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(The FASEB Journal. 2007;21:108-116.)
© 2007 FASEB

Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury

Kurtis I. Auguste*,{dagger}, Songwan Jin*, Kazunori Uchida{ddagger}, Donghong Yan{dagger}, Geoffrey T. Manley{dagger}, Marios C. Papadopoulos*,{ddagger} and A. S. Verkman*

Departments of
* Medicine and Physiology, and

{dagger} Neurological Surgery, University of California, San Francisco, California, USA; and

{ddagger} Academic Neurosurgery Unit, St. George’s University of London, London, UK

1Correspondence: 1246 Health Sciences East Tower, University of California, San Francisco, 505 Parnassus Ave., San Francisco, CA 94143-0521, USA. E-mail: alan.verkman{at}ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We reported previously that astroglia cultured from aquaporin-4-deficient (AQP4–/–) mice migrate more slowly in vitro than those from wild-type (AQP4+/+) mice (J. Cell Sci. 2005;118, 5691–5698). Here, we investigate the migration of fluorescently labeled AQP4+/+ and AQP4–/– astroglia after implantation into mouse brains in which directional movement was stimulated by a planar stab wound 3 mm away from the axis of the injection needle. Two days after cell injection we determined the location, elongation ratio, and orientation of labeled cells. Migration of AQP4+/+ but not AQP4–/– cells toward the stab was greater than away from the stab. AQP4+/+ astroglia moved on average 1.5 mm toward the stab compared with 0.6 mm for AQP4–/– cells. More than 25% of the migrating AQP4+/+ cells but <3% of AQP4–/– cells appeared elongated (axial ratio>2.5). In transwell assays, AQP4+/+ astroglia migrated faster than AQP4–/– cells in a manner dependent on pore size. At 8 h, ~50% of AQP4+/+ cells migrated through 8-µm diameter pores, whereas equivalent migration of AQP4–/– cells was found for 12-µm diameter pores. These results provide in vivo evidence for AQP4-dependent astroglial migration and suggest that modulation of AQP4 expression or function might alter glial scarring—Auguste, K. I., Jin S., Uchida K., Yan D., Manley G. T., Papadopoulos M. C., Verkman A. S. Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury.


Key Words: AQP4 • astrocyte • chemotaxis • reactive gliosis • water channel


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE AQUAPORINS (AQPS) ARE A FAMILY of water channel proteins expressed in multiple tissues (1 , 2) . AQPs are found primarily in plasma membranes and are organized as tetramers, with each monomer containing its own water pore. AQP4, the main water channel in the brain, is strongly expressed in astroglial cell plasma membranes (3 , 4) . AQP4 facilitates the flow of water into and out of the central nervous system (CNS) (5 , 6) and modulates neural signal transduction (7 8 9) . We recently discovered a new role for AQPs in cell migration. Targeted AQP1 deletion in mice impairs the migration of vascular endothelial cells (10) and proximal renal tubule cells (11) , and AQP3 deletion impairs the migration of corneal epithelial cells (12) . Chinese hamster ovary and Fisher rat thyroid cells transfected with AQP1 or AQP4 migrate faster than control, nontransfected cells (10) . AQPs are also expressed in many types of malignant tumor cells (13 14 15 16 17) , where they appear to enhance tumor spread and metastasis by increasing tumor cell migration (18) . AQPs polarize to protrusions at the leading edge of migrating cells, which may facilitate water transport across lamellipodia where rapid changes in ion fluxes and actin turnover occur.

We found that cultured AQP4 knockout and knockdown cortical astroglial cells migrate more slowly than AQP4-expressing astroglial cells in in vitro assays of cell migration (19) . However, it is not known whether AQP4 plays a role in astroglial cell migration in vivo. Several key components of intact brain cannot be reproduced in culture models, including the extracellular matrix (ECM) and extracellular space geometry, which profoundly influence astroglial cell migration. A major impediment in studying astroglial cell migration in vivo has been the lack of suitable animal models and measurement methods.

