N-methyl-D-aspartate (NMDA) receptor involvement in central nervous system prostaglandin production during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis

OR IG INAL

ART ICLE

N-methyl-D-aspartate (NMDA) receptorinvolvement in central nervous systemprostaglandin production during the relapsephase of chronic relapsing experimentalautoimmune encephalomyelitis (CR EAE)

Christopher Boltona, Elizabeth G. Woodb, Samir S. Ayoubc*aNeuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science,

St. Bartholomew’s and The London School of Medicine and Dentistry, 4, Newark Street, London, E12 AT, UKbCentre for Translational Medicine and Therapeutics, William Harvey Research Institute, St. Bartholomew’s and The

London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, EC1M

6BQ, UKcCentre for Biochemical Pharmacology, William Harvey Research Institute, St. Bartholomew’s and The London School

of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK

Keywords

(+) MK-801,

experimental autoimmune

encephalomyelitis,

NMDA receptor,

prostaglandins

Received 17 January 2012;

revised 23 April 2012;

accepted 25 May 2012

*Correspondence and reprints:

[email protected]

ABSTRACT

Our previous studies have established that major changes in central nervous sys-

tem (CNS) prostaglandin (PG) levels occur during the relapse phase of chronic

relapsing experimental autoimmune encephalomyelitis (CR EAE), an animal model

of the human demyelinating disease multiple sclerosis. PG production is controlled

through a series of enzymic pathways that, in EAE, are influenced by neuroanti-

gen-driven autoimmune events. In non-immune-based models of CNS disease,

endogenous glucocorticoids have been proposed as instigators of PG synthesis via

activation of the N-methyl-D-aspartate (NMDA) receptor. Glucocorticoids have an

important regulatory role in the pathogenesis EAE and the NMDA receptor is inti-

mately involved in many of the characteristic neuroinflammatory processes that

govern the disease. Therefore, the alterations in prostanoid concentrations during

the relapse stage of CR EAE may ultimately be governed by glucocorticoid-induced

NMDA receptor activation. The current investigation has examined the proposed

glucocorticoid–NMDA receptor link by determining the effects of the receptor

antagonist, (+) MK-801, on CNS PGE2 and PGD2 levels in Biozzi mice with relapse

symptoms of CR EAE. Prostanoid concentrations in the cerebral cortex were not

altered by drug administration, and in cerebellar tissues, a vehicle effect negated

any drug-induced changes. However, the level of PGD2 in spinal cords from (+)MK-801-dosed mice was significantly lower, compared to controls, but PGE2 con-

centrations remained unchanged. The results suggest that glucocorticoid–NMDA

receptor-linked events are not primarily responsible for PG generation in the brain

but may influence prostanoid production in discrete areas of the CNS.

INTRODUCT ION

Involvement of the N-methyl-D-aspartate (NMDA) recep-

tor in neuroinflammation and, specifically, experimental

autoimmune encephalomyelitis (EAE), the recognized

model of the human demyelinating disease, multiple

sclerosis (MS), has been demonstrated and confirmed

through the use of various targeted pharmacological

ª 2012 The Authors Fundamental and Clinical Pharmacology © 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 1

doi: 10.1111/j.1472-8206.2012.01050.x

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agents [1]. Moreover, the uncompetitive NMDA receptor

antagonist (+) MK-801 (dizocilpine maleate) and the

aminoadamantane, memantine (1-amino-3, 5-dimethyl-

adamantane) have been shown to prevent the cardinal

signs of EAE including ascending neurological deficits,

blood–brain barrier breakdown (BBB) and inflamma-

tory cell infiltration into the central nervous system

(CNS) in the apparent absence of effects on the immune

system [2–5].Clearly, the NMDA receptor occupies an important

position in the pathogenesis of EAE, and studies in other

models, where the receptor has been shown to operate,

strongly suggest a pivotal role in the control of experi-

mental neuroinflammation and neurodegeneration. In

particular, Nair and Bonneau [6] demonstrated that

NMDA receptor activation, by a stress-induced increase

in endogenous glucocorticoids, resulted in microglial

activation and proliferation. Furthermore, the use of (+)MK-801, to block NMDA receptor actions, dampened

the microglial response to elevated corticosteroid levels.

Interestingly, the studies suggest NMDA receptor-linked

microglial proliferation is regulated by intermediary

inflammatory mediators that include the prostaglandins

(PGs) and associated precursor enzymes. In vitro studies

by Jing et al. [7] have also shown that glucocorticoid

treatment for neuronal cells results in upregulated

transcriptional activity of the NMDA receptor together

with enhanced expression of the receptor protein. The

authors speculate that a steroid-induced increase in

NMDA receptor numbers, in vivo, may lead to excitotox-

icity and subsequent neurodegeneration such as occurs

in EAE and MS.

Several studies have demonstrated that circulating

endogenous glucocorticoids are dramatically increased,

prior to, and during the neurological phases of acute

and chronic relapsing (CR) EAE, and are closely related

to the recovery from symptoms [8–10]. Conversely, theglucocorticoids have the potential to hinder the recov-

ery process via the induction of neuronal damage with

release of the agonist, glutamate, thereby causing

excitotoxic activation of the NMDA receptor [11–15].NMDA receptor activation is achieved following the ini-

tial binding of glutamate to the neighbouring a-amino-

3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

receptor. Loss of Mg2+ from the NMDA receptor ion

channel is thereby triggered which, on the subsequent

binding of glutamate, activates the receptor, to cause

the eventual upregulation of enzyme systems that are

implicated in the pathogenesis of EAE [1,16,17].

