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The Journal of Neuroscience, May 1, 2003, 23(9):3826
Aberrant Chloride Transport Contributes to Anoxic/Ischemic White
Matter Injury
Sameh A.
Malek,
Elaine
Coderre, and
Peter K.
Stys
Ottawa Health Research Institute, Ottawa Hospital, University of
Ottawa, Canada K1Y 4K9
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ABSTRACT |
Rundown of ionic gradients is a central feature of white matter
anoxic injury; however, little is known about the contribution of
anions such as Cl . We used the in
vitro rat optic nerve to study the role of aberrant Cl transport in anoxia/ischemia. After 30 min of
anoxia (NaN3, 2 mM), axonal membrane
potential (Vm) decreased to 42 ± 11%
of control and to 73 ± 11% in the presence of tetrodotoxin (TTX)
(1 µM). TTX + 4,4'-diisothiocyanatostilbene-2,2'
disulfonic acid disodium salt (500 µM), a broad spectrum
anion transport blocker, abolished anoxic depolarization (95 ± 8%). Inhibition of the K-Cl cotransporter (KCC) (furosemide 100 µM) together with TTX was also more effective than TTX
alone (84 ± 14%). The compound action potential (CAP) area
recovered to 26 ± 6% of control after 1 hr anoxia. KCC blockade (10 µM furosemide) improved outcome (40 ± 4%), and
TTX (100 nM) was even more effective (74 ± 12%). In
contrast, the Cl channel blocker niflumic acid (50 µM) worsened injury (6 ± 1%). Coapplication of TTX
(100 nM) + furosemide (10 µM) was more
effective than either agent alone (91 ± 9%). Furosemide was also
very effective at normalizing the shape of the CAPs. The KCC3a isoform
was localized to astrocytes. KCC3 and weaker KCC3a was detected in
myelin of larger axons. KCC2 was seen in oligodendrocytes and within
axon cylinders. Cl gradients contribute to resting
optic nerve membrane potential, and transporter and channel-mediated
Cl fluxes during anoxia contribute to injury,
possibly because of cellular volume changes and disruption of axo-glial
integrity, leading to propagation failure and distortion of fiber
conduction velocities.
Key words:
anoxia; ischemia; axon; chloride; K-Cl
cotransporter; KCC
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Introduction |
Anoxia/ischemia induces cellular
ionic deregulation caused by failure of regulatory mechanisms such as
ATPases and coupled ion exchangers. As shown previously in CNS white
matter, with the fall in energy substrates, axonal
Na+ overload occurs that in turn initiates
Ca2+ accumulation in large part through
reverse operation of the
Na+/Ca2+
exchanger (Stys et al., 1992 ). In anoxic CNS axons,
Na+ overload is essentially balanced by an
equivalent efflux of K+ from the axoplasm
(LoPachin and Stys, 1995; Stys and LoPachin, 1998), thus maintaining an
electroneutral exchange of ions. One might therefore expect that
restraining Na+ influx into axoplasm
during anoxia (e.g., by applying tetrodotoxin (TTX) or replacing bath
Na+ with an impermeant cation) would
secondarily reduce K+ loss. Unexpectedly,
axoplasmic K+ depletion is not restrained
in anoxic optic nerve axons even when Na+
overload is blocked by TTX (Stys and LoPachin, 1998). Instead, K+ loss is balanced by efflux of
Cl and likely other organic anions (Stys
and LoPachin, 1998). Anion transporters including Na-K-2Cl (NKCC)
cotransporter, Na+-coupled
Cl/HCO3
exchange, and the KCl-cotransporter (KCC) play an important role in
intracellular Cl regulation in central
neurons (Kaila, 1994). Given its cotransport of
K+ and Cl,
KCC would be a plausible candidate for mediating the observed parallel
efflux of these ions during anoxia under conditions when Na+ overload is restrained (as may occur
during attempts at neuroprotection with
Na+ channel blockers or glutamate receptor
antagonists). The emergence of
K+-Cl
co-efflux under such conditions may have important implications for
cellular integrity, because osmotically obligated water loss may have
deleterious effects on cellular volume and therefore mechanical
integrity, and in addition, this loss of water may in turn
paradoxically concentrate remaining ions (such as
Na+) to toxic levels (see Discussion).
There are currently four known isoforms of the KCC (Gillen et al.,
1996; Payne et al., 1996; Mount et al., 1999), which are part of the
larger family of cation-coupled cotransporter proteins that also
includes the Na-K-2Cl cotransporter. Identified in 1996 by Payne and
colleagues, the neuronal isoform (KCC2) appears to mainly extrude
Cl out of the cell under physiological
conditions (Payne et al., 1996). KCC3 has been shown to have a robust
expression in the brain (Mount et al., 1999) with a cellular
localization to white matter tracts (Pearson et al., 2000, 2001). On
the basis of the fact that during anoxia and
Na+-channel inhibition there is persistent
decay in membrane potential (Leppanen and Stys, 1997), probably as a
result of K+ efflux that proceeds in
conjunction with Cl exit (Stys et al.,
1997), we hypothesized that blockade of
Cl loss during
anoxia/Na+-channel inhibition will impede
K+ efflux and protect white matter against
anoxia better than Na+-channel blockers
alone. We confirmed that combined inhibition of
Na+ influx and
K+ + Cl
efflux via the KCC reduced anoxic depolarization and improved compound
action potential (CAP) recovery to a greater extent than Na+ channel inhibitors alone. Moreover,
quantitative evaluation of the CAP wave-shape revealed that combined
treatment also greatly improved not only CAP area but also the shape,
suggesting a preservation of the underlying tissue architecture (e.g.,
maintenance of axo-glial relationships or myelin integrity that would
preserve conduction velocities of constituent fibers). We therefore
suggest that, at least for white matter, combined
Na+ channel inhibition and reduction of
secondary K+ and
Cl efflux represents an improved
neuroprotective strategy.
