Pathophysiology 14 (2007) 183–193
Disruption of ionic and cell volume homeostasis
in cerebral ischemia: The perfect storm
Alexander A. Mongin∗
Center for Neuropharmacology and Neuroscience, Albany Medical College,
47 New Scotland Avenue (MC-136), Albany, NY 12208, USA
The mechanisms of brain tissue damage in stroke are strongly linked to the phenomenon of excitotoxicity, which is defined as damage or
death of neural cells due to excessive activation of receptors for the excitatory neurotransmitters glutamate and aspartate. Under physiological
and cytoplasmic Ca2+overload. High cytoplasmic levels of Ca2+activate many degradative processes that, depending on the metabolic status,
cause immediate or delayed death of neural cells. This traditional view has been expanded by a number of observations that implicate Cl−
channels and several types of non-channel transporter proteins, such as the Na+,K+,2Cl−cotransporter, Na+/H+exchanger, and Na+/Ca2+
exchanger, in the development of glutamate toxicity. Some of these ion transporters increase tissue damage by promoting pathological cell
swelling and necrotic cell death, while others contribute to a long-term accumulation of cytoplasmic Ca2+. This brief review is aimed at
illustrating how the dysregulation of various ion transport processes combine in a ‘perfect storm’ that disrupts neural ionic homeostasis and
culminates in the irreversible damage and death of neural cells. The clinical relevance of individual transporters as targets for therapeutic
intervention in stroke is also briefly discussed.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Excitotoxicity; Cellular edema; Volume-regulated anion channels; Na+,K+,2Cl−cotransport; Na+/H+exchange; Na+/Ca2+exchange
1. Introduction: overview of ischemic brain damage
majority of strokes, such a reduction is restricted to isolated
regions of the brain and is caused by an occlusion of one of
the major brain arteries, either by an embolus or thrombosis.
In some cases, local perfusion deficits are associated with
hemorrhage. Cerebral blood supply may also be completely
almost exclusively dependent on oxidation of blood-derived
glucose, reductions in cerebral blood flow below a threshold
metabolism, which is followed by severe tissue damage (for
review see [1–3]).
∗Tel.: +1 518 262 9052; fax: +1 518 262 5799.
E-mail address: MonginA@mail.amc.edu.
The degree of brain tissue damage is determined by the
severity and duration of the perfusion deficit . In focal
strokes, ischemic tissue is divided into an ‘infarction core’
and ‘penumbra’. The core is the area of the brain where
blood flow is reduced below ∼15–20% of its normal levels.
In the core, rapid anoxic depolarization causes immediate
loss of membrane potential followed by the loss of mem-
brane integrity and rapid necrotic cell death. The penumbra
is the tissue surrounding the core where the blood flow is
perfusion to 20–50% of normal levels places tissue at severe
risk, while the higher rates of blood flow allow for survival.
Cells in penumbra may retain their viability for hours or per-
haps days, although penumbral neurons typically lose their
excitability within a few minutes after the onset of ischemia
Over time, anoxic depolarization in the core starts to
0928-4680/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
A.A. Mongin / Pathophysiology 14 (2007) 183–193
depolarizations in the penumbral tissue, referred to as ‘peri-
additional metabolic burden on the already compromised tis-
sue and promote propagation of the ischemic lesion, which
may grow as much as 30–100% compared to its initial size
The major cause of cell death in the ischemic tissue
is an uncontrolled elevation of intracellular [Ca2+] [6–8].
In the core, levels of extracellular Ca2+drop from ∼2
to 0.05–0.08mM, reflecting major Ca2+movement into
the intracellular space. Two main Ca2+permeability path-
ways, which contribute to this shift, are glutamate-gated
N-methyl-d-aspartate (NMDA) receptor-channels (NMDA
is the selective agonist for these receptors) and voltage-gated
Ca2+channels. Both NMDA and voltage-dependent Ca2+
channels require strong membrane depolarization in order
to be fully activated. In the ischemic tissue, membrane
depolarization is determined by a metabolic inhibition of
the Na+,K+-pump, but is greatly accelerated by Na+influx
via the voltage-gated Na+channels and glutamate-gated
(AMPA) receptor-channels (AMPA is the selective agonist
for these receptors). The resulting sustained and typically
irreversible increases in cytoplasmic [Ca2+] directly or
indirectly trigger numerous pathological processes. Because
acid neurotransmitters, glutamate and aspartate, they are
collectively termed ‘excitotoxicity’ [6,9,10].