Astroglial cell migration in the adult CNS is a fundamental component of glial scarring. In mammals, the glial scar has beneficial, as well as harmful properties: it enables repair of the blood-brain barrier, inhibits the entry of inflammatory cells into damaged brain, and limits neuronal death (20 21 22) , but also impairs axonal regeneration (23) and incorporation of neuronal grafts (24) . The aim of this paper was to determine whether AQP4-expressing (AQP4+/+) astroglial cells migrate faster than AQP4-null (AQP4–/–) astroglial cells in mouse brain in vivo. To achieve this, we developed a mouse model of astroglial cell migration that involves stereotactic injection of fluorescently labeled astroglial cells into brain parenchyma, in which a previously created stab wound induces directional migration. The in vivo experiments showed that AQP4 deletion produced an even more dramatic slowing of astroglial cell migration than suggested by prior in vitro data, which was further investigated by measurements of astroglial cell migration through pores of different sizes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
AQP4 null mice were generated by targeted gene disruption (25) in a CD1 genetic background and were bred to produce neonatal mice for cell culture. Adult wild type (WT) CD1 mice were used for cell implantations in migration studies. Protocols were approved by the University of California, San Francisco Committee on Animal Research.

Astroglial cultures
Astroglia were cultured from neocortex of WT and AQP4 null neonatal mice, as described previously (26) . Briefly, the cerebral hemispheres were isolated, minced with forceps, and incubated in minimal essential medium (MEM) plus 0.25% trypsin and 0.01% DNase. Dissociated cells were centrifuged, resuspended in MEM containing 10% FBS, seeded on poly-L-lysine-coated flasks and grown at 37°C in a 5% CO2 incubator with a change of medium every 2 days. At confluence, cultures were treated with 10 µM cytosine arabinoside for 48 h to prevent proliferation of other cell types, and the medium was replaced with MEM containing 3% FBS. Differentiation factors, such as dibutyryl cAMP, were not used.

Adhesion assay
Confluent cultured astroglia were trypsinized and suspended in MEM + 3% FBS. After determination of cell density using a hemocytometer, 104 cells were added to each well in 24-well plates (Corning Costar, Fisher Scientific, Pittsburgh, PA) coated with poly-L-lysine. Medium was exchanged 4 h after plating. Adhesion was defined as the percentage of plated cells remaining after medium exchange.

Astroglial cell fluorescent labeling
Cell cultures were washed with MEM and then incubated with 5 µM Cell Tracker Red dye CMTPX (Molecular Probes, Invitrogen, Carlsbad, CA) for 30–45 min at 37°C. Cells were washed 4 times with PBS and trypsinized for 5–10 min. Serum-containing medium was added, and the suspension was centrifuged at 1,000 rpm for 2–3 min. The pellet was resuspended in PBS to a concentration of 4–8 x 104 cells/µl and kept on ice until injection.

In vivo astroglial cell migration
WT mice (age 6–8 wk, ~30 g) were anesthetized with intraperitoneal (i.p.) 2,2,2-tribromoethanol, and the head was immobilized in a stereotactic frame (David Kopf Instruments, Tujunga, CA). The scalp was opened with a sagittal incision along the midline. A 3 x 2.5 x 0.1 mm razor blade was mounted to the stereotactic frame and used to create a coronal stab injury, located 2 mm posterior to bregma and 1 mm right lateral to midline. The bone at this site was thinned with a 0.7 mm bit on a high-speed drill (Foredom Micro Motor FM3545, Blackstone Industries, Bethel, CT). The blade was inserted through the skull to a depth of 2.5 mm and immediately removed. Two days after stab injury, fluorescently labeled cells (1 µl, 4 – 8x104 cells/µl) were injected using a 30-gauge needle attached to a Hamilton syringe through a burr hole 1 mm anterior to bregma, 2 mm right lateral to midline at a depth of 2.5 mm. Mice were killed 2 days after cell injection by anesthetic overdose and underwent transcardiac perfusion of PBS followed by 10% formalin. Brains were kept overnight at 4°C in formalin, transferred to 30% sucrose in PBS, and then embedded in OCT.

