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Memantine abrogates neurological deficits, but not CNS inflammation, in Lewis rat experimental autoimmune encephalomyelitis
JOURNAL
OF THE
NEUROLOGICAL SCIENCES
ELSEVIER
Journal
of the Neurological Sciences 137 (1996) 89-%
Memantine abrogates neurological deficits, but not CNS inflammation, in Lewis rat experimental autoimmune encephalomyelitis Erik Wallstriim a7* , Per Diener b, he Ljungdahl b, Mohsen Khademi a, Carl-Gustaf Nilsson ‘, Tomas Olsson a*d ’ Department of Medicine, Molecular Medicine Unit, Karolinskn b Department of Clinical Neuroscience and Family Medicine, Division ’ Clinical Research ’ Department of Neurology,
Institute. House Ll:OI. Karolinska Hospital, of Neurology, Karolinska Institute, Huddinge Sweden Center, Novum, Stockholm, Sweden Karolinrku Hospital, Stockholm, Sweden
171 76 Stockholm, Sweden University Hospital, Stockholm,
Received 19 June 1995; revised 16 October 1995; accepted 1 November 1995
Abstract Memantine, a clinically employed drug with iV-methyl-paspartate (NMDA) receptor antagonistic effects, dose-dependently ameliorates neurological deficits in Lewis rat experimental autoimmune encephalomyelitis (EAE). Interestingly, this therapeutic effect was not due to dampened CNS inflammation, as assessed by immunohistochemical evaluation of spinal cord tissue. Furthermore, numbers of interferon gamma (IFWy) mRNA expressing cells were not decreased, as assessed by in situ hybridization. Systemic immunity in terms of numbers of IPNy secreting cells in response to immunodominant myelin basic protein (MBP) peptides ex vivo was not reduced, and non-toxic doses of memantine did not affect lymphocyte proliferation or 1PN-y secretion in vitro. Considering these findings, we hypothesize that effector mechanisms responsible for reversible neurological deficits in EAE may involve NMDA receptors, and this highlights neurons as targets during autoimmune neuroinflammation. Memantiue; Experimental autoimmune encephalomyelitis (EAB); N-methyl+aspartate Interferon gamma (IFNy); Neurological deficit
Keywords:
1. Introduction In multiple sclerosis (MS), it is well known that there is often a poor correlation between clinical disease and degree of inflammation-demyelination, as assessed either by magnetic resonance imaging (MRI) (Jacobs et al., 1986) or
traditional histopathology (Gilbert and Sadler, 1983). Effector mechanisms responsible for reversible neurological deficits, without striking structural changes, are largely unknown. A number of inflammatory mediators have been implicated in this respect: cytokines such as tumor necrosis factor alpha (TNFa) and interferon gamma (IFNy) (Moreau et al., 1994), proteases (Opdenakker and van Damme, 1994) and nitric oxide (NO) (Chao et al., 1992). In this study we approached this question using Lewis rat experimental autoimmune encephalomyelitis (EAE) and memantine, a drug with potent N-methyl-o-aspartate Corresponding author. Tel.: + 46 8 72%246; Fax: + 46 8 72%248; E-mail: [email protected]. l
0022-510X/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0022-5 10X(95)00339-8
(NMDA)
receptor; Myelin basic protein (MBP);
(NMDA) receptor antagonistic effects (Bormann, 1989; Chen et al., 1992; Parsons et al., 1993). Memantine (l-amino-3,5-dimethyl-adamantane) is closely related to amantadine (1 -amino-adamantane), but memantine binds more efficiently to NMDA-receptors (Komhuber et al., 1991). NMDA-receptors have been associated with pathogenic events in a variety of neurological diseases; epilepsy, parkinsonism, motor neuron disease and ischemic stroke (Lipton and Rosenberg, 1993; Calne, 1994). Memantine is utilised as an antispastic and antiparkinsonistic agent (Rabey et al., 1992), and its use in experimental models have demonstrated protective effects against cerebral ischemia (Seif el Nasr et al., 1990) and quinolinic acid-induced hippocampal damage (Keilhoff and Wolf, 1992). Amantadine had significant positive effects in double-blind placebo-controlled studies on MS-associated fatigue (Rosenberg and Appenzeller, 1988; Cohen and Fisher, 1989). The aim of this study was to investigate possible effects of memantine on clinical signs of EAE (i.e. ascending paresis), and relate this to immunohistologi-
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cal findings in spinal cord and some key aspects of T-cell mediated myelin basic protein (MBP)-autoreactivity, especially cellular IFNy production.
2. Materials
and methods
2.1. Induction protocol
of EAE, clinical assessment
and treatment
Male Lewis rats (310-370 g and 200-260 g, Zentralinstitut fur Versuchtierzucht, Hannover, Germany) were immunized in both hind footpads with a total of 50 mg guinea pig spinal cord, 2 mg Mycobacterium tuberculosis (Strain H 37 RA, Difco laboratories, Detroit, USA), 100 ~1 Freund’s incomplete adjuvant (Sigma, St. Louis, USA) and 100 ~1 saline. Clinical disease severity was graded as follows: 0 = no illness, 1 = flaccid tail, 2 = moderate paraparesis, 3 = severe paraparesis, 4 = moribund state or death. Cumulative disease index, i.e. added scores for the whole observed period, was calculated for each rat and used for non-parametric statistics. Memantinehydrochloride (Akatinol Memantine, Merz + Co., Frankfurt/Main, Germany) at a low dose (10 mg/kg) or a high dose (20 mg/kg) was administered intraperitoneally (i.p.) on days 7, 9, I 1 and 13 post-immunization (p.i.). Control rats received 1 ml sterile saline at the same days pi. One group of animals were studied up to day 31 p.i. Another group of rats were sacrificed at day 13 or 14 p.i., for immunoassays as described below. Additional untreated rats were immunized and sacrificed at day 14-17 p.i., for studies of memantine effects on lymphoid cells in vitro. Animal experiments were approved by the local ethical committee. 2.2. Preparation
of lymph node mononuclear
cells (WC)
Popliteal and inguinal lymph nodes were dissected and crushed through sterile wire meshes. Cells obtained were washed twice and adjusted to 5 X lo6 viable LNC/ml in Dulbecco’s modified Eagles medium (Gibco, Paisley, UK) containing 1% (v/v) MEM Amino Acids solution (Gibco), 50 IU/ml respective 50 pg/ml Penicillin-Streptomycin (Gibco), 2 mM L-glutamine (Gibco) and 5% (v/v) fetal calf serum (Gibco). 2.3. Single cell assay for secretion of IFNy antigen
in response to
An immunospot assay that enables detection of single cells secreting IFNy (Czerkinsky et al., 1988), adapted for rat IFN-y (Mustafa et al., 1991) was employed. 96well nitrocellulose bottomed microtitre plates (Millipore, Bedford, USA) were coated overnight at 4°C with 100 ~1 (20 pg/ml) aliquots of monoclonal antibody directed against rat IFNy (DBl, kindly provided by Peter van der Meide, Biomedical Primate Research Centre, Rijswijk, The
