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Drowning stars: reassessing the roleof astrocytes in brain edemaAlexander S. Thrane1,2,3, Vinita Rangroo Thrane1,2,3, and Maiken Nedergaard1

1 Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, Department of Neurosurgery,

University of Rochester Medical Center, Rochester, New York 14642, USA2 Department of Ophthalmology, Haukeland University Hospital, Bergen 5021, Norway3 Letten Centre, Institute of Basic Medical Sciences, Department of Physiology, University of Oslo, 0317 Oslo, Norway

Opinion

Edema formation frequently complicates brain infarc-tion, tumors, and trauma. Despite the significant mor-tality of this condition, current treatment options areoften ineffective or incompletely understood. Recentstudies have revealed the existence of a brain-wideparavascular pathway for cerebrospinal (CSF) and inter-stitial fluid (ISF) exchange. The current review criticallyexamines the contribution of this ‘glymphatic’ system tothe main types of brain edema. We propose that incytotoxic edema, energy depletion enhances glymphaticCSF influx, whilst suppressing ISF efflux. We also arguethat paravascular inflammation or ‘paravasculitis’ playsa critical role in vasogenic edema. Finally, recentadvances in diagnostic imaging of glymphatic functionmay hold the key to defining the edema profile of indi-vidual patients, and thus enable more targeted therapy.

Unclear waters in brain research?Brain edema is a potentially fatal accumulation of fluidwithin the brain tissue, which can be caused by a range ofmedical conditions, including stroke, traumatic brain inju-ry (TBI), brain tumors or metastases, meningitis, brainabscesses, water intoxication, altitude sickness, malignanthypertension, hypoglycemia, and metabolic encephalopa-thies [1]. In the current review, we will primarily discussthe acute causes of brain edema and refer to other texts forcoverage on more chronic conditions such as peri-tumoredema [2,3].

Edema is a pathological phenomenon that may aggra-vate injury by either causing cellular dysfunction if fluidaccumulates intracellularly, or by increasing the distancethrough which oxygen, nutrients and wastes have to dif-fuse when it is extracellular. Fluid build-up is more dan-gerous in the brain than in peripheral tissues for severalmacro- and microscopic reasons. Macroscopically, thebrain is encased within a rigid skull causing any paren-chymal swelling to increase intracranial pressure (ICP)and potentially compress other fluid compartments, suchas the vasculature. This space limitation can set in motiona vicious cycle where elevated ICP compresses both capil-lary perfusion and venous drainage, which if unchecked,

0166-2236/

� 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tins.2014.08.010

Corresponding author: Thrane, A.S. (alexander.thrane@gmail.com).Keywords: cerebral edema; astrocyte; aquaporin-4; glymphatic; paravascular.

causes further edema, cerebral ischemia, brain herniation,and a lethal compression of brainstem cardiorespiratorycenters. Brain edema can, therefore, be thought of as anintracranial compartment syndrome, and this global un-derstanding forms the basis for core therapies such astrephination or surgical decompression, which have beenpracticed since ancient times [1].

Although several key molecular players that contributeto fluid accumulation have been identified in the lastdecade, our ‘microscopic’ understanding of brain edemais still incomplete. Key players likely include the waterchannel, aquaporin-4 (AQP4), the Na+-K+-Cl– cotranspor-ter 1 (NKCC1), sulfonylurea receptor 1 (SUR1)-regulatednon-selective cation channels (NCCa-ATP), matrix-metallo-proteinase 9 (MMP-9), thrombin, substance P, complementreceptors, chemokine receptors (e.g., CCR2), and vascularendothelial growth factor (VEGF) (see Glossary) [2,4–6]. However, inhibiting or deleting some of these putativemolecular targets can be both beneficial and detrimental,depending on when the treatment is initiated and thecause of the edema. We propose that these therapeuticheterogeneities can be at least partly explained by a pre-viously unrecognized contribution from a brain-wide sys-tem for cerebrospinal fluid (CSF) and interstitial fluid(ISF) exchange, called the glymphatic pathway.

Composition of major water compartments in brainTo understand the molecular mechanisms that underliebrain edema, we first need to examine physiological waterand ion homeostasis in the central nervous system (CNS)[3,7,8]. Water and solutes in the brain are distributed intofour distinct fluid compartments separated by specializedcellular barriers: the intracellular fluid (ICF); ISF; CSF;and vascular compartments (Figure 1). CSF composition isprimarily determined by the choroid plexus, and its pro-duction can be experimentally suppressed by inhibition ofNKCC1 or carbonic anhydrase [9]. CSF contains a rela-tively high concentration of sodium to compensate forits low protein content [9]. ICF composition is energy-dependent and set up by the Na+-K+-ATPase and severalco-transporters relying on the transmembrane Na+ gradi-ent that this pump generates, such as (Na+)-K+-Cl–, gluta-mate, glucose, Na+–H+ and Na+–Ca2+-transporters [10].The ICF composition in brain differs in several importantrespects between different cell types, broadly discussedhere as neurons and glia (Table 1) [11,12]. Neurons, for

Trends in Neurosciences xx (2014) 1–9 1

Glossary

Aquaporin-4 (AQP4): plasma membrane water channel, belonging to the

aquaporin family, and primarily expressed by astrocytes in brain on

paravascular processes or end-feet.

