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.
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).
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.
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).
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.
Control dorsal column slices displayed <5% change in mean peak CAP amplitude during 3 h of in-vitro monitoring at 37°C (Fig.1 B). 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).
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).
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).
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 seeStys 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 α-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.
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.
Address for reprint requests: P. K. Stys, Loeb Health Research Institute, Division of Neuroscience, 725 Parkdale Ave., Ottawa, Ontario K1Y 4K9, Canada (E-mail:).
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