Axonal protection achieved in a model of multiple sclerosis using ...

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tained across the axolemma by the ATP-dependent. Na+/K+-ATPase (sodium pump). As a consequence, events that deplete axonal energy stores, such as.

J Neurol (2006) 253: 1542–1551 DOI 10.1007/s00415-006-0204-1

David A. Bechtold Sandra J. Miller Angela C. Dawson Yue Sun Raju Kapoor David Berry Kenneth J. Smith

Received: 24 November 2005 Received in revised form: 3 January 2006 Accepted: 9 January 2006

D.A. Bechtold Æ S.J. Miller A.C. Dawson Æ Y. Sun Æ R. Kapoor K.J. Smith (&) Dept. of Clinical Neuroscience King’s College London Guy’s Campus London, UK Tel.: 44 (0) 207 848 6121 Fax: 44 (0) 207 848 6123 E-Mail: [email protected] R. Kapoor National Hospital for Neurology and Neurosurgery London, UK D. Berry Medical Toxicology Unit Guy’s and St Thomas’ Trust London, UK

ORIGINAL COMMUNICATION

Axonal protection achieved in a model of multiple sclerosis using lamotrigine

j Abstract Axonal degeneration is

a major cause of permanent disability in multiple sclerosis (MS). Recent observations from our and other laboratories suggest that sodium accumulation within compromised axons is a key, early step in the degenerative process, and hence that limiting axonal sodium influx may represent a mechanism for axonal protection in MS. Here we assess whether lamotrigine, a sodium channel-blocking agent, is effective in preventing axonal degeneration in an animal model of MS, namely chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE). When administered from 7 days post-inoculation, lamotrigine provided a small but significant reduction in the neurological deficit present at the termination of the experiments (averaged over three independent experiments; vehicle:

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Introduction Axonal degeneration is a major cause of permanent disability in a range of neuroinflammatory disorders, including multiple sclerosis (MS). Histological studies of MS lesions have shown that axonal degeneration can be substantial [17, 18, 59], and that its magnitude correlates with disability [7, 8, 16, 20, 28, 38, 39, 55, 60]. The mechanisms that underlie axonal degeneration in MS remain unclear, but recent observations

3.5 ± 2.7; lamotrigine: 2.6 ± 2.0, P < 0.05) and preserved more functional axons in the spinal cord (measured as mean compound action potential area; vehicle: 31.7 lVms ± 23.0; lamotrigine: 42.9 ± 27.4, P < 0.05). Histological examination of the thoracic spinal cord (n = 71) revealed that lamotrigine treatment also provided significant protection against axonal degeneration (percentage degeneration in dorsal column; vehicle: 33.5 % ± 38.5; lamotrigine: 10.4 % ± 12.5, P < 0.01). The findings suggest that lamotrigine may provide a novel avenue for axonal protection in MS.

j Key words Axonal loss Æ degeneration Æ EAE Æ sodium channel

have indicated that an accumulation of intra-axonal sodium ions is a key and early stage in the degenerative process [4, 29, 37, 50]. A strong sodium ion gradient is normally maintained across the axolemma by the ATP-dependent Na+/K+-ATPase (sodium pump). As a consequence, events that deplete axonal energy stores, such as ischemia, allow sodium to accumulate within the axon. While sodium ions are not toxic per se, excessive sodium accumulation within axons can drive calcium entry via the reverse operation of the

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Na+/Ca2+ exchanger, resulting in calcium-mediated axonal degeneration [58]. Intra-axonal sodium accumulation has now been implicated in a number of injury models, including anoxia [36, 57], ischemia [26, 56], spinal cord injury [13, 63], and recently experimental autoimmune encephalomyelitis (EAE) [4, 37] and experimental autoimmune neuritis (EAN) [6], a model of Guillain-Barre´ syndrome. During MS, axons are likely vulnerable to energy depletion and subsequent sodium loading due to the disruption of mitochondrial function by inflammatory mediators, such as nitric oxide (NO) [5]. Abnormal impulse activity and altered sodium channel expression, both observed in MS patients, could also amplify axonal sodium accumulation (see below and [5]). We and others have recently shown that the sodium channel blocking agents flecainide and phenytoin can provide significant axonal protection in chronic-relapsing [4], and progressive [37] forms of EAE in the rat and mouse respectively, suggesting that sodium channel inhibition may provide a therapy for axonal protection in patients with MS. In preparation for a potential clinical trial, and bearing in mind the possible drawback in this respect of cardiac side effects of flecainide, we have assessed the therapeutic potential of lamotrigine.