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Materials and Methods |
Electrophysiology. Compound resting membrane
potential was recorded from optic nerves in vitro dissected
free from adult Long-Evans rats. One nerve was recorded immediately
using a grease gap chamber at 37°C as described previously (Leppanen
and Stys, 1997), whereas the second was stored in oxygenated artificial
CSF (ACSF) containing (in mM): 126 NaCl, 3 KCl,
26 NaHCO3, 2 MgSO4, 1.25 NaH2PO4, 2 CaCl2, and 10 glucose, pH 7.45) at room
temperature for later study. No consistent differences were noted
between nerves recorded immediately and those held for later study. Raw
baseline gap potentials (Vg) varied
from nerve to nerve (typical range  45 to 50 mV) because of
differences in the short circuit factor (Stämpfli, 1954).
Therefore for display purposes all potentials were normalized (denoted
Vm) to the true resting potential of
CNS myelinated axons of 80 mV (Stys et al., 1997). Quantitative
comparisons over time were performed using ratios of recorded
potentials; therefore, this normalization had no effect on such
calculations. For technical reasons, we were unable to obtain
reproducible responses in the grease gap chamber using
N2/CO2 as a means of
inducing anoxia, even with the use of oxygen scavengers (data not
shown). This was likely because of the configuration of the chamber,
which prevented adequate isolation allowing some ambient
O2 to access the nerves, resulting in excessively
variable recordings. Instead we elected to induce anoxia chemically
(Leppanen and Stys, 1997) with either CN
or N3, which gave
similar results.
Propagated compound action potentials were recorded using suction
electrodes as described previously (Stys et al., 1991). Briefly, nerves
were placed in an interface perfusion chamber, perfused with ACSF (2 ml/min, 37°C), and gassed with either 95% O2
or 95% N2, balance CO2.
Supramaximal constant voltage stimuli were delivered and responses were
recorded using a pair of glass suction electrodes. Anoxia was achieved
by switching to N2/CO2, and
ischemia was simulated by exposure to anoxia with equimolar replacement
of glucose by sucrose [oxygen-glucose deprivation (OGD)].
Pharmacology. TTX (Alomone Labs) was prepared as a stock
solution in distilled water. 4,4'-Diisothiocyanatostilbene-2,2'
disulfonic acid disodium salt (DIDS), furosemide, bumetanide, and
niflumic acid were purchased from Sigma (St. Louis, MO).
DIDS was added directly to the desired volume of ACSF solution to make
up the required concentration. Furosemide was first dissolved in DMSO. Both bumetanide and niflumic acid were dissolved in ethanol. NaCN was
acquired from BDH (Toronto, Ontario, Canada).
NaN3 was purchased from Fisher
Scientific. All other salts were purchased from Sigma.
All errors are reported as SDs, and statistical significance was
assessed using a Student's t test. All experimental
protocols were approved by the institutional animal care committee.
Immunohistochemistry. Deeply anesthetized Long-Evans rats
(200-300 gm) were perfused transcardially with 0.9% saline followed by 2% paraformaldehyde containing 20 mM
L-lysine, 2.5 mM sodium periodate, and 2.5% potassium dichromate. The optic nerves were postfixed for 2 hr and immersed in 0.1 M PBS for
24 hr. The protocol was as follows: wash three times for 10 min each in
Tris buffer containing 1.5% NaCl and 0.3% Triton X-100 (TBS-T) and
incubate for 30 min at 4°C in methanol; wash three times for 10 min
each in TBS-T; block with 10% normal goat serum in TBS-T for 1 hr at room temperature; and incubate overnight with primary antibody diluted
in TBS-T containing 2% normal goat serum. All KCC primary antibodies
(Chemicon, Temecula, CA) were diluted at a concentration of 5 µg/ml and 16 µg/ml for neurofilament 160 (NF160; Sigma,
Oakville, Ontario, Canada). The next day, the optic nerves were
washed three times for 10 min each in TBS-T. Goat anti-rabbit Cy2
(1:200) and goat anti-mouse Texas Red (1:100; Jackson
ImmunoResearch, West Grove, PA) were used for secondaries.
Sections were imaged on a Bio-Rad 1024 or
Nikon C1 confocal with 60× oil immersion objective.
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Results |
Vg recordings in ACSF typically
stabilized 90 min after insertion into the grease gap chamber. Raw
control resting potentials ranged from 45 to 50 mV. No consistent
differences were noted between the first and second nerves studied
sequentially. To compare responses over time and between different
treatments, ratios of Vg values were
calculated at different time points (typically 30 and 60 min) with
respect to potentials at time 0 (defined as a stable potential baseline
before any experimental treatment).
Effects of Cl transport inhibition on membrane
potential (Vm) during anoxia
We elected to induce anoxia chemically, using 2 mM
either NaCN or NaN3, inhibitors of complex IV of
the respiratory chain (Kauppinen and Nicholls, 1986; Tadic, 1992).
Figure 1A shows a typical response to chemical anoxia induced by
CN. Resting membrane potential
depolarized within minutes, decaying to 44 ± 14 and to 34 ± 13% of control after 30 and 60 min.
N3 produced very
similar results (40 ± 6 and 30 ± 5% of control potential
remaining after 30 and 60 min). Results from both treatments were
therefore combined in subsequent analyses (Table
1). Chemical ischemia was induced by
combining NaN3 (2 mM) and
iodoacetic acid-Na+ salt (IAA) (1 mM), an irreversible blocker of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Sabri and Ochs, 1971).