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Netherlands). After washing, duplicate 200 ~1 aliquots LNC suspension (IO6 cells/well) were added, together with antigen or without antigen as background controls. The antigens used were the major encephalitogenic epitope in Lewis rat; guinea pig myelin basic protein (MBP) 63-88 peptide (Vandenbark et al., 1985) and the minor encephalitogenic epitope MBP 89-101 peptide (Offner et al., 1989) at final concentrations in medium of 10 pg/ml. MBP peptides were synthesized as described (Olsson et al., 1990). The lectin Concanavalin A (ConA; Sigma) at final concentration 5 pg/ml was used as positive control. After 24 h culture at 37°C and 7% CO, in humid atmosphere, cells were discarded and plates washed thoroughly. Secreted and bound IFNy was visualized by rabbit polyclonal anti-rat IFNy antibody (Van der Meide), biotinylated anti-rabbit IgG (Dakopatts, Denmark), avidin-biotin peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA) and carbazole staining, resulting in stained spots corresponding to single cells that had secreted IFNy. Spots were counted by use of a dissection microscope and standardized to numbers of spots per lo6 LNC originally applied in the well. The variation between wells within duplicates was less than 20%. 2.4. lmmunohistochemistty
and image analysis
Lumbar spinal cord was dissected and snap-frozen in liquid nitrogen. Cryostat sections (14 pm) were incubated with primary antibodies overnight at 4°C in a moist chamber. The following mouse monoclonal antibodies were used as primary antibodies: Ox 39 (anti-rat interleukin 2 (IL-2) receptor; Sedgwick et al., 1987), Ox 6 (anti-rat MHC class II; McMaster and Williams, 1979), Ox 18 (anti-rat MHC class I; Fukumoto et al., 1982), W3/25 (anti-rat CD4, T helper cells and macrophages; Williams et al., 1977) and Ox 8 (anti-rat CD8, T cytotoxic/supressor cells; Brideau et al., 1980). Ox 39 and Ox 18 were purchased from Seralab (Crawley Down, UK). Ox 6, W3/25 and Ox 8 were purified from culture supematants of hybridomas (Holmdahl et al., 1985). Sections were stained according to the avidin-biotin technique. Omission of the primary antibody served as negative control. Specificity of the staining was controlled on sections of peripheral lymphoid organs. Ox 39 stained cells were counted in whole spinal cord sections using X 20 magnification. Tissue areas were measured by image analysis (Sonata, Seescan, Cambridge, UK), and were typically 30 mm* per animal. Stained cells were standardized as number of cells per 100 mm2 tissue area. Ox 6 staining was measured as percentage of stained area in computer images covering most of the whole tissue area. One section per animal was analyzed. Threshold to detect positive staining was set to five standard deviations darker greyshade value than the non-stained background. Threshold was set individually for each section. The microscope lamp was switched on 30
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min before image analysis, for optimum stability. Ox 8, W3/25 and Ox 18 were semiquantitatively assessed by manual counting; - = 0, + = l-100, + + = 100-1000, + + + = > 1000 stained cells per 100 mm’ tissue area. 2.5. In situ hybridization
for IFNy
mRNA
Cryostat sections (14 pm> from the same part of the lumbar spinal cord as used for immunohistochemistry were thaw-mounted onto ProbeOn slides (ProbeOn, Fischer Scientific, Pittsburgh, USA). In situ hybridization was performed as previously described (Mustafa et al., 19931, with 35S-labelled synthetic oligonucleotide probes. Only cells with more than 14 autoradiographic grains over their cytoplasm were considered positive, and expressed as number of EN-y mRNA positive cells per 100 mm2 tissue area.
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2.6. Eflects proliferation
(19%)
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of memantine and secretion
on antigen induced of IFNy in vitro
lymphocyte
To assess lymphocyte proliferative responses triplicate aliquots of LNC (6 X lo5 cells per well) were cultured in round bottomed 96-well microtitre plates (Nunc, Nunclon, Denmark). Antigens were added as described above. Memantine was added at final concentrations in medium ranging 10m3 to lo-* M, or was omitted. After 60 h of incubation at 37°C 7% CO,, humid atmosphere, the cells were pulsed for 10 h with [3H]methyl-thymidine (final concentration 5 &i/ml; Amersham, Little Chalfront, UK). Cells were harvested and thymidine incorporation counted in a beta scintillation counter (Beckman). The same conditions were used to test effects of memantine on antigen induced IFNy secretion by immunospot (as above).
(a) 23 2-
2 ?! 8 3 1,5 .o .s 0 s 1
e! ii z 1,5.o 5 0 ls r” 0,5-
9 0,5
0
10
12
14
16
18
31
0
8
Days p.i. --e--s+
* -ew
Saline, n=8 Memantine 10 mg/kg, n=8 Memantine 20 mg/kg, n=7 (d)
10
10 12 Days p.i.
Saline, n=9 Memantine 10 mg/kg, n=9 Memantine 20 mg/kg, n=lO
on
2
$ 3 .” ‘O
-0 .E
6
z 33
A I-
0
8-
l 0
6-
0a
0
A00
Saline
Memantine
Memantine
10
20
mg/kg
mg/kg
14
2 .5
2
z 3
o-
mane
-
0
m 0 0 I Saline
0 I
non. 1
Memantine
Memantine
10
20
mg/kg
mgfkg
Fig. 1. (a and b) Mean clinical scores of EAE in rats treated with saline (controls), or 10 mg/kg respective 20 mg/kg memantine. Stars denote levels of significance comparing drug treated rats with control rats for the whole observed period, n.s., not significant (p > 0.05); * , p < 0.05; * * , p < 0.01; * * * , p < 0.001; n is number of rats. (c and d) Added clinical scores for the whole observed period (cumulative disease index). Each rat is represented by one tilled dot.
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2.7. Statistical evaluation
The non-parametric Mann-Whitney
U-test was used.
3. Results 3.1. Clinical course
The effects of memantine on clinical signs of EAE were similar in two different experimental sessions (mean clinical scores for groups in Fig. 1 a and b, individual cumulative disease index in Fig. 1 c and d). In the first experiment (Fig. 1 a and c) c1’mica1 ’ scores were followed to day 31 p.i. Treatment with the higher dose of memantine almost completely abolished neurological deficits. Among the rats treated with the low dose one heavily diseased rat died, which is reflected by a high mean clinical score day 1.5 p.i. Despite this, the rats treated with the low dose of memantine were less diseased than the controls ( p < 0.01). There were no late relapses. In the second experiment (Fig. 1 b and d), only the high dose of memantine resulted in a reduction of clinical scores that were statistically significant at the time of sacrifice. No apparent behavioral abnormalities were observed among the memantine treated rats. 3.2. Immunohistochemistry
Immunohistochemical evaluation revealed perivascular inflammation with infiltration of CD4+ and CD8+ T cells and upregulation of MHC Class I and II expressions both in saline- and memantine-treated rats. A thorough quantitative analysis of the number of IL-2 receptor positive cells (Fig. 2, Fig. 4a) and degree of MHC Class II expression (Fig. 3, Fig. 4b) revealed no differences between the groups. Semiquantitative assessment of MHC Class I expression, CD4+ and CD8+ cells also failed to demonstrate any major differences between controls and treatment groups (data not shown). 3.3. Antigen induced fFNy
Saline
IO mgikg
20 mg/kg
Memantine Fig. 2. IL-2 receptor expressing cells infiltrating lumbar spinal cord day 13-14 pi. Individual rats as tilled bars, mean values in groups + one standard deviation as open bars. Mark under empty column denotes technical error - data lost. No statistically significant difference was recorded between groups.
