Increased brain edema in aqp4-null mice in an experimental model of subarachnoid hemorrhage.

We investigated the role of the glial water channel protein aquaporin-4 in brain edema in a mouse model of subarachnoid hemorrhage in which 30 microl of blood was injected into the basal cisterns. Brain water content, intracranial pressure and neurological score were compared in wildtype and aquaporin-4 null mice. We also measured blood-brain barrier permeability, and the osmotic permeability of the glia limitans, one of the routes of edema elimination. Wildtype and aquaporin-4 null mice had comparable baseline brain water content, intracranial pressure and neurological score. At 6 h after blood injection, aquaporin-4 null mice developed more brain swelling than wildtype mice. Brain water content increased by 1.5+/-0.1% vs. 0.5+/-0.2% (Mean+/-Standard Error, P<0.0005) and intracranial pressure by 36+/-5 vs. 21+/-3 mm Hg (P<0.05) above pre-injection baseline, and neurological score was worse at 18.0 vs. 24.5 (median, P<0.05), respectively. Although subarachnoid hemorrhage produced comparable increases in blood-brain barrier permeability in wildtype and aquaporin-4 null mice, aquaporin-4 null mice had a twofold reduction in glia limitans osmotic permeability. We conclude that aquaporin-4 null mice manifest increased brain edema following subarachnoid hemorrhage as a consequence of reduced elimination of excess brain water.

Aquaporin-4 in brain and spinal cord oedema.

Brain oedema is a major clinical problem produced by CNS diseases (e.g. stroke, brain tumour, brain abscess) and systemic diseases that secondarily affect the CNS (e.g. hyponatraemia, liver failure). The swollen brain is compressed against the surrounding dura and skull, which causes the intracranial pressure to rise, leading to brain ischaemia, herniation, and ultimately death. A water channel protein, aquaporin-4 (AQP4), is found in astrocyte foot processes (blood-brain border), the glia limitans (subarachnoid cerebrospinal fluid-brain border) and ependyma (ventricular cerebrospinal fluid-brain border). Experiments using mice lacking AQP4 or alpha syntrophin (which secondarily downregulate AQP4) showed that AQP4 facilitates oedema formation in diseases causing cytotoxic (cell swelling) oedema such as cerebral ischaemia, hyponatraemia and meningitis. In contrast, AQP4 facilitates oedema elimination in diseases causing vasogenic (vessel leak) oedema and therefore AQP4 deletion aggravates brain oedema produced by brain tumour and brain abscess. AQP4 is also important in spinal cord oedema. AQP4 deletion was associated with less cord oedema and improved outcome after compression spinal cord injury in mice. Here we consider the possible routes of oedema formation and elimination in the injured cord and speculate about the role of AQP4. Finally we discuss the role of AQP4 in neuromyelitis optica (NMO), an inflammatory demyelinating disease that produces oedema in the spinal cord and optic nerves. NMO patients have circulating AQP4 IgG autoantibody, which is now used for diagnosing NMO. We speculate how NMO-IgG might produce CNS inflammation, demyelination and oedema. Since AQP4 plays a key role in the pathogenesis of CNS oedema, we conclude that AQP4 inhibitors and activators may reduce CNS oedema in many diseases.

AQP4 gene deletion in mice does not alter blood-brain barrier integrity or brain morphology.

The glial cell water channel aquaporin-4 (AQP4) plays an important role in brain edema, astrocyte migration, and neuronal excitability. Zhou et al. [Zhou J, Kong H, Hua X, Xiao M, Ding J, Hu G (2008) Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice. Neuroreport 19:1-5] recently reported that AQP4 deletion significantly altered blood-brain barrier integrity and glial fibrillary acidic protein (GFAP) immunoreactivity in their AQP4 null mice. Here we describe a detailed characterization of baseline brain properties in our AQP4 null mice, including gross appearance, neuronal, astrocyte and oligodendrocyte characteristics, and blood-brain barrier integrity. Gross anatomical measurements included estimates of brain and ventricle size. Neurons, astrocytes and oligodendrocytes were assessed using the neuronal nuclear marker NeuN, the astrocyte marker GFAP, and the myelin stain Luxol Fast Blue. The blood-brain barrier was studied by electron microscopy and the horseradish peroxidase extravasation technique. There were no differences in brain and ventricle sizes between wild type and AQP4 null mice, nor were there differences in the cerebral cortical density of NeuN positive nuclei, perimicrovessel and glia limitans GFAP immunoreactivity, or the thickness and myelination of the corpus callosum. The ultrastructure of microvessels in the frontal cortex and caudate nucleus of wild type vs. AQP4 null mice was indistinguishable, with features including intact endothelial tight junctions, absence of perimicrovessel astrocyte foot process edema, and absence of horseradish peroxidase extravasation. In contrast to the report by Zhou et al. (2008), our data show that AQP4 deletion in mice does not produce major structural abnormalities in the brain.

Aquaporins and cell migration.