Blood–brain barrier (BBB): a selective permeability barrier consisting of

vascular endothelial cells and endothelial tight junctions that separates the

central nervous system from the vascular compartment.

Convection: collective movement of water and solutes either as a result of

diffusion and/or advection. The latter refers to bulk motion of the fluid driven

by any combination of gradients (e.g., pressure, electrical, thermal, gravita-

tional). In rare instances where the flow of a system = 0, then total convection

�diffusion. However, in most biologically relevant situations where flow is

high, convection �advection.

Cushing reflex: severely elevated intracranial pressure (ICP) can cause patients to

develop bradycardia, irregular breathing, and increased blood-pressure.

Cytotoxic brain edema: swelling of brain cells due to a failure of cellular energy

metabolism (i.e., intact blood–brain barrier). Examples include ischemic stroke

and traumatic brain injury.

Diffusion: Redistribution of solutes from an area of high to an area of low

concentration as a result of Brownian or random molecule movement.

Diffusion flux increases as the concentration gradient becomes larger (Fick’s

first law) and decreases as a function of Hmolecular weight (Graham’s law).

Diffusion versus advection: diffusion is a slow passive process that does not

involve bulk movement of fluid, and is highly dependent on concentration

gradient and weight of diffusing molecules. Advection relates to rapid

directional movement of a fluid, which is largely independent of molecular

weight and concentration gradients. Advection is thought to be the main

mechanism governing interstitial fluid turnover in peripheral tissues, and likely

also the brain [31,108].

Glymphatic system: paravacular fluid exchange pathway that enables brain

interstitial and cerebrospinal fluid turnover and is facilitated by glial cells.

Hemorrhagic brain edema: brain swelling caused by a complete breakdown of

the BBB with leakage of all vascular contents including red blood cells, usually

in the context of a hemorrhagic stroke or traumatic brain injury.

Interstitial brain edema: an anatomical term used to describe brain swelling

caused by fluid accumulation in the interstitial space, which can occur during

both ionic and vasogenic edema.

Ionic brain edema: a functional term used to describe brain swelling caused by

net influx of salts (primarily NaCl) and water from the vasculature and/or CSF.

Examples include ischemic stroke and traumatic brain injury.

Na+-K+-Cl– cotransporter 1 (NKCC1): transmembrane cation-chloride trans-

porter widely expressed in secretory organs, the choroid plexus, and at a lower

level in both neurons and astrocytes.

Osmotic brain edema: a subtype of ionic edema where low blood osmolarity

forces net water influx to the brain; for example, water intoxication, syndrome

of inappropriate antidiuretic hormone secretion (SIADH).

Penumbra: a region of perfused and potentially salvageable tissue surrounding

the core of a brain infarct. The penumbra is often defined experimentally by

staining for hypoxic tissue with 2,3,5-triphenyltetrazolium chloride (TTC) or

clinically by examining the perfusion–diffusion mismatch (PDM) on MRI.

Starling’s equation: Jv = Kf [(Pcapillary – Pinterstitium) – (pcapillary –

pinterstitium)]; that is, net fluid movement (Jv) = filtration coefficient (Kf) �[(hydrostatic pressure gradient) – reflection coefficient (s) � (oncotic pressure

gradient)]. A recent reconsideration of this equation by Simard et al. has added

separate filtration coefficients for hydrostatic and osmotic forces, to better

reflect the unique properties of the BBB.

Sulfonylurea receptor 1 (SUR1)-regulated non-selective cation channels

(NCCa-ATP): transmembrane cation channels that becomes activated following

energy depletion and is linked to cytotoxic, ionic and vasogenic brain edema.

Vasogenic brain edema: a functional term used to describe brain swelling

caused by increase BBB permeability, causing leakage of protein, water and

salts. Examples include ischemic stroke, traumatic brain injury, brain tumor or

metastasis, subarachnoid hemorrhage, and meningitis.

Virchow–Robin space (VRS): a term used for macroscopically visible extension

of the subarachnoid space that ensheaths arteries as they penetrate the brain

parenchyma. The VRS represents the proximal extension of the peri-arterial

space implicated in the glymphatic system.

Gap width≈ 20 nm

Dura m.

Ast ro.

PVS

Neur.