3.4. In situ hybridization for IFNy mRNA
In spinal cord sections, cells expressing mRNA for IFN? were detected in all rats (Fig. 5). There were large inter-individual variations (Fig. 6) and no statistically significant difference was recorded between saline- and memantine-treated groups. No positive cells were recorded using negative control sense-probe. 3.5. Efects of memantine on antigen induced lymphocyte proliferation and secretion of IFNy in vitro
As shown in Table 2 and 3, the effects of memantine in short-term cultures of LNC from EAE rats were similar 16, 14
secretion
The number of cells which respond to MBP 63-88 antigen in Lewis rat EAE were dramatically increased compared to background control (0 Ag) day 13- 14 p.i. (Table 11, and such cells might represent in vivo primed T-cells (Mustafa et al., 1991). Memantine treatment in vivo apparently did not reduce their numbers; in fact the response to MBP 63-88 was slightly elevated (p = 0.05) in the low dose group. MBP 89-101 induced no IFNy secretion over background level, indicating that the response to MBP 63-88 was not due to unspecific peptide stimulation.
Saline
10 mdkcr
20 m&n
Memantine Fig. 3. MHC Class II expression in lumbar spinal cord day 13-14 p.i. Individual rats as tilled bars, mean values in groups + one standard deviation as open bars. No statistically significant difference was recorded between groups.
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Fig. 4. Immunostained cryosections of spinal cord tissue from EAE-rats day 13 p.i., (a) scattered Ox 39-stained magnification), (b) densely Ox 6-stained (MHC Class II expressing) perivascular infiltrates (X 180 magnification).
regarding two aspects of lymphocyte function; proliferative and IPNr secretory responses to antigen. Doses of 10e3 to 10m4 M memantine suppressed both these responses, while lo’-’ M partially suppressed MBP 63-88 induced lymphocyte proliferation ( p = 0.02). Lower concentrations of memantine gave no detectable effects in these experiments. These ranges of concentrations are relevant for in vivo conditions since treatment of SpragueDawley rats with memantine 20 mg/kg i.p. has been estimated to give a brain concentration of 2 /AM 1 h after injection (Spanagel et al., 1994). The mechanism of in
Fig. 5. Autoradiogram than 14 autoradiographic
93
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(IL-2
receptor
positive)
cells (X 180
vitro suppression with the high concentrations of memantine was most probably direct cytotoxicity, as judged by inability of cells exposed to high concentrations of memantine to exclude trypan blue.
4. Discussion We demonstrate here that memantine reduces neurological deficits in EAE without measurable effects on the degree of CNS inflammation, as assessed by immunohisto-
of in situ hybridization for 1lW-y mRNA expressing cells in spinal cord tissue from grains over the cytoplasm per cell) at X 280 magnification.
EAE-rats
day 13 p.i. Two positive
cells (more
E. Wallstriim
94 Table 1 Numbers vivo
of IFNy
secreting
Group
cells a in response
Stimulus
Saline
Memantine 10 mg/kg
Memantine
of the
et al./Journal
to antigen
or lectin
added to culture ConA
MBP 89-101
MBP 63-88
m b SDC nd e PI m
7.3 6.8 9
> 600
6.8 3.0 9 n.s. 9.9
93.4 35.1 9 < 0.002 135.1
SD n
6.1 9
PI P2 g m
ns. 8.8
9 < 0.002 >600
f
8.5 9 n.s. ns. 8.9
9 < 0.002 >600
Sciences
0
29.7 9 < 0.002 0.05 127.1
20mg/k
IO-*
M
lo-’
M
1O-6 M SD n
2.5 10
PI P2
n.s.
4.5 10 n.s. n.s.
9 < 0.002
34.8 10 < 0.002 n.s.
a Analyzed with an immunospot assay on LNC from in vivo day 13-14 p.i. b Values denote mean (m of IFNy spots per lo6 plated LNC) ’ Standard deviation d Number of rats analyzed e Levels of significance comparing numbers of IFNy spots cultures receiving stimulus with numbers of IFNy spots cultures receiving no stimulus (0) f not significant ( p > 0.05) g levels of significance comparing treated with saline treated (control) EAE rats.
treated
10-s
1O-4 M
in in
memantine
chemistry. Furthermore, the number of cells expressing IFNy mRNA were not reduced. In the systemic compartment, the number of LNC secreting IIWy in responseto MBP 63-88 ex vivo were not reduced in drug treated rats
“E t
M
rats
obtained obtained
250,
137 (1996)
Table 2 Effects of memantine tion a in vitro Memantine concentration
0
9.1
ex
Neurological
1O-3 M
89-96
on antigen Stimulus
b
induced
lymphocyte
prolifera-
added to culture
0
ConA
MBP 89-101
MBP 63-88
9484.8 1118.1
430310.9 52123.0 0.014 402057.2 7952 1.3 0.014 n.s. 465737.5 74765.8 0.014 n.s. 377827.9 39566.1 0.014
10528.1 2574.0 n.s. f 13098.9 859.2 0.014 n.s. 13574.5 285 1.9 n.s. n.s. 10174.7 2338.3 n.s. n.s. 5945.7 2765.9 n.s. n.s. 941.8 1329.2 n.s. 0.014 743.7 856.0 n.s. 0.014
20804.6 6542.6 0.014 22723.6 3837.7 0.014 n.s. 24817.6 2377.7 0.014 n.s. 20904.2 2806.6 0.014 n.s. 12631.9 23 14.8 0.014 0.020 353.2 315.8 n.s 0.014 731.7 587.5 n.s. 0.014
m ’ SDd c PI m SD Pi P2 g m SD
n.s. 9234.8 2308.8
PI P2 m SD
n.s. 8487.1 1922.4
Pi P2 m SD PI P2 m SD
or lcctin
8920.4 931.4
n.s. 4089.4 1635.9 0.014 236.1 19.7
PI P2 m SD
0.014 753.4 576.3
PI P2
0.014
ll.S.
