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Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema

MARIOS C. PAPADOPOULOS^*, GEOFFREY T. MANLEY, SANJEEV KRISHNA and A. S. VERKMAN^*,1

^* Departments of Medicine and Physiology, Cardiovascular Research Institute, and
Department of Neurological Surgery, University of California, San Francisco, California, USA; and
Department of Cellular and Molecular Sciences, St. George’s Hospital Medical School, London, UK

¹Correspondence: Cardiovascular Research Institute, University of California, San Francisco, CA, 94143-0521, USA. E-mail: verkman{at}itsa.ucsf.edu

SPECIFIC AIMS

Abnormal accumulation of brain water, termed cerebral edema, is a major clinical problem. The water channel aquaporin-4 (AQP4) has been proposed to play an important role in the formation of cytotoxic (cellular) brain edema. Using AQP4-deficient mice, we investigated whether AQP4 also plays a role in the pathophysiology of vasogenic (noncellular) brain edema.

PRINCIPAL FINDINGS

1. Brains of AQP4 null mice are normal
Brain morphology, baseline intracranial pressure (ICP), and bulk intracranial compliance of wild-type vs. AQP4 null mice did not differ significantly. RT-PCR of whole brain homogenates indicated expression of transcripts encoding AQPs 1, 4, and 9 in wild-type mice and only AQPs 1 and 9 in AQP4 null mice. Quantitative RT-PCR showed no significant difference in expression of transcripts encoding AQP1 and AQP9 in wild-type vs. AQP4 null mice.

2. AQP4 null mice develop more brain swelling than wild-type mice in response to intraparenchymal fluid infusion
To test whether AQP4 is involved in brain water exit, isotonic fluid (artificial cerebrospinal fluid, aCSF) was infused into the brain parenchyma at a rate of 0.5 µL/min for 60 min with continuous ICP recording. Evans blue dye added to the micropipette showed that over 60 min the infusate moved away from the injection site well into the brain parenchyma (Fig. 1 A, inset). As shown in representative ICP recordings in Fig. 1A , the increase in ICP during infusion was greater in AQP4 null mice. Figure 1B summarizes the incremental ICP (ICP) at 60 min (compared with 0 min baseline), showing a significantly greater ICP in AQP4 null mice (P<0.01). Water content of the infused hemisphere after a 60 min infusion was significantly elevated in AQP4 null mice (P<0.001) whereas water content of the noninfused contralateral hemisphere was not different (Fig. 1C ). In control experiments in five AQP4 null mice, drilling the burr hole and inserting the micropipette into the brain for 60 min without infusion caused no significant increase in ICP or hemispheric water content. The percent water content of uninjured mouse brain was not different in six wild-type vs. six AQP4 null mouse brains (78.6±0.2 vs. 79.2±0.3).

View larger version (32K): [in this window] [in a new window]   Figure 1. Increased ICP and brain water content in AQP4 null mice after intraparenchymal fluid infusion. A) Top: isotonic fluid containing Evans blue was infused slowly using a glass micropipette, inserted in the direction of the arrow into the striatum. Note staining of the lower tract and diffusion through the parenchyma. Bottom: representative ICP traces from 3 wild-type mice (left) and 3 AQP4 null mice (right) (starting ICP shown on the left of each trace). B) Increased ICP at 60 min (ICP) in response to isotonic fluid infusion (0.5 µL/min). Data shown for individual mice and mean ± SE, *P<0.01. C) Brain water content of ipsilateral (fluid-infused side) and contralateral hemispheres (computed from wet-to-dry weight ratios) from experiments in panel B. (mean±SE, *P<0.01 comparing ipsilateral hemispheres). D) Right: ICP at 10 min after infusing hypotonic fluid (1.5 µL/min, 100 mOsm) (*P<0.01). Left: ICP traces from two wild-type and two AQP null mice.

Infusion studies were done using hypotonic aCSF, which is predicted to be taken up rapidly into the brain cellular compartment. Infusion of hypotonic aCSF at 2.5 µL/min produced a rise in ICP within 10 min (Fig. 1D , left), which was significantly greater in AQP4 null mice (P<0.005) (Fig. 1D , right). ICP recording was discontinued after 11 min at which time some mice died.

3. AQP4 null mice develop more brain swelling than control mice after focal cortical freeze injury
Cortical freeze was produced using a cold copper probe making contact with the exposed skull. Mice were followed for 6 h postinjury to allow brain swelling to occur. In control experiments, the amount of Evans blue dye extravasation into the brain parenchyma was not different on wild-type compared with AQP4 null mice (n=5). Although some mice had mild neurological impairment initially, probably related to the anesthesia, no neurological impairment was detectable in wild-type mice at 6 h after freeze injury as judged by the neurological score whereas significant impairment was found in AQP4 null mice (P<0.002). The worse neurological outcome in AQP4 null mice is probably due to their significantly greater ICP (22±4 vs. 9±1 cm H2O) 6 h after freeze injury (P<0.02). In both groups of mice, freeze injury caused a greater increase in water content in AQP4 null vs. wild-type mice of the injured hemisphere (80.9±0.1 vs. 79.4±0.1%, P<0.001).