Vm decayed to values comparable with
those observed with chemical anoxia alone (41 ± 5 and 31 ± 6% at 30 and 60 min, respectively) (n = 10) (Fig.
1B).
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Figure 1.
Effect of chemical anoxia and ischemia on the
membrane potential of rat optic nerve. Vg
represents recorded gap potential and Vm
represents membrane potential. Time 0 in this and all subsequent
figures denotes application of insult. A, NaCN (2 mM) caused a rapid loss of membrane potential decaying to
~42 ± 11 and 32 ± 10% of control at 30 and 60 min,
respectively, of chemical anoxia. B, Chemical ischemia,
2 mM NaN3 + 1 mM IAA, caused a
decay of 41 ± 5 and 31 ± 6% after 30 and 60 min of
perfusion.
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Table 1.
Summary of different pharmacological manipulations and
their effects on membrane potential of rat optic nerve expressed as
means ± SD
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Previous reports have demonstrated the effectiveness of
Na+ channel inhibition as a
neuroprotective strategy in anoxic white matter (Stys et al.,1992; Fern
et al., 1993; Leppanen and Stys, 1997). The effects of TTX (1 µM) on optic nerve resting membrane potential during
anoxia or ischemia are illustrated in Figure 2 and quantitatively in Figure 4.
Application of TTX under normoxic conditions caused a hyperpolarization
as observed previously (Stys et al., 1993; Leppanen and Stys, 1997).
Depolarization was less pronounced in anoxic nerves exposed to TTX
(73 ± 11% of control Vm
remaining after 30 min in TTX + anoxia vs 42 ± 11% with anoxia alone, p < 0.01; 63 ± 18% after 60 min,
p < 0.001; n = 12). Replacement of
Na+ with an impermeant cation during
chemical anoxia resulted in a blunted depolarization to 77 ± 9%
of control membrane potential at 30 min (p < 0.01 vs chemical anoxia) and to 73 ± 10% after 60 min
(p < 0.01; n = 3) (see Fig. 4).
Because neither of the above manipulations completely prevented anoxic
depolarization, other non-Na+-dependent
pathways promoting loss of resting membrane potential in anoxic axons
may exist.
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Figure 2.
Effect of Na+-channel
inhibition on the membrane potential of rat optic nerve. TTX (1 µM) preapplied for 1 hr before chemical anoxia caused a
brief hyperpolarization of Vm, thus
indicating the presence of a Na+ conductance at
rest. During chemical anoxia, 1 µM TTX blunted the rapid
phase of the characteristic depolarization (73 ± 11% of control
Vm remaining after 30 min in TTX + anoxia vs
42 ± 11% with anoxia alone, p < 0.005;
63 ± 18% after 60 min, p < 0.001;
n = 12), suggesting that Na+
influx through TTX-sensitive channels constitutes one of the primary
events in producing the anoxic depolarization.
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The role of anion transporters on resting membrane potential during
anoxia was studied using DIDS, a broad-spectrum anion transport blocker
that acts on the
K+-Cl
cotransporter (Russell, 2000), volume-sensitive
Cl channels (Estevez et al., 1999),
Cl-HCO3
exchange (Clark et al., 1998; Sakai and Tosaka, 1999), and
hyperpolarization-activated Cl channels
(Clark et al., 1998). In contrast, DIDS has no reported effect on
Na+-K+-2Cl
or Na+-Cl
cotransporters (Russell, 2000). DIDS (500 µM) alone did
not alter the anoxic depolarization (43 ± 8%, p = 0.99 vs chemical anoxia at 30 min; 40 ± 9% at 60 min,
p = 0.45; n = 2) (see Fig. 4).
Previous results indicate that Na+ channel
inhibition promotes efflux of Cl in
parallel with K+ from anoxic optic axons
(Stys and LoPachin, 1998); thus concomitant blockade of
Na+-influx and
Cl-efflux pathways would be expected to
spare K+-efflux and further reduce anoxic
depolarization. Application of TTX (1 µM) together with
500 µM DIDS (Figs.
3A,
4) reduced the amount of
depolarization to a greater degree than TTX alone (95 ± 8 vs
73 ± 10% in TTX alone at 30 min, p < 0.001;
89 ± 6 vs 63 ± 18% at 60 min, p < 0.001;
n = 13) (Fig. 3A). DMSO (0.2% v/v used for
DIDS stock solutions) had no effect on the anoxia-induced membrane
potential changes (data not shown). Zero
Na+/choline/NMDG reduced anoxic
depolarization to a similar extent as did TTX, and addition of DIDS
(500 µM) to the
zero-Na+ perfusate was even more effective
(Fig. 4) (Vm maintained at 92 ± 3% of control after 30 min in zero-Na+
DIDS vs 77 ± 9% in zero-Na+
alone).
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Figure 3.
Effect of anion transport and
Na+-channel co-blockade on the membrane potential of
rat optic nerve during chemical anoxia. A, DIDS (500 µM), a broad-spectrum anion transport blocker, applied
during normoxic conditions, had no effect on 1 µM TTX
hyperpolarization. During chemical anoxia, the combined treatment
allowed for maximum maintenance of Vm
(95 ± 8 vs 73 ± 10% in TTX alone at 30 min,
p < 0.001; 89 ± 6 vs 63 ± 18% at 60 min, p < 0.001; n = 13),
suggesting an anion component for the depolarization. B,
Furosemide (100 µM), a relatively specific blocker for
KCC, produced a blunting of the residual depolarization seen with TTX + CN (84 ± 14% Vm
remaining after 30 min in TTX + furosemide vs 73 ± 10% in TTX
alone at 30 min, p < 0.05; 79 ± 16 vs
63 ± 18%, p < 0.05; n = 20). This suggests that the DIDS effect is mediated primarily, but not
exclusively, via KCC.