436102.5 18751.3 0.014 n.s. 115842.9 114236.6 0.014 0.014 3988.1 5493.6 n.s. 0.014
a Measured by [ 3H]thymidine uptake b Final molar concentrations in medium ’ Mean values (cpm) of four separate experiments d Standard deviation e Levels of significance comparing cultures receiving stimulus tures receiving no stimulus (0) f Not significant (p > 0.05) g levels of significance comparing tine exposed with unexposed (0) cultures
with culmeman-
I
I
Saline
10 mglkg
20 mglkg
Memantine Fig. 6. IFNy mRNA expressing cells in lumbar spinal cord day 13-14 p.i. Individual rats as tilled bars, mean values in groups + one standard deviation as open bars. No statistically significant difference was recorded between groups.
compared to saline treated rats. In fact there were even more peptide-induced IFN-y secreting cells in cultures from rats treated with the low dose of memantine (p = 0.05). T-cell responsesand levels of 1FN-y were investigated since Thl-cells, producing proinflammatory cytokines like 1FN-y, may be of central importance in neuroinflammatory disease(Olsson, 1995). IFNy induces major histocompatibility (MHC) class I and II antigens (Skoskiewicz et al., 1985), recognition molecules for T-cell homing (Duijvestijn et al., 1986) and promotes production of IL-I and TNFa (Collart et al., 1986). Furthermore, IFNy administered to MS-patients exacerbated disease(Panitch et al., 1987). However, there are some reports suggesting that IFNy, under certain conditions, may suppressEAE (Billiau et al., 1988; Voorthuis et al., 1990; Duong et al., 1992). In the present study, we observed increased num-
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bers of LNC secreting IFNy in responseto MBP 63-88 in the group receiving low dosesof memantine.However, we do not believe that this explains the drug effects on the neurological deficits, since one of the major effects of IFNy, the upregulation of MHC Class II expression, was not affected. Indeed, analysis of numbers of IFNr mRNA expressingcells in spinal cord tissue also failed to demonstrate a significant difference between controls and treatment groups. Furthermore, direct exposure of LNC to memantine in vitro failed to demonstrate stimulatory effects, both in the assay for detection of single cells secreting IFNy and in the proliferation assay. The finding of equally upregulated MHC Class II expression in spinal cord tissue of memantine treated rats and control rats argues against a drug effect on macrophage activation. However, we can not exclude more unorthodox drug-effects on the function of this cell type or glial cells, or effects on cellular production of cytokines other than IFNy, such as tumor necrosis factor alpha (TNFa). A dissociation between neurological deficits and CNSinflammation in EAE has previously been observed under a number of other experimental conditions. For example, Lewis-resistant and DA-rat F, hybrids had pronounced perivascular and submeningealcellinfiltration, but no clinical signs of EAE (Gasser et al., 1990). Hydroxymate matrix metalloprotease inhibitor treatment in SJL/J mice gave a significant decrease in the clinical expression of EAE, despite histopathological inflammation and demyelination, seemingly through restoration of the blood-brain barrier (Gijbels et al., 1994). Based on our findings, we speculate that the mode of action of memantine is late in the chain of pathogenic events leading to ascending paresis. Since demyelination alone seemsan insufficient explanation of all neurological deficits in neuroinflammatory disease, it is tempting to hypothesize that the NMDA-receptor antagonism by memantine might interfere with presumed soluble effector molecules important for the generation of nerve conduction block and paresis. Quinolinic acid is a potential mediator in this respect, since IFNr has been shown to induce its production in inflammed brain tissue (Heyes et al., 1993) and it is elevated in spinal cords during EAE (Flanagan et al., 1995). An other possiblemediator is glutamate, which is cytotoxic to NMDA-receptor positive neurons in supematants from cultured brain macrophages (Piani et al., 1991, Piani et al., 1992). Furthermore, our data are consistent with damage on neurons-axons as a basisfor neurological deficits during autoimmune neuroinflammation. In conclusion, we here demonstrate that memantine affects mechanismsimportant for neurological deficits in EAE. Firstly, these findings stimulate further study of whether NMDA-receptors are indeed involved in EAE pathogenesis. Secondly, memantine may be potentially useful as a treatment for neurological deficits in human neuroinflammatory diseases.
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Keilhoff, G. and Wolf, G. (1992) Memantine prevents quinolinic acid-induced hippocampal damage. Eur. J. Pharmacol., 219: 451-454. Komhuber, J., Bormann, J., Hiibers, M., Rusche, K. and Riederer, P. (1991) Effects of the I-amino-adamantanes at the MK-Sol-binding site of the NMDA-receptor-gated ion channel: a human postmortem brain study. Eur. J. Pharmacol., 206: 297-300. Lipton, S.A. and Rosenberg P.A. (1993) Excitatory ammo acids as a final common pathway for neurologic disorders. N. Engl. J. Med., 330: 613-622. McMaster, W.R. and Williams, A.F. (1979) Identification of Ia glycoprotein in rat thymus and purification from rat spleen. Eur. J. Immunol., 9: 426-433. Moreau, T., Wing, M., Waldman, H. and Compston, A. (1994) Cytokine release increases symptoms in patients with multiple sclerosis. Reported at 10th congress of the european committee for treatment and research in multiple sclerosis, Athens, 4-5 November 1994. Mustafa, MI., Diener, P., Hijjeberg, B., Van der Meide, P. and Olsson, T. (1991) T cell immunity and interferon-y secretion during experimental allergic encephalomyelitis in Lewis rats. J. Neuroimmunol., 31: 165-177. Mustafa. M.I., Vingsbo, C., Olsson, T., Ljungdahl, A., Hojeberg, B. and Holmdahl, R. (1993) The major histocompatibility complex influences myelin basic protein 63-88 induced T cell cytokine profile and experimental autoimmune encephalomyelitis. Eur. J. Immunol., 23: 3089-3095. Offner, H., Hashim, G.A., Cehtik, B., Galang, A., Li, X.B., Bums, F.R., Shen, N., Heber-Katz, E. and Vandenbark, A.A. (1989) T cell determinants of myelin basic protein include a unique encephalitogenic I-E restricted epitope for Lewis rats. J. Exp. Med., 170: 355-367. Olsson. T., Zhi, W.W., Hiijeberg, B., Kostulas, V., Jiang, Y.-P., Andersson, G., Ekre, H.-P. and Link, H. (19901 Autoreactive T lymphocytes in multiple sclerosis determined by antigen induced secretion of interferon-gamma. J. Clin. Invest., 86: 981-985. Olsson, T. (19951 Cytokine-producing cells in experimental autoimmune encephalomyelitis and multiple sclerosis. Neurology, 45, S 11-S 15. Opdenakker, G. and van Damme, J. (19941 Cytokine regulated proteases in autoimmune diseases. Immunol. Today, 15: 103- 107. Panitch, H.S., Hirsch, R.L., Schindler, J. and Johnston, K.P. (1987) Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system. Neurology, 37: 1097- 1102.