Methods j Induction of eAE and drug therapy Male dark agouti (DA) rats (150–200 g, Harlan, Bicester, UK) were inoculated using a subcutaneous injection of syngeneic spinal cord homogenate (100 mg) and complete Freund’s adjuvant (100 ll; Sigma, Dorset, UK) at the base of the tail. Animals were weighed and assessed daily for the magnitude of neurological deficit on a 15 point scale, receiving 1 point for each of the following signs: 5% weight loss over two days, piloerection, loss of tail tip muscle tone, loss of total tail muscle tone, tail paralysis, decreased toe spread, unsteady gait, 1 point/hindlimb dragged (maximum 3 pts.), 1 point/ limb paralysed (maximum 3 pts.), moribund, death. In addition, the peak deficit score (the maximum score recorded for each animal at any time during the experiment, with dead animals recorded as 15) and terminal deficit score (deficit score on the final day of the experiment - rats which died during the experiment were not included) were also recorded for each animal. All the experiments were approved by the local ethics committee (Kings College London) and were licensed under the Animals (Scientific Procedures) Act of the UK. The effects of lamotrigine were examined in three independent CR-EAE experiments (total n = 140) as summarized in Table 1. The animals were randomly assigned to receive lamotrigine (Experiment 1: 20 mg/kg/day; Experiments 2–3: 30 mg/kg/day) or vehicle (0.5% methylcellulose in isotonic saline; pH 7.4; Sigma), and drug was administered by subcutaneous injection (Experiment 1) or gavage (Experiments 2–3) twice daily on a 12:12 hour schedule from 7 days post-injection (dpi) until the end of the experiment. The experiments were terminated 27–29 dpi.

Table 1 Deficit scores, electrophysiology and axonal degeneration Vehicle n

Lamotrigine mean (SD)

Peak deficit score Experiment 1 20 8.4 (3.9) Experiment 2a 25 10.3 (2.9) Experiment 3a 25 9.3 (3.5) Mean 70 9.4 (3.4) Terminal Deficit Score Experiment 1 18 1.3 (1.4) 22 4.5 (2.7) Experiment 2a a 23 4.3 (1.5) Experiment 3 Mean 63 3.5 (2.4) CAP Area (lV.msec) Experiment 1 15 44.4 (21.7) 18 29.4 (21.3) Experiment 2a Experiment 3a 23 25.3 (22.6) Mean 56 31.7 (23.0) Axonal degeneration (% of area) – animals with peak score > 8 only Experiment 1 9 46.5 (35.8) 13 55.4 (42.6) Experiment 2a a 14 4.8 (6.1) Experiment 3 Mean 36 33.5 (38.5) Peak Deficit Score is the maximum deficit score received at any time during the experiment with lethal EAE as 15 Terminal Deficit Score is the deficit score received on the final day of the experiment a = lamotrigine given at 30 mg/kg/day * = P < 0.05 versus vehicle-treated rats ** = P < 0.01 versus vehicle-treated rats SD = standard deviation

n

mean (SD)

20 25 25 70

7.9 (3.4) 10.9 (2.9) 8.2 (3.1) 9.1 (3.4)