In vitro migration studies
Migration was assayed using a modified Boyden chamber (Corning Costar, Fisher Scientific, Pittsburgh, PA) containing a porous polycarbonate membrane filter (6.5 mm diameter) coated with poly-L-lysine between an upper and lower chamber (19) . Membrane filters containing pores of different diameters were used: 3, 5, 8, and 12 µm. The upper chamber was plated with 104 astroglial cells in Dulbecco’s modified Eagle medium (DMEM) + 1% FBS. The lower chamber contained DMEM + 10% FBS (chemoattractant) or 1% FBS (control). Cells were allowed to migrate for 8 h at 37°C in 5% CO2/95% air, and then stained with Coomassie blue for 1 h, photographed, and counted. The upper surface of the membrane was then scraped with a cotton swab, and cells migrating to the bottom surface of the porous filter were counted. Cells trapped in pores were counted separately from cells not associated with pores.

Immunocytochemistry
Cultured astroglial cells and axial brain sections were fixed in 10% formalin and incubated with 1:200 rabbit anti-AQP4 or 1:1,000 rabbit anti-GFAP antibody (Ab) (Chemicon, Temecula, CA), followed by FITC- or Texas Red-conjugated anti-rabbit secondary Ab (Vector Laboratories, Burlingame, CA) at 1:80 dilution. Nuclei were counterstained blue with 4,6-diamidino-2-phenylindole (4',6'-diamidino-2-phenylidole (DAPI)). Sections were visualized with an inverted fluorescence microscope equipped with a high-resolution Spot color cooled CCD camera (Diagnostic Instruments, Sterling Heights, MI).

Analysis of in vivo cell migration
Image analysis software was developed using Matlab (Mathworks). Injection sites and stab injuries were located in low magnification (x5) images (see Fig. 2 ). A virtual x-y coordinate plane was then applied to each section with the injection site as the origin (coordinate 0, 0). The migration x axis was oriented perpendicular to a best-fit line overlying the stab injury. Images at x50 magnification were used for determination of cell position, elongation, and orientation. The injection site location was defined as the center of a cluster of pixels with lower intensity than a local threshold (typically, 10–30 for 8-bit image). The locations of fluorescent cells were determined as bodies with pixels greater than a set threshold. Because of uneven background intensity, local (rather than global) thresholding was used, in which the threshold was interactively modified to be 1.2–1.5 times brighter than that of local background. The coordinates of the cell center (xi, yi) were determined as the mean position of cell body pixels. A user-directed rejection of identified cells was included as an option, as well as user-directed inclusion of additional labeled cells in the analysis. The major axis of each labeled cell was determined by linear regression analysis of cell body pixels, giving a cell orientation angle, {theta}cell, computed from the slope of cell axis (acell): {theta}cell = tan–1(acell). The angle between the x axis and a line connecting the injection site and the cell location was defined as the migration angle, {theta}mig. {theta}cell ranged from 0 to 180 , and {theta}mig ranged from –180 to 180 . Also, an elongation factor, eL, for each labeled cell was computed as the ratio of the long-to-short axes of the cell, defined as the longest length parallel and perpendicular, respectively, to the major cell axis.


Figure 1
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  		Figure 1. Stab injury/cell injection model of astroglial cell migration in brain. Two days before cell injection, a 3-mm length, 2.5-mm-deep stab was created 2 mm posterior to bregma and 1 mm right lateral to midline. Cells were injected through a 0.7-mm diameter needle 1 mm anterior to bregma, 2 mm right lateral to midline, and 2.5 mm deep. A) View from top. B) Three-dimensional diagram depicting planar section, showing stab and injection sites and x,y-axes. C) DAPI-stained low-magnification section of mouse brain at 2 days after cell injection, showing stab and injection sites.


Figure 2
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  		Figure 2. In vivo migration of fluorescently stained astroglial cells injected into mouse brain. A) Low- and higher-magnification images of mouse brain section at 2 days after injection of red fluorescently stained astroglial cells. Fluorescent cells shown as red dots in the low-magnification panel on the left. B) Schematic showing x,y coordinate system used to quantify locations of fluorescently stained cells in brain sections. C) Colocalization of red fluorescent cells with the astroglial cell marker GFAP, immunostained in green. D) Expression of AQP4, immunostained in green, in red fluorescent cells (cultured from AQP4+/+ mice).