Vein

CSF

Peri.Mic r.

Artery

CSF(10%)Blood(10%)Brain(80%)

ISF(12%)

ICF(68%)

(A) (B)

AQP4Key: CNXZOSLC NKA Solutes

Para-venous clea ran cePara-arterial influx Convec�on(C)

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Figure 1. The glymphatic system regulates cerebrospinal fluid (CSF) and

interstitial fluid (ISF) exchange in the brain. (A) Illustration of the main fluid

compartments in the brain. (B) Diagram of fluid influx via penetrating arteries and

efflux along a subset of large-caliber veins. (C) Diagram of proposed molecular

mechanisms governing paravascular CSF–ISF exchange. Abbreviations:

paravascular space, PVS; solute carrier, SLC; zonula occludens, ZO; connexin,

CNX; and Na+-K+-ATPase, NKA; intracellular fluid, ICF; aquaporin-4, AQP4.

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example, maintain a lower intracellular Cl– concentrationthan glia through expression of KCC2, which is importantfor the hyperpolarizing effect of the inhibitory neurotrans-mitter, g-aminobutyric acid (GABA) [13]. Glia have up tofour-times greater water permeability due to enrichmentwith water channel aquaporin-4 (AQP4) [14]. ISF compo-sition is dependent both on solutes exported from braincells and exchange with CSF, and is very similar incomposition to the latter. Conversely, the vascular

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compartment is largely independent from all the otherwater compartments in the CNS due to the blood–brainbarrier (BBB), which has a very low permeability to majorosmolytes like Na+, K+ and Cl–, and is impermeable toproteins [9,15].

Brain, CSF, and blood are separated by two concentricbarriersThe different elements of the neurovascular unit are closelyinter-dependent, and interrupting endothelial tight junc-tions, pericyte coverage, or astrocyte function alone cancompromise the entire BBB [16–20]. The barrier functionin most living vertebrates is thought to lie in the vascularendothelium, which expresses abundant tight junctionsthat prevent solute entry into brain [16,17,21]. Unlike inother organs, cerebral endothelial cells are entirely devoid ofwater channel aquaporin-1 (AQP1) and other aquaporins[3]. However, the cerebral endothelium has extensive trans-porter expression, along with a potential for selective vesic-ular transcytosis (pinocytosis) in pathological settings,which can flux large amounts of water along with ions,glucose and amino acids [9,22,23]. Most vertebrates alsopossess a second successive ‘glial barrier’ outside the bloodvessel wall [24], which has arisen multiple times duringevolution, yet its exact function has long been unclear[25]. In rodents, a recent electron micrographic 3D recon-struction found this second barrier consists primarily ofastrocyte end-foot processes, covering 99.7% of the vascula-ture, with the remaining area being made up of small 20-nminter-cellular clefts [26]. Pericytes and microglial processesare also scattered in between the vascular wall and astro-cytic end-feet [26]. The dimensions of the intercellular clefts

Table 1. Approximate composition of major water-containing compartments in cerebral cortex [10,31,109–111]

ECF ICF

Blood CSF ISF Glia Neurons

Volume fraction (%) 10% 10% 12% 41% 27%

Smallest diameter (mm) 8 5 0.04–0.06 0.5 (incl. gap junctions = 0.0015)

Water (%) 93% 99% 99% 77%

Sodium (mM) 138 155 143 17–20 4–10

Chloride (mM) 102 125 130 30–35 2–10

Potassium (mM) 4.5 3–4 3–4 100–140

Bicarbonate (mM) 23 28 10–20 10–15

Amino acids (mM) 9 1 1 138

Protein (mg/ml) 70.0 0.4 0.35 200

Glucose (mM) 5 3–4 3 3

Calcium (mM) 1.5 1.5–2.0 1.5–2.0 0.0001

Magnesium (mM) 0.5 0.9–1.3 0.9–1.3 9–11

pH 7.40 7.38 7.22 7.00

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would imply that the filter size of the glial barrier is largeenough for nearly all mammalian proteins (median size�35 kDa and diameter 2–3 nm; including serum albumin�70 kDa and 4–6 nm) [27]. Additionally, the ‘glial barrier’has a much higher water permeability than the BBB, asalmost 40% of its surface area is studded with AQP4 waterchannels, expressed as large rafts of tetramers called or-thogonal arrays of particles (OAPs) [8].

