Role of aquaporin-4 polarization in extracellular solute clearance.

Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid (CSF) enters the brain in perivascular spaces of arteries, crosses the astrocyte endfoot layer, flows through the parenchyma collecting waste that is subsequently drained along veins. Glymphatic clearance is dependent on astrocytic aquaporin-4 (AQP4) water channels that are highly enriched in the endfeet. Even though the polarized expression of AQP4 in endfeet is thought to be of crucial importance for glymphatic CSF influx, its role in extracellular solute clearance has only been evaluated using non-quantitative fluorescence measurements. Here we have quantitatively evaluated clearance of intrastriatally infused small and large radioactively labeled solutes in mice lacking AQP4 (Aqp4-/-) or lacking the endfoot pool of AQP4 (Snta1-/-). We confirm that Aqp4-/- mice show reduced clearance of both small and large extracellular solutes. Moreover, we find that the Snta1-/- mice have reduced clearance only for the 500 kDa [3H]dextran, but not 0.18 kDa [3H]mannitol suggesting that polarization of AQP4 to the endfeet is primarily important for clearance of large, but not small molecules. Lastly, we observed that clearance of 500 kDa [3H]dextran increased with age in adult mice. Based on our quantitative measurements, we confirm that presence of AQP4 is important for clearance of extracellular solutes, while the perivascular AQP4 localization seems to have a greater impact on clearance of large versus small molecules.

MAIN POINTS: Solute clearance is reduced in mice lacking AQP4 Polarization of AQP4 to the endfeet may have a greater impact on clearance of large versus small molecules Clearance of large but not small solutes is correlated with age within adult age.

Increased membrane Ca2+ permeability drives astrocytic Ca2+ dynamics during neuronal stimulation at excitatory synapses.

Astrocytes are intricately involved in the activity of neural circuits; however, their basic physiology of interacting with nearby neurons is not well established. Using two-photon imaging of neurons and astrocytes during higher frequency stimulation of hippocampal CA3-CA1 Schaffer collateral (Scc) excitatory synapses, we could show that increasing levels of released glutamate accelerated local astrocytic Ca2+ elevation. However, blockage of glutamate transporters did not abolish this astrocytic Ca2+ response, suggesting that astrocytic Ca2+ elevation is indirectly associated with an uptake of extracellular glutamate. However, during the astrocytic glutamate uptake, the Na+ /Ca2+ exchanger (NCX) reverse mode was activated, and mediated extracellular Ca2+ entry, thereby triggering the internal release of Ca2+ . In addition, extracellular Ca2+ entry via membrane P2X receptors further facilitated astrocytic Ca2+ elevation via ATP binding. These findings suggest a novel mechanism of activity induced Ca2+ permeability increases of astrocytic membranes, which drives astrocytic responses during neuronal stimulation of CA3-CA1 Scc excitatory synapses.

Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema.

Brain edema is a feared complication to disorders and insults affecting the brain. It can be fatal if the increase in intracranial pressure is sufficiently large to cause brain herniation. Moreover, accruing evidence suggests that even slight elevations of intracranial pressure have adverse effects, for instance on brain perfusion. The water channel aquaporin-4 (AQP4), densely expressed in perivascular astrocytic endfeet, plays a key role in brain edema formation. Using two-photon microscopy, we have studied AQP4-mediated swelling of astrocytes affects capillary blood flow and intracranial pressure (ICP) in unanesthetized mice using a mild brain edema model. We found improved regulation of capillary blood flow in mice devoid of AQP4, independently of the severity of ICP increase. Furthermore, we found brisk AQP4-dependent astrocytic Ca2+ signals in perivascular endfeet during edema that may play a role in the perturbed capillary blood flow dynamics. The study suggests that astrocytic endfoot swelling and pathological signaling disrupts microvascular flow regulation during brain edema formation.

Impaired astrocytic Ca2+ signaling in awake-behaving Alzheimer's disease transgenic mice.

Increased astrocytic Ca2+ signaling has been shown in Alzheimer's disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signaling in such mice. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signaling in 15-month-old plaque-bearing mice. Using the genetically encoded fluorescent norepinephrine sensor GRABNE, we demonstrate a reduced norepinephrine signaling during spontaneous running and startle responses in the transgenic mice, providing a possible mechanistic underpinning of the observed reduced astrocytic Ca2+ responses. Our data points to a dysfunction in the norepinephrine-astrocyte Ca2+ activity axis, which may account for some of the cognitive deficits observed in Alzheimer's disease.

