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Department of Neurology and Paralyzed Veterans Association/Eastern Paralyzed Veterans Association of America Neuroscience Research Center, Yale University School of Medicine, New Haven, 06510; and the Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare Systems, West Haven, Connecticut 06516
Submitted 6 May 2003; accepted in final form 29 July 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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; Trapp et al. 1998), and it has been suggested that axonal degeneration contributes to the acquisition of persistent (nonremitting) clinical deficits (Davie et al. 1995; Lee et al. 2000). The loss of axons in the spinal cord, in particular, appears to contribute significantly to neurological disability in animal models of MS and in MS (Bjartmar et al. 2000; Ganter et al. 1999; Lovas et al. 2000; Wujek et al. 2002). Thus neuroprotective interventions that maintain axonal integrity and preserve conduction might be expected to slow or halt the development of neurological disability.
Although a number of studies have shown that it is possible to use immunomodulatory interventions to ameliorate the clinical course of experimental allergic encephalomyelitis (EAE), an animal model of MS in which axonal degeneration is known to occur (Korneck et al. 2000; Raine and Cross 1989; White et al. 1992), neuroprotective strategies that directly target neurons have not been studied in EAE. Evidence suggesting that voltage-gated sodium channels might be tractable targets for neuroprotection of white matter axons was provided by early studies (Stys et al. 1992a). This work demonstrated that voltage-gated sodium channels can participate in the production of calcium-mediated degeneration of white matter axons after injury by providing a route for persistent sodium current that drives reverse operation of the Na+/Ca2+ exchanger which, in turn, results in a damaging influx of calcium. As suggested by this mechanism, sodium channel blockade with tetrodotoxin (TTX) and tertiary and quarternary anesthetics has been shown to prevent the development of irreversible dysfunction of axons within the anoxic optic nerve in vitro (Stys et al. 1992a,b). In vitro studies have also demonstrated that phenytoin, a drug that blocks sodium channels and inhibits persistent sodium currents (Chao and Alzheimer 1995; Segal and Douglas 1997), has a protective effect on axons within white matter that has been subjected to anoxia (Fern et al. 1993).
Although the molecular events leading to axonal degeneration in MS have not been delineated, it has been suggested that, as a result of inflammatory damage to small blood vessels, hypoxia may contribute to neuronal damage in MS (Lassmann 2003). In addition, a role for sodium channels is suggested by the protective effects of TTX (Garthwaite et al. 2002) and of low doses of lidocaine and flecainide (Kapoor et al. 2003) on experimental axonal injury triggered by nitric oxide (NO); NO is present at elevated concentrations in MS lesions (Bo et al. 1994; Brosnan et al. 1994; Smith et al. 1999) and, like anoxia, is thought to trigger mitochondrial failure with resultant energy depletion and an increase in intra-axonal sodium (Bolanos et al. 1997; Brown et al. 1995; Kapoor et al. 2003). Reasoning that a persistent sodium current might provoke reverse Na+/Ca2+ exchange that contributes to axonal injury in inflammatory disorders of the CNS, Lo et al. (2002) recently studied the optic nerve in EAE and observed an increased number of preserved axons after treatment with phenytoin. These initial results in the optic nerve suggest that phenytoin may have a neuroprotective effect in EAE as measured by axon counts.
In the present study, we have examined the effect of phenytoin on axonal degeneration within the dorsal columns (cuneate fasciculus) and dorsal corticospinal tracts of the spinal cord. Using axonal counting, electrophysiology, and behavioral assessment after induction of EAE, we show that treatment with phenytoin results in robust protection of the integrity of spinal cord axons, preservation of action potential conduction, and amelioration of neurological deficits.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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We measured the number of axons within the cervical spinal cord after staining them for neurofilaments. Mice were perfused with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer. Spinal cords were carefully excised from the brain stem to the lumbar region and cryoprotected with 30% sucrose in PBS. The cervical enlargement was identified and then transected at the exact midpoint of the cervical enlargement to standardize a site along the longitudinal axis of the cord, ensuring that the same cervical spinal cord regions were analyzed for all conditions. Transverse sections were cut and incubated with antibodies against phosphorylated neurofilamentsH (SMI-31, 1:20,000; Sternberger Monoclonals, Lutherville, MD) and nonphosphorylated neurofilaments (SMI-32, 1:20,000) overnight at 4°C on a rotating shaker. To count all axons, including axons with both phosphorylated and nonphosphorylated neurofilaments (Lo et al. 2002; Lovas et al. 2000; Wujek et al. 2002), we incubated sections with SMI-31 and -32 antibodies in combination or individually with SMI-32, which has been used as a marker for demyelinated or damaged white matter axons (Trapp et al. 1998), and with SMI-31. (As noted in RESULTS, SMI-32 immunostained axons accounted for <5% of the total axon population in each region studied, and analysis of tissue stained for SMI-32 and SMI-31 individually did not reveal any trends that differed from those for the total axon population stained by SMI-32 and -31 in combination). Sections reacted with SMI-31 and/or -32 were then incubated sequentially in PBS, rabbit anti-mouse IgG-biotin (1:1000, Sigma), PBS, avidin-HRP (1:1000, Sigma), PBS, and heavy metal-enhanced DAB (Pierce, Rockford, IL).
Image acquisition and analysis
Sections were examined with a Nikon E800 microscope equipped with a x100 oil objective, and digital images were captured with a Spot RT color camera (Spot Diagnostic Instruments, Sterling Heights, MI). Axonal densities were determined within preselected fields (500 µm2 in area) at specific sites within the dorsal column (cuneate fasciculus), lateral column, ventral column, and dorsal corticospinal tract white matter regions, using the borders of the gray matter as consistent landmarks (Fig. 1A). Neurofilament stained axons were manually counted from each 500-µm2 area using IPLab software (Scanalytics, Fairfax, VA) as described previously (Lo et al. 2002). The number of mice analyzed in each group were as follows: control, 4; control + phenytoin, 5; EAE, 6; and EAE + phenytoin, 5. Statistical analyses between control and experimental groups were performed using Student's t-test with Microsoft Excel software.
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Electrophysiological recordings
Fourteen adult male C57/BL6 mice were used for assessing spinal cord conduction. Untreated EAE (n = 6) and phenytoin-treated EAE (n = 4) mice were studied between days 24 and 30 and age-matched phenytoin-treated mice (n = 4) were used as controls. Mice were anesthetized with halothane, and deep anesthesia was maintained with 1.1% halothane through a tracheal cannula by artificial ventilation (75–80 strokes/min, 0.7 ml/stroke) and verified by the absence of a withdrawal reflex to noxious peripheral pinch. Heartbeats (
360 beats/min) and body temperature were carefully monitored. Mice were fixed in a stereotaxic apparatus (Kopf Instruments). A midline incision was made through the skin, a laminectomy was performed, and the dura was carefully cut to expose the lower thoracic and lumbar spinal cord regions.
Silver wire electrodes (0.01-in diam, A-M Systems) insulated except at the tips were used for stimulation of dorsal column axons at L4–5 and recording of compound action potentials (CAP) at T11–12 (Fig. 3A). Using stereotaxic measurements, the electrodes were positioned 8 mm apart at the surface of the midline spinal cord. Pancuronium bromide (0.3 mg/kg, Sigma) was injected ip to prevent further muscular contractions throughout the experiment. The signal from the recording electrode was amplified with filters set at 300–3,000 Hz (Dam 80, WPI) and collected (CED 1401+, Cambridge Electronic Design), and a computer was used for data analysis (Spike 2 program, Cambridge Electronic Design). Single-current pulses (0.05 ms) were applied through a stimulus isolation unit (A365, WPI, and Master-8, AMPI) in increments (0.2, 0.3, 0.6, 0.8, and 1 mA). The amplitude of CAP was calculated as the value between the positive and negative peaks of the biphasic wave (Fig. 3B). The area of the CAP was calculated by rectifying the negative component (full-wave rectification using Spike 2 script software, Cambridge Electronic Design) and measuring its area. Conduction velocity was estimated by measuring the latency to the peak of the negative component of the supramaximal CAP at 8 mm, then moving the recording electrode to 9 mm and measuring latency; conduction velocities measured in this manner were within 5% of conduction velocities measured between the stimulating electrode and the recording electrode at 8 mm. At the end of each experiment, the dorsal columns and corticospinal tract were transected between stimulating and recording electrodes to confirm that a CAP could not be detected.
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Induction of EAE
Experiments were carried out in accordance with National Institutes of Health guidelines for the use of laboratory animals; all animal protocols were approved by the Yale Institutional Animal Care and Use Committee. C57/BL6 mice 6–10 wk of age were injected subcutaneously with 200 µl of an emulsion of 300 µg of rat myelinoligodendrocyte glycoprotein (MOG) 35–55 peptide (Keck Biotechnology Center, Yale University) in incomplete Freund's adjuvant (IFA, Sigma, St. Louis, MO) supplemented with 250–500 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). The MOG injection, with mycobacterium supplemented IFA, was repeated in the opposite flank 1 wk later. The mice also received an injection of 250–500 ng pertussis toxin (Sigma) in 200 µl PBS ip immediately after the first immunization with MOG and then again 48 h later.
Immunized mice were observed daily for clinical signs and scored on a 0–6 scale, with 0.5 gradations for intermediate scores, as follows: 0, normal without clinical signs; 1, flaccid tail; 2, abnormal righting reflex; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, moribund; and 6, death.
Phenytoin treatment
Beginning on day 10 after the initial MOG injection, control and EAE-induced mice were fed pelleted mice chow incorporating phenytoin (5,5 diphenylhydantoin; Sigma). A separate group of EAE mice were fed the identical standard mice chow not containing phenytoin. Serum phenytoin levels were measured by homogeneous enzyme immunoassay (Pinus and Abraham 1991) from two mice in each group on day 14 and on the day of either perfusion (days 27–28) or electrophysiological study (days 24–30). Mean serum phenytoin levels were: day 14: control + phenytoin, 18.3 µg/ml; EAE + phenytoin, 18.0 µg/ml; and day 28 (perfusion): control + phenytoin, 19.0 µg/ml, EAE + phenytoin, 20.1 µg/ml. For mice used in spinal cord electrophysiological studies, phenytoin serum levels were 16.8 µg/ml. These serum phenytoin levels are within the therapeutic range determined in the clinical setting (10–20 µg/ml).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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We counted the number of cervical spinal cord axons via counts within standardized 500-µm2 fields at predetermined sites in the dorsal corticospinal tract, dorsal column (cuneate fasciculus), lateral column, and ventral column (Fig. 1A, Table 1). To identify all axons within these fields in the mid-cervical spinal cord, we labeled axons using a combination of antibodies directed against both phosphorylated (SMI-31) and non-phosphorylated (SMI-32) neurofilaments (Lo et al. 2002; Lovas et al. 2000). The density of axons labeled with neurofilament antibodies within the dorsal corticospinal tract was 162 ± 9/500 µm2 in untreated controls and 172 ± 8/500 µm2 for phenytoin-treated controls (not statistically significant; P > 0.05). The density of axons labeled with neurofilament antibodies within the dorsal columns (cuneate fasciculus) was 68 ± 8/500 µm2 in untreated controls and 74 ± 12/500 µm2 in phenytoin-treated controls (not significant; P > 0.05). For simplicity, we compare axon counts in untreated EAE and phenytoin-treated EAE to phenytoin-treated control mice in Fig. 2, but comparisons to untreated controls yielded similar results.
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There was significant loss of axons within the dorsal corticospinal tract and cuneate fasciculus of untreated EAE (Figs. 1B and 2A). The density of axons labeled with neurofilament antibodies in the corticospinal tract after 27–28 days of untreated EAE decreased substantially and was 64 ± 8/500 µm2 (P < 0.05 compared with phenytoin-treated control mice), representing a 62.5% dropout of axons (Figs. 1B and 2B). The density of axons in the cuneate fasciculus after 27–28 days of untreated EAE was also reduced and was 42 ± 8/500 µm2 (P < 0.05, compared with phenytoin-treated controls), a 43.1% decrease of axons. In contrast, lateral and ventral column axon densities (Fig. 1B) were only slightly decreased (lateral column: 61 ± 8/500 µm2 and ventral column: 64 ± 8/500 µm2) after 27–28 days of untreated EAE, compared with phenytoin-treated controls (lateral column: 74 ± 8/500 µm2; ventral column: 77 ± 12/500 µm2), representing a loss of 17% of axons in these regions of the spinal cord. These differences were not statistically significant.
Phenytoin was orally administered beginning on day 10 after the initial MOG induction of EAE. Phenytoin had a significant protective effect on dorsal corticospinal column and dorsal column (cuneate fasciculus) axons (Fig. 2A). Axon loss in the corticospinal tract of phenytoin-treated EAE was only 27.7% (125 ± 22/500 µm2) compared with 62.5% loss (P < 0.05; see preceding text) in the same region in untreated EAE. Axon loss in the dorsal column (cuneate fasciculus) was also reduced by treatment with phenytoin and was 16.7% (62 ± 6/500 µm2) compared with a dropout of 43.1% (P < 0.05) in the same location of untreated EAE (Fig. 2B). In the lateral and ventral columns, where axonal loss was less pronounced (see preceding text), axon densities were maintained near normal in phenytoin-treated EAE (76 ± 6/500 and 72 ± 8/500 µm2, respectively), but the protective trend in phenytoin-treated EAE compared with untreated EAE did not reach statistical significance in these regions.
Because neurofilaments exist in phosphorylated and non-phosphorylated forms, which may be individually expressed or co-expressed in axons (Sloan and Stevenson 1987), we also immunoreacted cervical spinal cord separately with SMI-32 or -31 antibodies and analyzed the axon densities in the same regions (dorsal corticospinal tract, dorsal column (cuneate fasciculus), lateral column, and ventral column) to determine whether there was a preferential effect of phenytoin on SMI-32 versus SMI-31 positive axons. This analysis did not reveal any trends that differed from those for the total axon population stained with SMI-32 and -31 in combination. The percentage of SMI-32 positive axons (i.e., SMI-32 positive/SMI-31 positive + SMI-32 positive) within the corticospinal tract in untreated EAE was 4.4%; the percentage of SMI-32 positive axons in the CST in phenytoin-treated controls was 0.5% and in phenytoin-treated EAE was 1.0%. Similarly, within the dorsal columns (cuneate fasciculus) the percentage of SMI-32 positive axons was 4.5% in untreated EAE, 0.3% in phenytoin-treated controls, and 0.9% in phenytoin-treated EAE.
Spinal cord electrophysiology
The average compound action potential (CAP) amplitude (Fig. 3, B and C) and CAP area (Fig. 3, D and E) in the untreated EAE group were significantly smaller than in the phenytoin-treated control group (P < 0.01). In fact, a biphasic CAP could not be recorded in four of six mice in the untreated EAE group. In addition, the average threshold for evoking a CAP in two mice in the untreated EAE group, in which a CAP could be elicited, was higher (0.3–0.6 mA) than in the phenytoin-treated control group (0.2–0.3 mA; Fig. 3C). In contrast, robust CAPs with a normal positive-negative configuration were observed in phenytoin-treated EAE, with a threshold similar to that in controls (Fig. 3C), and the average CAP amplitude and area in the phenytoin-treated EAE group were not significantly different from in the phenytoin-treated control group (Fig. 3, C, D, and E). Mean ± SE conduction velocity in the EAE group (2.28 ± 1.65 m/s) was substantially slower than in controls (11.58 ± 2.10 m/s) but was maintained close to normal levels (13.15 ± 1.06 m/s; not significant, P > 0.05 compared with controls) in phenytoin-treated EAE (Fig. 3F).
Neurological status
We assessed the clinical score of each mouse in each experimental condition using a rating scale (see METHODS) that is sensitive to motor function, particularly in the hindlimbs. Phenytoin-treated control mice did not display any pathological clinical signs (clinical score = 0). Untreated EAE mice manifested progressive clinical impairment, with an average clinical score of 3.8 ± 0.18 (n = 6) on day 27–28. In contrast, phenytoin-treated EAE mice exhibited a less severe clinical course than untreated mice, with an average clinical score of 1.5 ± 0.26 on day 27–28 (Fig. 4). The clinical scores in the untreated EAE and phenytoin-treated EAE mice were significantly different (P < 0.05) at all times starting on day 21 onward.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Our observations of substantial loss of axons within the spinal cord in EAE are consistent with earlier studies (Kornek et al. 2000; Raine and Cross 1989; White et al. 1992), which demonstrated axonal dropout within EAE; however, the present study is the first to document substantial loss of corticospinal tract axons. The robust nature of axonal degeneration in our EAE mice provided an animal model of loss of spinal cord axons, which is known to contribute to the progression of neurological disability in MS (Bjartmar et al. 2000; Losseff et al. 1996; Wujek et al. 2002), against which we could assess the effects of phenytoin. Consistent with the conclusion that axon loss in the spinal cord contributes to the degree of neurological disability in EAE, we found that the loss of spinal cord axons in untreated EAE is accompanied by hind-limb paralysis and that preservation of spinal cord axons in phenytoin-treated EAE is paralleled both by preservation of action potential conduction and by reduced neurological disability (i.e., reduced clinical score).
In rodents, a major component of the corticospinal tract mediating motor behavior is located adjacent to sensory axons within the dorsal columns (Brosamle and Schwab 1997, 2000). Therefore we expected spinal cord conduction to parallel clinical behavior. To functionally assess conduction in the axons of the dorsal columns and corticospinal tract, CAPs were studied in vivo. The CAPs recorded in this study were abolished by transection of the dorsal columns and the dorsal corticospinal tract at the end of each experiment, indicating that they were evoked in axons coursing through the dorsal column and the dorsal corticospinal tract and not in other (lateral or ventral column) axons. The results suggest that conduction in these axons is severely impaired in EAE and shows a protective effect of phenytoin with relatively preserved CAP amplitude and area in phenytoin-treated EAE, in agreement with the histological data and behavioral scores. We observed a substantial reduction in conduction velocity in EAE, with conduction velocities in our control and EAE spinal cords very close to those previously reported (Imaizumi et al. 1998) for control and demyelinated rodent spinal cord. Interestingly, conduction velocities were maintained at values close to normal in phenytoin-treated EAE, suggesting that treatment with phenytoin may have prevented demyelination in addition to protecting axons from degeneration.
The sodium-channel-blocking actions of phenytoin are well established (Ragsdale and Avoli 1998; Ragsdale et al. 1996) and its protective effect on axons within the anoxic optic nerve in vitro has been attributed to the block of axonal sodium channels (Fern et al. 1993; Stys et al. 1993). Imaizumi et al. (1997) demonstrated that a significant component of axonal loss, after anoxic injury to the dorsal columns in vitro, is a result of reverse operation of the sodium Na+/Ca2+ exchanger that is triggered by sodium influx via voltage-gated sodium channels. Tetrodotoxin (Agrawal and Fehlings 1996; Rosenberg et al. 1999; Teng and Wrathall 1997), procaine (Agrawal and Fehlings 1996; Rosenberg et al. 1999), and QX-314 (Agrawal and Fehlings 1997) have been shown to have a protective effect on spinal cord axons in in vitro and in vivo models of traumatic injury, suggesting that sodium channels may be more generally involved in the injury-induced cascade that produces degeneration of spinal cord axons. In addition, it has been demonstrated that TTX (Garthwaite et al. 2002) and low concentrations of lidocaine and flecainide (Kapoor et al. 2003) protect axons from degeneration induced by NO, which is present at high levels in MS lesions (Bo et al. 1994; Brosnan et al. 1994; Smith et al. 1999), where it has been postulated to trigger mitochondrial failure and hypoxia-like energy depletion (Kapoor er al. 2003). Moreover, Lassmann (2003) has suggested that hypoxic injury due to inflammatory damage to small blood vessels may contribute to tissue damage in MS.
Phenytoin was originally developed as an antiepileptic medication and is also used clinically for the treatment of some pain syndromes, presumably on the basis of its sodium-channel-blocking properties. Nonetheless, phenytoin is not a specific sodium channel blocker. Phenytoin acts to stabilize membranes in excitable cells and (at concentrations higher than those required for block of sodium channels) blocks calcium channels (McLean and MacDonald 1993; Narahashi 1988) and potassium channels (Yaari et al. 1986). In the anoxic optic nerve model, phenytoin has a protective effect at concentrations of 1 µM, lower than the concentration required for block of sodium, calcium, and potassium channels; it has been suggested that the protective effect of phenytoin in the anoxic optic nerve might be due to the potentiation of sodium channel block by phenytoin by depolarization (Fern et al. 1993). Because phenytoin is not a specific sodium channel blocker, the present results do not provide conclusive evidence that sodium channels contribute to the degeneration of axons in EAE. However, consistent with this suggestion, Bechtold et al. (2002) have observed that systemically administered flecainide has a protective effect on dorsal column axons in a rat model of chronic-relapsing EAE, and TTX has been shown to protect axons so that they do not degenerate after a spectrum of insults (Agrawal and Fehlings 1996; Garthwaite et al. 2002; Stys et al. 1992a; Teng and Wrathall 1997).
In summary, the present results demonstrate that phenytoin has a protective effect on axons in vivo, reducing the degeneration of spinal cord axons in EAE. Our results also demonstrate that the preservation of axons in phenytoin-treated EAE is paralleled by preservation of axonal conduction within the spinal cord and by amelioration of clinical deficits. These results may provide a basis for novel therapeutic approaches that will preserve axons and axonal function in neuroinflammatory disorders such as MS.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. G. Waxman, Dept. of Neurology LCI 707, Yale School of Medicine, 333 Cedar Street, P.O. Box 208018, New Haven, CT 06520-8018 (E-mail: stephen.waxman{at}yale.edu).
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
Agrawal SK and Fehlings MG. The effect of the sodium channel blocker QX-314 on recovery after acute spinal cord injury. J Neurotrauma 14: 81-88, 1997.[ISI][Medline]
Bechtold DA, Kapoor R, and Smith KJ. Axonal protection mediated by flecainide therapy in experimental inflammatory demyelinating disease. J Neurol 249: 204, 2002.
Bjartmar C, Kidd G, Mork S, Rudick R, and Trapp BD. Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl-aspartate in chronic multiple sclerosis patients. Ann Neurol 48: 893-901, 2000.[ISI][Medline]
Bo L, Dawson TM, Wesselingh S, Mork S, Choi S, Kong PA, Hanley D, and Trapp BD. Induction of nitric oxide synthetase in demyelinating regions of multiple sclerosis. Ann Neurol 36: 78-786, 1994.
Bolanos JP, Almeida A, Stewart V, Peuchen S, Land JM, Clark JB, and Heales SJ. Nitric-oxide mediated mitochondrial damage in the brain: mechanism and implications for neurodegenerative diseases. J Neurochem 68: 2227-2240, 1997.[ISI][Medline]
Brosamle C and Schwab ME. Cells of origin, course, and temination patterns of the ventral, uncrossed component of the mature rat corticospinal tract. J Comp Neurol 386: 293-303, 1997.[ISI][Medline]
Brosamle C and Schwab ME. Ipsilateral, ventral corticospinal tract of the adult rat: ultrastructure, myelination and synaptic connections. J Neurocytol 29: 499-507, 2000.[ISI][Medline]
Brosnan CF, Battistini L, Raine C, Dickson DW, and Casadevall A. Reactive nitrogen intermediates in human neuropathology: an overview. Dev Neurosci 16: 152-161, 1994.[ISI][Medline]
Brown GC, Bolanos JP, Heales SJ, and Clark JB. Nitric oxide produced by actived astrocytes rapidly and reversibly inhibits cellular respiration. Neurosci Lett 193: 201-204, 1995.[ISI][Medline]
Chao TI and Alzheimer C. Effects of phenytoin on the persistent Na+ current of mammalian CNS neurons. Neuroreport 6: 1778-1780, 1995.[ISI][Medline]
Davie C, Barker G, Webb S, Tofts P, Thompson A, Harding A, McDonald W, and Miller D. Persistent functional deficit in multiple sclerosis and autosomal dominant cerebellar ataxia is associated with axon loss. Brain 118: 1583-1592, 1995.
Ferguson B, Matyszak MK, Esiri MM, and Perry VH. Axonal damage in acute multiple sclerosis lesions. Brain 120: 393-399, 1997.
Fern R, Ransom BR, Stys PK, and Waxman SG. Pharmacological protection of CNS white matter during anoxia: actions of phenytoin, carbamazepine, and diazepam. J Pharmacol Exp Ther 266: 1549-1555, 1993.
Ganter P, Prince C, and Esiri MM. Spinal cord axonal loss in multiple sclerosis: a post-mortem study. Neuropathol Appl Neurobiol 25: 459-467, 1999.[ISI][Medline]
Garthwaite G, Goodin DA, Batchelor AM, Leeming K, and Garthwaite J. Nitric oxide toxicity in CNS white matter: an in vitro study using rat optic nerve. Neuroscience 109: 145-155, 2002.[ISI][Medline]
Imaizumi T, Kocsis JD, and Waxman SG. Anoxic injury in the rat spinal cord: pharmacological evidence for multiple steps in Ca2+-dependent injury of the dorsal columns. J Neurotrauma 14: 299-311, 1997.[ISI][Medline]
Imaizumi T, Lankford KL, Waxman SG, Greer CA, and Kocsis JD. Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the demyelinated dorsal columns of the rat spinal cord. J Neurosci 18: 6176-6185, 1998.
Kapoor R, Davies M, Blaker PA, Hall SM, and Smith KJ. Blockers of sodium and calcium entry protect axons from nitric-oxide mediated degeneration. Ann Neurol 53: 174-180, 2003.[ISI][Medline]
Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, Linington C, Schmidbauer M, and Lassmann H. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am J Pathol 157: 267-276, 2000.
Lassmann H. Hypoxia-like tissue injury as a component of multiple sclerosis lesions. J Neurol Sci 206: 187-191, 2003.[ISI][Medline]
Lee MA, Blamire AM, Pendlebury S, Ho KH, Mills KR, Styles P, Palace J, and Matthews PM. Axonal injury or loss in the internal capsule and motor impairment in multiple sclerosis. Arch Neurol 57: 65-70, 2000.
Lo AC, Black JA, and Waxman SG. Neuroprotection of axons with phenytoin in experimental allergic encephalomyelitis. Neuroreport 13: 1909-1912, 2002.[ISI][Medline]
Losseff NA, Webb SL, O'Riordan JI, Page R, Wang L, Barker GJ, Tofts PS, McDonald WI, Miller DH, and Thompson AJ. Spinal cord atrophy and disability in multiple sclerosis. Brain 119: 701-708, 1996.
Lovas G, Szilagyi N, Majtenyi K, Palkovits M, and Komoly S. Axonal changes in chronic demyelinated cervical spinal cord plaques. Brain 123: 308-317, 2000.
Mclean MJ and MacDonald RL. Multiple actions of phenytoin on mouse spinal cord neurons in cell culture. J Pharmacol Exp Ther 227: 779-789, 1983.
Narahashi T. Drugs acting on calcium channels. Handbk Exp Phramacol 83: 255-274, 1988.
Pinus MR and Abraham NZ Jr. Toxicology and therapeutic drug monitoring (18th ed.). In: Clinical Diaganosis and Management by Laboratory Methods, edited by Henry JB. Phildelphia, PA: Saunders, 1991, p. 349-384.
Ragsdale DS and Avoli M. Sodium channels as molecular targets for anti-epileptic drugs. Brain Res Rev 26: 16-28, 1998.[Medline]
Ragsdale DS, McPhee JC, Scheuer T, and Catterall WA. Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels. Proc Natl Acad Sci USA 93: 9270-9275, 1996.