Excitotoxic cell damage and death involve activation of
numerous damaging cascades, which are set in motion,
directly or indirectly, by elevations in cytosolic Ca2+. Some
key damaging events include activation of Ca2+-dependent
proteases and phospholipases, alterations in the plasma
membrane permeability, depolarization of mitochondria and
release of mitochondrial pro-apoptotic proteins, production
Depending on their metabolic status, neural cells may die
via necrotic or apoptotic mechanisms. The complex biology
of the ischemic cell death is discussed in detail in several
comprehensive reviews [1,3,5].
This review focuses on the ischemic dysregulation of
several ion transporters, which are not directly involved in
transmembrane Ca2+fluxes and therefore receive little atten-
tion in the ischemia literature. Failure to maintain proper
ionic gradients plays a major role in determining the fate
of the neural cells in the ischemic brain and contributes
to spatial propagation of the ischemic infarction. The main
emphasis here is placed on the in vivo data obtained in
evant. However, because many of the molecular mechanisms
involved in ischemic cell damage have been deciphered in
vitro, the important in vitro experiments are also briefly dis-
cussed. For more extensive coverage of the relevant topics
the reader is encouraged to consult several comprehensive
reviews [11–14], and particularly .
2. Transmembrane Na+and Cl−fluxes are
underappreciated contributors to the excitotoxic
tissue damage: an osmotic connection
Although excitotoxicity is frequently considered synony-
mous with Ca2+-dependent cell death, early studies clearly
distinguished two neurotoxic effects of glutamate: rapid tox-
icity determined mainly by the influx of Na+and Cl−and
osmotic swelling, and a more delayed Ca2+-dependent cell
death [6,7,9]. The first form of excitotoxicity is reliant on
the presence of extracellular Na+and Cl−, but not Ca2+,
and manifests as rapidly forming dendritic varicosities fol-
lowed by generalized somatic swelling and necrotic cell
death [6,16–18]. Replacement of extracellular Na+or Cl−
with impermeant ion species prevents glutamate-induced
cell swelling and strongly reduces cell death. Pharmaco-
logical evidence suggests that Na+enters the cell mainly
via the glutamate-gated AMPA and NMDA channels, while
the identity of the Cl−influx pathway is less clear. Several
studies proposed that Cl−influx may be at least partially
mediated by GABAAreceptor-channels (GABA stands for
gamma-aminobutyric acid, a glutamate-derived inhibitory
neurotransmitter), as GABAA receptor blockers partially
prevent excitotoxic cell injury [19,20]. However, a recent
study by Inoue and Okada demonstrated that, in cortical
slices and cultured neurons, glutamate-induced Cl−fluxes
are largely mediated by the volume-sensitive outwardly rec-
literature as volume-sensitive organic osmolyte-anion chan-
nel (VSOAC) or volume-regulated anion channel (VRAC)
[22–24]. In this review the acronym VRAC will be used.
Application of VRAC blockers prevents both gluta-
mate agonist-induced dendritic swelling and neuronal death
to excitotoxins, as well as for cell volume recovery when
excitotoxins are removed . In vivo, two VRAC blockers,
Merck compound L644,711 and tamoxifen, potently protect
brain tissue against ischemic damage in animal models of
focal transient and permanent ischemia [26–29]. However,
as discussed in the next section, the protective actions of
VRAC blockers are not restricted to preventing pathological
cell swelling, but also involve other mechanisms.
Uncontrolled cell swelling is frequently associated with
necrosis and may be harmful to neuronal and glial cells for
multiple reasons [13,30]. The simplest mechanism of cell
of surface membranes and can increase their volume by 2-
to 3-fold without subjecting their plasma membranes to sub-
stantial mechanical stress [31,32]. Furthermore, animal cells
possess volume regulatory mechanisms serving to protect
activates Cl−efflux via the above mentioned VRAC in con-
junction with K+loss via swelling-activated K+channels.