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Figure 4.
Summary of different pharmacological manipulations
and their effects on Vm of rat optic nerve.
Both chemical anoxia and ischemia caused comparable decay profiles.
Anion transport blockers, 500 µM DIDS and 10 µM furosemide, when used alone during chemical anoxia
were ineffective in maintaining Vm at
pre-anoxic levels. Na+-channel blockade, 1 µM TTX, was more effective in preserving
Vm to a large extent during chemical anoxia.
Nevertheless, there existed a residual portion of the depolarization,
which was Na+ independent as seen from inhibition of
either Na+ channels or Na+
replacement. Co-blockade of Na+ channel (1 µM TTX) and anion transport (500 µM DIDS)
or Na+ replacement (NMDG) in combination with 500 µM DIDS produced a robust maintenance of
Vm. Furosemide (100 µM), KCC
blocker, coapplied with 1 µM TTX, improved
Vm maintenance as compared with TTX alone.
However, the levels were not as comparable as those of DIDS, presumably
because there are other pathways involved in mediating residual
Vm depolarization during TTX/chemical anoxia
perfusion.
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Of the many anion transporters inhibited by DIDS, one potential route
that could mediate both Cl and
K+ flux is the KCC. More selective
inhibition of this transporter with furosemide (100 µM),
a relatively specific blocker of KCC at this concentration (for review,
see Cabantchik and Greger, 1992; Payne, 1997) reduced anoxic
depolarization more than TTX alone (Figs. 3B, 4) (84 ± 14% Vm remaining after 30 min in TTX + furosemide vs 73 ± 10% in TTX alone at 30 min,
p < 0.01; 79 ± 16 vs 63 ± 18% at 60 min,
p < 0.05; n = 20). However, the fact that DIDS reduced anoxic depolarization more than furosemide suggests that additional Cl transport pathways
were operating in parallel (TTX + DIDS vs TTX + furosemide;
p < 0.05 at both 30 and 60 min).
Effects of Cl transport inhibition on the
propagated compound action potential
Figure 5A shows a typical
control CAP recorded from optic nerve under normoxic conditions and 3 hr in normoxia after 1 hr of anoxic insult. The area of the CAP
recovered to 24 ± 12% (n = 46) of control after
1 hr anoxia/reoxygenation, in agreement with previous reports (Stys et
al., 1992). In vitro ischemia (1 hr of OGD) (Figs.
5B, 9) allowed mean CAP area recovery of only 8 ± 3%
of control after 3 hr of reperfusion (n = 9).
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Figure 5.
Effects of anoxia versus oxygen/glucose
deprivation (OGD) on the compound action potential of the rat optic
nerve. A, Recovery of the CAP area after 60 min anoxic
insult in normal ACSF was 26 ± 6% of control at 3 hr after
reoxygenation. B, With the stronger insult of 60 min
OGD, the recovery was only 8 ± 4% of control, also at 3 hr after
reoxygenation.
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Previous reports have shown that axoplasmic
Na+ overload, a primary event in the
injury cascade, occurs mainly through TTX-sensitive channels during
anoxia/ischemia and trauma (for review, see Stys, 1998). Nerves
subjected to 1 hr of anoxia in the presence of TTX 100 nM
(Fig. 6A) recovered to
74 ± 12% of control CAP area versus 26 ± 6% without TTX
after 3 hr of reoxygenation and wash in TTX-free ACSF
(p < 0.001; n = 12) (Figs.
6A, 9). This prolonged wash period was necessary to
maximize removal of the blocker, which was still incomplete (control
CAPs recovered to only 80% after a similar exposure to TTX/wash period
without anoxia). As expected with the more severe ischemic insult (1 hr
of OGD) (Figs. 5B, 9), mean CAP area recovered to only
8 ± 3% of control. Nevertheless TTX (100 nM) partially rescued the nerves from ischemia as
well (Figs. 6B, 9), allowing 56 ± 4% CAP area
recovery after 3 hr of reperfusion/wash (p < 0.01; n = 3).
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Figure 6.
Effect of Na+-channel blockers
on CAP recovery during either anoxia or OGD. A, TTX (100 nM) significantly improved recovery. A 1 hr pre-anoxic
application abolished CAP. TTX was then continued for 30 min after
anoxic insult of 60 min. CAP area recovered to 75 ± 12%
(p < 0.001; n = 12) of
control at the 3 hr post-anoxia mark. B, A 30 min
pre-OGD application was continued for another 30 min after a 60 min OGD
insult. CAP area recovered significantly to 56 ± 4% in
comparison with OGD alone (8 ± 3%; p < 0.001; n = 3).
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Anion transport blockade using 500 µM DIDS caused an
irreversible depression of the CAP to 16 ± 17% after 1 hr
normoxic perfusion (Figs. 7A,
9). At 2 hr after anoxia, the CAP recovered to only 6 ± 5%
(p < 0.001 vs anoxia; n = 8).
Hence we could not assess the effect of DIDS on CAP (in contrast to
Vm). The effect of KCC inhibition on
CAP recovery was assessed during normoxic, anoxic, and ischemic
conditions. Furosemide (10 µM), a relatively
specific KCC inhibitor at these concentrations (Alvarez-Leefmans,
1990; Jarolimek et al., 1999), had no effect on the control CAP
(see Fig. 9). However, this blocker partially prevented anoxic (mean CAP area recovery 40 ± 4 vs 26 ± 6% without furosemide;
p < 0.01; n = 8) (Figs. 7B,
9) and ischemic (20 ± 14 vs 8 ± 3%; p < 0.05; n = 9) injury as measured by CAP area.