The glymphatic system facilitates rapid interstitial fluidturnoverAs an organ, the brain combines a densely cellular tissuewith a disproportionally high metabolic rate, indicating aneed for rapid fluid turnover within a tight space (Figure 1)[28]. However, the brain is entirely devoid of a lymphaticdrainage system, which normally facilitates ISF turnoverin other tissues of the body [29]. Until recently, the rapidISF turnover observed experimentally in brain (0.15–0.29 ml min–1 g–1) was thought to occur mainly via passiveintercellular diffusion [30]. However, the extracellularcompartment in the brain is so narrow and tortuous thatit would take albumin-sized molecules 10 h to diffuse 1 mm[31]. Recent evidence may help answer this puzzle. Datafrom our group suggests that two distinct barriers (endo-thelial and glial) at the blood–brain interface may haveevolved to delineate a separate paravascular ‘highway’ forfluid exchange [32–36]. Since the brain is also denselyvascularized, with an inter-capillary distance of 17–58 mm in grey matter [37], a slow paravascular circulationprovides unique access to all areas. Our results indicatethat the paravascular compartment between glial end-footprocesses and vascular cells is continuous all the way downalong penetrating arterioles to capillaries and continuesalong veins; permitting ISF influx along almost all pene-trating arteries (called the Virchow–Robin space), andefflux along a select population of large-caliber ‘deep veins’[32].

Due to the lymphatic-like function of this system andits dependence on convective water movement facilitatedby glial cells it was termed the ‘glymphatic’ system[32]. Deleting the glial water channel AQP4 decreasesglymphatic solute movement by more than 60% [32]. Theglymphatic system thus provides a novel explanation tothe paradoxical localization of AQP4, which is only

expressed on astroglial end-feet abutting the vessel wall,whilst the endothelial barrier is entirely devoid of aqua-porins [8]. Several factors have been identified that coulddrive glymphatic bulk-flow, such as arterial pulse-pres-sure, which, when either pharmacologically or surgicallyaltered, can increase paravascular tracer movement by20–30% [34]. Glymphatic ISF turnover might also beindirectly regulated by other parameters such as neuro-nal activity, sleep, and anesthesia, which all alter thedimensions of the ISF compartment [31,35]. Glial controlof extracellular matrix tension, and thus hydrostatic ISFpressure via cytoskeletal remodeling during physiologyand in reactive gliosis, could also influence glymphaticfunction [38]. Finally, the glymphatic system may notonly be important for fluid transport, but recent evidencealso suggests this system facilitates the movement oflipids, signaling molecules, and immune cells[33,39,40]. The glymphatic system may, therefore, repre-sent not only a missing link in our understanding ofphysiological water and ion homeostasis, but also brainedema [32,41,42].

Current concepts regarding brain edema formationTo explain the heterogeneous features of brain edema indifferent pathologies and the different response to anti-edema therapies most authors distinguish between at leasttwo different types: cytotoxic and vasogenic [43]. All cellsrequire energy to maintain volume homeostasis, and en-ergy depletion can, therefore, cause brain cell swelling,termed cytotoxic edema [44]. If energy supply is not re-stored, this process will inevitably lead to cell lysis withspillage of all intracellular content, also termed necroticcell death [43]. However, because there is no net entry offluid into the brain from the vasculature, there should, intheory, be no overall tissue swelling with ‘pure’ cytotoxicedema, merely a fluid redistribution. As we will discussnext, this is not the case, and most authors use the term‘ionic edema’ for the net entry of water and ions into thebrain that accompanies cytotoxic edema [43]. We wouldalso argue that the term cytotoxic should not be used forintracellular fluid accumulation unrelated to energy deple-tion (e.g., osmotic), because the mechanisms differ signifi-cantly [45]. Vasogenic brain edema is traditionally thoughtto represent the net extravasation of protein-rich fluid into

3

Box 1. Outstanding questions

� How is glymphatic function affected by common brain insults such

as injury, infarction, hemorrhage, infection and tumors? Several

parameters have been identified to date that can increase or decrease

glymphatic function, including size of the interstitial space, AQP4

expression/localization, and arterial pulse-pressure. Resistance to

CSF–ISF exchange, for instance, was shown to decrease rapidly

during sleep due to an expansion of ISF compartment [35]. Similarly,

ISF expansion in the area surrounding an injury or infarct would

increase glymphatic flux, whilst cytotoxic ISF shrinkage at the core

would compromise glymphatic function. Could astrocytic regulatory

volume decrease also represent a protective mechanism to regulate

interstitial size, and therefore, glymphatic flow [44]?

� What is the relative contribution of fluid diffusion and convection

at the blood–brain and CSF–brain interfaces? Because of low

hydraulic and osmotic permeability at the BBB, we would argue

that most fluid movement occurs at the paravascular CSF–ISF

interface. When convection of interstitial fluid has been examined in

other organs, the contribution of this mechanism relative to

diffusion correlates closely with molecular size [112]. Therefore,

larger molecules, such as immunoglobulins, complement factors or

extravasated albumin, would be most sensitive to the function of

glymphatic convection.