Water permeability measurements
As described previously (26) , astroglial cells cultured on round coverglasses were loaded with calcein by incubation for 15 min with 5 µM calcein-AM (Molecular Probes) at room temperature. After rinsing in PBS (pH 7.4), coverglasses were mounted in a perfusion chamber. Osmotic water permeability was measured from the kinetics of cytoplasmic calcein fluorescence on perfusate exchange between PBS and hypotonic saline (diluted 1:1 with distilled water). Measurements were made on a Nikon inverted epifluorescence microscope equipped with halogen light source, calcein filter set, x40 oil immersion lens, and photomultiplier detector.

Statistical analysis
Statistical analysis was performed with a two-tailed indirect Student’s t test, Mann-Whitney-U test, or Student-Newman-Keuls test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As diagrammed in Fig. 1 A, a 2.5-mm-deep stab injury was created 2 mm posterior to bregma and 1 mm right lateral to midline. Two days later, fluorescently labeled astroglia were injected 1 mm anterior to bregma, 2 mm right lateral to midline, and 2.5 mm deep from the brain surface. These locations and the time between stab injury and cell injection were chosen based on pilot studies and prior work (6 , 27) , to give a maximal stimulus for astroglial cell migration in a region of brain devoid of major structures, such as large vessels, ventricles, and major white matter tracts. The cut view in Fig. 1B shows the locations of the stab injury and injection site, as well as a planar section of brain cut perpendicular to the stab injury and the axis of the injection needle. We defined (x, y) coordinates in the plane as indicated, with (0, 0) as the injection site. Figure 1C shows a low magnification fluorescence micrograph of DAPI-stained mouse brain, with injection site, stab injury, and x,y-axes indicated.

Brains were fixed two days after injection of fluorescently labeled cells, at which time substantial astroglial cell migration toward the stab injury was seen in pilot experiments. Figure 2 A shows a low-magnification brain section, with locations of fluorescent cells indicated. The red, fluorescently stained cells were visualized at higher magnification in the fluorescence micrographs on the right, which were used to determine cell coordinates. Figure 2B depicts labeled cells at coordinates (xi, yi), with the site of injection at (0, 0), and the stab injury at (xstab, y), with xstab ~3 mm.

Immunostaining was done to verify that the red fluorescent migrating cells were astroglia. Figure 2C shows colocalization of red fluorescent cells with the astroglial marker GFAP, which was immunostained green. Examination of multiple sections in brains injected with AQP4+/+ or AQP4–/– cells indicated that >85% of red fluorescent cells were GFAP positive. The red fluorescent AQP4+/+ cells strongly expressed AQP4 protein (Fig. 2D ). Lower AQP4 staining was seen in endogenous astroglial cells. No AQP4-positive cells were in brain sections from AQP4–/– mice (not shown), as expected.

Experiments were done to test whether migration of AQP4–/– cells is impaired in brain in vivo. Initial studies, where brains were coinjected with a mixture of red fluorescently stained AQP4+/+ cells and green fluorescently stained AQP4–/– cells, showed remarkably more AQP4+/+ than AQP4–/– cells far from the injection site (Fig. 3 A). For quantitative experiments, to avoid potential confounding effects of fluorescent dye brightness and other properties, we injected (wild-type) mice with red fluorescently stained AQP4+/+ or AQP4–/– cells.


Figure 3
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  		Figure 3. Impaired migration of AQP4–/– astroglial cells in vivo. A) Fluorescence micrograph of brain section at 2 days after injection of a mixture of an equal number of red-stained AQP4+/+ and green-stained AQP4–/– astroglial cells. Arrowheads denote fluorescent cells. Injection site shown as an open circle. Scale bar, 100 µm. B) Locations of migrating fluorescently stained AQP4+/+ cells (green) and AQP4–/– cells (orange). The x axis is shown as percentage of distance between injection and stab injury sites (xrel). Data obtained from 15 different mice. C) Histogram analysis (particle density function) summarizing number of fluorescent cells along the xrel (top) and yrel (bottom) axes.