19 19 24 62

0.5 (0.7) 3.1 (1.7) 3.8 (1.7) 2.6 (2.0)*

15 17 24 56

48.9 (25.1) 41.9 (17.3) 39.9 (34.2) 42.9 (27.4)*

8 15 12 35

16.9 (17.4) 11.8 (11.2) 4.3 (7.5) 10.4 (12.5)**

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j Electrophysiological examination At the termination of the experiments (27–29 dpi) the animals were anaesthetised (2% halothane in oxygen) and examined electrophysiologically. The skin of the back was incised and a stimulating cathode was inserted in the paravertebral muscle at the T9/T10 vertebral junction, with the anode positioned over the right shoulder blade. Active’ and indifferent’ recording electrodes were positioned percutaneously at the base of the tail and the tail tip, respectively. A ground electrode was inserted subcutaneously over the caudal end of the pelvis. Electrical stimuli (80 V, 20 lsec) were applied at 1Hz and averaged (n = 16) compound action potentials (CAPs) were recorded digitally. Each record was quantitatively analysed using our own purpose-written software, and CAP area used as a measure representative of the number of axons able to conduct from the stimulating electrode to the active recording electrode. Over the three experiments, 28 rats were not assessed electrophysiologically or histologically because the animals either showed only a very mild or absent neurological deficit (vehicle: n = 2; lamotrigine: n = 4), or were killed on ethical grounds due to severe expression of disease (vehicle: n = 12; lamotrigine: n = 10). j Histological examination Following the electrophysiological recording, the rats were perfused via cardiac puncture with rinse (0.9% saline containing 10 mM HEPES, 0.05% lignocaine, 2 U/ml heparin, 0.02% NaNO2), followed by 3.5% glutaraldehyde in 0.15 M phosphate buffer (PB). Spinal cords were excised and stored in the same fixative at 4 C. A 0.5 mm block of tissue was taken from the mid-thoracic (T9/T10) region of the spinal cords and embedded into TAAB resin (TAAB laboratories, UK) as previously described [46]. Following resin embedding, semi-thin sections (0.8 lm) were cut, collected onto slides, and stained with toluidine blue and pyronin Y. Previous work has demonstrated that axonal degeneration in the thoracic spinal cord tends to be aggregated in a region located superficially and medially within the dorsal column [4], and so our histological studies focused on this region. High-resolution images of the dorsal columns were obtained with a digital camera and the extent of axonal degeneration was quantified. First, the borders of the counting region (approximately the fasciculus gracilis) were established by triangulating from the base of the dorsal column to the visible margins of the tract on the superior surface of the dorsal column. Second, areas within this tract were comprised of degenerating axons were identified and measured using SigmaScan digital analysis software. Axonal loss was expressed as the percentage of the counting area, which exhibited a pronounced loss of axons. All of the stages of the experiment and analysis were performed blind to the treatment. j Lamotrigine concentration Serum was collected from each of the CR-EAE animals at the termination of experiment 3 and the concentration of lamotrigine determined by fully validated HPLC techniques. In brief, following the addition of an internal standard to small (200 ll) serum samples the pH was adjusted prior to extraction with solvent to remove the drug from the biomatrix. After evaporating the solvent to dryness the residue was reconstituted in HPLC mobile phase prior to injection onto a reversed phase column with monitoring of the eluent by UV (wavelength varies according to the drug). Quantification was by reference to calibrators that were spiked with lamotrigine across the range of values encountered during normal therapy. These calibrators were carried through the procedure with each batch of samples and every analytical run was subjected to internal quality control at three concentrations.

Fig. 1 The progression of neurological deficit in CR-EAE. The course and severity of CR-EAE was not substantially altered by treatment with lamotrigine (A). In panel A, rats which died during the experiment received a score of 15, and were subsequently removed from the histogram. No significant differences were detected between the mean peak neurological deficit scores of the CREAE rats (B), but lamotrigine did cause a significant reduction in neurological deficit at the termination of the experiments (C). Peak deficit = the maximum score received on the final day of the experiment, with dead animals recorded as 15. Terminal deficit = deficit score on the final day of the experiment. The mean terminal deficit scores shown in panel C are not identical to that shown in panel A due to the fact that the experiments were terminated over a 3 day period Statistics Differences measured between rats treated with lamotrigine and those treated with vehicle were tested for significance using MannWhitney U test (neurological deficit scoring) or an unpaired student’s t-test (electrophysiological and histological results).