� What is the role of the interstitial and paravascular extracellular

matrix during physiology and disease? Similar to the interstitium in

other organs, both the ISF and paravascular compartments are

likely to be more of a gel than liquid phase [38,108]. The properties

of extracellular matrix proteins are, therefore, important to under-

standing the real pore size and charge selectivity of the glymphatic

system. The extracellular matrix is known to protect against

excessive ISF expansion during edema in other tissues by causing

interstitial hydrostatic pressure to build up [108]. Could attachments

of the extracellular matrix to glial end-feet open inter-end-foot clefts

when ISF pressure increases? How does extensive protein leakage,

basal lamina remodeling and reactive gliosis at the gliovascular

interface alter the extracellular matrix? Might widening of inter-

cellular clefts between adjacent glial end-foot processes contribute

to edema or alter glymphatic function?

� Is it possible to modulate the function of the glymphatic system by

interfering with water or solute transporters? Would inhibiting this

system represent a double-edged therapeutic sword similar to

AQP4 deletion, being beneficial in early cytotoxic edema, but

detrimental in vasogenic edema and delay edema resorption [8]?

What roles do other solute transporters such as those for Na+-K+-

Cl–, glucose, glutamate, amino acids and the Na+-K+-ATPase play?

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brain following a breakdown of the BBB [46]. This processis believed to involve a widening of the inter-cellular cleftsbetween endothelia and a loss of tight junctions[47,48]. Vasogenic edema will, therefore, per definitionrequire vascular perfusion, and should be more importantwhen blood flow is increased or normal (e.g., brain tumorsand metastases), than, for instance, at the core of a braininfarct [49]. However, insults that generate mainly cyto-toxic and ionic edema early on (<24 h), such as TBI andstroke, are also known to develop a second peak of vaso-genic edema after 2–4 days; termed the biphasic edemaresponse [43,50,51].

Reassessing the role of interstitial fluid dynamics inbrain edemaThe current literature leaves several important questionsunanswered about the formation and resorption of brainedema (Box 1). To explore these issues, we will next discussthe potential contribution of the glymphatic system to thedifferent stages of brain edema, using focal ischemia as anexample. Cytotoxic cell swelling begins only minutes afteran acute infarct [52], whilst overall brain swelling typicallyoccurs more slowly, with intracranial pressure reaching a

Core:cytotoxic

Penumbra:Ionic

Focal edema Core

ICFISFCSFBlood

Na+ K+

BBB

E.f.m.

P.m.K+ Na +

Penumbra

(A) (B)

TRENDS in Neurosciences

Figure 2. Focal brain edema is caused by the interplay of cytotoxic changes in the

core of an infarct or injury, and ionic mechanisms in the surrounding tissue or

penumbra. (A) Illustration of focal brain edema following an ischemic stroke. (B)

Diagram showing how net influx of Na+ into dead or dying cells in the core can set

up an ionic gradient for water influx into the penumbra, which is incompletely

counterbalanced by K+ efflux. Abbreviations: plasma membrane, P.m.; end-foot

membrane, E.f.m.; blood–brain barrier, BBB; cerebrospinal fluid, CSF; interstitial

fluid, ISF; intracellular fluid, ICF.

4

peak within the first 24 h for rats and 48–72 h for humans[3,43,46]. How does this acute energy depletion and cyto-toxic fluid redistribution into cells cause net fluid influxinto a tissue that has a severely compromised blood perfu-sion and microcirculation [53]? The answer is likely thatmost fluid accumulation occurs outside the infarct core, in amuch larger perfused region called the penumbra, wherecytotoxic edema plays a lesser role [4,54–56] (Figure 2).Previous studies exposing entire brain slices or cell cul-tures to oxygen and glucose deprivation (OGD) thereforeprovide a poor model of brain edema. To understand focalbrain edema, we need to examine the interplay betweencytotoxic fluid redistribution in an infarcted core and netfluid entry into a better-perfused penumbra [57].

Glymphatic fluid influx could drive ionic brain edemaformationIf overall brain swelling or ionic edema requires net fluidentry into the tissue surrounding an acute infarct, wheredoes this fluid come from? Arguably, it can only come fromblood or CSF [43]. Current theories were developed prior tothe discovery of the glymphatic system and suggest thationic edema originates from the vasculature [3]. An inter-esting recent theory for instance suggests that Na+ andwater influx across the BBB could occur via NKCC1 andNCCa-ATPexpressed on endothelial cells in the ATP-depletedcore of an infarct [4]. However, we would argue that fluidinflux from across the BBB is less likely for several reasons.First, the core of the injury or infarct is poorly perfused, andwould, therefore, exhibit mainly cytotoxic fluid redistribu-tion [58]. Second, the BBB, or more precisely endothelialtight junctions, are relatively intact in the perfused penum-bra of an infarct or injury [9,15]. An intact BBB would implya low osmotic, hydrostatic, and ion permeability (includingNa+, Cl– and K+) [9]. However, altered endothelial expres-sion of sodium transporter following ischemia could increaseionic and osmotic permeability somewhat [4,59,60]. Third,transcriptome data suggest that vascular endothelial cellsexpress low levels of NKCC1 and NCCa-ATP during basalconditions in the mouse brain [61].