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Parsons, C.G., Gruner, R., Rozental, J., Millar, J. and Lodge, D. (1993) Patch clamp studies on the kinetics and selectivity of N-methyl-r> aspartate receptor antagonism by memantine (I-amino-3,5-dimethyladamantan). Neuropharmacol., 32: 1337-1350. Piani D., Frei K., Do K.Q., Cuenod M., Fontana A. (1991) Murine brain macrophages induced NMDA receptor mediated neurotoxicity in vitro by secreting glutamate. Neurosci. Lett., 133, 159- 162. Piani D., Spranger M., Frei K., Schaffner A., Fontana A. (1992) Macrophage-induced cytotoxicity of N-methyl-Daspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur. J. Immunol., 22.2429-2436. Rabey, J.M., Nissipeanu, P. and Korczyn, A.D. (1992) Efficacy of memantine, an NMDA receptor antagonist, in the treatment of Parkinson’s disease. J. Neural Transm. [P-D Sect.], 4: 277-282. Rosenberg, G.A. and Appenzeller, 0. (1988) Amantadine, fatigue, and multiple sclerosis. Arch. Neurol., 45: 1104- 1106. Sedgwick, J., Brostoff, S. and Mason, D. (1987) Experimental allergic encephalomyelitis in the absence of a classical delayed type hypersensitivity reaction. Severe paralytic disease correlates with the presence of interleukin 2 receptor-positive cells infiltrating the central nervous system. J. Exp. Med., 165: 1058-1075. Seif el Nasr, M., Peruche, B., Ropberg, C., Memrel, H.-D. and Krieglstein. J. (1990) Neuroprotective effects of memantine demonstrated in vivo and in vitro. Eur. J. Pharmacol., 185: 19-24. Skoskiewicz, M.J., Colvin, R.B., Schneeberger, E.E. and Russel, P.S. (1985) Widespread and selective induction of major histocompatibility complex determined antigens in vivo by gamma interferon. J. Exp. Med., 162: 1645-1664. Spanagel R., Eilbacher B., Wilke R. (19941 Memantine-induced dopamine release in the prefrontal cortex and striatum of the rat-a pharmacokinetic microdialysis study. Eur. J. Pharmacol., 262, 21-26. Vandenbark, A.A., Offner, H., Reshef, T., Fritz, R., Chou, C.H. and Cohen, I.R. (1985) Specificity of T lymphocyte line for peptides of myelin basic protein. J. Immunol., 135: 229-233. Voorthuis, J.A., Uitdenhaag, B.M., de Groot, C.J., Goede, P.H., van der Meide, P.H. and Dijkstra, C.D. (1990) Supression of experimental allergic encephalomyelitis by intraventricular administration of interferon-gamma in Lewis rat. Chn. Exp. Immunol., 81: 183-188. Williams, A.F., Galfre, G. and Milstein, C. (19771 Analysis of cell surfaces by xenogeneic myeloma hybrid antibodies: differentiation antigens of rat lymphocytes. Cell, 12: 663-673.
OF THE
NEUROLOGICAL SCIENCES
ELSEVIER
Journal
of the Neurological Sciences 137 (1996) 89-%
Memantine abrogates neurological deficits, but not CNS inflammation, in Lewis rat experimental autoimmune encephalomyelitis Erik Wallstriim a7* , Per Diener b, he Ljungdahl b, Mohsen Khademi a, Carl-Gustaf Nilsson ‘, Tomas Olsson a*d ’ Department of Medicine, Molecular Medicine Unit, Karolinskn b Department of Clinical Neuroscience and Family Medicine, Division ’ Clinical Research ’ Department of Neurology,
Institute. House Ll:OI. Karolinska Hospital, of Neurology, Karolinska Institute, Huddinge Sweden Center, Novum, Stockholm, Sweden Karolinrku Hospital, Stockholm, Sweden
171 76 Stockholm, Sweden University Hospital, Stockholm,
Received 19 June 1995; revised 16 October 1995; accepted 1 November 1995
Abstract Memantine, a clinically employed drug with iV-methyl-paspartate (NMDA) receptor antagonistic effects, dose-dependently ameliorates neurological deficits in Lewis rat experimental autoimmune encephalomyelitis (EAE). Interestingly, this therapeutic effect was not due to dampened CNS inflammation, as assessed by immunohistochemical evaluation of spinal cord tissue. Furthermore, numbers of interferon gamma (IFWy) mRNA expressing cells were not decreased, as assessed by in situ hybridization. Systemic immunity in terms of numbers of IPNy secreting cells in response to immunodominant myelin basic protein (MBP) peptides ex vivo was not reduced, and non-toxic doses of memantine did not affect lymphocyte proliferation or 1PN-y secretion in vitro. Considering these findings, we hypothesize that effector mechanisms responsible for reversible neurological deficits in EAE may involve NMDA receptors, and this highlights neurons as targets during autoimmune neuroinflammation. Memantiue; Experimental autoimmune encephalomyelitis (EAB); N-methyl+aspartate Interferon gamma (IFNy); Neurological deficit
Keywords:
1. Introduction In multiple sclerosis (MS), it is well known that there is often a poor correlation between clinical disease and degree of inflammation-demyelination, as assessed either by magnetic resonance imaging (MRI) (Jacobs et al., 1986) or
traditional histopathology (Gilbert and Sadler, 1983). Effector mechanisms responsible for reversible neurological deficits, without striking structural changes, are largely unknown. A number of inflammatory mediators have been implicated in this respect: cytokines such as tumor necrosis factor alpha (TNFa) and interferon gamma (IFNy) (Moreau et al., 1994), proteases (Opdenakker and van Damme, 1994) and nitric oxide (NO) (Chao et al., 1992). In this study we approached this question using Lewis rat experimental autoimmune encephalomyelitis (EAE) and memantine, a drug with potent N-methyl-o-aspartate Corresponding author. Tel.: + 46 8 72%246; Fax: + 46 8 72%248; E-mail: [email protected]. l
0022-510X/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0022-5 10X(95)00339-8
(NMDA)
receptor; Myelin basic protein (MBP);
(NMDA) receptor antagonistic effects (Bormann, 1989; Chen et al., 1992; Parsons et al., 1993). Memantine (l-amino-3,5-dimethyl-adamantane) is closely related to amantadine (1 -amino-adamantane), but memantine binds more efficiently to NMDA-receptors (Komhuber et al., 1991). NMDA-receptors have been associated with pathogenic events in a variety of neurological diseases; epilepsy, parkinsonism, motor neuron disease and ischemic stroke (Lipton and Rosenberg, 1993; Calne, 1994). Memantine is utilised as an antispastic and antiparkinsonistic agent (Rabey et al., 1992), and its use in experimental models have demonstrated protective effects against cerebral ischemia (Seif el Nasr et al., 1990) and quinolinic acid-induced hippocampal damage (Keilhoff and Wolf, 1992). Amantadine had significant positive effects in double-blind placebo-controlled studies on MS-associated fatigue (Rosenberg and Appenzeller, 1988; Cohen and Fisher, 1989). The aim of this study was to investigate possible effects of memantine on clinical signs of EAE (i.e. ascending paresis), and relate this to immunohistologi-
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cal findings in spinal cord and some key aspects of T-cell mediated myelin basic protein (MBP)-autoreactivity, especially cellular IFNy production.
