfat milk, and incubated with rabbit anti-AQP4 (1:1000 dilution) or rabbit anti-β-actin (1:2000 dilution, Santa Cruz Biotechnology). Detection was done using the ECL Plus Western blotting detection system (GE Healthcare). Reverse Transcription-Polymerase Chain Reaction—Brain tissues were stored in RNA Later™ solution (Ambion). Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was reverse-transcribed from mRNA using random primers (SuperScript III first-strand synthesis system, Invitrogen). Fluorescence-based real-time PCR was carried out using the LightCycler with LightCycler FastStart DNA MasterPLUS SYBR kit using and for and and for sequences were from GenBank accession and PCR was carried out according to the using as gene and wild-type cDNA as the are as to the were by intraperitoneal of and in a was and at °C. In the was using to blood A was mm to the of the the a of was mm to and mm to A pressure was inserted through the and the between the of the and the was with tissue ICP was at using a GFAP-AQP4 transgenic mice with wild-type mice and AQP4 knock-out mice were used for functional with of water accumulation across the blood-brain barrier was using the acute water intoxication model (12Manley G.T. Fujimura M. Ma T. Noshita N. Filiz F. Bollen A.W. Chan P. Verkman A.S. Nat. Med. 2000; 6: 159-163Crossref PubMed Scopus (1286) Google Scholar, M. R. Neely J.D. A. Agre P. S. FASEB J. 2004; 18: PubMed Scopus Google Scholar), in which water is through producing In mice for ICP as a of water was with to water The water from the into the cerebral parenchyma through the blood-brain barrier, which is by To the of following water intoxication, concentration was determined at following intraperitoneal water Blood were through a protein was removed by and concentration was determined by after of ICP were to ICP, at 10 and 20 min after water intoxication, the of ICP increase at 10 min, and to and of AQP4-overexpressing Transgenic transgenic mice, C57 BL/6JxCBA injected with the transgene construct in Fig. were into C57 BL/6JxCBA producing F0 generation mice. of the F0 mice carried the GFAP-AQP4 transgene as determined by genomic PCR analysis using primers for the transgene sequence are in Fig. analysis of from of F0 generation GFAP-AQP4 mice with wild-type mice F1 generation mice with carrying the GFAP-AQP4 transgene GFAP-AQP4 mice, wild-type indicating The mouse producing with the brain AQP4 protein expression immunoblot was used as the to establish an AQP4-overexpressing transgenic mouse The GFAP-AQP4 mice had normal survival, appearance, and and and bred was significant in mouse of GFAP-AQP4 versus wild-type mice the 10 of not was however, that in of wild-type brain AQP4 expression the of was the of mice carried the GFAP-AQP4 indicating of the GFAP-AQP4 transgene from the in However, the genetic of the mouse for a number of AQP4-overexpressing mice could be generated for and brain swelling PCR analysis of RNA from brain homogenates that the GFAP-AQP4 mice had expression of mRNA AQP4 than wild-type mice AQP4 mRNA was not in AQP4 knock-out mice, as Fig. immunoblot analysis of brain homogenates from mice at The protein which was in AQP4-null mice, was increased ± using in the GFAP-AQP4 mice. AQP4 protein expression was not different in of GFAP-AQP4 mice and wild-type mice not as cells do not express These data that AQP4 transgene expression by the GFAP promoter increases AQP4 protein expression in glial To that GFAP-AQP4 transgene expression had on glial cell or brain and GFAP were compared in GFAP-AQP4 and wild-type mice. The of major cerebral as as the of membranes and cerebral was in wild-type versus GFAP-AQP4 mice AQP4 a AQP4 protein in the GFAP-AQP4 and wild-type mice, with expression in the GFAP-AQP4 mice. AQP4 and GFAP protein were in the cells with AQP4 and antibody the of AQP4 protein in in the GFAP-AQP4 mice not These glial AQP4 overexpression in brain in GFAP-AQP4 mice. ICP in a of mice using an pressure ICP did not in GFAP-AQP4 versus wild-type mice or in AQP4 knock-out mice. Fig. an ICP a GFAP-AQP4 in which a of water was as by the a ICP ICP increased with increases at min, to brain herniation and death. Fig. the of following intraperitoneal water in a of mice. Fig. ICP for mice of ICP increases were in GFAP-AQP4 mice than in wild-type mice or AQP4-null