Results j Neurological disability DA rats inoculated with spinal cord homogenate and complete Freund’s adjuvant followed a relapsing EAE disease course (Fig. 1), typically exhibiting two peaks in disease activity within the 28 day test period. Lamotrigine treatment did not appreciably alter the magnitude of peak disease activity, or the disease course (Fig. 1A, B). However, lamotrigine did reduce the magnitude of the deficit present at the end of the experiments by a small but significant margin, when compared with vehicle-treated rats (vehicle: 3.5 ± 2.4;

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lamotrigine: 2.6 ± 2.0, P < 0.05, Fig. 1C). The improvement observed in the deficit scores of rats treated with lamotrigine at the termination of the experiments suggests that persistent disability may be reduced in these animals. However, a longer observation period would be required to confirm this possibility. Increasing the dosage of lamotrigine from 20 to 30 mg/kg/day did not have a noticeable effect on neurological deficit scores. Drug treatment did not have significant effect on weight loss experienced by rats with CR-EAE (data not shown). Also, lamotrigine treatment did not significantly alter the incidence (vehicle: 11.1%; lamotrigine: 11.0%) or timing of animal mortality due to CR-EAE.

j Preservation of axonal conduction Terminal electrophysiological recordings were used as a measure of axonal conduction between the thoracic spinal cord and the base of the tail in normal rats and rats with CR-EAE (Fig. 2A). In normal rats, supramaximal stimulation of the thoracic spinal cord evoked monophasic compound action potentials (CAPs) with a mean area of 55.6 lVÆmsec ± 5.7 (Fig. 2B, dashed line). This area was substantially reduced in rats with CR-EAE treated with vehicle in each of the three experiments (mean CAP area: 30.8 lVÆmsec ± 22.2). However, a greater number of functional axons were present in rats with CR-EAE treated with lamotrigine, as evidenced by a significantly greater mean area of the CAP evoked upon CNS stimulation (lamotrigine: 42.9 ± 27.4 lVÆmsec; P < 0.05).

j Axonal protection High-resolution digital images of the dorsal columns allowed the clear identification of intact as well as degenerating axons. Areas exhibiting axonal degeneration were identified and measured. A pronounced loss of axons and evidence of ongoing axonal degeneration was often observed in the spinal cords of rats with CR-EAE treated with vehicle (Fig. 3A and C), but damage was reduced in corresponding spinal cord sections collected from rats with CR-EAE treated with lamotrigine (Fig. 3B and D). In the present study we introduced a new method for assessing the magnitude of axonal loss, namely tracing around, and then measuring the regions of the dorsal column that exhibited pronounced axonal degeneration. The adoption of this method reduced the time required to assess axonal loss, and its validity was tested by comparing such area of degeneration’ measurements with counts of the actual number of surviving axons using spinal cord sections collected

Fig. 2 Electrophysiological examination of axonal conduction. A: Representative records from normal rats, and rats with CR-EAE treated with vehicle or lamotrigine following thoracic spinal cord stimulation. Shading in the upper record denotes the area measured. B: The area of the CAP recorded from vehicle-treated rats with CR-EAE was significantly reduced from normal (dashed line). Lamotrigine treatment resulted in a significantly greater mean CAP area. Bars = standard error. * = P < 0.05

during previous EAE studies (work published in [4]). Axon number was generated by individually marking and counting all axons within the triangulated region of the dorsal column. Comparison of these two methods demonstrated that although area of degeneration’ slightly underestimated axonal loss (likely due to some diffuse loss of axons), the two measures were strongly correlated (r = )0.806; P < 0.001, spearman’s rank correlation; Fig. 4A). Area measurements were used for the three lamotrigine experiments presented here. We have previously shown that substantial axonal degeneration does not occur in DA rats with CR-EAE unless the disease is sufficiently severe (peak neuro-