Edema

Blood flowCa�on cont.

Protein cont.

2 Time (days)4 8

↓ATP

↓ISF

↑Na+

0.5

ISF

1

Cytotoxic Vasogenic Resorp�onIonic

Peri.

TGF-β

IFN-γ

IL-6

MMP-9

VEGF

Micr.

Mono.

Astr.

Paravenous

Protein

(A)

(B)

↔ATP

↑Na+ ↑ISF

TNF-α

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Figure 3. Phases of brain edema formation and resorption after an acute ischemic stroke. (A) Graph showing the likely changes in key parameters such as blood flow and

water content after an acute infarct (units and scale are not listed on the Y-axis as this graph represents an illustration of the most likely changes in key parameters, and not

real experimental data). (B) Diagram of suggested mechanisms that might be involved during the different phases of brain edema.

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An alternative explanation might be that edema fluid isentering the brain parenchyma as CSF via the low-resistancepara-arterial space. Arguably, this can only happen as aresult of increased CSF influx into the parenchyma, de-creased ISF efflux or a combination of the two. Glymphaticremoval of excess ISF is likely decreased following injury orinfarction [62,63], but the exact mechanisms will be discussedin subsequent sections, as these mechanisms are also impor-tant for edema resorption. With regards to CSF influxinto theparenchyma, continuous CSF secretion by the choroid plexusis likely the primary source of this fluid [9]. To facilitate thissecretion, choroid plexus cells have NKCC1 expression levelsthat are several orders of magnitude higher than endothelialcells [61], and this expression is further increased after injury[64]. Previous studies showing that NKCC1 inhibitiondecreases brain edema could, therefore, at least in theory,relate to lower choroid plexus production CSF, reducingglymphatic influx [65]. During basal conditions, para-arterialmovement of CSF is also thought to be driven by the pulsa-tility of the vascular wall [34], which is generally increasedafter a stroke, and can become further increase if intracranialpressure rises as a result of the Cushing reflex [66]. Finally,pericyte constriction and microvascular collapse could forcean expansion of the paravascular space, decreasing resis-tance to fluid influx by this route [67] (Figure 3).

Ultimately, edema build-up from either a vascular orparavascular source requires net solute entry into the brain.As is evident from Table 1, the Na+ concentration in CSF isgreater than blood, to compensate for CSF having almost noproteins to exert osmotic pressure. ATP depletion withconsequent Na+-K+-ATPase and Na+-cotransporter shut-down and cytotoxic cell swelling in the infarct core wouldcause an extensive redistribution of Na+ and Cl– from theISF to the ICF [49,68]. NCCa-ATP channels activated by ATPdepletion and expressed on parenchymal cells might

facilitate this process [4]. Gap junction connections betweenastrocytes might also rapidly distribute this Na+-influxacross the glial syncytium [19,69]. Na+ and Cl– redistribu-tion would in turn make ISF slightly hypo-osmolar relativeto CSF [49]. As opposed to the BBB, the perivascular glialend-foot barrier provides almost no osmotic resistance todiffusion by virtue of its AQP4 expression, and this osmoticISF–CSF gradient would, in theory, pull CSF into the ISFcompartment [32]. Influx of Na+ into the brain parenchymais usually accompanied by net K+ efflux that could offset theosmotic Na+ load [43]. However, in brain edema K+ efflux islikely to be much smaller than the Na+ influx for severalreasons [4,15,58], including negatively charged intracellu-lar proteins retaining ions in the ICF (see Table 1).

Astrocytes: swollen glue or drowning stars?Having examined the origins of edema fluid; we next wantto discuss where it might accumulate. Because of theirproximity to the vascular compartment and high-waterpermeability, astrocytes have long been thought to accu-mulate most of the edema fluid intracellularly [5]. At firstglance, many histological and cell culture studies seem tosupport this model [5,70]. However, more recent slice andin vivo imaging studies have revealed more contradictoryresults, with some studies finding that astrocytes swellreadily [71–74], whilst other reports finding that astro-cytes regulate volume tightly [44,75–77]. What could ex-plain this discrepancy? Technical limitations such as lowimage resolution or fluorochrome dilution upon swellingcould cause imaging results to falsely underestimate oroverlook cell volume changes [78]. Conversely, experiencefrom our lab also suggests that it is possible to overesti-mate volume changes, as ‘unhealthy’ astrocytes subject tophototoxicity, with inadequate energy supply, or in dam-aged tissues, swell much more readily [44,76].