Neurodegenerative conditions such as Parkinson's or Alzheimer's disease are characterized by neurons dying and being damaged. Yet neurons are only one type of brain actors; astrocytes, for example, are star-shaped 'companion' cells that have recently emerged as being able to fine-tune neuronal communication. In particular, they can respond to norepinephrine, a signaling molecule that acts to prepare the brain and body for action. This activation results, for instance, in astrocytes releasing chemicals that can act on neurons. Certain cognitive symptoms associated with Alzheimer's disease could be due to a lack of norepinephrine. In parallel, studies in anaesthetized mice have shown perturbed astrocyte signaling in a model of the condition. Disrupted norepinephrine-triggered astrocyte signaling could therefore be implicated in the symptoms of the disease. Experiments in awake mice are needed to investigate this link, especially as anesthesia is known to disrupt the activity of astrocytes. To explore this question, Åbjørsbråten, Skaaraas et al. conducted experiments in naturally behaving mice expressing mutations found in patients with early-onset Alzheimer's disease. These mice develop hallmarks of the disorder. Compared to their healthy counterparts, these animals had reduced astrocyte signaling when running or being startled. Similarly, a fluorescent molecular marker for norepinephrine demonstrated less signaling in the modified mice compared to healthy ones. Over 55 million individuals currently live with Alzheimer's disease. The results by Åbjørsbråten, Skaaraas et al. suggest that astrocyte­norepinephrine communication may be implicated in the condition, an avenue of research that could potentially lead to developing new treatments.

Astrocytic Ca2+ signaling is reduced during sleep and is involved in the regulation of slow wave sleep.

Astrocytic Ca2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca2+ activity, brain rhythms and behavior. We show that astrocytic Ca2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca2+ signaling in regulating slow wave sleep.

Impaired glymphatic function and clearance of tau in an Alzheimer's disease model.

The glymphatic system, that is aquaporin 4 (AQP4) facilitated exchange of CSF with interstitial fluid (ISF), may provide a clearance pathway for protein species such as amyloid-ß and tau, which accumulate in the brain in Alzheimer's disease. Further, tau protein transference via the extracellular space, the compartment that is cleared by the glymphatic pathway, allows for its neuron-to-neuron propagation, and the regional progression of tauopathy in the disorder. The glymphatic system therefore represents an exciting new target for Alzheimer's disease. Here we aim to understand the involvement of glymphatic CSF-ISF exchange in tau pathology. First, we demonstrate impaired CSF-ISF exchange and AQP4 polarization in a mouse model of tauopathy, suggesting that this clearance pathway may have the potential to exacerbate or even induce pathogenic accumulation of tau. Subsequently, we establish the central role of AQP4 in the glymphatic clearance of tau from the brain; showing marked impaired glymphatic CSF-ISF exchange and tau protein clearance using the novel AQP4 inhibitor, TGN-020. As such, we show that this system presents as a novel druggable target for the treatment of Alzheimer's disease, and possibly other neurodegenerative diseases alike.

Pathological mitochondria in neurons and perivascular astrocytic endfeet of idiopathic normal pressure hydrocephalus patients.

BACKGROUND: A growing body of evidence suggests that the accumulation of amyloid-ß and tau (HPτ) in the brain of patients with the dementia subtype idiopathic normal pressure hydrocephalus (iNPH) is associated with delayed extravascular clearance of metabolic waste. Whether also clearance of intracellular debris is affected in these patients needs to be examined. Hypothetically, defective extra- and intra-cellular clearance of metabolites may be instrumental in the neurodegeneration and dementia characterizing iNPH. This study explores whether iNPH is associated with altered mitochondria phenotype in neurons and astrocytes. METHODS: Cortical brain biopsies of 9 reference (REF) individuals and 30 iNPH patients were analyzed for subcellular distribution and morphology of mitochondria using transmission electron microscopy. In neuronal soma of REF and iNPH patients, we identified normal, pathological and clustered mitochondria, mitochondria-endoplasmic reticulum contact sites and autophagic vacuoles. We also differentiated normal and pathological mitochondria in pre- and post-synaptic nerve terminals, as well as in astrocytic endfoot processes towards vessels. RESULTS: We found a high prevalence of pathological mitochondria in neuronal soma and pre- and post-synaptic terminals, as well as increased mitochondrial clustering, and altered number of mitochondria-endoplasmic reticulum contact sites in iNPH. Non-fused autophagic vacuoles were more abundant in neuronal soma of iNPH patients, suggestive of cellular clearance failure. Moreover, the length of postsynaptic densities was reduced in iNPH, potentially related to reduced synaptic activity. In astrocytic endfoot processes, we also found increased number, area and area fraction of pathological mitochondria in iNPH patients. The proportion of pathological mitochondria correlated significantly with increasing degree of astrogliosis and reduced perivascular expression of aquaporin-4 (AQP4), assessed by light microscopy immunohistochemistry. CONCLUSION: Our results provide evidence of mitochondrial pathology and signs of impaired cellular clearance in iNPH patients. The results indicate that iNPH is a neurodegenerative disease with close similarity to Alzheimer's disease.