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Figure 7.
Effect of anion transport blockade on compound
action potential during in vitro anoxia.
A, DIDS (500 µM) applied for 1 hr during
normoxic conditions and continued up to 30 min after anoxia caused a
severe depression of CAP after a 1 hr normoxic drug application.
Furthermore, CAP recovered to 6 ± 5% compared with 24 ± 13% at 2 hr after anoxia (p < 0.001 vs
anoxia; n = 8). Such an effect is presumably caused
by the interaction of DIDS with Na+ channel (Liu et
al., 1998). B, Furosemide (10 µM) applied
for 1 hr and also continued up to 30 min after anoxia had no effect on
normoxic CAP but improved CAP area recovery to 40 ± 4%
(p < 0.01 vs anoxia; n = 8). C, Effect of combined
Na+-channel and K-Cl cotransport blockade on the
compound action potential of the rat optic nerve during anoxia. TTX
(100 nM) and furosemide (10 µM) was more
protective than either agent alone (TTX + furosemide 91 ± 8% vs
74 ± 12% TTX alone; p < 0.001;
n = 11).
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The axoplasmic anoxia-induced Cl loss
observed only in the presence of Na+
channel inhibition (Stys and LoPachin, 1998) suggests that a combination of Na+ channel and KCC
blockade may be more protective than either agent alone. Mean CAP area
recovery was significantly improved after anoxia after the addition of
furosemide to TTX (Figs. 7C, 9), compared with TTX alone
(91 ± 9 vs 74 ± 12% TTX alone; p < 0.001; n = 11). Moreover, furosemide substantially normalized
the shapes of the post-anoxic CAPs (see next section). However, this
drug combination was not more effective in OGD, producing a recovery of
only 50 ± 19% (vs 56 ± 4% in TTX alone; n = 14; p > 0.05) (see Fig. 9).
Axoplasmic Ca2+ is known to increase
during anoxia (Stys and LoPachin, 1998); thus it was of interest to
study potential Ca2+-sensitive anion
transporters such as the Ca2+-activated
Cl channel during anoxia/ischemia.
Inhibition of Ca2+-activated
Cl channels with 50 µM
niflumic acid (Scott et al., 1988; Currie et al., 1995), during 1 hr
normoxic perfusion, caused a minor insignificant depression of the CAP.
Niflumic acid unexpectedly worsened post-anoxic CAP recovery (6 ± 1 vs 26 ± 6% in ACSF alone; p < 0.001;
n = 6) (Figs.
8A,
9), in contrast to KCC inhibition, which improved outcome (see above). Similarly, during in
vitro ischemia, CAP recovery was also worse with niflumic acid
treatment (3 ± 1 vs 8 ± 3% in ACSF alone;
p < 0.01; n = 3) (Figs.
8B, 9).
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Figure 8.
Evaluation of Ca2+-activated
Cl channel during in vitro anoxia
and ischemia. A, Niflumic acid (NFA; 50 µM) caused a minor but statistically insignificant
depression of normoxic CAP when perfused for 1 hr. NFA (50 µM) worsened CAP recovery compared with anoxia alone
(6.0 ± 0.7 vs 26 ± 6% at 3 hr after anoxia;
p < 0.01; n = 6).
B, Similarly, with OGD, the recovery worsened to 3 ± 1 vs 8 ± 3% at 3 hr (p < 0.05;
n = 3). These results suggest that niflumic
acid-sensitive Cl channels (e.g.,
Ca2+-activated or volume-regulated
Cl channels) may play a protective role during
anoxic/ischemic conditions.
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Figure 9.
Quantitative effects of different pharmacological
manipulations on CAP. Normoxia, TTX (100 nM) caused a
reversible depression of CAP. Neither furosemide (10 µM)
nor NFA (50 µM) had any effect on CAP during normoxia.
Anoxia, A marked improvement of CAP was seen with TTX (100 nM) perfusion with a maximum recovery of 74 ± 12% at
3 hr after anoxia. KCC blockade allowed for 40 ± 4%
recovery versus 26 ± 6% with anoxia
(p < 0.01). This reflects the aberrant
activity of the transporter during anoxia. DIDS (500 µM),
on the other hand, because of its unspecific effect, caused depression
of the CAP during normoxic application (16 ± 17%). After 2 hr of
reoxygenation, the CAP recovered partially to only 6 ± 5%, which
is far below the value observed with anoxia alone at this time point
(23 ± 13%; p < 0.001; n = 8). The combination of Na+ channel and KCC
blockade was the most effective manipulation in producing maximal
recovery of CAP after anoxia (91 ± 9 vs 74 ± 12% TTX
alone; p < 0.001; n = 11). On
the other hand, Ca2+-activated
Cl-channel blockade caused a deterioration of the
CAP with a recovery of 5 ± 2, 5 ± 2, and 6.0 ± 0.9%
at 1, 2, and 3 hr after anoxia (p < 0.01;
n = 6; at 3 hr reoxygenation vs anoxia alone). OGD,
Under this paradigm, only 8 ± 3% of control CAP area was rescued
when measured at 3 hr after OGD normoxic perfusion
(n = 9). As with anoxia, a robust recovery was seen
with Na+-channel blockade with TTX (100 nM) (56 ± 4 vs 8 ± 3% 3 hr reoxygenation;
p < 0.01; n = 3). Similarly,
KCC inhibition by furosemide (10 µM) recovered 20 ± 14% of control CAP area measured at 3 hr after OGD
(p < 0.05; n = 9).