The locations of red fluorescent AQP4+/+ and AQP4–/– cells in brain sections from a series of mice are summarized in Fig. 3B . The x-axis is shown as the relative distance between the injection site (xrel=0) and stab injury site (xrel=1). Negative x-values correspond to cells that migrated from the injection site in a direction opposite to the stab injury. The data summary indicates increased cell movement toward vs. away from the stab injury for AQP4+/+ cells, though nearly comparable movement toward vs. away from the stab for AQP4–/– cells. Most remarkably, there was greater movement of AQP4+/+ vs. AQP4–/– cells toward the stab. Figure 3C shows histograms of x and y cell coordinates of the migrating fluorescent cells. At 2 days after cell injection, there was marked movement of AQP4+/+ cells toward the stab, with averaged xrel of 0.50 (Fig. 3C , top). There was also marked asymmetry in x-direction, with a ratio of 5.6:1 cells moving toward:away from the stab. The x-direction ratio was near unity (1.1:1) for the AQP4–/– cells, with averaged xrel of 0.20. The yrel histogram (Fig. 3C , bottom) showed no significant y-asymmetry, as anticipated. Also, there was greater movement of AQP4+/+ vs. AQP4–/– cells in the y-direction.

Figure 4 A shows a gallery of AQP4+/+ and AQP4–/– red fluorescent cells at high magnification. Inspection of red fluorescent cells revealed a significant fraction of elongated AQP4+/+ cells, particularly cells far from the injection site, which was not seen for AQP4–/– cells. To quantify cell shape, image processing was done as depicted in Fig. 4B . After establishing the cell boundary, an elongation factor, eL, was computed. An eL of 1 indicates a round, nonelongated cell. Figure 4C (left) summarizes eL measured for AQP4+/+ and AQP4–/– cells in sections from 15 mice. Remarkably more elongated AQP4+/+ vs. AQP4–/– cells were seen. Because 2-dimensional planar sections were analyzed, the absolute fraction of elongated cells is likely to be underestimated, as out-of-plane projections would appear shortened. Figure 4C (right) shows the relationship between eL and distance away from the migration site. Correlation was seen for AQP4+/+, but not AQP4–/– cells, with a greater propensity for elongated AQP4+/+ cells far from the injection site.


Figure 4
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  		Figure 4. Analysis of shape and orientation of migrating astroglial cells. A) Gallery of high-magnification fluorescence micrographs of migrating AQP4+/+ and AQP4–/– cells. Scale bar, 5 µm. B) High-magnification image of fluorescently stained cell (left, scale bar, 5 µm), with schematic (right) showing cell axis (dashed red line) with long and short cell dimensions depicted (whose ratio gives the elongation factor, eL). The angle between the cell axis and a line connecting the injection site and cell position shown as {Delta}{theta} (see text for explanations). C) left: Distribution of eL for AQP4+/+ and AQP4–/– cells (from 15 mice). Difference in mean eL significant at P < 0.001. C) right: Correlation between distance from injection site, xrel, and eL. D) Histogram of particle density function vs. {Delta}{theta} binned in 10° intervals. Correlations of density function vs. {Delta}{theta} not significant.

Figure 4D shows the relationship between the cell orientation angle, {theta}cell (angle between cell long axis and x axis), and the migration angle, {theta}mig (angle between x axis and line connecting injection site and cell location) (see Methods). Histograms of {Delta}{theta} are plotted, where {Delta}{theta} represents the absolute value of difference between {theta}cell and {theta}mig. A {Delta}{theta} of 0° indicates perfect alignment of cells with their hypothetical direction of movement directly away from the injection site, whereas {Delta}{theta} of 45° indicates random alignment. The mean values of {Delta}{theta} were 49 ± 26° and 45 ± 28° for AQP4+/+ and AQP4–/– cells, respectively (SD, ~400 cells analyzed per genotype, differences from 45° not significant). The lack of correlation of {theta}cell and {theta}mig indicates that the local direction of cell movement in the crowded ECS is random, as might be predicted.

