Axonal protection using flecainide in experimental autoimmune encephalomyelitis.код для вставкиСкачать
ORIGINAL ARTICLES 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 2 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 (www.interscience.wiley.com). DOI: 10.1002/ana.20045 © 2004 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services 607 geneic spinal cord homogenate (100mg) and complete Freund’s adjuvant (100l; 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 records. 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 (30m) 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 Treatment 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 n Mean (⫾SD) n Mean (⫾SD) 20 20 16 56 6.3 (⫾3.9)a 7.9 (⫾2.7)a 5.8 (⫾2.7)a 6.7 (⫾3.2) 20 8.8 (⫾3.7) 16 36 8.4 (⫾3.1) 8.7 (⫾3.4) 20 20 16 56 0.4 (⫾1.1)c 1.4 (⫾3.4)c 0.7 (⫾1.4)c 0.82 (⫾2.3) 20 2.3 (⫾2.8) 16 36 3.8 (⫾4.8) 2.7 (⫾3.7) Flecainide ⫺3 dpi Vehicle ⫺3 dpi n Mean (⫾SD) n Mean (⫾SD) 20 14 34 9.8 (⫾1.5) 8.2 (⫾3.0) 9.1 (⫾2.3) 20 14 34 11.6 (⫾2.7) 10.4 (⫾4.2) 11.1 (⫾3.4) 20 14 34 1.4 (⫾3.3)c 1.5 (⫾4.1)c 1.4 (⫾3.6) 20 14 34 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 a b c CR-EAE ⫽ chronic relapsing experimental autoimmune encephalomyelitis; dpi ⫽ days post-innoculation; SD ⫽ standard deviation. 608 Annals of Neurology Vol 55 No 5 May 2004 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 (16m) 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. Statistics 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. Results 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 disability.17 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 ⬍ 0.001. Bechtold et al: Axonal Protection in EAE 609 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). 610 Annals of Neurology Vol 55 No 5 May 2004 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 ⫾ 30V 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 ⫾ 20V 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 ⫾ 27V 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 ⬍ 0.01. 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. Discussion 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 611 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 ⫽ 100m in A, C, and E; 10m 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 612 Annals of Neurology Vol 55 No 5 May 2004 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 613 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 ⫽ 100m. 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 614 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 Conclusions 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. 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