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Fig. 3 Degeneration of dorsal column axons in rats with CR-EAE. Pronounced axonal degeneration was evident in the spinal cords of rats with CR-EAE, and the extent of axonal pathology in the dorsal column was quantified by measuring areas of severe degeneration (A, outline). Whereas axon loss and ongoing degeneration were often prominent in rats with CR-EAE treated with vehicle (A and C), axonal pathology was greatly reduced in rats treated with lamotrigine (B and D). Arrows indicate areas of degeneration. FG = area assessed for axonal loss, essentially the fasciculus gracilis. Bar = 100 lm in A-B, and 40 lm in C-D

logical deficit score of 8 or above)[4]. Such rats typically exhibit paralysis of the tail, and some loss of hind limb function. A similar association between axonal degeneration and peak deficit score was observed in the present study, with severe axonal degeneration occurring only in animals which achieved a peak deficit score above 8 (Fig. 4B). If the quantification of axonal degeneration in the current study was restricted to rats with CR-EAE that exhibited a severe level of disease (peak score ‡ 8), then lamotrigine administration provided significant axonal protection (degeneration as a percentage of the area examined: 10.4% ± 12.5), when compared with vehicle (33.5% ± 38.5, P < 0.01; Fig. 4C), resulting in a 68% reduction in the area exhibiting profound axonal loss. The protection provided by lamotrigine remained significant when comparisons were made using the 15 most severely affected animals per treatment per experiment (lamotrigine: 9.0% ± 12.9; vehicle: 31.1% ± 37.9; P < 0.01), or when all animals, regardless of deficit score were compared. This precludes the possibility that the significant finding in the severe group was misleading, due to the possibility

that lamotrigine may have exacerbated neurological scores by blocking conduction in axons conducting with low safety factor, thereby moving animals with pathologically mild or moderate disease into the severe category. The extent of axonal degeneration varied substantially between the three experiments; however the cause of this difference in disease severity is unknown.

j Lamotrigine concentration Serum collected at the termination of experiment 3 was assayed for lamotrigine content (Fig. 5). Animals sampled at approximately the mid-point of the dosing schedule (~6 hr post-dose), exhibited a mean lamotrigine concentration of 11.5 lg/ml. The accepted therapeutic range for lamotrigine, based on the treatment of patients with epilepsy, is 1–15 lg/ml. Lamotrigine decayed slowly over the 10 hr sampling period, suggesting that it was maintained at a therapeutically relevant concentration over the entire 12 hr dosing period.

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Fig. 5 Serum drug concentration. Graph showing the concentration of lamotrigine in the serum of rats with CR-EAE after 28 days of drug administration. Lamotrigine remained at a therapeutically relevant serum concentration (1–15 lg/ml) throughout the dosing period. Shaded area represents drug concentration considered therapeutically relevant in the treatment of patients with epilepsy

Fig. 4 Protection of axons in severely diseased rats by lamotrigine. A: Plot comparing total axon number with area of degeneration’ measurements, demonstrating the strong correlation between the two measures of axonal loss. B: Histogram demonstrating that substantial axonal degeneration is typically limited to animals which experience severe disease (peak score above 8). C: Histogram showing the magnitude of axonal degeneration in severely diseased rats with CR-EAE. Lamotrigine treatment significantly reduced axonal degeneration in rats with CR-EAE, when compared with vehicle treated rats. Bars = standard error. * = P < 0.05

