Loeb Health Research Institute, Ottawa HospitalCivic Campus,
University of Ottawa, Ottawa, Ontario K1Y 4K9, Canada
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ABSTRACT |
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Li, Shuxin,
Qiubo Jiang, and
Peter K. Stys.
Important Role of Reverse Na+-Ca2+
Exchange in Spinal Cord White Matter Injury at Physiological
Temperature.
J. Neurophysiol. 84: 1116-1119, 2000.
Spinal cord injury is a devastating condition in which most of the
clinical disability results from dysfunction of white matter tracts.
Excessive cellular Ca2+ accumulation is a common phenomenon
after anoxia/ischemia or mechanical trauma to white matter, leading to
irreversible injury because of overactivation of multiple
Ca2+-dependent biochemical pathways. In the present study,
we examined the role of Na+-Ca2+ exchange, a
ubiquitous Ca2+ transport mechanism, in anoxic and
traumatic injury to rat spinal dorsal columns in vitro. Excised tissue
was maintained in a recording chamber at 37°C and injured by exposure
to an anoxic atmosphere for 60 min or locally compressed with a force
of 2 g for 15 s. Mean compound action potential amplitude
recovered to 25% of control after anoxia and to
30% after
trauma. Inhibitors of Na+-Ca2+ exchange (50 µM bepridil or 10 µM KB-R7943) improved functional recovery to
60% after anoxia and
70% after traumatic compression. These
inhibitors also prevented the increase in calpain-mediated spectrin
breakdown products induced by anoxia. We conclude that, at
physiological temperature, reverse Na+-Ca2+
exchange plays an important role in cellular Ca2+ overload
and irreversible damage after anoxic and traumatic injury to dorsal
column white matter tracts.
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INTRODUCTION |
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In excitable cells
the Na+-Ca2+ exchanger
helps to maintain intracellular Ca2+ homeostasis.
These antiporters utilize the energy stored in the electrochemical
gradient of Na+ to export
Ca2+ from the cytosol against a steep
concentration gradient typically exceeding 104.
The brain Na+-Ca2+
exchangers transport three Na+ ions for each
Ca2+ ion, resulting in electrogenic operation,
and are able to operate in either the Ca2+ import
or export modes depending on the prevailing ion gradients and membrane
potential (for review see Blaustein and Lederer 1999). Under pathophysiological conditions such as anoxia, ischemia, or
trauma, intracellular Na+ concentration rises and
membranes depolarize as a result of energy failure, which leads to
impaired ion pumping. Such conditions could lead to reverse
Na+-Ca2+ exchange, which
would cause Ca2+ to accumulate in the
intracellular compartment.
Spinal cord injury is a devastating condition in which the bulk
of the clinical disability is caused by dysfunction of ascending and
descending white matter tracts. Although reverse
Na+-Ca2+ exchange has been
shown to mediate significant cellular Ca2+ influx
in white matter during anoxia (Imaizumi et al. 1997;
Stys and LoPachin 1998
), its role in traumatic white
matter injury is controversial (Agrawal and Fehlings
1996
). Given the key role of this transporter in
anoxic/ischemic white matter injury, we decided to examine its
contribution to the pathogenesis of traumatic injury in white matter in
vitro. We found that, at physiological temperature, the
Na+-Ca2+ exchanger plays a
significant role in white matter trauma. These results were presented
in abstract form (Stys and Li 1999
).
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METHODS |
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Electrophysiology
Adult Long-Evans male rats were deeply anesthetized with sodium
pentobarbital and sections of dorsal columns were gently excised. Tissue was gradually warmed to 37°C in an interface recording chamber
and perfused with artificial cerebrospinal fluid (aCSF) containing (in
mM) 126 NaCl, 3.0 KCl, 2.0 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 2.0 CaCl2, and 10 dextrose, pH 7.4, bubbled with 95% O2/5% CO2. Control
recordings were taken 30 min later. Propagated compound action
potentials (CAPs) were evoked using a bipolar silver-wire stimulating
electrode placed on one end of the dorsal column slice and a constant
voltage pulse (50 µs and, typically, 70 V) delivered once every 30 min. CAPs were recorded extracellularly at the opposite end using
large-tipped glass microelectrodes filled with 150 mM NaCl (Li
et al. 1999). Evoked CAPs were digitized, stored, and analyzed
using WaveTrak software (Stys 1994
). Anoxia was achieved
by switching to a 95% N2/5%
CO2 atmosphere, with solution bottles bubbled
with this same mixture. The functional integrity of the dorsal column
was quantitated by measuring peak CAP amplitude.