2. Materials
and methods
2.1. Induction protocol
of EAE, clinical assessment
and treatment
Male Lewis rats (310-370 g and 200-260 g, Zentralinstitut fur Versuchtierzucht, Hannover, Germany) were immunized in both hind footpads with a total of 50 mg guinea pig spinal cord, 2 mg Mycobacterium tuberculosis (Strain H 37 RA, Difco laboratories, Detroit, USA), 100 ~1 Freund’s incomplete adjuvant (Sigma, St. Louis, USA) and 100 ~1 saline. Clinical disease severity was graded as follows: 0 = no illness, 1 = flaccid tail, 2 = moderate paraparesis, 3 = severe paraparesis, 4 = moribund state or death. Cumulative disease index, i.e. added scores for the whole observed period, was calculated for each rat and used for non-parametric statistics. Memantinehydrochloride (Akatinol Memantine, Merz + Co., Frankfurt/Main, Germany) at a low dose (10 mg/kg) or a high dose (20 mg/kg) was administered intraperitoneally (i.p.) on days 7, 9, I 1 and 13 post-immunization (p.i.). Control rats received 1 ml sterile saline at the same days pi. One group of animals were studied up to day 31 p.i. Another group of rats were sacrificed at day 13 or 14 p.i., for immunoassays as described below. Additional untreated rats were immunized and sacrificed at day 14-17 p.i., for studies of memantine effects on lymphoid cells in vitro. Animal experiments were approved by the local ethical committee. 2.2. Preparation
of lymph node mononuclear
cells (WC)
Popliteal and inguinal lymph nodes were dissected and crushed through sterile wire meshes. Cells obtained were washed twice and adjusted to 5 X lo6 viable LNC/ml in Dulbecco’s modified Eagles medium (Gibco, Paisley, UK) containing 1% (v/v) MEM Amino Acids solution (Gibco), 50 IU/ml respective 50 pg/ml Penicillin-Streptomycin (Gibco), 2 mM L-glutamine (Gibco) and 5% (v/v) fetal calf serum (Gibco). 2.3. Single cell assay for secretion of IFNy antigen
in response to
An immunospot assay that enables detection of single cells secreting IFNy (Czerkinsky et al., 1988), adapted for rat IFN-y (Mustafa et al., 1991) was employed. 96well nitrocellulose bottomed microtitre plates (Millipore, Bedford, USA) were coated overnight at 4°C with 100 ~1 (20 pg/ml) aliquots of monoclonal antibody directed against rat IFNy (DBl, kindly provided by Peter van der Meide, Biomedical Primate Research Centre, Rijswijk, The
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Netherlands). After washing, duplicate 200 ~1 aliquots LNC suspension (IO6 cells/well) were added, together with antigen or without antigen as background controls. The antigens used were the major encephalitogenic epitope in Lewis rat; guinea pig myelin basic protein (MBP) 63-88 peptide (Vandenbark et al., 1985) and the minor encephalitogenic epitope MBP 89-101 peptide (Offner et al., 1989) at final concentrations in medium of 10 pg/ml. MBP peptides were synthesized as described (Olsson et al., 1990). The lectin Concanavalin A (ConA; Sigma) at final concentration 5 pg/ml was used as positive control. After 24 h culture at 37°C and 7% CO, in humid atmosphere, cells were discarded and plates washed thoroughly. Secreted and bound IFNy was visualized by rabbit polyclonal anti-rat IFNy antibody (Van der Meide), biotinylated anti-rabbit IgG (Dakopatts, Denmark), avidin-biotin peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA) and carbazole staining, resulting in stained spots corresponding to single cells that had secreted IFNy. Spots were counted by use of a dissection microscope and standardized to numbers of spots per lo6 LNC originally applied in the well. The variation between wells within duplicates was less than 20%. 2.4. lmmunohistochemistty
and image analysis
Lumbar spinal cord was dissected and snap-frozen in liquid nitrogen. Cryostat sections (14 pm) were incubated with primary antibodies overnight at 4°C in a moist chamber. The following mouse monoclonal antibodies were used as primary antibodies: Ox 39 (anti-rat interleukin 2 (IL-2) receptor; Sedgwick et al., 1987), Ox 6 (anti-rat MHC class II; McMaster and Williams, 1979), Ox 18 (anti-rat MHC class I; Fukumoto et al., 1982), W3/25 (anti-rat CD4, T helper cells and macrophages; Williams et al., 1977) and Ox 8 (anti-rat CD8, T cytotoxic/supressor cells; Brideau et al., 1980). Ox 39 and Ox 18 were purchased from Seralab (Crawley Down, UK). Ox 6, W3/25 and Ox 8 were purified from culture supematants of hybridomas (Holmdahl et al., 1985). Sections were stained according to the avidin-biotin technique. Omission of the primary antibody served as negative control. Specificity of the staining was controlled on sections of peripheral lymphoid organs. Ox 39 stained cells were counted in whole spinal cord sections using X 20 magnification. Tissue areas were measured by image analysis (Sonata, Seescan, Cambridge, UK), and were typically 30 mm* per animal. Stained cells were standardized as number of cells per 100 mm2 tissue area. Ox 6 staining was measured as percentage of stained area in computer images covering most of the whole tissue area. One section per animal was analyzed. Threshold to detect positive staining was set to five standard deviations darker greyshade value than the non-stained background. Threshold was set individually for each section. The microscope lamp was switched on 30
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et al./Journd
min before image analysis, for optimum stability. Ox 8, W3/25 and Ox 18 were semiquantitatively assessed by manual counting; - = 0, + = l-100, + + = 100-1000, + + + = > 1000 stained cells per 100 mm’ tissue area. 2.5. In situ hybridization
for IFNy
mRNA
Cryostat sections (14 pm> from the same part of the lumbar spinal cord as used for immunohistochemistry were thaw-mounted onto ProbeOn slides (ProbeOn, Fischer Scientific, Pittsburgh, USA). In situ hybridization was performed as previously described (Mustafa et al., 19931, with 35S-labelled synthetic oligonucleotide probes. Only cells with more than 14 autoradiographic grains over their cytoplasm were considered positive, and expressed as number of EN-y mRNA positive cells per 100 mm2 tissue area.