The primary astroglial cell cultures used for in vivo injection and to be used for in vitro migration studies below were further characterized. Figure 5 A shows GFAP and AQP4 immunostaining of AQP4+/+ and AQP4–/– cultures. More than 90% of red fluorescent cells were GFAP-positive. More than 90% of red fluorescent cells from AQP4+/+ cultures were AQP4 positive. Figure 5B shows osmotic water permeability, as measured by calcein fluorescence quenching. Water permeability was ~5-fold higher for AQP4+/+ than for AQP4–/– cultures, similar to prior data on astroglial cells cultured for >26 days and subjected to dibutryl-cAMP treatment (26) . The residual water permeability in AQP4–/– cultures is likely through the lipid bilayer.


Figure 5
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  		Figure 5. Characterization of AQP4+/+ and AQP4–/– astroglial cell cultures. A) GFAP (red) and AQP4 (green, inset) immunostaining of astroglial cells after 12 days in culture. B) left: Osmotic water permeability measured by calcein fluorescence quenching. Perfusate solutions changed rapidly between osmolalities of 300 and 150 mOsm. B) right: Data summary of osmotic equilibration rates 1/{tau}. Data from separate measurements shown along with average (SE, *P<0.001).

We postulated that the substantial impairment in migration of AQP4–/– astroglial cells in vivo is in part related to their difficulty in passing through narrow clefts in brain extracellular space. To assess astroglial cell migration through small spaces, in vitro studies of migration were done in which cells were induced by a serum gradient to migrate through transwell pores of different diameters. As depicted in Fig. 6 A (left), cells were added to the upper compartment of a transwell Boyden chamber. Photographs of stained cells on filters with 8-µm diameter pore size are shown in Fig. 6A (right). A similar number of adherent cells were seen (upper panels, "before scrape"), whereas fewer AQP4–/– than AQP4+/+ cells migrated through the filter (lower panels, "after scrape"). Figure 6B shows no significant differences in the numbers of adherent AQP4+/+ vs. AQP4–/– cells to filters of different pore sizes. However, as summarized in Fig. 6C (top) there were fewer migrating AQP4–/– than AQP4+/+ cells, with much reduced migration at smaller pore sizes. The cells counted in Fig. 6C (top) were not associated with pore, indicating complete migration through pores. Substantial reduction in migration of AQP4+/+ cells was found, as pore size decreased from 8 to 5 µm, whereas the reduction in migration of AQP4–/– cells was found at larger pore sizes of 12 to 8 µm. Another interesting observation was that many cells were apparently stuck in the smaller pores. As shown in Fig. 6C (bottom), nearly all cells remaining after scraping the filter containing 3-µm diameter pores were associated with pores. Significantly more AQP4–/– than AQP4+/+ cells were pore-associated for filters containing 8-µm and 5-µm diameter pores.


Figure 6
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  		Figure 6. Migration of AQP4+/+ and AQP4–/– astroglial cells in vitro. A) left: Schematic of in vitro migration assay showing migrating cells from top-to-bottom through a porous transwell filter. A) right: Representative photos of transwell migration assay using 10% FBS as chemoattractant, showing astroglial cells that adhered to the (8-µm diameter pore size) filter ("before scrape") and cells that migrated through the filters ("after scrape"). Cells were stained with Coomassie blue. Arrows in bottom panels indicate migrated cells. Scale bar = 50 µm. B) Migration data summary (32 AQP4+/+ and 32 AQP4–/– transwells, SE). Cell adherence was determined by counting all stained cells on filters before scraping. Differences not significant. C) top: Migration efficiency shown as percentage of adherent cells that migrated completely through the filter at 8 h. (C, bottom) Percentage of cells "stuck" in pores. *P < 0.05, **P < 0.01.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that AQP4 deletion in astroglial cells markedly impairs their ability to migrate in adult mouse brain toward a stab wound. Several key aspects of in vivo migration differed significantly in AQP4–/– compared with AQP4+/+ astroglial cells, including their migration speed, direction, and cell shape. These results significantly extend our initial finding that AQP4 knockout and knockdown impairs astroglial cell migration in vitro and slows the turnover of plasma membrane protrusions (19) .