Under physiological conditions these two independent per-
meability pathways mediate electrically coupled loss of K+
A.A. Mongin / Pathophysiology 14 (2007) 183–193
and Cl−, which is accompanied by an efflux of osmotically
obligated water, and regulatory volume decrease, or RVD
[30,33,34]. However, in pathology, anoxic and/or glutamate-
driven depolarization disrupts cell volume regulation due to
an inhibition of the Na+,K+-pump, dissipation of K+and
Cl−electrochemical gradients, and blockade of volume reg-
ulatory channels [13,21,25,35]. Na+begins to accumulate
uncontrollably in the cells via leak mechanisms, voltage-
gated Na+channels, and other Na+-transporting pathways,
which are considered in following sections. In glial cells,
ischemia additionally opens the unique ATP-dependent non-
selective cation channels that greatly increase Na+influx and
promote astroglial swelling .
The cell swelling can be detrimental due to activa-
tion of various ion transporting pathways even when K+
gradients and intracellular ATP levels are relatively well pre-
served (as it would be expected on periphery of penumbra).
The above mentioned regulatory loss of K+and Cl−pro-
tects cells against swelling, but at the same time decreases
the transmembrane K+gradient and membrane potential,
enhances the activity of the Na+,K+-pump in both neurons
and glia [37–39]. In electrically active neuronal cells, the
Na+,K+-pump represents the main ATP-consuming enzyme.
In the face of falling ATP content, the Na+,K+-ATPase starts
to compete for ATP with the plasma membrane Ca2+pump
(PMCA). One recent report indicates that such competition
may compromise the activity of the PMCA and promote the
necrotic Ca2+overload .
Another potential link between cell volume and tissue
damage is via modulation of the NMDA channels, which,
in cortical neurons, are potently activated by pathological
receptors are insensitive to cell volume changes . Thus,
NMDA channel activation may trigger cell swelling, which,
a pathological feed-forward mechanism. Cell swelling also
activates several other membrane Ca2+permeability path-
ways, such as the transient receptor potential cation channel
TRPV4 [42,43], and triggers intracellular Ca2+release from
the IP3-sensitive stores . On one hand, all of the above
mentioned processes contribute to the Ca2+overload; on the
other hand, they accelerate ATP depletion via stimulating the
activity of the Na+,K+- and Ca2+-ATPases.
In summary, glutamate toxicity is strongly promoted by
Na+and Cl−accumulation and cell swelling. Cell swelling
activates a number of ion transport pathways. Normally such
activation is beneficial as it mediates regulatory volume
restoration via the RVD process. However, in energetically
compromised cells, substantial transmembrane ion fluxes
cannot be sustained, and therefore cell swelling becomes
continuous and diverts cells to necrotic cell death. Impor-
tantly, cell swelling activates or strongly potentiates several
Ca2+transporting pathways that likely also contribute to
delayed forms of excitotoxic cell injury. Although swelling
ischemic cell and tissue damage in vitro and in vivo (see next
3. Volume-regulated anion channels and the reversed
mode of glutamate transporters are two major
sources of pathological glutamate release in vivo
Pathological swelling found in vitro, in neural cells sub-
jected to glutamate, anoxia, or chemical ischemia, has an in
vivo equivalent named cellular or cytotoxic edema. Cellular
and has been found in human stroke and animal ischemia
in the brain is detected indirectly by means of magnetic
resonance imaging (MRI). Using diffusion-weighted imag-
ing (DWI) clinicians generate maps of apparent diffusion
coefficient (ADC) of water in the brain. Upon movement
from the extracellular to the intracellular space, the mobil-
ity of H2O molecules is hindered by high concentration of
organic macromolecules; a corresponding decrease in the
ADC reflects cellular swelling. In human stroke, cellular
edema can be registered as early as measurements are practi-
In animal models, changes in ADC signal correspond very
(EM) in perfusion-fixed brain sections . The final vol-
ume of the brain damage in stroke strongly correlates with
changes in the ADC [49–51]. Therefore, cellular edema is
closely associated with ischemic brain damage.