Western blotting
Frozen dorsal column strips were homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, containing 0.01% Triton, 10 mM EGTA, and 0.5 mM
phenylmethylsulfonyl fluoride) and centrifuged; supernatants were then
extracted and stored at 80°C. Samples containing identical total
protein contents (typically 10 or 20 µg per lane) were dissolved in
sample buffer, run on an 8% gel, and transferred to nitrocellulose membranes. Membranes were probed with anti-spectrin breakdown product
antibody (Ab38) at 4°C overnight and then treated with secondary antibody conjugated with horseradish peroxidase
(HRP). Chemiluminescent detection was performed using an ECL
kit (Amersham).
Pharmacological agents
Bepridil (RBI) and KB-R7943 (2-[2-[4-(4 nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) were first dissolved in DMSO (for anoxia) or ethanol (for spinal cord injury) and then added to aCSF to the desired final concentration. Vehicle concentrations never exceeded 0.1% vol/vol. All salts were from Sigma. All data are expressed as mean ± SD.
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RESULTS |
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Control dorsal column slices displayed <5% change in mean peak
CAP amplitude during 3 h of in-vitro monitoring at 37°C (Fig. 1B). After 60 min of anoxia in
aCSF, mean CAP amplitude was reduced to 27 ± 4% (SD) and
26 ± 6% of control after 1 or 2 h of reoxygenation, respectively (Fig. 1). Slices were exposed to 50 µM bepridil or 10 µM KB-R7943, two well-known
Na+-Ca2+ exchange
inhibitors (Garcia et al. 1988; Hoyt et al.
1998
; Iwamoto et al. 1996
). Both agents
significantly improved recovery of CAP amplitude following 60 min of
anoxia. For example, after 2 h of reoxygenation, mean CAP
amplitude recovered to 56 ± 5% and 63 ± 7% in bepridil or
KB-R7943, respectively, versus
26% without drug (P < 0.01), indicating that
Na+-Ca2+ exchange partially
contributes to white matter injury during anoxia (Fig. 1).
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Recent studies suggest that traumatic axonal injury may share
many pathophysiological mechanisms with anoxic injury. For example, the
cellular damage caused by both anoxic and mechanical injury to the
spinal cord is heavily dependent on abnormal Na+
influx through voltage-gated Na+ channels,
intracellular Ca2+ overload, and glutamate
excitotoxicity (Agrawal and Fehlings 1996; Li et
al. 1999
; Stys et al. 1992b
; Wrathall et
al. 1997
). We hypothesized that, as with anoxic injury,
Na+-Ca2+ exchange
contributes to traumatic damage. Our in-vitro spinal white matter
injury model consisted of excised dorsal column slices subjected to a
2-g compression for 15 s (Agrawal and Fehlings 1996
; Li et al. 1999
), which resulted in mean
CAP amplitude recovery to 32 ± 10% of preinjury values 1 h
after trauma (Fig. 2). Both Na+-Ca2+ exchange
inhibitors, 50 µM bepridil and 10 µM KB-R7943, were significantly
neuroprotective against in-vitro SCI, increasing the recovery of mean
CAP amplitudes to 68 ± 5% and 68 ± 2% of preinjury
amplitudes, respectively, 1 h after compression (P < 0.01 compared with aCSF or vehicle controls; Fig. 2).
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Western blots were performed on control and anoxic dorsal column slices, in the absence or presence of 50 µM bepridil or 10 µM KB-R7943, using an antibody specific for calpain-mediated spectrin breakdown fragments. Significant spectrin breakdown, which almost doubled after 1 h of anoxic exposure, was seen in control tissue. This increase was prevented by Na+-Ca2+ exchange inhibitors, paralleling the improvement in functional recovery (Fig. 3).
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DISCUSSION |
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White matter damage is a common feature of many CNS
disorders such as stroke, spinal cord injury, and multiple sclerosis. A
series of studies elucidated many of the basic mechanisms of white
matter injury resulting from anoxia, ischemia, and trauma (Agrawal and Fehlings 1997; Fern et al.
1995
; Garthwaite et al. 1999
; Li et al.
1999
; LoPachin et al. 1999
; Stys et al.