4. AQP4 null mice develop more brain swelling than control mice after melanoma tumor cell implantation
Melanoma cells were injected into the right striatum as diagrammed (Fig. 2 A, top). The injected cells produced a rapidly growing, well-demarcated dark tumor in both wild-type and AQP4 null mice (Fig. 2A , middle). Tumor volume was assessed 4 and 7 days postimplantation by optical imaging of 1 mm-thick sections of formalin-fixed brain parenchyma (Fig. 2A , right). Tumor volumes did not differ significantly in wild-type vs. AQP4 null mice (Fig. 2A , bottom). However, AQP4 null mice had a significantly worse neurological score on days 6–8 (P<0.001, Fig. 2B ) and higher ICP (P<0.02, Fig. 2C ).

View larger version (57K): [in this window] [in a new window]   Figure 2. Increased ICP and worse clinical outcome in AQP4 null mice with melanoma brain tumor. A) Top: site of injection of melanoma cells. Middle: tumor size 4 and 7 days after implantation showing similar-sized tumors in wild-type and AQP4 null mice. Right: coronal sections (1 mm thick) of brain used to determine brain total tumor volume. Bottom: tumor volume measured 4 and 7 days after implantation; differences not significant. B) Neurological score; C) ICP measured at 7 days (mean±SE, *P<0.001 for neurological scores, *P<0.02 for ICP).

CONCLUSIONS AND SIGNICANCE

Our results provide direct evidence for the involvement of AQP4 in the resolution of "vasogenic" brain edema. Intraparenchymal fluid infusion produced greater elevation in ICP and increased brain water content in AQP4 null mice, as did freeze injury, a model of vasogenic edema caused by opening of the blood-brain barrier. Because hydrostatic driving forces producing brain edema are identical in wild-type and AQP4 null mice, the greater brain water accumulation in AQP4 mice is a consequence of reduced transfer of water from the brain parenchyma to the vascular, intraventricular, and subarachnoid compartments. Although AQP4 deletion did not affect brain tumor growth over a 7-day period, there was greater elevation in ICP and accelerated neurological deterioration in the AQP4 null mice as a likely consequence of impaired removal of brain water. The normal brain phenotype of AQP4 null mice and the protective effect of AQP4 deletion in the early phase of "cytotoxic" edema raised questions of why a highly conserved brain protein could lack vital function in normal mice and confer a survival disadvantage in injury. Our findings help resolve this issue and relate a molecular-level mechanism, bidirectional AQP4-dependent water transport, to the clinically relevant pathophysiology of vasogenic brain edema.

Vasogenic edema is thought to be cleared primarily by bulk flow of fluid through extracellular space and the glia limitans into the ventricles and subarachnoid space, and to a lesser extent through astrocyte foot processes and capillary endothelium into the blood (Fig. 3 ). Figure 3 also shows the expression of AQP4 at barriers across which edema fluid is absorbed. In mammals, AQP4-rich astrocyte processes of the glia limitans interna and glia limitans externa form a dense mesh at the brain/cerebrospinal fluid (CSF) boundaries. Ultrastructural studies show that these long astrocytic processes are separated by narrow (<20 nm) intercellular clefts, interconnected by gap junctions, and lack transcytotic vesicles. Because of the long diffusion path, the presence of gap junctions between adjacent astrocytes, and the absence of transcytotic transport between clefts, the glia limitans is a significant permeability barrier to the extracellular flow of water and solutes. The impaired clearance of brain edema fluid in AQP4 null mice after intraparenchymal fluid infusion, freeze injury, and brain tumor suggests that AQP4 provides a low-resistance, transcellular route that allows edema fluid to move across the astrocyte cell membranes of the glia limitans into the CSF.

View larger version (64K): [in this window] [in a new window]   Figure 3. Schematic of AQP4-dependent fluid absorption in brain. AQP4 expression (green circles) shown at barriers in the major fluid absorption pathways (blue arrows). Edema fluid crosses the external glial-limiting membrane and pia to enter the subarachnoid space (top left), the internal glial-limiting membrane and ependyma into the ventricles (bottom left), and astrocyte foot processes and capillary endothelium to enter the bloodstream (top right).

There is a considerable body of experimental evidence for altered AQP4 expression in brain edema, providing indirect evidence for a role of AQP4 in the pathophysiology of brain edema. In humans, AQP4 is up-regulated in astrocytes around edematous brain tumors and in brain contusions, bacterial meningitis, and infarcted brain. In rodents, AQP4 expression in astrocytes is increased after hyponatremia, around infarcts, in brain injury, and after mannitol administration. However, AQP4 down-regulation has been reported in rats after traumatic brain injury and hyponatremia combined with brain contusion. Our data here suggest that increased AQP4 expression/function should accelerate the elimination of edema fluid from the brain parenchyma whereas decreased AQP4 expression/function would slow edema formation, particularly by cytotoxic mechanisms. The direction of change in AQP4 expression in different regions of the brain may be a compensatory response to edema fluid formation or absorption.

In conclusion, our data provide strong evidence that brain AQP4 not only participates in edema formation, as previously shown, but also in the absorption of excess brain water. AQP4 inhibitors would reduce cytotoxic brain swelling only if administered early to slow the entry of edema fluid into the brain parenchyma. Administering AQP4 inhibitors late after the onset of cytotoxic edema or in vasogenic edema is predicted to increase brain swelling. Augmentation in AQP4 expression and/or function thus may be beneficial in reducing brain swelling in vasogenic edema and in the resolution of phase of cytotoxic edema.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1723fje; doi: 10.1096/fj.04-1723fje

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