Cell swelling in ischemia is mainly seen in one cell type,
astrocytes, and is most pronounced in their perivascular pro-
cesses called astrocytic endfeet. Some dendritic swelling
is also found, but neuronal soma are not changed or even
shrunken. The precise reasons for such selective susceptibil-
ity of astrocytes to ischemic swelling are unknown; several
hypothetical mechanisms are discussed elsewhere [13,45].
A key study that proposed a mechanistic link between
astrocytic swelling and brain damage has been done by
Kimelberg and co-workers, who found that exposure of
cultured astrocytes to hypoosmotic media triggers massive
release of several cytosolic amino acids, including gluta-
mate and aspartate . The swelling-activated amino acid
release permeability pathway is sensitive to a number of
anion channel blockers, and has strong similarities to the
already mentioned swelling-activated anion channel VRAC
[22,24,52–54]. Since electrophysiological studies confirmed
that VRAC possesses measurable permeability to glutamate
and aspartate [55–57], this channel has long been considered
a hypothetical pathway for pathological release of excitotox-
ins from swollen cells [13,45,46].
In spite of the extensive search in the field, the
molecular identity of VRAC remains unknown [24,58].
Therefore any studies designed to explore the physiolog-
A.A. Mongin / Pathophysiology 14 (2007) 183–193
ical and pathological roles for this channel have to rely
on pharmacological tools. Several commonly used VRAC
blockers are 5-nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB, IC50∼8–25?M, depending on a cell type), 4,4?-
at negative potentials, but up to 150?M at positive poten-
tials), and tamoxifen (IC50<1?M) [23,59]. The major
problem with these compounds is that they do not dis-
criminate well between VRAC and other Cl−channels.
Tamoxifen, which is frequently described as the potent and
selective inhibitor of the VRAC, is completely ineffective
in neuronal cells and blocks a number of other anion and
cation channels [60–62]. Other VRAC blockers include 1,9-
dideoxyforskolin, niflumic acid, calaxarenes, and phloretin
[23,24]. The most selective VRAC inhibitor identified so far
is the ethacrynic-acid derivative DCPIB (IC50=4?M) .
At the concentrations of 10–20?M, DCPIB nearly com-
pletely blocks Cl−currents via VRAC, but does not affect
ClC-1, -2, -4, and -5), nor voltage gated K+, Na+and Ca2+
In the rat global ischemia model, using a cortical cup
technique, Phillis et al. found 30–70% inhibition of gluta-
mate and aspartate release with the anion channel blockers
tamoxifen [65,66]. In a microdialysis study, also done in
the global ischemia model, Seki et al. found that another
anion channel inhibitor 4,4?-dinitrostilbene-2,2?-disulfonic
acid (DNDS) reduced ischemic release of excitotoxins by
50–70%, when added at 1 or 10mM in microdialysis per-
fusate . In the penumbra of the rat focal ischemia model,
50?M tamoxifen reduced ischemic glutamate and aspartate
and such contribution is likely much higher in the penumbra.
Because cell swelling in penumbra is limited, the impor-
tant question is whether it is sufficient to activate the VRAC.
Several recent in vitro studies have found that a number
of substances, which are produced or released in ischemia,
such as reactive oxygen species H2O2, reactive nitrogen
species peroxynitrite, thrombin, and ATP, all drastically
increase swelling-activated glutamate release from moder-
ately swollen astrocytes [69–72].
A therapeutical relevance of the VRAC pathway has
been tested in animal neuroprotection studies. Anion chan-
and strongly decreases the infarction size in several ischemia
is comparable with tissue plasminogen activator, the only
selective VRAC blockers because it may protect brain tissue
In addition to the release via a VRAC-like pathway, ele-
vated levels of excitatory amino acids in ischemia may occur
due to reversal of Na+-dependent glutamate transporters.
Under physiological conditions such transporters maintain
low-extracellular glutamate levels via a process of [Na+]o-
and [K+]i-dependent uptake. If the Na+and K+gradients
are collapsed, the transporters start to function in a reversed
mode, and pump glutamate outside of the cell [75,76]. In
vivo, 1mM dihydrokainate (DHK), a relatively selective
inhibitor of the glia-specific glutamate transporter GLT-1,
blocks ∼50% of ischemic glutamate release in the rat global
ischemia model  and approximately the same effect was
found in the core of the focal ischemia model . However,
when perfused in the penumbra of the focal ischemia model,
release . These data indicate that a complete collapse of
ionic gradients triggers glutamate release via reversal of the
served (as in the penumbra) transporters continue to remove
glutamate from the extracellular space.
