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First published online 22 November 2005
doi: 10.1242/jcs.02680

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Involvement of aquaporin-4 in astroglial cell migration and glial scar formation

Samira Saadoun^1,3,*, Marios C. Papadopoulos^1,3,*, Hiroyuki Watanabe², Donghong Yan², Geoffrey T. Manley² and A. S. Verkman^1,

¹ Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, 505 Parnassus Avenue, San Francisco, CA 94143-0521, USA
² Department of Neurological Surgery, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA
³ Academic Neurosurgery Unit, St George's University of London, Cranmer Terrace, London, SW17 0RE, UK

View larger version (90K): [in a new window]   Fig. 1. Characterization of cultured astroglia. (A) Confluent wild type (+/+) and AQP4-null (–/–) astroglial cell cultures showing similar morphology by phase-contrast microscopy (left panel) and similar GFAP expression (in red, middle panel). Right panel shows AQP4 immunofluorescence (green). Nuclei are counterstained blue using DAPI. (B) Proliferation of wild-type (AQP4+/+) and AQP4-null (AQP4–/–) astroglia. Cells were counted (eight wells per time point) on alternate days after plating 2.8x104 astroglia/cm2. (C) Percentage of astroglia adhering to poly-L-lysine at 4 hours after plating (ten wells per genotype). B and C show mean±s.e.m.; differences were not significant. Bar, 200 µm.

View larger version (67K): [in a new window]   Fig. 2. Slowed migration of AQP4-deficient astroglia in vitro. (A) Representative images (left) of Transwell migration assay using 10% FBS as chemoattractant, showing wild-type astroglia (+/+) and AQP4-null astroglia (–/–) before (pre) and after (post) scraping the non-migrated cells. Cells were stained with Coomassie Blue. Data summary (right) of migration experiments towards 10% FBS (15 wild-type vs. 13 AQP4-null Transwells; *P<0.001) or 1% FBS (1 wild-type vs. 1 AQP4-null Transwell). (B) Phase-contrast micrographs (left) showing astroglial cultures immediately after (0 h) and 48 hours after scratch. Dashed yellow lines indicate wound edges. Summary (right) of astroglial migration data at 24 and 48 hours after scratch (24 wild-type vs. 24 AQP4-null astroglial cultures per time point). Data are mean±s.e.m. Differences are significant at 24 hours (*P<0.01) and 48 hours (*P<0.001). Bar, 100 µm (A); 400 µm (B).

View larger version (50K): [in a new window]   Fig. 3. Reduced AQP4 expression, water permeability and migration in AQP4 RNAi-treated wild-type astroglia. (A) Phase-contrast micrographs of wild-type astroglial cell cultures after exposure to AQP9 (control) RNAi (left) or AQP4 RNAi (right). (B) Immunoblot of AQP4 expression in wild-type astroglia (+/+) and AQP4-null astroglia (–/–) without pre-treatment (blank), or with pre-treatment (arrow) with AQP4 RNAi, AQP9 RNAi or oligofectamine (+ Oligo). Positions of molecular size markers in kDa are indicated. (C) Osmotic water permeability of astroglial cells with and without AQP4 RNAi treatment. Changes in calcein fluorescence signal (left) in response to hypo-osmolar medium (150 mOsm) and return to iso-osmolarity (300 mOsm). Summary (right) of relative rates of water transport (mean±s.e.m.; n=3-4 cultures/condition; *P<0.01). (D) Summary of Transwell migration data for 1% or 10% serum, labeled as in B. Data are the mean±s.e.m. The number of Transwells used for migration is shown over each data bar in parentheses. *P<0.05 compared with migration of untreated wild-type astroglial cells. Bar, 200 µm.

View larger version (60K): [in a new window]   Fig. 4. Evidence for AQP4-faciliated water transport at the leading edge of migrating cells. (A) Cultured astroglia at the edge of a wound (left) immunostained for AQP4 (green) and the plasma membrane marker WGA (red), with nuclei counterstained with DAPI. Arrow indicates direction of migration. Green:red fluorescence ratios (right) are shown for each of the corresponding pairs of squares shown on the left. Squares were placed at the leading edge (yellow, front) and away from the wound (purple, back). (B) Phase-contrast micrographs (top) and outline (bottom) of the leading end of a migrating wild-type and AQP4-null astroglial cell. Arrows indicate direction of migration. (Numbers are fractal dimensions.) (C) Experimental set-up (top) used for studying the effect of extracellular osmotic gradient on cell migration. Percentage of astroglia that migrated to the bottom surface of the Transwell within 10 hours (bottom), as a function of the difference in osmolality (top chamber minus bottom chamber). Data are mean±s.e.m.; *P<0.005 front vs back. Bar, 25 µm (A); 10 µm (B).

View larger version (79K): [in a new window]   Fig. 5. Astroglial cell migration after stab injury in vivo. (A) Reactive astroglia (arrowheads) stain strongly for GFAP (brown) and have multiple processes. (B-C) Stab injury produces a front of reactive astroglia (blue line) that migrates towards (arrows) the margin of injured brain (red line). Representative data for wild-type mice at 3 days (B) and 7 days (C) after stab injury. (D-E) Astroglial front (blue line) and margin of stab injury (red line) in wild-type (D) and AQP4-null (E) mouse at 3 days. Arrows indicate direction of migration of the reactive astroglial front. (F) Average distance of reactive astroglial front from the edge of the stab wound at 3 days after stab injury. Data are mean±s.e.m. *P<0.001. Bar, 50 µm (A); 100 µm (B-C); 400 µm (D-E).

View larger version (86K): [in a new window]   Fig. 6. AQP4 immunostaining of astroglia in vivo. (A) Control (uninjured) wild-type (+/+) mouse brain parenchyma, showing AQP4 expression in pericapillary astroglial foot processes. (B) Stab-injured wild-type mouse brain showing strong AQP4 immunoreactivity in the astroglial scar. (C) Absence of AQP4 expression in stab-injured AQP4-null (–/–) mouse brain. The blue line demarcates the edge of the glial scar; red lines indicate the margin of injured brain. Each micrograph is representative of three mice. Bar, 100 µm.

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