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It is interesting to speculate whether the extensiveastrocytic expression of salt and water transporters hasbeen misinterpreted as a pathway for intracellular fluidaccumulation, and might instead represent a pathway forparavascular fluid elimination [32]. If ‘energized’ astro-cytes are capable of actively regulating their volume acrossthe broad range of osmotic and ionic challenges somestudies suggest (>20% osmolarity drop and >50 mM[K+]o increase) [44,76,77], edema fluid might at least ini-tially accumulate in the interstitial space of the penumbra;‘drowning’ the astrocytes by increasing the distance fornutrient and waste diffusion. Consistent with this idea,increasing the resistance to glymphatic circulation bydeleting glial AQP4 not only increases ISF volume duringbasal conditions [14], but also worsens experimental inter-stitial edema and causes a higher rate of spontaneoushydrocephalus in knock-out animals [3]. Conversely, ische-mia-related cytokines released from the core of an infarctcan alter astrocytic solute transporter expression in thepenumbra, and potentially compromise astrocyte volumeregulation [59]. Unfortunately, no studies have directlyexamined penumbral ISF volume in vivo during brainedema to help us resolve this important point. A moredetailed discussion of astrocyte volume regulation is be-yond the remit of the current review, we refer the reader toother overview articles on this topic [3,8,79].

Cerebral paravasculitis is the hallmark of vasogenicedemaAlthough the early stages of brain edema formation afteran infarct are characterized by salt and water influx (0–3 h), the later stages (from >3 h to 14 days) also involveBBB opening and significant extravasation of plasma pro-teins [43,46,62,80]. Traditionally, extensive BBB openinghas been thought to cause edema by increased hydraulicconductivity, causing blood-pressure-dependent proteinextravasation, with osmotic ‘fluid-drag’ [81]. However, ex-perimental measurements have revealed that the directosmotic effects of albumin extravasation are not temporal-ly correlated with edema formation and that the estimated‘fluid-drag’ can only account for a fraction of the total waterinflux (<10%) [58]. Extravasated proteins are also rapidlytaken up and subjected to lysosomal degredation by micro-glia and astrocytes, rather than being deposited in theinterstitial space [82]. Therefore, the osmotic effects ofprotein extravasation are unlikely to explain vasogenicedema. Instead, radioisotope studies indicate that, similarto ionic edema, vasogenic edema also correlates best with22Na+ influx into the brain. Since the core of an infarct orinjury has decreased 22Na+permeability during the laterstages (>3 h), most of this salt and water entry must alsooccur in the penumbra [58,83,84].

What alternative mechanisms might mediate vasogenicedema? We would argue that the delayed onset of vaso-genic edema most closely mirrors the paravascular im-mune response, or paravasculitis, which can be initiated bynecrotic debris, plasma proteins, cytokines, and/or tumorcells. Inflammation is a potent trigger and potentiator ofbrain edema through several mechanisms including hy-peremia, BBB opening, leukocyte influx, osmolyte produc-tion, immune complex accumulation, reactive oxygen

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species generation, and cytokine-related cell swelling[6,82]. Several recent lines of evidence support this im-mune response being primarily paravascular, rather thanacross the BBB. Firstly, CSF represents a major reservoirfor complement factors and immune cells in the CNS,containing up to 3000 leukocytes per ml during normalconditions [85,86]. Second, perivascular astrocytes andmicroglia can produce most major complement factorsand cytokines, secreting them via the paravascular spacedirectly into CSF [86]. Third, cytokine secretion and leu-kocyte infiltration is primarily concentrated in the para-vascular region of brain parenchyma and in the choroidplexi [87–90]. Finally, paravascular immune complex ac-cumulation has long been recognized in a variety of braindisorders, including stroke, TBI, and even neurodegenera-tive disorders like Alzheimer’s disease [40]. Conversely,blood-borne leukocytes would have limited interactionwith both CSF and ISF in the perfused regions surround-ing an injury or infarct due to the (at least initially) intactBBB discussed above [47]. The only exception to this rule isintracerebral hemorrhage, where BBB integrity iscompletely lost causing a spillage of all intravascular cellsand mediators [43].

We would, therefore, suggest that the paravascularspace acts as a crucial immune compartment during vaso-genic edema formation where astrocytes, microglia, peri-cytes, endothelial cells, and leukocytes can interactdirectly [33,91]. Peri-arterial influx and peri-venous effluxof CSF from the parenchyma could drive antigen andleukocyte recirculation to T-helper lymphocytes and den-dritic cells in the choroid plexi, which may act similarly toregional lymph nodes [92]. Following an acute brain insult,perivascular astrocytes and resident microglial immunecells are known to rapidly produce pro-inflammatory cyto-kines such as VEGF-A, interleukin-1beta (IL-1b), tumornecrosis factor-alpha (TNF-a) and interferon-gamma (IFN-g) [51,93,94]. Genetic variants related to these inflamma-tory mediators are strongly associated with worse out-comes following TBI or stroke [95–97].