In summary, a disruption of transmembrane ionic gra-
dients causes pathological glutamate and aspartate release
anisms. Swelling-independent release is mediated by the
reversal of glutamate transporters. This mechanism is likely
of limited therapeutic value, because glutamate transporters
are functionally important in non-ischemic tissue. Second,
swelling-activated mechanism of release likely involves
anion channel VRAC. Since VRAC activity is restricted to
cological inhibitors of VRAC are highly protective in several
animal models of ischemia. The VRAC blocker tamoxifen
is particularly effective because it is permeable through the
blood–brain barrier and therefore may be given systemically.
However, the specificity of the neuroprotective effects of
tamoxifen has to be verified using more selective VRAC
blockers, such as DCPIB.
4. Na+,K+,2Cl−cotransport is involved in NaCl
overload and pathological cell swelling
family, NKCC1 (coded by SLC12A2) and NKCC2
(SLC12A1), mediate the electroneutral co-transport of Na+,
K+, and 2Cl−. NKCC1 is ubiquitously expressed and can be
found in the CNS in both neuronal and non-neuronal cells,
while the NKCC2 expression is limited to kidney cells in
the loop of Henle [78,79]. The major biological roles for
els determined by the Cl−electrochemical equilibrium, cell
volume regulation, and epithelial NaCl reabsorption. In the
A.A. Mongin / Pathophysiology 14 (2007) 183–193
ronal function . High expression levels of NKCC1 in
immature CNS neurons and in peripheral sensory neurons
increase [Cl−]iand make GABA an excitatory neurotrans-
mitter due to outwardly directed Cl−fluxes via the GABAA
channels. On the contrary, in mature CNS neurons, NKCC1
KCC2, which drives Cl−out of the cell, thereby turning
GABA into the inhibitory neurotransmitter . In shrunken
in its phosphorylation levels. Activated transporter causes a
net movement of ions inside the cell that, at least partially,
mediates the process of regulatory volume increase, or RVI
result in cell swelling (see below).
Both NKCC isoforms are potently blocked by the ‘loop
diuretics’ furosemide and bumetanide (IC50<1?M, but the
diuretic actions of these compounds are related to inhibition
tion. When used at the concentration of 10?M, bumetanide
inhibits NKCC, but not other ion transporters. Furosemide
has much lower affinity towards NKCC and inhibits several
other anion transport pathways. Therefore, furosemide is not
used as the NKCC inhibitor in experimental studies.
artery (MCAo), continuous delivery of 100?M bumetanide
in the cortical tissue significantly reduced infarction volume
by ∼25% and tissue edema by >70% . This shows
that pathological activation of the cortical NKCC1 is a
substantial contributor to neuronal death and edema for-
mation. Interestingly, systemic delivery of 7.4–30.4mg/kg
bumetanide also reduced infarction volume by >50% and
. To exclude renal effects of bumetanide, this latter study
has been performed in nephroectomized animals. Since
bumetanide has limited permeability across the blood–brain
barrier, these results point to protective endothelial effects
of the diuretic. The pathological significance of NKCC1 in
animals. NKCC1−/−mice show 30–45% reduction in the
infarction volume, as well as ∼30–50% reduction in water
accumulation (brain edema) in the infarcted hemisphere in
a transient ischemia model . As previously mentioned,
ischemic cell swelling is largely restricted to astroglial cells,
where it is mainly seen in the perivascular processes [13,45].