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2.6. Eflects proliferation
(19%)
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of memantine and secretion
on antigen induced of IFNy in vitro
lymphocyte
To assess lymphocyte proliferative responses triplicate aliquots of LNC (6 X lo5 cells per well) were cultured in round bottomed 96-well microtitre plates (Nunc, Nunclon, Denmark). Antigens were added as described above. Memantine was added at final concentrations in medium ranging 10m3 to lo-* M, or was omitted. After 60 h of incubation at 37°C 7% CO,, humid atmosphere, the cells were pulsed for 10 h with [3H]methyl-thymidine (final concentration 5 &i/ml; Amersham, Little Chalfront, UK). Cells were harvested and thymidine incorporation counted in a beta scintillation counter (Beckman). The same conditions were used to test effects of memantine on antigen induced IFNy secretion by immunospot (as above).
(a) 23 2-
2 ?! 8 3 1,5 .o .s 0 s 1
e! ii z 1,5.o 5 0 ls r” 0,5-
9 0,5
0
10
12
14
16
18
31
0
8
Days p.i. --e--s+
* -ew
Saline, n=8 Memantine 10 mg/kg, n=8 Memantine 20 mg/kg, n=7 (d)
10
10 12 Days p.i.
Saline, n=9 Memantine 10 mg/kg, n=9 Memantine 20 mg/kg, n=lO
on
2
$ 3 .” ‘O
-0 .E
6
z 33
A I-
0
8-
l 0
6-
0a
0
A00
Saline
Memantine
Memantine
10
20
mg/kg
mg/kg
14
2 .5
2
z 3
o-
mane
-
0
m 0 0 I Saline
0 I
non. 1
Memantine
Memantine
10
20
mg/kg
mgfkg
Fig. 1. (a and b) Mean clinical scores of EAE in rats treated with saline (controls), or 10 mg/kg respective 20 mg/kg memantine. Stars denote levels of significance comparing drug treated rats with control rats for the whole observed period, n.s., not significant (p > 0.05); * , p < 0.05; * * , p < 0.01; * * * , p < 0.001; n is number of rats. (c and d) Added clinical scores for the whole observed period (cumulative disease index). Each rat is represented by one tilled dot.
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2.7. Statistical evaluation
The non-parametric Mann-Whitney
U-test was used.
3. Results 3.1. Clinical course
The effects of memantine on clinical signs of EAE were similar in two different experimental sessions (mean clinical scores for groups in Fig. 1 a and b, individual cumulative disease index in Fig. 1 c and d). In the first experiment (Fig. 1 a and c) c1’mica1 ’ scores were followed to day 31 p.i. Treatment with the higher dose of memantine almost completely abolished neurological deficits. Among the rats treated with the low dose one heavily diseased rat died, which is reflected by a high mean clinical score day 1.5 p.i. Despite this, the rats treated with the low dose of memantine were less diseased than the controls ( p < 0.01). There were no late relapses. In the second experiment (Fig. 1 b and d), only the high dose of memantine resulted in a reduction of clinical scores that were statistically significant at the time of sacrifice. No apparent behavioral abnormalities were observed among the memantine treated rats. 3.2. Immunohistochemistry
Immunohistochemical evaluation revealed perivascular inflammation with infiltration of CD4+ and CD8+ T cells and upregulation of MHC Class I and II expressions both in saline- and memantine-treated rats. A thorough quantitative analysis of the number of IL-2 receptor positive cells (Fig. 2, Fig. 4a) and degree of MHC Class II expression (Fig. 3, Fig. 4b) revealed no differences between the groups. Semiquantitative assessment of MHC Class I expression, CD4+ and CD8+ cells also failed to demonstrate any major differences between controls and treatment groups (data not shown). 3.3. Antigen induced fFNy
Saline
IO mgikg
20 mg/kg
Memantine Fig. 2. IL-2 receptor expressing cells infiltrating lumbar spinal cord day 13-14 pi. Individual rats as tilled bars, mean values in groups + one standard deviation as open bars. Mark under empty column denotes technical error - data lost. No statistically significant difference was recorded between groups.
3.4. In situ hybridization for IFNy mRNA
In spinal cord sections, cells expressing mRNA for IFN? were detected in all rats (Fig. 5). There were large inter-individual variations (Fig. 6) and no statistically significant difference was recorded between saline- and memantine-treated groups. No positive cells were recorded using negative control sense-probe. 3.5. Efects of memantine on antigen induced lymphocyte proliferation and secretion of IFNy in vitro
As shown in Table 2 and 3, the effects of memantine in short-term cultures of LNC from EAE rats were similar 16, 14
secretion
The number of cells which respond to MBP 63-88 antigen in Lewis rat EAE were dramatically increased compared to background control (0 Ag) day 13- 14 p.i. (Table 11, and such cells might represent in vivo primed T-cells (Mustafa et al., 1991). Memantine treatment in vivo apparently did not reduce their numbers; in fact the response to MBP 63-88 was slightly elevated (p = 0.05) in the low dose group. MBP 89-101 induced no IFNy secretion over background level, indicating that the response to MBP 63-88 was not due to unspecific peptide stimulation.
Saline
10 mdkcr
20 m&n
Memantine Fig. 3. MHC Class II expression in lumbar spinal cord day 13-14 p.i. Individual rats as tilled bars, mean values in groups + one standard deviation as open bars. No statistically significant difference was recorded between groups.
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et al./Journd
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Fig. 4. Immunostained cryosections of spinal cord tissue from EAE-rats day 13 p.i., (a) scattered Ox 39-stained magnification), (b) densely Ox 6-stained (MHC Class II expressing) perivascular infiltrates (X 180 magnification).
regarding two aspects of lymphocyte function; proliferative and IPNr secretory responses to antigen. Doses of 10e3 to 10m4 M memantine suppressed both these responses, while lo’-’ M partially suppressed MBP 63-88 induced lymphocyte proliferation ( p = 0.02). Lower concentrations of memantine gave no detectable effects in these experiments. These ranges of concentrations are relevant for in vivo conditions since treatment of SpragueDawley rats with memantine 20 mg/kg i.p. has been estimated to give a brain concentration of 2 /AM 1 h after injection (Spanagel et al., 1994). The mechanism of in
Fig. 5. Autoradiogram than 14 autoradiographic
93
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(IL-2
receptor
positive)
cells (X 180
vitro suppression with the high concentrations of memantine was most probably direct cytotoxicity, as judged by inability of cells exposed to high concentrations of memantine to exclude trypan blue.
4. Discussion We demonstrate here that memantine reduces neurological deficits in EAE without measurable effects on the degree of CNS inflammation, as assessed by immunohisto-
of in situ hybridization for 1lW-y mRNA expressing cells in spinal cord tissue from grains over the cytoplasm per cell) at X 280 magnification.
EAE-rats
day 13 p.i. Two positive
cells (more
E. Wallstriim
94 Table 1 Numbers vivo
of IFNy
secreting
Group
cells a in response
Stimulus
Saline
Memantine 10 mg/kg
Memantine
of the
et al./Journal
to antigen
or lectin
added to culture ConA
MBP 89-101
MBP 63-88
m b SDC nd e PI m
7.3 6.8 9
> 600
6.8 3.0 9 n.s. 9.9
93.4 35.1 9 < 0.002 135.1
SD n
6.1 9
PI P2 g m
ns. 8.8
9 < 0.002 >600
f
8.5 9 n.s. ns. 8.9
9 < 0.002 >600
Sciences
0
29.7 9 < 0.002 0.05 127.1
20mg/k
IO-*
M
lo-’
M
1O-6 M SD n
2.5 10
PI P2
n.s.