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Compound action potential wave-shape recovery
Combined treatment with furosemide + TTX not only improved CAP
area recovery after anoxia, but also appeared to significantly improve
the shape of the response, which in part reflects the preservation of
normal conduction velocities of the constituent fibers. To quantify
these observations we devised a measure of the shape of the CAP
compared with the control wave-shape before injury, independent of CAP
magnitude. This "wave-shape fidelity index" was calculated by first
normalizing the areas between the control CAP before injury and
response after treatment, by scaling the smaller waveform so that areas
are equal. Next a point-by-point squared difference was calculated
between the two CAPs, and these squared differences averaged to yield a
single numerical index. In mathematical terms:
where F is the "wave-shape fidelity index,"
w0 and
w1 are control and post-treatment CAP
waveforms, respectively, A0 and A1 are control and post-treatment CAP
areas, and n is the number of points in each waveform.
An index of zero denotes a post-treatment wave-shape that is identical
to control, regardless of its size. An increasing index indicates a
wave-shape that differs more and more from control (again independent
of magnitude). Selected normalized waveforms are shown in Figure
10A-D, and indices
for various treatments are summarized in Fig 10E. Of
the various treatments tested, combined application of TTX and
furosemide was the most successful in restoring CAP shape after anoxic
exposure; indeed, wave-shapes were not statistically different between
this treatment group and time-matched normoxic controls, indicating
that not only did this combination of drugs allow recovery of CAP area,
but the configuration of the CAP was restored to near normal. This is
in contrast to TTX alone, which was very effective at protecting CAP
area but resulted in distorted wave-shapes reflected by a higher
wave-shape fidelity index.
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Figure 10.
Panels A-D, Representative tracings (normalized
with respect to CAP area) of normoxic, untreated anoxic,
post-anoxic/washed nerves treated with TTX (100 nM) and TTX + furosemide (10 µM), respectively. Black and gray curves
denote control and post-anoxic recordings, respectively. As expected
with normoxia, there is very little change in the wave-shape over time;
however, with anoxia/reoxygenation, the wave-shape becomes very
distorted. TTX results in a significant recovery of CAP area, but the
wave-shapes (particularly latencies of the slower peaks) remained
significantly distorted. With addition of the KCC blocker furosemide,
the wave-shape is improved further, and the fidelity index is not
different from time-matched normoxic controls
(p = 0.20). The peak latencies of the
pre-anoxic and post-anoxic waves are similar, indicating a return of
not only the number of fibers able to conduct, but also normalization
of the conduction velocities of constituent fibers. E,
Summary of wave-shape fidelity indices. The lowest index was obtained
with time-matched normoxic controls indicating little change in
wave-shape with control incubation. Although TTX provided a robust
recovery of CAP area, the wave-shape remained noticeably distorted
(prolonged peak 2 latency, absence of peak 3), reflected by an index
approaching that for anoxic nerves alone (without TTX). In contrast, furosemide not only improved CAP area
recovery but substantially improved wave-shapes as well, reducing the
mean index to a value not different from normoxic controls. Niflumic
acid worsened CAP area recovery and wave-shape distortion.
*p < 0.05; **p < 0.01.
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Immunolocalization of KCC
Figure 11 shows representative
confocal microscopic images of rat optic nerve immunostained for
different KCC isoforms. The red channel outlines axons stained with
neurofilament, and green shows KCC stained with isoform-specific
antibodies. Strong KCC3a signal was found on optic nerve astrocytes and
their processes (Fig. 11A,B).
Weaker KCC3a signal was found in the myelin of many larger axons (Fig.
11A, inset).
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Figure 11.
Laser scanning confocal micrographs showing
immunohistochemical localization of different KCC isoforms (green) in
rat optic nerve (axons stained with NF160 in red). A,
B, There was strong KCC3a signal in astrocytes, and
occasional signal in the myelin of larger axons (inset, arrowhead).
C, D, Less intense but reproducible,
often punctate, KCC3 signal was observed in the myelin sheath of larger
axons. Because all axons are myelinated in mature optic nerve,
fluorescence immediately adjacent to axon cylinders as seen here is
consistently localized to the sheath rather than glial processes.
E, KCC2 was clearly present in cell bodies of
oligodendrocytes (arrows) and also diffusely in the optic nerve,
including signal within axon cylinders (appearing as a more orange hue
because of colocalization with red NF160 signal; compare with a more
pure red neurofilament signal in the other panels). F,
Controls with 1° antisera omitted were clean.
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There was more modest but reproducible, often punctate, KCC3 signal in
the myelin sheaths of larger axons (Fig. 11C,D).
KCC2 appeared more widespread, with signal in cell bodies of
oligodendrocytes, and also within axon cylinders (hence the orange
hue in Fig. 11E, representing colocalization of green
KCC2 fluorescence and red NF160). KCC1 staining was very weak,
diffusely localized, and not consistently stronger than controls, so no
conclusions about its presence or localization could be drawn (data not shown).
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Discussion |
In adult mammalian neurons, the Cl
gradient is influenced by a number of systems, including the KCC and
NKCC cotransporters (for review, see Alvarez-Leefmans, 1990). KCC2, the
neuronal isoform, appears to mainly extrude
Cl out of the cell (Payne et al., 1996).
By lowering the internal [Cl] during
development, there is a switch in the GABA response from depolarizing
to hyperpolarizing (Rivera et al., 1999). In immature rat optic nerve
axons, a significant proportion of resting conductance depends on
Cl (Connors and Ransom, 1984). Indeed,
three types of Cl channels were found on
myelinated peripheral Xenopus sciatic nerve axons by single
channel recordings (Wu and Shrager, 1994), although
direct evidence of such channels in CNS axons is lacking. Stys et al.