Notably, astrocytic perivascular endfeet express high levels
of NKCC1, which is co-localized with the water channel
Molecular mechanisms involved in neuroprotection by
bumetanide have been explored in several in vitro studies. In
cultured neurons, 10?M bumetanide strongly reduces cell
death triggered by oxygen–glucose deprivation or by appli-
cation of NMDA . Such protective effects of bumetanide
are absent in cultured neurons prepared from NKCC1−/−
long-term decreases in neuronal [Cl−]i that are associ-
ated with neuronal death after oxygen–glucose deprivation
. In cultured astrocytes, bumetanide and the deletion of
the NKCC1 gene potently inhibit high [KCl]-induced cell
swelling, which is used to mimic pathological swelling in
the ischemic brain, and partially suppress swelling-activated
release of excitatory amino acids [76,86,87]. In the acute
preparation of the optic nerve, oxygen–glucose deprivation
triggers degeneration of immature oligodendroglial cells,
and such degeneration is prevented by the application of
an AMPA receptor blocker or bumetanide . Since in
this in situ preparation NKCC is expressed in astrocytes
only, the authors concluded that ischemia triggers NKCC-
dependent swelling and death of astrocytes, which release
excitotoxins and cause a downstream damage to oligoden-
droglia [88,89]. Importantly, in cultured astrocytes and C6
glioma cells, but not in cultured cerebellar granule neurons,
swelling strongly stimulates NKCC activity [39,90]. There-
fore, glial cell swelling and NKCC activation are mutually
moting the release of excitotoxins in vitro and in vivo.
In summary, NKCC1 contributes to pathological NaCl
accumulation, cell death and formation of brain edema
in animal ischemia models. The selective NKCC inhibitor
bumetanide is protective when given systemically or deliv-
ered locally in brain tissue. Neuroprotective properties of
bumetanide likely involve several cellular mechanisms, as it
of the blood–brain barrier. In vivo, NKCC activation is one
of the probable causes for pronounced astrocytic swelling.
Because of its potent diuretic actions, usage of bumetanide
for human stroke treatment is not practical. However, if
developed, novel pharmacological compounds discriminat-
5. Pathological roles for Na+/H+exchange: impact on
[Na+]i, and cell volume
The family of Na+/H+antiporters (NHE) includes nine
human proteins NHE1–NHE9 (coded by SLC9A1–SLC9A9)
that catalyze an electroneutral exchange of Na+and H+
[91,92]. Five of these, NHE1–5, are plasmalemmal trans-
porters whose activity is driven by inwardly directed Na+
is ubiquitously expressed and plays a major role in the regu-
lation of cytoplasmic pH and cell volume, while NHE2–5
participate in additional tissue-specific processes [33,91].
NHE6–9 are intracellular Na+(K+)/H+exchangers in the
secretory and endocytic pathways; they mediate pH regu-
lation in various organelles [91,92].
There are several groups of pharmacological compounds
that are employed to potently and selectively inhibit mem-
brane NHEs in vitro and in vivo (reviewed in [91,93]).
A.A. Mongin / Pathophysiology 14 (2007) 183–193
Amiloride derivatives, such as ethylisopropylamiloride
(EIPA) and dimethylamiloride (DMA), benzoylguanidines,
such as HOE-642 and HOE-694, and a number of ‘bicyclic’
inhibitors, such as cariporide, zoniporide, eniporide, SM-
20220 and SM-20550, all have Kior IC50in the 1–100nM
range, but their inhibitory potency may be reduced by
physiological concentrations of Na+[91,93]. Typically, the
to the NHE blockers.
potential for the NHE inhibitors has been initially proposed
and explored in animal models of myocardial ischemia and
reperfusion injury [91,93,94]. Several large-scale clinical
trials using the NHE blockers eniporide and cariporide pro-
duced mixed, but encouraging, results [93,95].