1992b
; Wrathall et al. 1997
; for review see
Stys 1998
). As with injury to neurons, whether caused by
anoxia or trauma, a common theme is the excessive accumulation of
Ca2+ ions in intracellular white matter
compartments. During anoxic injury to optic nerve or spinal white
matter tracts, reverse
Na+-Ca2+ exchange, driven
by membrane depolarization and axoplasmic Na+
accumulation, is an important route of axonal
Ca2+ accumulation leading to injury
(Imaizumi et al. 1997
; Stys et al.
1992b
), and is further supported by immunohistochemical
localization of the NCX-1 exchanger isoform in central white
matter tracts (Steffensen et al. 1997
). The present
results on anoxic dorsal columns are in agreement with these reports
(Fig. 1).
The role of this transporter in mechanical white matter trauma,
despite similar membrane potential and ionic deregulation, was
questionable. In a detailed study using in-vitro traumatic compression
of spinal cord white matter slices, Agrawal and Fehlings (1996) concluded that the
Na+-Ca2+ exchanger, in
contrast to anoxia, did not play an important role in trauma, which is
at variance with our findings. The Agrawal and Fehlings
(1996)
study, however, was performed at subphysiological temperatures (25-33°C). Even modest reductions of temperature (as
little as 2.5-5°C) have dramatic effects on postanoxic recovery in
CNS white matter (Stys et al. 1992a
). Indeed, in
absolute terms, the degree of functional injury after identical
traumatic insults was significantly less at hypothermic temperatures
[recovery of CAP amplitude was
70% of control (Agrawal and
Fehlings 1996
) vs.
30% at physiological temperature (the
present study)]. Given that the Q10 of the
Na+-Ca2+ exchanger is quite
high (
3-4) (Kimura et al. 1987
), it is likely that
the reduced degree of injury and the apparent lack of
Na+-Ca2+ exchange
contribution in Agrawal and Fehlings (1996)
were at least partially a result of the lower temperatures.
Cellular Ca2+ overload resulting from
anoxia/ischemia or trauma activates calpain, which induces proteolysis
of a number of structural proteins, including spectrin (Buki et
al. 1999; Li and Stys 2000
; Roberts-Lewis
et al. 1994
). Semiquantitative evaluation of the degree of
spectrin breakdown can thus serve as a marker for
Ca2+-dependent, calpain-mediated tissue
injury. Western analysis of calpain-cleaved spectrin breakdown
products showed a significant baseline level ("control" in Fig. 3)
that was markedly higher than that in a related study using optic nerve
(Jiang and Stys 2000
). This difference is probably
artifactual, reflecting a much larger transected surface in dorsal
columns created during excision as compared with optic nerve, where
only the ends need to be cut. Physiologically the dorsal column slices
remained very healthy for hours in vitro at 37°C. The amount of
spectrin breakdown increased during anoxia and, in parallel with an
improvement in functional recovery, this rise was prevented by
Na+-Ca2+ exchange
inhibitors, most likely because of a reduction of axonal Ca2+ overload. Notably, function was not
completely restored by inhibitors of
Na+-Ca2+ exchange whereas
Ca2+-depleted conditions are virtually completely
neuroprotective (Stys et al. 1990
), indicating alternate
parallel routes of cellular Ca2+overload such as
voltage-gated Ca2+ channels (Imaizumi et
al. 1999
) and Ca2+-permeable
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
(Li and Stys 2000
). Taken together, our data unequivocally indicate that, at 37°C, the
Na+-Ca2+ exchanger
contributes significantly to structural and functional dorsal column
injury. Inhibitors of this ion transporter may therefore be useful
neuroprotective agents in conditions where white matter is prominently involved.
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ACKNOWLEDGMENTS |
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Ab38 was a gift from Dr. R. Siman, University of Pennsylvania. KB-R7943 was a gift from Kanebo Ltd., Osaka, Japan.
This work was supported in part by Ontario Neurotrauma Foundation Grant ONRO-31 and Heart and Stroke Foundation of Ontario Grant B-3743. S. Li is supported by a scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). P. K. Stys is supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario.
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FOOTNOTES |
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Address for reprint requests: P. K. Stys, Loeb Health Research Institute, Division of Neuroscience, 725 Parkdale Ave., Ottawa, Ontario K1Y 4K9, Canada (E-mail: pstys{at}lri.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 February 2000; accepted in final form 3 May 2000.
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REFERENCES |
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