(1997) showed that Cl is not passively
distributed but instead has a concentration significantly above its
predicted passive level of 7 mM; resting axoplasmic [Cl]i
is in the range of 40-50 mM, indicating active
accumulation into fibers, likely mediated at least in part by Na-K-2Cl
cotransport that favors a predicted resting
[Cl]i of 55 mM (Stys et al., 1997) (Fig.
12). Using immunohistochemistry, Alvarez-Leefmans et al. (2001) confirmed the presence of Na-K-2Cl cotransporter on the membranes of both axons and Schwann cells in
peripheral nerves. Using in situ hybridization, others have demonstrated Na-K-2Cl cotransporter mRNA in both gray and white matter
areas in rat CNS, indicating that this
Cl regulator appears widely distributed
in the mammalian nervous system (Kanaka et al., 2001). In central
axons, [Cl] appears to be determined
by a coordinated interplay of accumulating (e.g., Na-K-2Cl cotransport)
systems passive and coupled efflux, mediated in part by channels and
other Cl regulatory pathways such as the
KCC.
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Figure 12.
Graph shows calculated theoretical
Clequilibrium concentrations that would be achieved by
the KCC and Na-K-2Cl cotransport (NKCC) as a function of axoplasmic
[K+] under various conditions. In normal axons,
the high transmembrane K+ gradient favors a low
[Cl]i maintained by KCC and ~55
mM Cl by NKCC (solid arrows). During
anoxia, with collapse of the K+ gradient, both KCC
and NKCC will attempt to import Cl to very high
concentrations (double solid arrow). Anoxia in the presence of TTX sees
residual K+i concentrated back up to
~55 mM because of water loss, thereby maintaining the KCC
in a Cl efflux mode (open arrow). Bottom panels
illustrate hypothetical Cl fluxes in anoxic optic
nerve axons under various conditions. i, In normal
axons, an equilibrium between Cl efflux (KCC and
various Cl channels) and influx (NKCC) maintains
[Cl] above what would be expected from passive
distribution. ii, The collapse of the
K+ gradient during anoxia will drive the KCC to
accumulate Cl, which is mostly compensated for by
activation of Cl channels; blocking these with
niflumic acid worsens outcome (see Results). Na+ and
K+ exchange across the axolemma in an electroneutral
manner through various channels. The drop in ATP levels may reduce NKCC
activity. iii, Blocking Na+ influx
into anoxic axons with TTX prevents Na+ from
entering; instead, electroneutrality is maintained by
Cl egress, through KCC [the loss of water
concentrates axoplasmic Na+ such that KCC remains
biased in the Cl-efflux direction (open arrow,
graph)] and Cl channels (Cl
is also concentrated such that ECl remains
much more positive than the resting membrane potential favoring
Cl loss), resulting in shrinkage and mechanical
disruption. iv, Addition of furosemide to block KCC
reduces Cl loss and tissue damage, possibly by
helping preserve volume and mechanical integrity. Anoxic depolarization
is also reduced, perhaps by decreasing K+ loss
because of the lower capacity for electroneutral co-efflux of
Cl anions.
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Previous studies on white matter anoxia, ischemia, and trauma
established the importance of axonal Na+
influx as a major event in the injury cascade (for review, see Stys,
1998). Our results agree with others whose general finding was that
blockade of TTX-sensitive Na+ channels
during injury was protective as assessed by electrophysiological, biochemical, and structural methods (Fern et al., 1993; Agrawal and
Fehlings, 1996; Imaizumi et al., 1997; Leppanen and Stys, 1997; Teng
and Wrathall, 1997; Jiang and Stys, 2000). The normal adult optic nerve
CAP configuration arises from an ordered segregation of fiber
conduction velocities, in turn dependent on fiber diameters and
myelination in the maturing animal (Foster et al., 1982). One might
expect the partially protective effects of TTX to be manifested in
elements that possess substantial densities of
Na+ channels, i.e., axons rather than glia
or the myelin sheath. Ultrastructural examination of anoxic optic nerve
suggests that glial damage may be attributable more to volume
disruption rather than cytoskeletal dissolution as occurs in the axon
cylinder (Waxman et al., 1994). This implies that volume changes in
glial elements, potentially including the myelin sheath, may play an
important role in causing serious alterations in fiber conduction
velocities, which would adversely affect information coding in white
matter tracts or result in complete propagation failure in fibers with more severely disrupted axo-glial architecture. Our immunolocalization data suggest that the KCC may contribute to such
Cl-dependent volume alterations under
pathological conditions in both axons and glia and the myelin sheath
(Fig. 11). Because Cl has been shown to
participate in various regulatory volume processes (for review, see
O'Neill, 1999), modulation of such pathways during injury might reduce
such deleterious volume changes, helping to preserve normal conduction
velocity distributions. In our study, the ability of furosemide to
normalize CAP wave-shape after anoxia and ischemia is consistent with
the idea that KCC mediates pathological Cl flux; the resultant osmotic and water
shifts are likely responsible for mechanical perturbation of
subcellular architecture and disturbances of action potential
propagation. We attempted to determine whether other
Cl transporters contributed to this
process by applying DIDS, a broad-spectrum anion transport blocker.
Unfortunately this compound caused a severe and irreversible depression
of CAP amplitude, possibly because of its blocking effect on
voltage-gated Na+ channels (Liu et al.,
1998), so we were unable to assess any putative protective effect on
CAP recovery. However, the incremental sparing of anoxic depolarization
observed with DIDS compared with furosemide (both in the presence of
TTX) (Fig. 4) suggests that additional anion transporters may play a role.