In a rat transient ischemia model, the NHE blocker SM-
20220 (1mg/kg, delivered i.v. immediately after initiation
of ischemia) reduced total infarction area by ∼50%, and
partially prevented formation of brain edema . These
effects may be relevant to preservation of blood–brain bar-
rier function and improved reperfusion rates , as well as
to reduced accumulation of leukocytes in damaged brain tis-
sue . In the same model, another selective NHE blocker,
sabiporide (3mg/kg, i.v. 20min before ischemia), reduced
both infarction volume and brain edema by ∼40% and had
a therapeutic window of 1h . EIPA (5mg/kg, i.p. 30min
neurological deficits in the gerbil global ischemia model
rat global ischemia (25?M, applied topically in the cortex)
. In a mouse transient focal ischemia model, inhibition
prior ischemia), or a decrease in the NHE1 expression in
heterozygous NHE1+/−animals, both significantly reduced
brain infarction by ∼30% and tended to suppress the devel-
opment of brain edema . The NHE1−/−animals have
very high mortality rates and therefore could not be used in
In cultured cortical neurons, oxygen–glucose depriva-
tion followed by the reoxygenation causes steep rises in
[Na+]iand [Ca2+]ithat is potently inhibited by the selective
NHE1 blocker 1?M HOE-642 . In the same paradigm
HOE-642 also potently suppressed neuronal death. Cytoso-
lic Na+and Ca+overload and the effects of HOE-642 were
blunted in the neurons prepared from NHE1−/−animals
. Cultured astrocytes typically do not die when sub-
jected to hypoxia–glucose deprivation. Nevertheless, similar
induces cell swelling . As in cultured neurons, HOE-
642 or the genetic deletion of NHE1 both reduce elevation in
[Na+]iand cellular swelling . Since astrocytic swelling
has been linked to the release of glutamate and aspartate and
ischemic tissue damage, these findings may be of patholog-
ical relevance. A comprehensive analysis of the in vitro data
on the role of NHE in ischemic cell damage can be found in
Ref. . Overall, there is a similarity between NKCC1 and
NHE1 in terms of their contributions to hypoxic changes in
cytosolic Na+, Ca2+, and cellular volume. Perhaps this is not
surprising. Despite differences in their transport stoichiome-
to establish the effects of NHE blockers on brain endothe-
lial cells. Some of the above mentioned protective effects
seem to be mediated by protection at the blood–brain barrier
The therapeutic potential for the NHE inhibitors in stroke
is not clear. On one hand, a number of highly selective and
clinically tested compounds are available, and the results of
animal studies are encouraging [93,95]. However, already
conducted cardioprotective trials limit the enthusiasm. In
the EXPEDITION trial involving coronary artery bypass
surgery, the cardioprotective effects of the NHE inhibitor
cariporide were offset by an unexpected increase in the
incidence of stroke. Furthermore, another NHE blocker, eni-
poride, was ineffective when applied during reperfusion,
suggesting a limited therapeutic window for this class of
6. Pathological reversal of the Na+/Ca2+exchange
may contribute to the cytoplasmic Ca2+overload
The Na+/Ca2+exchanger (NCX) transporter family
includes three mammalian gene products, NCX1–3 (coded
by SLC8A1–SLC8A3), which in a majority of cell types cat-
alyze the electrogenic extrusion of one cytoplasmic Ca2+
in exchange for three Na+taken from the extracellular
space [12,104]. Four additional related proteins, NKCX1–4,
move four Na+from the extracellular space in exchange for
while NCX2 and 3 are found nearly exclusively in muscle
and neural cells (i.e., neurons and glia) . The kinetics of
NCX are primarily governed by the Na+gradient and trans-
membrane potential, but are also affected by non-transported
Ca2+, protons, ATP, and diverse signaling molecules .
In the muscle and nerve cells, NCX (and NKCX) proteins
serve as an important “high capacity” Ca2+-extruding mech-
anism, complementing the Ca2+-ATPase in pumping Ca2+
against its electrochemical gradient. However, dissipation of
the Na+gradient and/or depolarization reverses the mode of
cytoplasmic Ca2+overload [12,104].
Several groups of inhibitors have been designed to
block NCX pharmacologically (reviewed in Ref. ).
The most commonly used blockers are the amiloride
derivatives 2?4?-dimethylbenzamyl (DMB, IC50=10?M)
and [N-(4-chlorobenzyl)]2,4-dimethylbenzamyl (CB-DMB,
IC50=7.3?M), isothiourea derivative KB-R7943 (IC50
∼1?M), and the derivative of diarylaminopropylamine,
bepridil (IC50=8.1?M). The more recently synthesized
5-ethoxyaniline) is the most potent NCX blocker with IC50
A.A. Mongin / Pathophysiology 14 (2007) 183–193
∼10–90nM, but it is effective predominantly against the
NCX1 isoform. Importantly, two of the NCX inhibitors, KB-
R7943 and SEA0400, block the reverse (i.e., “pathological”)
mode of Na+/Ca2+exchange.