Discussion Axonal degeneration is a major cause of permanent disability in MS [8, 39, 43], but there is no recognised therapy that is effective in axonal protection. The current findings show that lamotrigine can provide significant protection against axonal degeneration in CR-EAE, an experimental model of MS. Importantly,

the protection of spinal cord axons in CR-EAE by lamotrigine included the preservation of axonal function, as demonstrated by the electrophysiological examination. Specifically, rats with CR-EAE that were treated with lamotrigine exhibited a significantly larger mean area of the CAP in response to spinal cord stimulation than did rats with CR-EAE treated with vehicle. The improvement in axonal conduction occurred despite the fact that lamotrigine is a sodium channel blocking agent and so may be expected to impair conduction. The beneficial effects of lamotrigine were shown consistently in each of the three independent experiments. Lamotrigine was effective in reducing axonal degeneration even though administration of the drug was delayed until 7 dpi, a time at which animals begin to exhibit signs of neurological deficit. This finding suggests that the current findings are relevant to clinical application of the therapy, and indicate that lamotrigine may protect axons from degeneration in patients with MS. The mechanisms which cause axons to degenerate in MS have not been clearly identified, however studies over the past few years suggest that the intraaxonal accumulation of sodium ions may be a triggering event in the degenerative process [4, 29, 37, 50]. Lamotrigine blocks voltage-gated sodium channels in a use-dependent manner [31, 32, 34, 35], and we propose that the ability of lamotrigine to limit sodium influx protects axons that have been compromised by inflammation and/or demyelination. It is known that neurons and their axons are easily damaged by some inflammatory factors, such as NO [51], and pathological studies have correlated the severity of axonal loss with the magnitude of inflammation in MS lesions [19, 59]. We have previously suggested that NO, and possibly additional factors associated with the inflammatory response, can disrupt axonal energy production and initiate a

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cascade of intra-axonal sodium and calcium accumulation, ultimately resulting in axonal degeneration [5]. NO is known to impair mitochondrial metabolism and limit ATP production [10] and failure of the ATPdriven Na+/K+-pump [56] can allow axoplasmic sodium concentrations to rise sufficiently high that the Na+/Ca2+-exchanger operates in reverse, resulting in calcium-mediated axonal degeneration [22, 50]. This cascade has been implicated in axonal degeneration and white matter injury in anoxia [58, 62]. It is known that NO is produced within inflammatory MS lesions, and several studies have suggested that it contributes to the axonal degeneration observed in MS and EAE [51]. Our group and others have shown that compounds which block axonal sodium channels can attenuate this Na+)Ca2+ cascade, and protect axons against NO-mediated degeneration [22, 29]. Here we show that lamotrigine is effective in protecting axons from degeneration in vivo, during neuroinflammatory disease. Demyelinated axons may be particularly vulnerable to excessive sodium loading due to their mode of impulse conduction and their altered expression of sodium channels. Thus demyelinated axons can generate continuous and triggered ectopic discharges, and mechanosensitivity [52], both of which can greatly increase impulse activity, and hence sodium loading. Also, some neurons and axons exhibit abnormal sodium channel expression in EAE and MS. For example, Nav1.8 expression, normally limited to the PNS in the adult nervous system, has been detected in Purkinje cells in both EAE and MS [9], and increases in axolemmal Nav1.2 has been demonstrated in axons in both these diseases [14]. An altered sodium conductance resulting in an increase in the persistent sodium current can also precipitate injurious axonal sodium accumulation. It is known that persistent sodium currents can be increased during hypoxia [24, 27] and upon NO exposure [2, 25], and there is evidence for hypoxia-like conditions and NO production in MS lesions [1, 23, 51]. Furthermore, Nav1.6 has been shown to co-localise with the sodium-calcium exchanger along extended lengths of demyelinated axons in animals with EAE [13], as well as in MS tissue [15]; this combination may be pathophysiologically significant as Nav1.6 can produce a substantial persistent current [47]. Interestingly, persistent sodium currents are often particularly sensitive to the actions of sodium channel blocking agents. In addition to inhibiting sodium currents, there is evidence that lamotrigine can reduce the release of glutamate at central excitatory synapses [11] and block voltage-gated calcium channels [54, 61], both of which may contribute to the neuroprotective effects of the drug during CR-EAE. A role for glutamate-mediated toxicity in EAE, and thus possibly in MS, is