Blood-Brain Barrier Dysfunction in Idiopathic Intracranial Hypertension.

Idiopathic intracranial hypertension (IIH) is traditionally considered benign and characterized by symptoms related to increased intracranial pressure, including headache and impaired vision. We have previously demonstrated that brains of IIH patients exhibit patchy astrogliosis, increased perivascular expression of the water channel aquaporin-4 (AQP4) as well as degenerating pericyte processes and capillary basement membranes. Given the established association between pericyte degeneration and blood-brain barrier (BBB) dysfunction, we investigated blood protein leakage by light microscopic immunohistochemistry. We also assessed perivascular AQP4 expression by immunogold transmission electron microscopy. The study included 14 IIH patients and 14 reference (REF) subjects undergoing neurosurgery for epilepsy, aneurysm, or tumor. Evidence of BBB dysfunction, measured as area extravasated fibrinogen/fibrin, was significantly more pronounced in IIH than REF individuals. The extent of extravasated fibrinogen was positively correlated with increasing degree of astrogliosis and vascular AQP4 immunoreactivity, determined by light microscopy. Immunogold transmission electron microscopy revealed no overall changes in AQP4 expression at astrocytic vascular endfeet in IIH (n = 8) compared to REF (n = 11) individuals. Our results provide evidence of BBB leakage in IIH, signifying that IIH is a more serious neurodegenerative disease than previously considered.

Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions.

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood-brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca2+ indicator GCaMP6f to study Ca2+ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two-photon microscopy. Ca2+ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca2+ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau-type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5-triphosphate receptor (IP3R2) severely suppressed the Ca2+ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca2+ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin-4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca2+ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca2+ signals only in subpial and perivascular endfeet.

Aquaporin-4-independent volume dynamics of astroglial endfeet during cortical spreading depression.

Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of gray matter. This phenomenon is believed to underlie the migraine aura and similar waves of depolarization may exacerbate injury in a number of neurological disease states. CSD is characterized by massive ion dyshomeostasis, cell swelling, and multiphasic blood flow changes. Recently, it was shown that CSD is associated with a closure of the paravascular space (PVS), a proposed exit route for brain interstitial fluid and solutes, including excitatory and inflammatory substances that increase in the wake of CSD. The PVS closure was hypothesized to rely on swelling of astrocytic endfeet due to their high expression of aquaporin-4 (AQP4) water channels. We investigated whether CSD is associated with swelling of endfeet around penetrating arterioles in the cortex of living mice. Endfoot cross-sectional area was assessed by two-photon microscopy of mice expressing enhanced green fluorescent protein in astrocytes and related to the degree of arteriolar constriction. In anesthetized mice CSD triggered pronounced endfoot swelling that was short-lasting and coincided with the initial arteriolar constriction. Mice lacking AQP4 displayed volume changes of similar magnitude. CSD-induced endfoot swelling and arteriolar constriction also occurred in awake mice, albeit with faster kinetics than in anesthetized mice. We conclude that swelling of astrocytic endfeet is a robust event in CSD. The early onset and magnitude of the endfoot swelling is such that it may significantly delay perivascular drainage of interstitial solutes in neurological conditions where CSD plays a pathophysiological role.

Loss of perivascular aquaporin-4 in idiopathic normal pressure hydrocephalus.