When observed, the effects of NCX blockers should be
analyzed with caution. Bepridil  and KB-R7943 
have been reported to directly block the NMDA channels in
hippocampal neurons. KB-R7943 may also exert its actions
by blocking L-type Ca2+channels  or, in vivo, via low-
ering the body temperature .
model, SEA0400 (3mg/kg bolus followed by 2-h infusion of
3mg/kg/h) reduced infarction volume by ∼50% in the cor-
tex and by ∼25% in the striatum . In the rat permanent
focal ischemia model, another group found potent protection
with KB-R7943 (10?g/kg delivered by osmotic minipump
over 24h) . However, in the same study three other
NCX blockers, bepridil, CB-DMB, and the NCX inhibitory
ume. The protective effect of KB-R7943 has been explained
by its potent hypothermic actions . Importantly, in
vivo downregulation of NCX1 and NCX3, but not NCX2,
increased infarction volumes and neurological deficits in a
rat permanent focal ischemia model . Consistent with
the latter data, two NCX blockers, bepridil and the more
selective CB-DMB, produced an irreversible loss of elec-
trical activity in striatal spiny neurons in the penumbra of
focal ischemia, as compared to untreated ischemic controls
. Overall, in vivo data suggest a beneficial role for
NCX in maintaining ionic homeostasis in the peri-infarct
The neuroprotective and damaging properties of the NCX
blockers have been explored in more detail in vitro and
in situ. Several groups reported that NCX blockers res-
cue both neurons and glia from hypoxia/reoxygenation or
glutamate-induced injury [112–114]. However, the others
found increased cell death upon NCX inhibition [115,116].
white matter tracks are functionally protected against anoxic
injury by removal of extracellular Ca2+or by NCX inhibitors
benzamil, bepridil, DCB, and KN-R7943 [117–119]. These
data are of particular importance, as ischemic white matter
injury is less understood and may contribute substantially to
neurological deficits in human stroke . However, some
of the previously reported effects of NCX inhibitors have to
be re-evaluated because bepridil and KB-R7943 also inhibit
NMDA receptors [105,106] and L-type Ca2+channels ,
two pathways that may also contribute to the anoxic injury
of various white matter components [14,121–124].
In summary, reversal of NCX may contribute to Ca2+
overload in some, but not all, forms of ischemic cell death.
This transporter may be of particular importance for axonal
damage in white matter. However, NCX is beneficial for the
survival and functional recovery of neurons in the ischemic
ers increase, rather that decrease, brain damage in animal
ischemia models. Therefore, the inhibition of NCX does not
currently seem to be a viable strategy for pharmacological
intervention in stroke.
7. Conclusions and perspectives
The failure of ionic homeostasis is a hallmark of ischemic
focused on transmembrane Ca2+fluxes and severe distur-
bances in cytoplasmic and mitochondrial Ca2+homeostasis.
In contrast, the pathological dysregulation of Na+and Cl−
transport receives relatively little attention. The present
analysis of experimental data suggests that a pathological
accumulation of Na+and Cl−plays a very important role in
effects of high cytosolic [NaCl] are realized via pathological
cell swelling, the impact of Na+overload on Ca2+home-
ostasis, and through an increased metabolic burden that is
placed on the cell due to enhanced work of the Na+,K+- and
least three cellular sites: (1) tested compounds may preserve
the function of the blood-barrier and prevent formation of
vasogenic brain edema by acting on endothelial cells; (2)
they may directly reduce necrotic and apoptotic death of
neuronal cells by preserving their ion homeostasis; (3) the
inhibitors may also prevent the pathological release of exci-
and the dissipation of ion gradients. Additionally, ion trans-
axonal fibers and their myelinating oligodendroglial cells).
it is important to take into consideration the blood–brain per-
meability of individual compounds and their effects at each
of the above mentioned sites.
A comprehensive understanding of the complex patho-
biology of ischemic tissue damage will greatly benefit the
development of future therapeutic treatments in stroke. The
failure of so many neuroprotectants in clinical trials repre-
sents a significant challenge and calls for novel treatment
approaches, perhaps involving multiple therapeutic targets
(for recent discussion, see [3,125,126]).
National Institute for Neurological Disorders and Stroke. I
gratefully acknowledge T.J. Harrigan for critical reading of
the manuscript and numerous helpful comments.
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