Aquaporin (AQP) water channels are expressed primarily in cell plasma membranes. In this paper, we review recent evidence that AQPs facilitate cell migration. AQP-dependent cell migration has been found in a variety of cell types in vitro and in mice in vivo. AQP1 deletion reduces endothelial cell migration, limiting tumor angiogenesis and growth. AQP4 deletion slows the migration of reactive astrocytes, impairing glial scarring after brain stab injury. AQP1-expressing tumor cells have enhanced metastatic potential and local infiltration. Impaired cell migration has also been seen in AQP1-deficient proximal tubule epithelial cells, and AQP3-deficient corneal epithelial cells, enterocytes, and skin keratinocytes. The mechanisms by which AQPs enhance cell migration are under investigation. We propose that, as a consequence of actin polymerization/depolymerization and transmembrane ionic fluxes, the cytoplasm adjacent to the leading edge of migrating cells undergoes rapid changes in osmolality. AQPs could thus facilitate osmotic water flow across the plasma membrane in cell protrusions that form during migration. AQP-dependent cell migration has potentially broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring, and other events requiring rapid, directed cell movement. AQP inhibitors may thus have therapeutic potential in modulating these events, such as slowing tumor growth and spread, and reducing glial scarring after injury to allow neuronal regeneration.

Molecular mechanisms of brain tumor edema.

Despite their diverse histological types, most brain tumours cause brain oedema, which is a significant cause of patient morbidity and mortality. Brain tumour oedema occurs when plasma-like fluid enters the brain extracellular space through impaired capillary endothelial tight junctions in tumours. Under-expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumour endothelial tight junctions. Recent evidence suggests that the membrane water channel protein aquaporin-4 (AQP4) also plays a role in brain tumour oedema. AQP4-deficient mice show remarkably altered brain water balance after various insults, including brain tumour implantation. AQP4 expression is strongly upregulated around malignant human brain tumours in association with reduced extracellular volume, which may restrict the flow of extracellular fluid from the tumour bed into the brain parenchyma. Elimination of excess fluid leaking into brain parenchyma requires passage across three AQP4-rich barriers: a) the glia limitans externa, b) the glia limitans interna/ependyma, and c) the blood-brain barrier. Modulation of the expression and/or function of endothelial tight junction proteins and aquaporins may provide novel therapeutic options for reducing brain tumour oedema.

Water transport becomes uncoupled from K+ siphoning in brain contusion, bacterial meningitis, and brain tumours: immunohistochemical case review.

Specimens of normal human brain, contused brain, brain with bacterial meningitis, and brain tumours were immunolabelled for aquaporin 4 (AQP4) and Kir4.1. In normal brain tissue, AQP4 and Kir4.1 were detected around the microvessels. In pathological brain tissue, AQP4 was upregulated in astrocytes in oedematous regions and Kir4.1 was upregulated in astrocytes in damaged brain. Changes in alpha syntrophin expression paralleled those of AQP4 and Kir4.1. The following hypothesis is proposed: in astrocytes, under normal conditions, AQP4 couples water transport with Kir4.1 mediated K+ siphoning, but in pathological states, AQP4 facilitates the flow of brain oedema fluid, and Kir4.1 buffers increased extracellular K+.

Increased aquaporin 1 water channel expression in human brain tumours.

Aquaporin 1 is a water channel protein. There was little aquaporin 1 immunoreactivity in normal brain parenchyma. In astrocytomas, aquaporin 1 was expressed in microvessel endothelia and neoplastic astrocytes. In metastatic carcinomas, aquaporin 1 was present in microvessel endothelia and reactive astrocytes. Aquaporin 1 may participate in the formation of brain tumour oedema.

Aquaporin-4 expression is increased in oedematous human brain tumours.

Aquaporin-4 (AQP4) is a highly conserved water channel protein. In rats, AQP4 is expressed in astrocyte foot processes and is important in brain water homeostasis. AQP4 expression has not been investigated in non-neoplastic human brain or oedematous brain tumours, where water homeostasis is disrupted. Therefore, immunohistochemistry was used to study AQP4 expression in non-neoplastic and neoplastic human brain and blood-brain barrier permeability was assessed using contrast enhanced computed tomograms. AQP4 was present around microvessels in five specimens of non-neoplastic brain and five low grade (Daumas-Duport I or II) astrocytomas. AQP4 was massively upregulated in four and absent in one high grade (Daumas-Duport III or IV) astrocytoma. Massive upregulation of AQP4 was also found in reactive astrocytes in five metastatic adenocarcinomas. There was significant (p<0.0001) correlation between blood-brain barrier opening and upregulated AQP4 expression. Increased AQP4 expression in high grade astrocytomas and adenocarcinomas may facilitate the flow of oedema fluid.

Occludin expression in microvessels of neoplastic and non-neoplastic human brain.