Under pathophysiological conditions such as anoxia/ischemia,
[Cl]i often
increases in gray matter (Jiang et al., 1992; Taylor et al., 1999).
Moreover, CA1 pyramidal cells subjected to hypoxia displayed a delayed
hypoxic depolarization in the presence of Cl transport inhibitors (Muller, 2000).
Figure 12 summarizes theoretical calculations of equilibrium
Cl concentrations under normal and
anoxic conditions on the basis of data from optic nerve (Stys et al.,
1997). Under normal conditions (Fig. 12, single solid arrowheads), KCC
attempts to maintain
[Cl]i at low
levels, well below 10 mM, whereas Na-K-2Cl cotransport will
accumulate Cl toward an equilibrium
concentration of  55 mM. Actual resting axonal
[Cl] lies between these two values,
reflecting a balance between these two opposing
Cl transport systems. During anoxia,
with a significant reduction of
[K+]i (LoPachin
and Stys, 1995; Stys and LoPachin, 1998) and a parallel rise in
[K+]o (Ransom et
al., 1992), both transporters will be biased toward strong
Cl accumulation (Fig. 12, double arrow)
and likely cause significant cellular volume deregulation.
Surprisingly, in contrast to gray matter, there is little change in
axonal or glial
[Cl]i in anoxic
white matter (LoPachin and Stys, 1995; Stys and LoPachin, 1998). One
Cl efflux mechanism that may have
compensated for such expected Cl
accumulation is the opening of a Cl
channel, such as
ClCa (for review,
see Scott et al., 1995; Frings et al., 2000). Calculations (LoPachin
and Stys, 1995) reveal that axonal Vm
remains more negative than ECl, even
at the end of a 60 min anoxic insult (Fig. 1) (axonal
Vm 44 mV vs
ECl 30 mV). Glial
Vm is also estimated to be more
negative than ECl. This indicates that
Cl flux through an uncoupled transporter
such as a channel would be continuously directed outward. For this
reason, ClCa would
be well poised to serve as a compensatory system because a rise in free
[Ca2+] will almost always be a feature
of a pathological state such as anoxia/ischemia. Consistent with this
hypothesis was the finding of Duchen (1990) who showed an enhancement
of ClCa current
during anoxia in DRG neurons. This would also explain the deleterious
effects of ClCa
channel inhibition by niflumic acid, which likely hindered the ability
of the optic nerve to compensate for an abnormal influx of
Cl through coupled transporters, as
assessed by CAP area recovery (Figs. 8, 9) and wave-shape (Fig. 10).
Other types of Cl channels blocked by
niflumic acid such as volume-regulated channels (Leaney et al., 1997)
could also participate in this mechanism.
Stys and LoPachin (1998) suggested that KCl cotransport may act as a
parallel pathway for K+ and
Cl loss during anoxia during concomitant
Na+ channel blockade. Under these
conditions, axonal volume was noted to decrease markedly (Waxman et
al., 1994) along with water content (LoPachin and Stys, 1995; Stys and
LoPachin, 1998). With the major Na+ influx
route blocked by TTX, it is likely that ionic rundown may switch from
Na+-K+
exchange to a parallel efflux of K+ and
Cl (and likely other anions); both modes
will maintain electroneutrality, but in contrast, the latter will drag
water out of the cytosol, causing cell shrinkage and possibly
mechanical damage (Waxman et al., 1994). Because the water loss will
substantially concentrate remaining intracellular ions (axoplasmic
[K+] estimated at ~55 mM
at the end of 60 min of anoxia in TTX-treated optic nerves vs ~15
mM in untreated anoxic nerves), despite a loss of 90% of
total axoplasmic K+ under both conditions
(LoPachin and Stys, 1995; Stys and LoPachin, 1998), the KCC will remain
biased in the Cl efflux mode (Fig. 12,
open arrowhead) and would be positioned to remove
K+ and Cl
from the cytoplasm. Indeed, whereas the Na-K-2Cl cotransporter would
attempt to accumulate Cl back into cells
under such conditions, its activity will be reduced by a fall in ATP
levels (Russell, 2000), whereas an ATP decrease will activate KCC
(Ortiz-Carranza et al., 1996); therefore under anoxic conditions the
transport rate of the Na-K-2Cl cotransporter may be greatly diminished,
unmasking the KCC- and Cl
channel-mediated Cl extrusion, precisely
what is observed with direct axonal
[Cl] measurements (Stys and LoPachin,
1998). This scenario might explain why blocking KCC with furosemide was
protective during anoxia: with concomitant
Na+ channel blockade, KCC inhibition
reduced the excessive Cl export while
decreasing abnormal Cl influx under
conditions in which Na+ channels were not
blocked. Therefore, excessive Cl
movements in either direction may have deleterious effects on volume
regulation resulting in mechanical injury. These findings may have
implications for the design of neuroprotective strategies, whereby
concomitant inhibition of Na+ channels and
KCC could result in better outcome than with blockade of either pathway alone.
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FOOTNOTES |
Received Dec. 2, 2002; revised Jan. 23, 2003; accepted Feb. 27, 2003.
This work was supported in part by National Institute of Neurological
Disorders and Stroke Grant R01 NS40087-01. S.A.M. is supported by a
studentship from the Ontario Neurotrauma Foundation. P.K.S. is
supported by a Career Investigator Award from the Heart and Stroke
Foundation of Ontario.
Correspondence should be addressed to Dr. Peter K. Stys, Ottawa Health
Research Institute, Division of Neuroscience, 725 Parkdale Avenue,
Ottawa, Ontario, Canada K1Y 4K9. E-mail:
pstys{at}ohri.ca.
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Expres
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TOP
ABSTRACT
Introduction