Idiopathic normal pressure hydrocephalus (iNPH) is a subtype of dementia that may be successfully treated with cerebrospinal fluid (CSF) diversion. Recently, magnetic resonance imaging (MRI) using a MRI contrast agent as a CSF tracer revealed impaired clearance of the CSF tracer from various brain regions such as the entorhinal cortex of iNPH patients. Hampered clearance of waste solutes, for example, soluble amyloid-ß, may underlie neurodegeneration and dementia in iNPH. The goal of the present study was to explore whether iNPH is associated with altered subcellular distribution of aquaporin-4 (AQP4) water channels, which is reported to facilitate CSF circulation and paravascular glymphatic drainage of metabolites from the brain parenchyma. Cortical brain biopsies of 30 iNPH patients and 12 reference individuals were subjected to AQP4 immunogold cytochemistry. Electron microscopy revealed significantly reduced density of AQP4 water channels in astrocytic endfoot membranes along cortical microvessels in patients with iNPH versus reference subjects. There was a significant positive correlation between density of AQP4 toward endothelial cells (perivascular) and toward parenchyma, but the reduced density of AQP4 toward parenchyma was not significant in iNPH. We conclude that perivascular AQP4 expression is attenuated in iNPH, potentially contributing to impaired glymphatic circulation, and waste clearance, and subsequent neurodegeneration. Hence, restoring normal perivascular AQP4 distribution may emerge as a novel treatment strategy for iNPH.

Non-invasive MRI of brain clearance pathways using multiple echo time arterial spin labelling: an aquaporin-4 study.

There is currently a lack of non-invasive tools to assess water transport in healthy and pathological brain tissue. Aquaporin-4 (AQP4) water channels are central to many water transport mechanisms, and emerging evidence also suggests that AQP4 plays a key role in amyloid-ß (Aß) clearance, possibly via the glymphatic system. Here, we present the first non-invasive technique sensitive to AQP4 channels polarised at the blood-brain interface (BBI). We apply a multiple echo time (multi-TE) arterial spin labelling (ASL) MRI technique to the mouse brain to assess BBI water permeability via calculation of the exchange time (Texw), the time for magnetically labelled intravascular water to exchange across the BBI. We observed a 31% increase in exchange time in AQP4-deficient (Aqp4-/-) mice (452 ±â€¯90 ms) compared to their wild-type counterparts (343 ±â€¯91 ms) (p = 0.01), demonstrating the sensitivity of the technique to the lack of AQP4 water channels. More established, quantitative MRI parameters: arterial transit time (δa), cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) detected no significant changes with the removal of AQP4. This clinically relevant tool may be crucial to better understand the role of AQP4 in water transport across the BBI, as well as clearance of proteins in neurodegenerative conditions such as Alzheimer's disease.

Ca2+ Signals in Astrocytes Facilitate Spread of Epileptiform Activity.

Epileptic seizures are associated with increased astrocytic Ca2+ signaling, but the fine spatiotemporal kinetics of the ictal astrocyte-neuron interplay remains elusive. By using 2-photon imaging of awake head-fixed mice with chronic hippocampal windows we demonstrate that astrocytic Ca2+ signals precede neuronal Ca2+ elevations during the initial bout of kainate-induced seizures. On average, astrocytic Ca2+ elevations preceded neuronal activity in CA1 by about 8 s. In subsequent bouts of epileptic seizures, astrocytes and neurons were activated simultaneously. The initial astrocytic Ca2+ elevation was abolished in mice lacking the type 2 inositol-1,4,5-trisphosphate-receptor (Itpr2-/-). Furthermore, we found that Itpr2-/- mice exhibited 60% less epileptiform activity compared with wild-type mice when assessed by telemetric EEG monitoring. In both genotypes we also demonstrate that spreading depression waves may play a part in seizure termination. Our findings imply a role for astrocytic Ca2+ signals in ictogenesis.

Cerebral microvascular abnormalities in patients with idiopathic intracranial hypertension.