mice. herniation and were by min in of GFAP-AQP4 mice, of wild-type mice, and 30% of AQP4 knock-out mice. were deduced from ICP data from a of mice. Fig. the increase in ICP from at 10 and 20 min after water the of ICP increase at 10 min, the of ICP and the at which the of ICP increase was AQP4 overexpression increased at 10 and 20 min, as as at 10 min and and the to in the opposite were AQP4-null and wild-type mice. Fig. between ICP with brain AQP4 protein at 10 min following intraperitoneal of water and Fig. a between brain AQP4 expression and the increase in brain water at 10 min, deduced from and brain in is by the following is the increase in brain water at 10 min, is ICP, and is ICP at 10 min A. K. R. J. Neurosurg. PubMed Scopus Google Scholar). between brain water at 10 min of water of the blood-brain and AQP4 protein expression, as found in Fig. is glial AQP4 is rate-limiting for brain water accumulation across the blood-brain barrier AQP4 In contrast, a is the endothelial cell is rate-limiting occurs on the endothelial cell versus glial water The is that glial cell-targeted, AQP4-overexpressing mice accelerated brain water accumulation in a water intoxication model of cytotoxic brain edema. water acute an for water into the brain across an intact blood-brain barrier. The accelerated increase in ICP and water into the brain in AQP4-overexpressing mice indicates that the AQP4 expression in control mice increases the water of the blood-brain barrier, that AQP4 and glial water are of is that altered AQP4 expression in various brain can be functionally significant by increasing water movement into the brain. and in studies show altered AQP4 expression in to various AQP4 and protein expression were reduced by in to D. 2007; PubMed Scopus Google Scholar). AQP4 expression was increased in with inflammation produced by J. H. T. K. H. 2007; PubMed Scopus Google Scholar). In mice, greatly increased AQP4 expression was found following M.C. Manley G.T. Krishna S. Verkman A.S. FASEB J. 2004; 18: 1291-1293Crossref PubMed Scopus (626) Google Scholar), brain (10Bloch O. Papadopoulos M.C. Manley G.T. Verkman A.S. J. Neurochem. 2005; 95: 254-262Crossref PubMed Scopus (159) Google Scholar), and bacterial meningitis (9Papadopoulos M.C. Verkman A.S. J. Biol. Chem. 2005; 280: 13906-13912Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). AQP4 expression correlated with brain water content in a mouse model of cerebral ischemia (8Badaut J. FASEB J. 2006; 20: 2423-2424Crossref PubMed Scopus (3) Google Scholar). water content from correlated with AQP4 expression in the tumor tissue (5Saadoun S. Papadopoulos M.C. Davies D.C. Krishna S. Bell B.A. J. Neurol. Neurosurg. Psychiatry. 2002; 72: 262-265Crossref PubMed Scopus (322) Google Scholar, 6Warth A. Simon P. Capper D. Goeppert B. Tabatabai G. Herzog H. Dietz K. Stubenvoll F. Ajaaj R. Becker R. Weller M. Meyermann R. Wolburg H. Mittelbronn M. J. Neurosci. Res. 2007; 85: 1336-1346Crossref PubMed Scopus (101) Google Scholar). AQP4 expression was also found in brain in C.J. Lee E.Y. Gyorgy A. Ecklund J.M. Agoston D.V. Ling G.S. J. Neurotrauma. 2007; 24: 1609-1617Crossref PubMed Scopus (30) Google and in (3Ghabriel M.N. Thomas A. Vink R. Acta Neurochir. 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A. 2003; 100: PubMed Scopus Google Scholar). have used to brain water accumulation, including in mice, ICP, brain water glial cell swelling, and water accumulation occurs by an in which reduced water into the brain through an intact blood-brain barrier. intraperitoneal water injection, water moves into the blood which with an for brain water in Fig. following intraperitoneal water We intraperitoneal water injection and ICP for studies of cytotoxic brain water accumulation it is an model that brain water or of fluid more control of of fluid would to be which would with water and in the of the model as the limited of AQP4-overexpressing mice for these the of a model with and In analysis of glial cell AQP4-overexpressing mice the that AQP4 expression in glial cell foot processes at the blood-brain barrier is and thus, that altered expression of AQP4 under is to brain water be to the of AQP4 overexpression on of cytotoxic edema, as and injury, as as on various of vasogenic edema and We the Transgenic for DNA and for mouse and