The formation and retraction of cell membrane protrusions at the leading edge of a migrating cell is an essential component of cell migration (28 29 30) . These changes in cell shape are associated with rapid actin depolymerization and repolymerization, as well as altered ion fluxes at the front end of the cell, which produce rapid changes in intracellular osmolality that drive transmembrane water flow (28 29 30 31 32) . Our experiments revealed that AQP4+/+ astrocytes are more elongated than AQP4–/– astrocytes during their migration through the brain, supporting a key role for AQP4 in facilitating changes in cell shape during migration.

The transwell assay revealed a strong dependence of cell migration on pore size. The difference in migration efficiency between AQP4+/+ and AQP4–/– astroglial cells was more pronounced for migration through 5 and 8 µm, compared with 12-µm diameter pores. Significant differences were also seen in the percentage of AQP4+/+ vs. AQP4–/– astroglial cells that were "stuck" in the smaller pores. Astroglial movement through small cylindrical pores in the transwell filter, or the geometrically complex brain extracellular space, probably requires rapid changes in cell volume to squeeze through barriers smaller than their normal size. Our data suggest that these cell volume changes are facilitated by AQP4. The width of brain extracellular space is <100 nm (33) , too small if static to support cell migration. During migration the astroglia probably extend processes that widen the extracellular space. The formation and retraction of such processes is also likely to be facilitated by AQP4.

We developed an experimental model of astroglial cell migration in adult mouse brain to test the involvement of AQP4 in astroglial cell migration in vivo. In previous attempts at producing rodent models of in vivo astroglial cell migration, injected astroglial cells failed to migrate in adult brain parenchyma (34 35 36) . From the literature, we concluded that the use of astroglial differentiating factors (35) and the lack of chemoattractants in normal brain parenchyma are two major factors restricting astroglial cell entry into and migration through brain parenchyma. Therefore, to overcome the problem of limited migration, we did not treat astroglial cell cultures with dibutyryl cAMP, and we created a planar stab injury 2 days before cell injection to provide a strong chemotactic stimulus. Under these conditions, the injected AQP4+/+ astroglial cells preferentially migrated in the direction of the wound. This mouse model allowed us to quantify several aspects of in vivo cell migration, including migration extent and direction, as well as cell shape and orientation.

We previously reported impairment of glial scarring in AQP4-null vs. WT mice (19) . However, it could not be determined whether the impaired glial scarring was due to slowed astroglial cell migration in the AQP4-null mice, to differences in brain extracellular space in AQP4-deficiency (37) , or other properties of WT vs. AQP4-null brain, such as the difference in local brain swelling after injury (5 , 6) . A key advantage of the in vivo model here is that astroglial cell migration is measured in the same brain background, which eliminates the confounding effects of extracellular space geometry and other factors.

Our findings suggest that inhibitors of AQP4 expression or water permeability would reduce astroglial cell migration and hence inhibit glial scar formation. Reduced glial scarring might augment neuronal plasticity after brain injury and enhance the integration of neural implants into adult CNS. On the basis of recent data showing AQP-dependent tumor cell infiltration and spread (18) , AQP4 inhibition might also limit the infiltration of malignant astrocytomas, which strongly express AQP4 (17) .


   ACKNOWLEDGMENTS
 
We thank Liman Qian for mouse breeding and genotype analysis and Dr. Mariko Hara-Chikuma for help with in vitro migration assays. This work was supported by grants DK35124, EB00415, EY13574, HL59198, DK72517, and HL73856 from the National Institutes of Health, and Research Development Program and Drug Discovery grants from the Cystic Fibrosis Foundation (to ASV) and by a Wellcome Trust Clinician Scientist Fellowship (to MCP).

Received for publication July 9, 2006. Accepted for publication August 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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