AIM: Idiopathic intracranial hypertension (IIH) is characterized by symptoms indicative of increased intracranial pressure (ICP), such as headache and visual impairment. We have previously reported that brain biopsies from IIH patients show patchy astrogliosis and increased expression of the water channel aquaporin-4 (AQP4) at perivascular astrocytic endfeet. METHODS: The present study was undertaken to investigate for ultrastructural changes of the cerebral capillaries in individuals with IIH. We examined by electron microscopy (EM) biopsies from the cortical parenchyma of 10 IIH patients and 8 reference subjects (patients, not healthy individuals), in whom tissue was retrieved from other elective and necessary brain surgeries (epilepsy, tumors or vascular diseases). IIH patients were diagnosed on the basis of typical clinical symptoms and abnormal intracranial pressure wave amplitudes during overnight ICP monitoring. RESULTS: All 10 IIH patients underwent shunt surgery followed by favorable clinical outcome. EM revealed abnormal pericyte processes in IIH. The basement membrane (BM) showed more frequently evidence of degeneration in IIH, but neither the BM dimensions nor the pericyte coverage differed between IIH and reference tissue. The BM thickness increased significantly with increasing age. Reference individuals were older than IIH cases; observations may to some extent be age-related. CONCLUSION: The present study disclosed marked changes of the cerebral cortical capillaries in IIH patients, suggesting that microvascular alterations are involved in the evolvement of IIH.

Brain Capillary Ultrastructure in Idiopathic Normal Pressure Hydrocephalus: Relationship With Static and Pulsatile Intracranial Pressure.

Idiopathic normal pressure hydrocephalus (iNPH) is a neurodegenerative disease of unknown cause. We investigated the morphology of capillaries in frontal cortex biopsies from iNPH patients and related the observations to overnight intracranial pressure (ICP) scores. A biopsy (0.9×10 mm) was taken from where the ICP sensor subsequently was inserted. Brain capillaries were investigated by electron microscopy of biopsies from 27 iNPH patients and 10 reference subjects, i.e. patients (not healthy individuals) without cerebrospinal fluid circulation disturbances, in whom normal brain tissue was removed as part of necessary neurosurgical treatment. Degenerating and degenerated pericyte processes were identified in 23/27 (85%) iNPH and 6/10 (60%) of reference specimens. Extensive disintegration of pericyte processes were recognized in 11/27 (41%) iNPH and 1/10 (10%) reference specimens. There were no differences in basement membrane (BM) thickness or pericyte coverage between iNPH and reference subjects. The pulsatile or static ICP scores did neither correlate with the BM thickness nor with pericyte coverage. We found increased prevalence of degenerating pericytes in iNPH while the BM thickness and pericyte coverage did not differ from the reference individuals. Observations in iNPH may to some extent be age-related since the iNPH patients were significantly older than the reference individuals.

Deletion of Aquaporin-4 Curtails Extracellular Glutamate Elevation in Cortical Spreading Depression in Awake Mice.

Cortical spreading depression (CSD) is a phenomenon that challenges the homeostatic mechanisms on which normal brain function so critically depends. Analyzing the sequence of events in CSD holds the potential of providing new insight in the physiological processes underlying normal brain function as well as the pathophysiology of neurological conditions characterized by ionic dyshomeostasis. Here, we have studied the sequential progression of CSD in awake wild-type mice and in mice lacking aquaporin-4 (AQP4) or inositol 1,4,5-triphosphate type 2 receptor (IP3R2). By the use of a novel combination of genetically encoded sensors that a novel combination - an unprecedented temporal and spatial resolution, we show that CSD leads to brisk Ca2+ signals in astrocytes and that the duration of these Ca2+ signals is shortened in the absence of AQP4 but not in the absence of IP3R2. The decrease of the astrocytic, AQP4-dependent Ca2+ signals, coincides in time and space with a decrease in the duration of extracellular glutamate overflow but not with the initial peak of the glutamate release suggesting that in CSD, extracellular glutamate accumulation is extended through AQP4-dependent glutamate release from astrocytes. The present data point to a salient glial contribution to CSD and identify AQP4 as a new target for therapy.

Human and mouse cortical astrocytes differ in aquaporin-4 polarization toward microvessels.