The tight junction protein occludin 'glues' normal, adjacent brain microvessel endothelial cells together. Malignant brain tumours cause cerebral oedema because they have leaky endothelial tight junctions, which allow plasma fluid to enter the brain from the microvessel lumen. In order to identify molecular abnormalities in tumour endothelial tight junctions, we investigated occludin expression in microvessels from adult human non-neoplastic brain tissue using immunohistochemistry and immunoblotting. The proportions of microvessels immunolabelling for occludin were >2/3 in 5/5 non-neoplastic brain tissue samples, >1/3 in 5/5 low grade (Daumas-Duport I or II) astrocytomas and <1/3 in 5/5 high grade (III or IV) astrocytomas and 6/6 metastatic adenocarcinomas. Six non-neoplastic brain tissue immunoblots gave a 55-kDa occludin band, three low-grade astrocytomas gave 55-kDa and 60-kDa bands, 13 high-grade astrocytomas gave 60-kDa or no band and four adenocarcinomas did not give an occludin band. Expression of 55-kDa occludin inversely correlated with the presence of contrast enhancement on computed tomograms (P < 0.001). Electron microscopy showed open endothelial tight junctions in 0/2 non-neoplastic human brain specimens and 2/2 high-grade astrocytomas. We suggest that loss of 55-kDa occludin expression in human brain tumours may contribute to endothelial tight junction opening. Characterizing the molecular pathology of brain endothelial tight junctions may facilitate the design of novel drugs against cerebral oedema.

Emerging molecular mechanisms of brain tumour oedema.

A common property of brain tumours is their ability to cause oedema in the surrounding brain. Oedema forms as a result of a leaky blood-tumour barrier and persists when the brain fails to clear the excess fluid. It is a significant source of morbidity and mortality. The principal anatomical component of the blood-brain barrier is the endothelial tight junction which opens in glioma microvessels. Multiple tight junction proteins have recently been identified, such as occludin, claudin, ZO-1, ZO-2 and ZO-3. We propose a model to explain tight junction opening in gliomas based on vascular endothelial growth factor secretion and loss of tight junction inducing factor production by tumour cells. The level of expression of the water channel aquaporin-4 in peritumoural astrocytes may determine the rate of oedema fluid clearance. The identification of the molecular mechanisms of brain tumour oedema may allow the design of novel anti-oedema medications.

Endothelin stimulates nitric oxide-dependent cyclic GMP formation in rat cerebellar astroglia.

Endothelins (ETs) elicit a diversity of cellular responses in cultured astrocytes that suggest an important role of these peptides in glial cell function. Stimulation of astroglial ET receptors induces phosphoinositide (PI) hydrolysis and intracellular calcium mobilization, but little is known about the signalling events that occur downstream of this system. Here we show that in rat cerebellar astroglia in culture ETs produce a receptor-mediated stimulation of cyclic GMP (cGMP) formation that is rapid and totally dependent on nitric oxide synthase (NOS) activity. The effect is blocked by an inhibitor of PI phospholipase C, compound U73122, and by depletion of intracellular calcium stores with thapsygargin. These results indicate that calcium released by inositol trisphosphate is responsible for NOS activation and subsequent cGMP formation.

Extracellular acidification modifies Ca2+ fluxes in rat brain synaptosomes.

We examined the influence of external acidification on Ca2+ fluxes (45Ca2+ influx and 45Ca2+ efflux) in rat brain synaptosomes. A change on external pH (pHe) from 7.5 to 6.5 linearly decreased the 45Ca2+ uptake (5nmoles/mg protein/pHunit) and increased the 45Ca2+ efflux (1.5 fold/pH unit). These changes were both Na+ dependent and amiloride sensitive suggesting that the Na+/Ca2+ exchanger could be involved. The addition of the Ca2+ channel blockers (diltiazem, verapamil, nifedipine) did not prevent the decrease of the 45Ca2+ uptake evoked by acid pHe and so the involvement of the voltage-sensitive Ca2+ channels could be discarded. In order to determine whether the Na+/Ca2+ exchanger was directly activated by H+ or was indirectly activated by an internal mobilization of Ca2+ from intrasynaptosomal stores we examined the effect of pHe variation on phophoinositide hydrolisis. An increase on phosphoinositide hydrolisis was observed at acid pHe values (7 and 6.5). The hydrolisis was amiloride insensitive. On the other hand 1mM neomycin did inhibit the effect of acidic pHe on Ca2+ fluxes. Taken together, the results of our study provide evidence that external acidification stimulates phospholipase C leading to an increase in phosphoinositide hydrolisis and Ca2+ mobilization. The increase in intracellular Ca2+ would stimulate the Na+/Ca2+ exchanger, increasing Ca2+ efflux and reducing the global Ca2+ influx.

Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture.

Ca2+ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca2+-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca2+ entry could also down-regulate intracellular cGMP by activating a Ca2+/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 microM) and the Ca2+ ionophore A23187 (10 microM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca2+ and is prevented by the calmodulin inhibitor W7 (100 microM). Membrane depolarization, manipulations of the Na+ gradient, and intracellular Ca2+ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca2+ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia.