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Axonal protection using flecainide in experimental autoimmune encephalomyelitis.

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Axonal Protection Using
Flecainide in Experimental
Autoimmune Encephalomyelitis
David A. Bechtold, MSc,1 Raju Kapoor, MD, PhD,2 and Kenneth J. Smith, PhD1
Axonal degeneration is a major cause of permanent neurological deficit in multiple sclerosis (MS), but no current
therapies for the disease are known to be effective at axonal protection. Here, we examine the ability of a sodium
channel–blocking agent, flecainide, to reduce axonal degeneration in an experimental model of MS, chronic relapsing
experimental autoimmune encephalomyelitis (CR-EAE). Rats with CR-EAE were treated with flecainide or vehicle from
either 3 days before or 7 days after inoculation (dpi) until termination of the experiment at 28 to 30 dpi. Morphometric
examination of neurofilament-labeled axons in the spinal cord of CR-EAE animals showed that both flecainide treatment
regimens resulted in significantly higher numbers of axons surviving the disease (83 and 98% of normal) compared with
controls (62% of normal). These findings indicate that flecainide and similar agents may provide a novel therapy aimed
at axonal protection in MS and other neuroinflammatory disorders.
Ann Neurol 2004;55:607– 616
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), which
typically displays a relapsing course characterized by
episodes of neurological disability followed by periods
of partial or complete clinical remission.1 Many patients later enter a progressive phase characterized by a
steady decline in neurological function,1 and recent evidence strongly suggests that axonal degeneration underlies the development of such permanent disability.2– 4 Moreover, neurological disability in patients
with MS has been correlated with the magnitude of
degeneration of spinal cord axons, and the magnitude
of brain and spinal cord atrophy as demonstrated by
pathological and magnetic resonance imaging studies.3,4 Axonal degeneration is evident even in the early
stages of the disease,5–7 and it can reduce axonal numbers to only 14% of normal in spinal cord lesions from
patients with long-standing disease.8
The mechanisms that underlie the axonal degeneration are not known, but postmortem and biopsy studies
of MS lesions suggest that axon loss is correlated with
the magnitude of inflammation.5,6 We have hypothesized that inflammatory mediators such as nitric oxide
(NO) may initiate a cascade of intraaxonal Na⫹ and
Ca2⫹ accumulation, which ultimately leads to axonal
degeneration,9 –11 and that sodium channel blockade
may attenuate such a cascade. In support of this hypothesis, we have demonstrated that exposure of electrically
active axons to NO can result in their degeneration12
and that this degeneration can be prevented with flecainide.13 In addition, Lo and colleagues recently have
shown that the sodium channel–blocking agent phenytoin can reduce axonal loss in a progressive form of experimental autoimmune encephalomyelitis (EAE).14,15
These findings suggest that sodium channel blockade
may provide a novel avenue to achieve axonal protection
in neuroinflammatory diseases, including MS. Toward
that goal, this study examined whether flecainide can reduce axonal degeneration in chronic relapsing (CR)–
EAE, an animal model of MS. The findings demonstrate
that flecainide administration provides significant protection against axonal degeneration, irrespective of
whether flecainide administration is initiated before inoculation, or close to the onset of neurological deficit.
From the 1Department of Neuroimmunology, King’s College; and
National Hospital for Neurology and Neurosurgery, London,
United Kingdom.
Address correspondence to Dr Smith, Department of Neuroimmunology, 2nd Floor Hodgkin Building, Guy’s Campus, King’s College, London, United Kingdom.
Materials and Methods
Induction of Experimental Autoimmune
Encephalomyelitis and Flecainide Therapy
Male dark agouti (DA) rats (150 –200gm; Harlan, Bicester,
UK) were inoculated using a subcutaneous injection of syn-
Received Sep 2, 2003, and in revised form Dec 22. Accepted for
publication Dec 22, 2003.
Published online Mar 21, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20045
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
geneic spinal cord homogenate (100mg) and complete
Freund’s adjuvant (100␮l; Sigma, Dorset, UK) at the base of
the tail. Animals were weighed and assessed daily for the extent of neurological deficit on a 15-point scale, receiving 1
point for each of the following signs: 5% weight loss over 2
days, piloerection, loss of tail-tip muscle tone, loss of total
tail muscle tone, tail paralysis, decreased toe spread, unsteady
gait, 1 point/hind limb dragged, 1 point/limb paralyzed,
moribund, death.
The effect of flecainide administration on CR-EAE was
examined over three individual trials (total n ⫽ 160) as summarized in the Table. For each trial, animals were randomly
assigned to receive either flecainide acetate (Tambocor injection; 3M, Loughborough, UK; in 2.5% glucose containing
20mM HEPES, pH 7.4, at 30mg/kg/day) or vehicle. Drug
was administered by subcutaneous injection twice a day from
either 7 days after inoculation (dpi) or 3 days before inoculation (⫺3 dpi), until the end of the experiment (28 –
30 dpi). During trial 2, rats designated to receive flecainide
from 7 dpi were given vehicle from 3 days before inoculation
and switched to flecainide at 7 dpi. An observer blinded to
the treatment groups performed all drug dosing, and the assessment of neurological deficit. All experiments detailed
herein have been approved by the local ethics committee and
have been licensed under the Animals (Scientific Procedures)
Act of the UK Home Office.
blocking agent to remove muscle action potentials from the
Neurofilament Immunohistochemistry
After the electrophysiological examination, the vasculature
was rinsed by transcardiac perfusion with 0.9% saline (containing 10mM HEPES, 0.05% lignocaine, 2,000U/L heparin, 0.002% NaNO2) followed by 4% paraformaldehyde in
0.1M phosphate-buffered saline (PBS). Spinal cords were removed and postfixed overnight in paraformaldehyde at 4°C.
Spinal cords then were rinsed, equilibrated with 30% sucrose
(in PBS), embedded in OCT (RA LAMB, Eastbourne, UK),
and stored at ⫺80°C until use. Transverse frozen sections
(30␮m) were collected, blocked in 5% horse serum in
PBS-T buffer (0.1M PBS, pH 7.4, 0.2% Triton X-100,
0.1% bovine serum albumin) for 1 hour, and incubated
overnight with neurofilament-160 (NF-160)–specific antibody (1:2,500; Sigma). After washing, sections were incubated in biotinylated anti–mouse IgG diluted in blocking
buffer (1:400) and processed using the Vectastain Elite ABC
kit (Vector, Peterborough, UK). Immunoreaction was visualized using 3,3⬘-diaminobenzidine (Vector). Omission of either the primary or secondary antibody resulted in no deposition of reaction product.
Analysis of Axon Number
Electrophysiological Examination
At the termination of the trials (28 –30 dpi), the animals
were anesthetised (2% halothane in oxygen) and examined
electrophysiologically. An assessment of axonal conduction in
the sacrococcygeal tract was obtained by measuring the area
of the compound action potential (CAP) recorded from the
base of the tail in response to supramaximal stimulation
(80 V; 0.02-millisecond duration) of the dorsal column at
the T9/T10 and L6/S1 vertebral junctions. In some experiments, suxamethonium was administered as a neuromuscular
Assessment of axonal degeneration in CR-EAE rats was performed on the medial dorsal column at the L3-4 and T10
levels of the spinal cord using NF-160 –labeled spinal cord
sections (n ⫽ 70). The region to be studied was reconstructed using digital photographs taken at high magnification and the counting area was defined by triangulating from
the dorsal borders of the fasciculus gracilis to the base of the
dorsal column (see Fig 4C). This counting area was selected
because the triangular shape can adjust for tissue edema
and/or atrophy. Immunopositive axons within this area were
Table. Neurological Deficit Scores of CR-EAE Rats
Flecainide 7 dpi
Peak deficit score
Trial 1
Trial 2b
Trial 3
Weighted mean
Terminal deficit score
Trial 1
Trial 2b
Trial 3
Weighted mean
Vehicle 7 dpi
Mean (⫾SD)
Mean (⫾SD)
6.3 (⫾3.9)a
7.9 (⫾2.7)a
5.8 (⫾2.7)a
6.7 (⫾3.2)
8.8 (⫾3.7)
8.4 (⫾3.1)
8.7 (⫾3.4)
0.4 (⫾1.1)c
1.4 (⫾3.4)c
0.7 (⫾1.4)c
0.82 (⫾2.3)
2.3 (⫾2.8)
3.8 (⫾4.8)
2.7 (⫾3.7)
Flecainide ⫺3 dpi
Vehicle ⫺3 dpi
Mean (⫾SD)
Mean (⫾SD)
9.8 (⫾1.5)
8.2 (⫾3.0)
9.1 (⫾2.3)
11.6 (⫾2.7)
10.4 (⫾4.2)
11.1 (⫾3.4)
1.4 (⫾3.3)c
1.5 (⫾4.1)c
1.4 (⫾3.6)
6.7 (⫾6.2)
5.9 (⫾6.4)
6.3 (⫾6.2)
Peak Deficit Score is the maximum deficit score received at any time during the trial (1–28 dpi) with lethal EAE as 15. Terminal Deficit Score
is the deficit score received on the final day of the trial with lethal EAE recorded as 15. Trials 1: Mann–Whitney U test; Trials 2, 3: One-way
ANOVA with Dunn’s post hoc test.
Statistically significant (p ⬍ 0.05) reduction of mean deficit scores compared with matched vehicle-treated CR-EAE rats
Flecainide (7 dpi) animals were treated with vehicle from ⫺3 dpi then switched to flecainide at 7 dpi
Statistically significant (p ⬍ 0.01) reduction of mean deficit scores compared with matched vehicle-treated CR-EAE rats
CR-EAE ⫽ chronic relapsing experimental autoimmune encephalomyelitis; dpi ⫽ days post-innoculation; SD ⫽ standard deviation.
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marked and counted using SigmaScan digital analysis software. The number of axons in the CR-EAE rats have been
expressed as a percentage of the number counted in the same
site in normal DA rats (n ⫽ 7). This calculation allowed the
results from the two levels of the spinal cord to be combined.
Myelin Histochemistry
Transverse frozen sections (16␮m) of the spinal cord were
collected and dried onto electrostatic slides for 2 hours. Sections then were washed in cold acetone for 10 minutes, hydrated in ddH2O, and stained with eriochrome cyanine R
for 30 minutes. Sections were rinsed for 30 seconds in running tap water and differentiated in aqueous ammonium ferric sulphate (10% wt/vol). Stained sections were washed for
5 minutes, dehydrated, cleared, and coverslipped. The areas
of myelin loss in the dorsal column were measured and divided by the total dorsal column area to determine the percentage of myelin loss per section. Values presented reflect
mean demyelination measured on four spinal cord sections
per animal, each separated by greater than 5mm.
Analysis of variance (ANOVA) was used to compare data
involving deficit scoring, electrophysiology, axonal degeneration, and myelin loss. When multiple comparisons were
made using ANOVA, a post hoc Dunn’s test was used. No
assumptions of parametric distribution were made. Fisher’s
exact test was used to compare mortality rates and Spearman’s rank correlation test was used to examine the correlation between myelin loss and axonal degeneration.
Reduction of Neurological Disability
The EAE model used in this study was chosen because
it exhibits similar pathology to that seen in MS, including demyelination and axonal degeneration.16 As
shown by the vehicle-treated CR-EAE animals (Fig 1),
DA rats immunized with syngeneic spinal cord and
complete Freund’s adjuvant demonstrated a chronicrelapsing disease course in which an initial attack was
typically followed by a relapse, separated by a period of
incomplete recovery of neurological function. The effects of flecainide on the outcome of CR-EAE were
examined in three independent trials (total n ⫽ 160),
two of which included two different timings for the
initiation of drug administration, namely, 7 dpi, a time
at which symptoms of the disease began to appear, or 3
days before inoculation (⫺3 dpi; summarized in the
Table). CR-EAE rats treated with flecainide from 7 dpi
or ⫺3 dpi exhibited a modest reduction in the severity
of disease symptoms compared with rats treated with
vehicle (see Fig 1A, B, respectively). The beneficial effect of flecainide on neurological deficit was most evident during the relapse (17–20 dpi) and persistent
(⬎23 dpi) phases of the disease, the stage at which axonal degeneration is likely to contribute to neurological
For statistical purposes, the effect of flecainide ad-
Fig 1. Disease progression in chronic relapsing experimental
autoimmune encephalomyelitis (CR-EAE) rats. (A) Graph
showing that the progression of CR-EAE in DA rats followed
a relapsing disease course, the severity of which was reduced by
flecainide treatment initiated at 7 dpi. (B) A similar reduction in disease severity was observed when flecainide treatment
was initiated 3 days before inoculation. (solid lines) Neurological deficit scores with animals that died during the trials
receiving a score of 15 for that day and subsequently removed
from the plot; (dashed lines) disease course when these animals were maintained at a score of 15 from the time of death
until the termination of the trial. (C) Histogram comparing
the mean peak deficit scores of the different groups of CR-EAE
rats. In comparison with vehicle-treated CR-EAE rats, the
mean peak neurological deficit scores were reduced in rats
treated with flecainide from either 7 or ⫺3 dpi. (D) Histogram comparing the mean terminal deficit scores of the CREAE rats. A significantly higher terminal deficit was evident
in CR-EAE rats treated with vehicle compared with animals
treated with flecainide. Error bars ⫽ 95% confidence interval
(A, B), standard deviation (C, D). *p ⬍ 0.05, ***p ⬍
Bechtold et al: Axonal Protection in EAE
ministration on the severity of neurological deficit in
CR-EAE was assessed using two criteria: mean peak
deficit (see Fig 1C) and mean terminal deficit scores
(see Fig 1D). These scores represented the maximum
deficit score received at any time during the trial, and
the final deficit score recorded for each animal, respectively. CR-EAE animals treated with vehicle from 7
dpi demonstrated a mean peak deficit score of 8.7 ⫾
3.4 (indicative of tail and/or hind limb paralysis). Flecainide treatment from 7 dpi resulted in a significant
reduction in the mean peak deficit score (6.7 ⫾ 3.2,
p ⬍ 0.05). A similar effect was observed in CR-EAE
rats treated from ⫺3 dpi (vehicle: 11.1 ⫾ 3.4, flecainide: 9.1 ⫾ 2.3), and it is of interest that both of the
flecainide treatment regimens lowered the peak deficit
scores by the same amount (approximately two points)
relative to vehicle-treated animals.
CR-EAE rats treated with vehicle from 7 dpi or ⫺3
dpi exhibited continuing neurological disability (typically persistent weakness of hind limbs and/or tail) at
the end of the trials, resulting in mean terminal deficit
scores of 2.7 ⫾ 3.7 and 6.3 ⫾ 6.2, respectively (see Fig
1D). The high terminal score observed in control animals treated from ⫺3 dpi with vehicle reflected the
high mortality rate (29.4%) among these unprotected
rats. In contrast, both flecainide treatment regimens
significantly reduced the terminal deficit scores of CREAE rats (flecainide 7 dpi: 0.8 ⫾ 2.3, p ⬍ 0.001, flecainide ⫺3 dpi: 1.4 ⫾ 3.6, p ⬍ 0.001) when compared with the vehicle-treated groups. The reduction of
terminal disability in CR-EAE rats treated with flecainide suggests that these animals have less persistent damage to the CNS, as would be expected from a reduction in axonal degeneration.17
A difference in peak scores was observed between the
CR-EAE rats treated with flecainide from 7 and ⫺3
dpi ( p ⬍ 0.05). A similar difference in peak deficit
score was observed between the two vehicle regimens
(7 and ⫺3 dpi) in the CR-EAE rats (see Fig 1C; p ⬍
0.05), suggesting that the onset of the dosing during
disease development (7 dpi) affected the course of the
disease. This may be because of stress experienced by
the rats during the first few days of the dosing, caused
by the extra handling. In an attempt to avoid this effect, CR-EAE animals designated to receive flecainide
from 7 dpi in the second trial were given vehicle from
⫺3 dpi, and then switched to flecainide at 7 dpi. The
peak deficit score of these rats were slightly higher than
those recorded for CR-EAE rats treated with flecainide
from 7 dpi during the first and third trials (see Table);
however, no significant difference was observed between the three trials.
Importantly, flecainide treatment significantly lowered the incidence of animal death as a result of CREAE from 20.3% in vehicle-treated groups to 3.4% in
flecainide-treated groups ( p ⬍ 0.01).
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Preservation of Axonal Conduction
At the termination of the trials, electrophysiological recordings were made to obtain a measure of axonal conduction between the thoracic spinal cord and the base
of the tail in normal and CR-EAE rats. Stimulation
sites at the T9/T10 and L6/S1 vertebral junctions were
used to assess CNS and peripheral nervous system
(PNS) conduction pathways, respectively (Fig 2A, B).
In normal DA rats, supramaximal stimulation of the
thoracic spinal cord evoked monophasic compound action potentials (CAPs) with an area of 50 ⫾ 30␮V 䡠
msec at the base of the tail (see Fig 2C). The area was
significantly reduced in CR-EAE rats treated with vehicle from 7 dpi (20 ⫾ 20␮V 䡠 msec, p ⬍ 0.01), indicating a considerable reduction in the number of
functional axons in these animals. CR-EAE rats treated
with flecainide from 7 dpi exhibited a significantly
greater mean CAP area after CNS stimulation than rats
treated with vehicle (flecainide: 37 ⫾ 27␮V 䡠 msec,
p ⬍ 0.05). The mean CAP area was also greater in
CR-EAE rats treated with flecainide from ⫺3 dpi compared with that observed in rats treated with vehicle
from ⫺3 dpi (flecainide: 28 ⫾ 20, vehicle: 21 ⫾ 22).
In comparison with normal DA rats, CR-EAE rats exhibited only a minor reduction in the area of the CAP
when the conduction pathway was limited to the PNS
(see Fig 2D), in accord with the observation that CREAE primarily affects the CNS in this model. Flecainide administration did not affect the CAP area evoked
following CNS or PNS stimulation in normal animals
(see Fig 2C, D).
Axonal Protection
At the termination of the trials, the extent of axonal
degeneration was determined in the medial dorsal columns of the spinal cord. Immunohistochemistry for
neurofilament (NF-160) resulted in the clear identification of axons within the spinal cord (Fig 3). Spinal
cord sections collected from CR-EAE rats were
marked by areas of axon loss (absence of NF-160
staining) in the dorsal, ventral, and lateral columns of
white matter. Quantification of axonal number was
restricted to an area located in the medial portion of
the dorsal column, approximately the fasciculus gracilis, because the axonal degeneration was most consistently exhibited in this area. Pronounced axonal loss
and evidence of ongoing axonal degeneration was often observed in the spinal cords of CR-EAE rats
treated with vehicle (see Fig 3A, B), but such axonal
pathology was greatly reduced in animals treated with
flecainide (see Fig 3C–E).
Interestingly, when the magnitude of axonal loss in
the fasciculus gracilis of vehicle-treated animals was
compared with their peak deficit score (Fig 4A), it was
clear that substantial axonal degeneration was restricted
to animals that had a peak deficit score above 8, the
Fig 2. Electrophysiological assessment of
chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) rats 28
to 30 dpi. (A, B) Representative records
from normal DA rats and CR-EAE rats
treated with vehicle or flecainide from 7
dpi, after central nervous system (CNS)
(A) and peripheral nervous system (PNS)
(B) stimulation. (C) The area of the CAP
recorded from rats with CR-EAE was significantly reduced from normal, but this
reduction was largely prevented by flecainide treatment. This protection was statistically significant with therapy from 7 dpi.
(D) As expected (because CR-EAE primarily affects central axons), mean CAP areas
recorded after PNS stimulation showed no
significant difference between normal and
CR-EAE rats. (dashed line) CAP area
recorded in normal DA rats; error bars ⫽
standard deviation. *p ⬍ 0.05, **p ⬍
score at which the disease was sufficiently severe to
cause temporary paralysis. Examination of the mean
number of surviving axons (relative to normal animals)
in the spinal cord of CR-EAE rats showed that vehicletreated CR-EAE animals that had suffered mild disease
(peak score ⬍8) retained a normal number of axons in
the counting area (see Fig 4B), whereas those rats that
had exhibited severe disease during the trial (peak score
ⱖ8) showed approximately a 40% reduction in axon
number (vehicle: 62% ⫾ 29% of normal). Treatment
of severely diseased CR-EAE rats with flecainide from
either 7 dpi or ⫺3 dpi, provided near complete axonal
protection, such that these animals had 98% (⫾ 20%)
and 83% (⫾ 14%) of the normal number of axons,
respectively. Importantly, flecainide administration
provided significant axonal protection in CR-EAE
whether initiated before inoculation (⫺3 dpi, p ⬍
0.05) or close to disease onset (7 dpi, p ⬍ 0.01).
Myelin Loss
The extent of myelin loss was assessed in CR-EAE rats
to examine whether flecainide treatment might affect
CR-EAE disease pathology outside of axonal degeneration. The degree of myelin loss was measured in the
dorsal column of CR-EAE rats (n ⫽ 58) at the termination of the trials (28 –30 dpi). Large areas of myelin
loss (arrows in Fig 5) were evident in most CR-EAE
animals treated with vehicle, with the fasciculus gracilis
being most severely affected (see Fig 5A). Myelin loss
was significantly reduced in CR-EAE rats treated with
flecainide from either 7 dpi or ⫺3 dpi (vehicle: 24 ⫾
14% loss; flecainide 7 dpi: 11 ⫾ 4%, p ⬍ 0.05; flecainide ⫺3 dpi: 13 ⫾ 9%, p ⬍ 0.05; see Fig 5B–D).
The loss of myelin along the dorsal column of the CREAE rats correlated strongly with the extent of axonal
degeneration (see Fig 5E, r2 ⫽ 0.81, p ⬍ 0.001); however, it was not possible to determine whether the myelin loss observed 28 to 30 dpi resulted from primary
demyelination or occurred secondary to axonal degeneration.
The findings show that a sodium channel blocking
agent, flecainide, provides significant protection against
axonal degeneration in an experimental model of MS,
CR-EAE. The effect of flecainide (30mg/kg/day) was
shown consistently over independent trials in which
drug administration reduced the severity of neurological disability and significantly increased the number of
surviving axons, irrespective of whether the drug was
given before inoculation (⫺3 dpi) or near the onset of
disease expression (7 dpi). By the termination of the
trials, CR-EAE rats that had been treated with flecainide exhibited little persistent disability (despite the
presence of the sodium channel–blocking agent),
whereas those animals treated with vehicle often were
Bechtold et al: Axonal Protection in EAE
Fig 3. Neurofilament immunoreactivity in the dorsal columns of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) rats. The dorsal column sections immunolabeled for neurofilament-160 shown are typical of the thoracic spinal cord of CREAE rats treated with vehicle (A, B), flecainide from 7 dpi (C, D), and flecainide from ⫺3 dpi (E, F). A substantial loss of dorsal
column axons was evident in the CR-EAE rats treated with vehicle (B). This loss was greatly reduced in CR-EAE rats treated with
flecainide from either 7 or ⫺3 dpi, although some evidence of axonal degeneration was still observed (C, E, arrows). fg ⫽ area
selected for axonal counting, based on the location of the fasciculus gracilis. Arrows identify regions of pronounced axonal degeneration. Bar ⫽ 100␮m in A, C, and E; 10␮m in B, D, and F.
left with considerable deficit, suggesting that flecainide
treatment reduces irreversible damage to the CNS in
CR-EAE. To demonstrate that the protection of axons
by flecainide during CR-EAE was not caused simply by
a reduction in the disease severity, we determined the
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number of axons surviving the disease separately in rats
that had only mild deficit (peak score ⬍8) and those
that exhibited a severe neurological disability (peak
score ⱖ8). Flecainide therapy provided significant axonal protection. Indeed, the protection was most obvi-
Fig 4. Preservation of axons in the dorsal columns of
flecainide-treated chronic relapsing experimental autoimmune
encephalomyelitis (CR-EAE) rats. (A) Graph comparing the
peak deficit score received by vehicle-treated CR-EAE rats with
the percentage of surviving axons determined at the end of the
trial. The number of axons in the counting area of CR-EAE
rats is expressed as a percentage of the number counted in
normal DA rats (n ⫽ 7). A substantial loss of axons was observed only in those animals that had reached a peak deficit
score above 8, the score at which paralysis becomes evident.
(B) Histogram showing the number of axons in the counted
region of the dorsal column of CR-EAE rats (n ⫽ 70). A
decrease in axon number was not detected in CR-EAE rats
that exhibited only mild signs of neurological deficit. In contrast, vehicle-treated animals, which showed evidence of severe
neurological deficit during the trial, were found to have a
38% reduction in axon number in the counting area (62%
⫾ 29% of normal). Flecainide administration provided near
complete axonal protection in CR-EAE rats, such that these
animals had a similar number of axons as normal rats when
treatment was initiated 7 dpi (98% ⫾ 20% of normal) or
⫺3 dpi (83% ⫾ 14% of normal). Importantly, flecainide
treatment provided significant axonal protection whether initiated before inoculation (⫺3 dpi, *p ⬍ 0.05) or close to disease onset (7 dpi, **p ⬍ 0.01) in CR-EAE rats when compared with animals treated with vehicle. Error bars ⫽
standard deviation.
ous in the severely affected group. It is now well established that axonal degeneration is an underlying cause
of permanent disability in multiple sclerosis,2– 4 and
therefore the current findings suggest that flecainide
and similar agents might provide a therapy for axonal
protection and the prevention of permanent disability
in patients with MS.
Flecainide is a well-characterized sodium channel–
blocking agent18 –20 commonly used to treat cardiac arrhythmias.21 We propose that the partial blockade of
Na⫹ currents by flecainide averts a deleterious axoplasmic accumulation of Na⫹ at sites of inflammation,
which, in turn, prevents a damaging increase in intraaxonal calcium. This concept is supported by studies
involving the anoxic optic nerve, which demonstrate a
role for Na⫹ accumulation, and particularly noninactivating Na⫹ currents, in the injury of white matter axons.22,23 Such studies detail a cascade initiated by the
depletion of energy stores within the axon, in which
failure of the Na⫹/K⫹-ATPase allows axoplasmic sodium levels to increase sufficiently high to stimulate
reverse operation of the Na⫹/Ca2⫹ exchanger resulting
in calcium-mediated axonal degeneration.22 We have
proposed that axons exposed to inflammatory mediators such as NO may quickly become depleted of energy, because NO is known to impair mitochondrial
metabolism and limit ATP production.24 Indeed, exposure of optic nerve to the NO donor, PAPA NONOate, causes a rapid and profound reduction in ATP levels
and ultimately leads to axonal degeneration.25 NO is
produced within inflammatory MS lesions, and several
studies have suggested that it contributes to the axonal
degeneration observed in MS and EAE (reviewed in
Smith and Lassmann26). This hypothesis is supported by
the observation that the severity of axonal injury in MS
correlates with the degree of inflammation.5,6
Additional aspects of the pathophysiology of multiple sclerosis, such as altered sodium channel expression,
also may contribute to axonal degeneration in MS. Increased expression of sodium channels in demyelinated
segments of the axon can contribute to the restoration
of action potential conduction27,28 and remission of
clinical symptoms in MS patients (reviewed in Smith
and McDonald29). However, it may also drastically increase the sodium load experienced by these axons and,
as a result, increase the risk of degeneration. Furthermore, experimentally demyelinated axons demonstrate
atypical electrical impulse activity in vivo, including increased ectopic firing, mechanosensitivity, and impulse
bursting (reviewed in Smith and McDonald29), all of
which would be expected to amplify the entry of sodium ions into the axon. There is also evidence for an
altered expression pattern of sodium channels in MS
and EAE. Nav1.8 expression, normally limited to the
PNS in the adult nervous system, has been detected in
Purkinje cells in both EAE and MS30 and nodal
Nav1.6 shifts toward Nav1.2 in EAE.31
The protection of spinal cord axons in CR-EAE by
flecainide treatment extended to a preservation of axonal function, as demonstrated by the electrophysiolog-
Bechtold et al: Axonal Protection in EAE
Fig. 5. Myelin loss in the dorsal columns of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) rats 28 to 30
dpi. Photomicrographs showing transverse sections of the dorsal columns of the spinal cord of animals with CR-EAE (n ⫽ 58)
stained for the presence of myelin. Myelin loss was evident in most CR-EAE animals treated with vehicle, with the fasciculus gracilis being most severely affected (A). Although, myelin loss also was observed in CR-EAE rats treated with flecainide from either 7
dpi or ⫺3 dpi (B and C, respectively), it was significantly reduced when compared with that measured in vehicle-treated rats (D,
one-way ANOVA with Dunn’s post hoc test). Myelin loss was strongly correlated with axon loss in the CR-EAE rats (E, r2 ⫽
0.81; p ⬍ 0.001, Spearman’s correlation test). Arrows indicate areas of myelin loss. Bar ⫽ 100␮m. Error bars ⫽ standard deviation.
ical examination of the CR-EAE rats at the termination
of the trials. Compared with normal DA rats, the CAP
area elicited from CNS stimulation was reduced by
more than 50% in CR-EAE animals treated with vehicle, demonstrating a significant impairment of impulse
conduction in these animals. The area of the CAP was
Annals of Neurology
Vol 55
No 5
May 2004
greater in the flecainide-treated animals compared with
controls, despite the fact that flecainide is a sodium
channel blocking agent and so would be expected to
diminish impulse activity. The concern that agents
such as flecainide may exacerbate neurological deficits
in MS may not be a problem in practice.
The observation that there was an apparent beneficial effect of delaying flecainide treatment, from ⫺3
dpi until 7 dpi, was unexpected. The possibility that
this effect was caused by a change in stress was explored, but we could not detect any associated increase
in serum corticosterone levels (data not shown). The
effect of delaying the treatment was reproducible but
not significant for the terminal deficit scores, CAP
area, or axonal protection.
The finding that flecainide administration reduced
the severity of neurological symptoms early in CR-EAE
(10 –13 dpi) was unexpected, and it raises the possibility that the drug may have some immunomodulatory
effects. It is possible, for example, that sodium channel
blockade may limit the severity of CR-EAE in DA rats
by altering an early stage in the development of EAE,
such as T-cell activation. A role for Na⫹ currents in
T-cell activation and costimulation has been suggested,32–34 and fast transient Na⫹ currents were obtained in several T-cell and B-cell lines (reviewed in
Gallin34). Furthermore, microglia are implicated in the
pathology of MS and EAE35 and voltage-gated Na⫹
channels have been described in rat36 and human37 microglia. It has been suggested that the activation of a
Na⫹ current might be necessary to trigger microglial
activation and facilitate a subsequent immune response.38
Apart from the current findings, the disruption of
Na⫹ homeostasis has been implicated as a key step in
axonal degeneration in several injury models including
anoxia,22,39 ischemia,22,40 axotomy,41 compression injury,42 and NO exposure.13,25 Based on these studies,
voltage-gated Na⫹ channels and the Na⫹/Ca2⫹ exchanger should be considered as targets for therapeutic
intervention in a wide range of CNS and PNS injury
models. This report demonstrates that sodium channel
blockade with flecainide can provide significant axonal
protection in a chronic-relapsing form of EAE. Together with the demonstration that phenytoin can protect axons in progressive EAE14,15 the current findings
suggest that flecainide and similar agents may offer
substantial therapeutic benefit in both relapsingremitting and progressive forms of MS.
This work was supported by the Multiple Sclerosis Society of Great
Britain and Northern Ireland (K.J.S.).
We thank M. Davies for expert technical help.
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