supported by the finding that specific AMPA-receptor antagonists can ameliorate the magnitude of neurological deficit and tissue damage in EAE [45, 53]. Despite reducing axonal degeneration, lamotrigine did not improve the scores for peak neurological deficit in the rats with CR-EAE. This observation may be partly due to the fact that the scoring criteria used to assess neurological deficit in the present study are weighted towards motor function, whereas the spinal cord tract used to quantify axonal loss primarily contains sensory axons. It is possible that motor axons remain unprotected in the lamotrigine-treated rats, but this seems unlikely since the terminal deficit scores are low. Alternatively, the lack of improvement in the peak deficit scores of rats treated with lamotrigine may arise because axonal degeneration is probably not the major cause of neurological disability at the peak of disease expression in animals affected by EAE. It has been shown that neurological disability in other models of EAE correlates more closely with the magnitude of spinal cord inflammation, rather than with axonal loss, at least in the early stages of the disease [64]. In line with this belief, it is worth noting that lamotrigine treatment reduced the terminal neurological deficit scores, even though this benefit was only achieved at the very end of the experiment. A experiment of longer duration would be required to confirm whether lamotrigine-treated rats would continue to improve. Nonetheless, the value of protecting axons from degeneration seems indisputable, and it is assumed that the beneficial effects of such protection would become most apparent in the long term, especially in patients with MS where it may delay or even prevent the development of permanent and severe disability. It is also possible that sodium channel blockade by lamotrigine may have contributed to the presence of neurological dysfunction by disrupting conduction in axons already compromised by inflammation and/or demyelination. A concern that the use of sodium channel blocking agents in patients with MS could exacerbate disability is raised by reports of an unmasking’ of clinically silent lesions in MS patients by the administration of lignocaine or mexiletine [48, 49]. The degree to which clinical doses of lamotrigine may suppress axonal conduction in EAE and MS is uncertain, although lamotrigine has been used in the treatment of positive symptoms and pain in MS patients without incurring significant impairment [40, 42, 44]. In addition, in the present study lamotrigine increased axonal conduction in rats affected by CREAE at the termination of the experiments when compared with animals treated with vehicle, indicating that if neurological function is compromised by lamotrigine, its beneficial action in preserving axonal function outweighs this effect.

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Recent work by our group and that of Stephen Waxman’s has demonstrated that sodium channel blocking agents can ameliorate spinal cord inflammation during EAE [3, 12]. Administration of flecainide to rats with CR-EAE reduced the number of activated microglia and macrophages in the spinal cord, as well as attenuating the expression of iNOS, the inducible form of nitric oxide synthase [3]. Similarly, Craner et al. have recently demonstrated a pronounced expression of sodium channel Nav1.6 in activated microglia and macrophages in EAE and MS, and have shown that phenytoin administration can reduce the spinal cord infiltrates in mice with EAE by 75% [12]. Furthermore, initial work in our laboratory has suggested that lamotrigine can inhibit T cell activation and proliferation. Specifically, therapeutically relevant doses of lamotrigine reduced lymph node cell proliferation in response to PHA [41]. A role for sodium currents in T cell activation and costimulation has been suggested [21, 30, 33]. These findings suggest that the improvements in neurolog-

ical function and increased axonal survival may be due to a reduction in disease severity. Therefore, it is possible that some anti-inflammatory actions of lamotrigine may play a role in the protection of axons during CR-EAE. However, it is difficult to assess the direct impact of lamotrigine treatment on spinal cord inflammation since histological examination was performed only at the termination of the experiments. The current findings demonstrate that lamotrigine can provide effective protection from axonal degeneration in a recognised animal model of MS, and support their use in clinical trials of neuroprotection in this disorder. j Acknowledgements We would like to thank Mr. Meirion Davies, Ms. Clare Farmer, Mr. Matthew Smith and Dr. Marija Sajic for technical assistance relating to this work. Lamotrigine was kindly provided by GlaxoSmithKline. The work was supported by grants from the Multiple Sclerosis Society of Great Britain and Northern Ireland, a PhD studentship (to DAB) from King’s College, and a bursary (to ACD) from the Health Foundation.

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