Aquaporin-4 (AQP4), the predominant water channel in the brain, is expressed in astrocytes and ependymal cells. In rodents AQP4 is highly polarized to perivascular astrocytic endfeet and loss of AQP4 polarization is associated with disease. The present study was undertaken to compare the expression pattern of AQP4 in human and mouse cortical astrocytes. Cortical tissue specimens were sampled from 11 individuals undergoing neurosurgery wherein brain tissue was removed as part of the procedure, and compared with cortical tissue from 5 adult wild-type mice processed similarly. The tissue samples were immersion-fixed and prepared for AQP4 immunogold electron microscopy, allowing quantitative assessment of AQP4's subcellular distribution. In mouse we found that AQP4 water channels were prominently clustered around vessels, being 5 to 10-fold more abundant in astrocytic endfoot membranes facing the capillary endothelium than in parenchymal astrocytic membranes. In contrast, AQP4 was markedly less polarized in human astrocytes, being only two to three-fold enriched in astrocytic endfoot membranes adjacent to capillaries. The lower degree of AQP4 polarization in human subjects (1/3 of that in mice) was mainly due to higher AQP4 expression in parenchymal astrocytic membranes. We conclude that there are hitherto unrecognized species differences in AQP4 polarization toward microvessels in the cerebral cortex.

Removal of aquaporin-4 from glial and ependymal membranes causes brain water accumulation.

There is a constitutive production of water in brain. The efflux routes of this excess water remain to be identified. We used basal brain water content as a proxy for the capacity of water exit routes. Basal brain water content was increased in mice with a complete loss of aquaporin-4 (AQP4) water channels (global Aqp4-/- mice), but not in mice with a selective removal of perivascular AQP4 or in a novel mouse line with a selective deletion of ependymal AQP4 (Foxj1-Cre:Aqp4flox/flox mice). Unique for the global Aqp4-/- mice is the loss of the AQP4 pool subjacent to the pial membrane. Our data suggest that water accumulates in brain when subpial AQP4 is missing, pointing to a critical role of this pool of water channels in brain water exit.

Cortical astrogliosis and increased perivascular aquaporin-4 in idiopathic intracranial hypertension.

The syndrome idiopathic intracranial hypertension (IIH) includes symptoms and signs of raised intracranial pressure (ICP) and impaired vision, usually in overweight persons. The pathogenesis is unknown. In the present prospective observational study, we characterized the histopathological changes in biopsies from the frontal brain cortical parenchyma obtained from 18 IIH patients. Reference specimens were sampled from 13 patients who underwent brain surgery for epilepsy, tumors or acute vascular diseases. Overnight ICP monitoring revealed abnormal intracranial pressure wave amplitudes in 14/18 IIH patients, who underwent shunt surgery and all responded favorably. A remarkable histopathological observation in IIH patients was patchy astrogliosis defined as clusters of hypertrophic astrocytes enclosing a nest of nerve cells. Distinct astrocyte domains (i.e. no overlap between astrocyte processes) were lacking in most IIH biopsy specimens, in contrast to their prevalence in reference specimens. Evidence of astrogliosis in IIH was accompanied with significantly increased aquaporin-4 (AQP4) immunoreactivity over perivascular astrocytic endfeet, compared to the reference specimens, measured with densitometry. Scattered CD68 immunoreactive cells (activated microglia and macrophages) were recognized, indicative of some inflammation. No apoptotic cells were demonstrable. We conclude that the patchy astrogliosis is a major finding in patients with IIH. We propose that the astrogliosis impairs intracranial pressure-volume reserve capacity, i.e. intracranial compliance, and contributes to the IIH by restricting the outflow of fluid from the cranium. The increased perivascular AQP4 in IIH may represent a compensatory mechanism to enhance brain fluid drainage.

Dynamics of Ionic Shifts in Cortical Spreading Depression.

Cortical spreading depression is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity. Accumulating evidence indicates that cortical spreading depression underlies the migraine aura and that similar waves promote tissue damage in stroke, trauma, and hemorrhage. Cortical spreading depression is characterized by neuronal swelling, profound elevation of extracellular potassium and glutamate, multiphasic blood flow changes, and drop in tissue oxygen tension. The slow speed of the cortical spreading depression wave implies that it is mediated by diffusion of a chemical substance, yet the identity of this substance and the pathway it follows are unknown. Intercellular spread between gap junction-coupled neurons or glial cells and interstitial diffusion of K(+) or glutamate have been proposed. Here we use extracellular direct current potential recordings, K(+)-sensitive microelectrodes, and 2-photon imaging with ultrasensitive Ca(2+) and glutamate fluorescent probes to elucidate the spatiotemporal dynamics of ionic shifts associated with the propagation of cortical spreading depression in the visual cortex of adult living mice. Our data argue against intercellular spread of Ca(2+) carrying the cortical spreading depression wavefront and are in favor of interstitial K(+) diffusion, rather than glutamate diffusion, as the leading event in cortical spreading depression.