Raine CS and Cross AH. Axonal dystrophy as a consequence of long-term demyelination. Lab Invest 60: 714-725, 1989.[ISI][Medline]
Rosenberg LJ, Teng YD, and Wrathall JR. Effects of the sodium channel blocker tetrodotoxin on acute white matter pathology after experimental contusive spinal cord injury. J Neurosci 19: 6122-6133, 1999.
Segal MM and Douglas AF. Late sodium channel openings underlying epileptiform activity are preferentially diminished by the anticonvulsant phenytoin. J Neurophysiol 77: 3021-3034, 1997.
Sloan KE and Stevenson JA. Differential distribution of phosphorylated and non-phosphorylated neurofilaments within the retina and optic nerve of hamsters. Brain Res 437: 365-368, 1987.[ISI][Medline]
Smith KJ, Kappor R, and Felts PA. Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathol 9: 69-92, 1999.[ISI][Medline]
Stys PK, Ransom BR, and Waxman SG. Tertiary and quaternary local anesthetics protect CNS white matter from anoxic injury at concentrations that do not block excitability. J Neurophysiol 67: 236-240, 1992b.
Stys PK, Sontheimer H, Ransom BR, and Waxman SG. Noninactivating, tetrodotoxin-sensitive Na+ conductance in rat optic nerve axons. Proc Natl Acad Sci USA 90: 6976-6980, 1993.
Stys PK, Waxman SG, and Ransom BR. Ionic mechanisms of anoxic injury in mammalian CNS white matter: role of Na+ channels and Na+-Ca2+ exchanger. J Neurosci 12: 430-439, 1992a.[Abstract]
Teng YD and Wrathall JR. Local blockade of sodium channels by tetrodotoxin ameliorates tissue loss and long-term functional deficits resulting from experimental spinal cord injury. J Neurosci 17: 4359-4366, 1997.
Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, and Bo L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med 338: 278-285, 1998.
White SR, Black PC, Samathanam GK, and Paros KC. Prazosin suppresses development of axonal damage in rats inoculated for experimental allergic encephalomyelitis. J Neuroimmunol 39: 211-218, 1992.[ISI][Medline]
Wujek JR, Bjartmar C, Richer E, Ransohoff RM, Yu M, Tuohy VK, and Trapp BD. Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis. J Neuropathol Exp Neurol 61: 23-32, 2002.[ISI][Medline]
Yaari Y, Selzer ME, and Pincus JH. Phenytoin: mechanism of its anticonvulsive action. Ann Neurol 20: 171-184, 1986.[ISI][Medline]
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, regions from which neurofilament-labeled axons were quantified. B: neurofilament immunostaining of spinal cord axons in the dorsal corticospinal tract (CST), dorsal columns (cuneate fasciculus), lateral columns and ventral columns demonstrates a significant reduction of axons in the CST and dorsal columns in untreated experimental allergic encephalomyelitis (EAE) compared with the same regions in controls. Scale bar = 5 µm.
indicates stimulus artifact). The typical biphasic wave seen in controls is highly attenuated in untreated EAE, but is restored in phenytoin-treated EAE. C: average CAP amplitudes (±SE, mV) in phenytoin-treated controls, untreated EAE, and phenytoin-treated EAE at different stimulating current intensities (*, P < 0.01, phenytoin-treated EAE compared with untreated EAE). D: average CAP area (±SE, mV x ms) at different stimulus intensities in phenytoin-treated control, untreated EAE, and phenytoin-treated EAE (*, P < 0.05, phenytoin-treated EAE compared with untreated EAE). E: average supramaximal CAP area (±SE, mV x ms) in phenytoin-treated control, untreated EAE, and phenytoin-treated EAE (*, P < 0.05 phenytoin-treated EAE compared with untreated EAE). F: average conduction velocity (±SE, m/s) in phenytoin-treated control, untreated EAE, and phenytoin-treated EAE (*, P < 0.05, phenytoin-treated EAE compared with untreated EAE).
) and for phenytointreated EAE (
). Oral adminstration of phenytoin was started on day 10, as indicated by the horizontal bar.