4.5 10 n.s. n.s.
9 < 0.002
34.8 10 < 0.002 n.s.
a Analyzed with an immunospot assay on LNC from in vivo day 13-14 p.i. b Values denote mean (m of IFNy spots per lo6 plated LNC) ’ Standard deviation d Number of rats analyzed e Levels of significance comparing numbers of IFNy spots cultures receiving stimulus with numbers of IFNy spots cultures receiving no stimulus (0) f not significant ( p > 0.05) g levels of significance comparing treated with saline treated (control) EAE rats.
treated
10-s
1O-4 M
in in
memantine
chemistry. Furthermore, the number of cells expressing IFNy mRNA were not reduced. In the systemic compartment, the number of LNC secreting IIWy in responseto MBP 63-88 ex vivo were not reduced in drug treated rats
“E t
M
rats
obtained obtained
250,
137 (1996)
Table 2 Effects of memantine tion a in vitro Memantine concentration
0
9.1
ex
Neurological
1O-3 M
89-96
on antigen Stimulus
b
induced
lymphocyte
prolifera-
added to culture
0
ConA
MBP 89-101
MBP 63-88
9484.8 1118.1
430310.9 52123.0 0.014 402057.2 7952 1.3 0.014 n.s. 465737.5 74765.8 0.014 n.s. 377827.9 39566.1 0.014
10528.1 2574.0 n.s. f 13098.9 859.2 0.014 n.s. 13574.5 285 1.9 n.s. n.s. 10174.7 2338.3 n.s. n.s. 5945.7 2765.9 n.s. n.s. 941.8 1329.2 n.s. 0.014 743.7 856.0 n.s. 0.014
20804.6 6542.6 0.014 22723.6 3837.7 0.014 n.s. 24817.6 2377.7 0.014 n.s. 20904.2 2806.6 0.014 n.s. 12631.9 23 14.8 0.014 0.020 353.2 315.8 n.s 0.014 731.7 587.5 n.s. 0.014
m ’ SDd c PI m SD Pi P2 g m SD
n.s. 9234.8 2308.8
PI P2 m SD
n.s. 8487.1 1922.4
Pi P2 m SD PI P2 m SD
or lcctin
8920.4 931.4
n.s. 4089.4 1635.9 0.014 236.1 19.7
PI P2 m SD
0.014 753.4 576.3
PI P2
0.014
ll.S.
436102.5 18751.3 0.014 n.s. 115842.9 114236.6 0.014 0.014 3988.1 5493.6 n.s. 0.014
a Measured by [ 3H]thymidine uptake b Final molar concentrations in medium ’ Mean values (cpm) of four separate experiments d Standard deviation e Levels of significance comparing cultures receiving stimulus tures receiving no stimulus (0) f Not significant (p > 0.05) g levels of significance comparing tine exposed with unexposed (0) cultures
with culmeman-
I
I
Saline
10 mglkg
20 mglkg
Memantine Fig. 6. IFNy mRNA expressing cells in lumbar spinal cord day 13-14 p.i. Individual rats as tilled bars, mean values in groups + one standard deviation as open bars. No statistically significant difference was recorded between groups.
compared to saline treated rats. In fact there were even more peptide-induced IFN-y secreting cells in cultures from rats treated with the low dose of memantine (p = 0.05). T-cell responsesand levels of 1FN-y were investigated since Thl-cells, producing proinflammatory cytokines like 1FN-y, may be of central importance in neuroinflammatory disease(Olsson, 1995). IFNy induces major histocompatibility (MHC) class I and II antigens (Skoskiewicz et al., 1985), recognition molecules for T-cell homing (Duijvestijn et al., 1986) and promotes production of IL-I and TNFa (Collart et al., 1986). Furthermore, IFNy administered to MS-patients exacerbated disease(Panitch et al., 1987). However, there are some reports suggesting that IFNy, under certain conditions, may suppressEAE (Billiau et al., 1988; Voorthuis et al., 1990; Duong et al., 1992). In the present study, we observed increased num-
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et al./Journal
of the Neurological
bers of LNC secreting IFNy in responseto MBP 63-88 in the group receiving low dosesof memantine.However, we do not believe that this explains the drug effects on the neurological deficits, since one of the major effects of IFNy, the upregulation of MHC Class II expression, was not affected. Indeed, analysis of numbers of IFNr mRNA expressingcells in spinal cord tissue also failed to demonstrate a significant difference between controls and treatment groups. Furthermore, direct exposure of LNC to memantine in vitro failed to demonstrate stimulatory effects, both in the assay for detection of single cells secreting IFNy and in the proliferation assay. The finding of equally upregulated MHC Class II expression in spinal cord tissue of memantine treated rats and control rats argues against a drug effect on macrophage activation. However, we can not exclude more unorthodox drug-effects on the function of this cell type or glial cells, or effects on cellular production of cytokines other than IFNy, such as tumor necrosis factor alpha (TNFa). A dissociation between neurological deficits and CNSinflammation in EAE has previously been observed under a number of other experimental conditions. For example, Lewis-resistant and DA-rat F, hybrids had pronounced perivascular and submeningealcellinfiltration, but no clinical signs of EAE (Gasser et al., 1990). Hydroxymate matrix metalloprotease inhibitor treatment in SJL/J mice gave a significant decrease in the clinical expression of EAE, despite histopathological inflammation and demyelination, seemingly through restoration of the blood-brain barrier (Gijbels et al., 1994). Based on our findings, we speculate that the mode of action of memantine is late in the chain of pathogenic events leading to ascending paresis. Since demyelination alone seemsan insufficient explanation of all neurological deficits in neuroinflammatory disease, it is tempting to hypothesize that the NMDA-receptor antagonism by memantine might interfere with presumed soluble effector molecules important for the generation of nerve conduction block and paresis. Quinolinic acid is a potential mediator in this respect, since IFNr has been shown to induce its production in inflammed brain tissue (Heyes et al., 1993) and it is elevated in spinal cords during EAE (Flanagan et al., 1995). An other possiblemediator is glutamate, which is cytotoxic to NMDA-receptor positive neurons in supematants from cultured brain macrophages (Piani et al., 1991, Piani et al., 1992). Furthermore, our data are consistent with damage on neurons-axons as a basisfor neurological deficits during autoimmune neuroinflammation. In conclusion, we here demonstrate that memantine affects mechanismsimportant for neurological deficits in EAE. Firstly, these findings stimulate further study of whether NMDA-receptors are indeed involved in EAE pathogenesis. Secondly, memantine may be potentially useful as a treatment for neurological deficits in human neuroinflammatory diseases.
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