Although inflammation and edema might be detrimen-tal in the short term due to space constraints, this processmay also be necessary for removal of cellular debris andmay paradoxically help seal the BBB faster by forming agliotic scar prior to re-establishment of tight-junctions[98]. It is perhaps, therefore, unsurprising that both unse-lective (e.g., dexamethasone) and selective (e.g., blockingIFN-g, chemokine receptor 2, MMP9, VEGF, IL-6, TNF-a,and TGF-b) anti-inflammatory therapies can both improveand worsen brain edema depending on when the treatmentis instituted and what the underlying cause is (e.g., infarc-tion, trauma, tumors) [89,92,99–103]. In conclusion, vaso-genic edema seems to represent a delayed and long-lastingincrease in glymphatic fluid influx of salt and water thatmay have evolved to facilitate paravascular leukocyte andcytokine delivery.

Brain edema is absorbed into paravascular CSF ratherthan bloodTo restore normal function brain edema needs to be clearedalong with excess ions and proteins that have leakedacross the BBB in a process that can take many weeks.

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Traditionally, this is thought to occur by clearance via thevascular compartment [42]. However, hydrostatic and os-motic forces would instead favor fluid egress from thevasculature into the brain so long as the BBB is open,and BBB closure often precedes edema resorption[9,49]. This discrepancy has led some authors to suggestdrainage via subarachnoid CSF, but it was unclear howfluid from deep in the parenchyma reaches the brainsurface [42]. The recently discovered glymphatic systemprovides an elegant explanation to this problem [32]. Ourdata suggest that excess parenchymal fluid and solutes canbe effectively cleared from the interstitial space via theparavascular space along a subset of large veins [32]. Thisglymphatic model may also better explain the long-termconsequences of surviving brain edema, such as chronicpost-traumatic encephalopathy, syndrome of the trephineand vascular dementia. Structural changes to the glym-phatic system, such as perivascular reactive gliosis andAQP4 mislocalization, can persist for almost 1 month afterinjury or infarction [63,62]. Both TBI and stroke can,therefore, cause a long-lasting impairment of glymphaticclearance of waste products such as b-amyloid [62,63], andmay be responsible for a chronically enlarged Virchow–Robin space often seen in these patient groups [104].

Concluding remarksIn summary, most brain disorders generate a complexspatial and temporal pattern of brain edema, involvingseveral distinct mechanisms, which respond very differ-ently to therapies (Box 1). Patients who, for instance, sufferan ischemic stroke or TBI likely experience some immedi-ate cytotoxic edema at the core of the injury, whilst thesurround tissue may first develop ionic and later vasogenicedema (see Figure 2) [43,46]. Carefully characterizing thetype of brain edema and choosing the time-point at whichto intervene might, therefore, be crucial. Decompressivecraniectomy can, for example, be helpful for the earlycytotoxic and ionic edema phase, whilst it can be detrimen-tal if a vasogenic component is present, or during theresorption phase [43]. Similarly, recently suggested thera-pies targeting water movement via glial AQP4 have beenshown to be beneficial in conditions associated with pri-marily cytotoxic and ionic edema by limiting water influx(e.g., large ischemic strokes, water intoxication) [5,14]. Con-versely, AQP4 inhibition or deletion adversely affects out-come in some (e.g., tumors, abscesses), but not all (e.g.,meningitis), diseases associated with vasogenic edema, orwhen given during the recovery phase of edema [3].

Current tools have limited sensitivity and specificity toaccurately determine what ‘type’ of edema a patient has[43]. Brain edema is frequently examined in patients usingdiffusion-tensor weighted (DWI) magnetic resonance im-aging (MRI) looking at the apparent diffusion coefficient(ADC) of isotropic water movement. However, a decreasedADC occurs both in cytotoxic edema (ISF shrinkage) and inisolated ISF expansion, making this tool less useful indistinguishing types of brain edema and guiding therapy[105,106]. It is, therefore, interesting to speculate whethera recently characterized MRI tracer imaging techniqueexamining glymphatic function might better guide thechoice and timing of treatment [41,107]. In conclusion,

further exploration of the critically important paravascu-lar region may hold the key to developing new edematherapies, correctly staging different types of edema andreducing long-term morbidity.

AcknowledgmentsWe thank E.A. Nagelhus and H.E. Fossum for discussions on the topic.This work was supported by the US National Institutes of Health grantsNS075177 and NS078304 to M.N. and the Fulbright Foundation.

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