Cyclo-oxygenase (COX) enzymes control PG produc-

tion, via the catalysis of arachidonate, and, interest-

ingly, one member of the group, COX-2 is upregulated

by NMDA receptor stimulation [18,19]. We have

recently shown profound changes in the expression of

COX enzymes, including COX-2, together with marked

alterations in PG levels in CNS tissues during the

course of chronic relapsing experimental autoimmune

encephalomyelitis (CR EAE) [20]. Earlier studies by us

have also described significant variations in CNS PG

concentrations with the development of the acute and

chronic forms of the disease [21–23]. Therefore, in

models of EAE, the involvement of COX enzymes and

subsequent PG formation may be closely allied with

prior NMDA receptor activation by glutamate, released

from neuronal tissue, as a result of raised glucocorti-

coid levels. The present study, prompted by the work of

Nair and Bonneau [6], has investigated a proposed glu-

cocorticoid–NMDA receptor-linked relationship, which

thereby accounts for the alterations in CNS PG levels

during the relapse phase of CR EAE. Evidence is pro-

vided for NMDA receptor participation in prostanoid

formation but only within specific areas of the CNS

affected by the disease.

MATER IALS AND METHODS

Animals and the induction of CR EAE

Male ABH Biozzi mice (H-2dql) were obtained from

Harlan Olac (Oxford, UK), at 7–9 weeks of age, housed

individually, under 12 : 12 h light/dark cycle, with

free access to standard diet and water. CR EAE was

induced in the aged-matched mice, after a 2-week

acclimatization period, as previously described [10],

and in accordance with the Animals Scientific Proce-

dures Act of 1986. Briefly, on day 0 and 7 days later,

each mouse received, in both flanks, 0.15 mL of inoc-

ulum containing pooled, lyophilized syngeneic spinal

cord, solubilized in sterile phosphate-buffered saline

(PBS) (Sigma-Aldrich, Dorset, UK), and incomplete Fre-

und’s adjuvant, supplemented with Mycobacterium

tuberculosis H37Ra and Mycobacterium butyricum. (Dif-

co, West Moseley, UK). Mice were weighed daily and

assessed for neurological disease, from day 13 post-

inoculation, using the following scoring system, (1)

partial flaccid tail, (2) complete flaccid tail, (3)

impaired righting reflex, (4) hind limb hypotonia, (5)

partial hind limb paralysis, (6) complete hind limb

paralysis.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

2 C. Bolton et al.

(+) MK-801 treatment regime

The dose of (+) MK-801 used was based on previous

studies [4] where the drug was prepared, in sterile PBS

vehicle, to a final concentration of 0.03 mg/mL. (+)MK-801 was administered, once daily and intraperito-

neally (ip), at a dose of 0.3 mg/kg body weight, from

the first day of body weight loss preceding the appear-

ance of relapse symptoms and for a further two consec-

utive days. An identical dosing regime was used for the

administration of vehicle.

Sampling of CNS tissues

Our recent studies have shown that the levels of PGE2and PGD2 were significantly, and exclusively, raised in

defined areas of the CNS, during the relapse phase of

CR EAE [20]. Therefore, CNS tissues were sampled, for

prostanoid determination, in the relapse stage of CR

EAE, 2 days after disease-associated body weight loss

and during the presence of neurological symptoms.

Mice were selected and randomly assigned to each

group, on the first day of disease-associated body

weight loss, which occurred in CR EAE-sensitized ani-

mals between days 30 and 46 post-inoculation. All

mice were sampled on the fourth day after the initial

loss of body weight. Specifically, samples from undosed

mice were dissected 33 days to 49 days post-inocula-

tion (mean day of sampling: 42 ± 7) and between

40 days to 49 days post-inoculation from vehicle- and

(+) MK-801-treated mice (mean day of sampling:

45 ± 4 and 44 ± 4, respectively). The cerebral cortex,

cerebellum and whole spinal cord were dissected from

CO2-asphyxiated, CR EAE-diseased undosed, vehicle-

or (+) MK-801-treated mice, as previously described

[20]. Each tissue was washed, in situ, with chilled

PBS, containing 10 lg/mL of the PG inhibitor, indo-

methacin (Sigma-Aldrich), and, after dissection, was

snap-frozen and stored at �80 °C. A minimum of 5

animals were used/treatment.

Isolation of PGs from CNS tissues

PGs were extracted from snap-frozen CNS tissues as

described by Ayoub et al. [24]. The cerebral cortex,

cerebellum and spinal cord, from individual mice, were

pulverized, using a nitrogen bomb (Biospec Products,

Bartlesville, OK, USA), suspended in 15% (v/v) ethanol

(pH 3.0), incubated for 10 min at 4 °C, and centri-

fuged, at 375 g, for 10 min at 4 °C. Supernatants were

collected and applied to ethanol/distilled water-condi-

tioned C-18 Sep-Pak separation columns (Waters Cor-

poration, Elstree, UK) and washed through with

ethanol/distilled water at a flow rate of 5 mL/min.

Sample fractions from each column were eluted with

ethyl acetate, at a flow rate of 5 mL/min and stored,

freeze-dried, at �80 °C until assayed for PG content.

Quantitation of PGE2 and PGD2 in CNS tissueextracts

PGE2 and PGD2 were measured, in extracted samples,

using commercially available immunoassay kits (Amer-

sham Biosciences, Amersham, UK, and Cayman Chemi-

cals, Ann Abor, MI, USA, respectively), according to

the manufacture’s instructions and as previously

described [24]. Briefly, each sample for PGE2 assay was

solubilized in sample buffer, incubated with goat anti-

mouse IgG plus antibodies against unlabelled and

horseradish peroxidase-labelled PGE2, followed by the

development of product with 3,3′,5,5′tetramethylbenzi-

dine substrate. The PGE2 content of each sample was

determined using a Tecan GENios microplate reader

(Jencons, Leicestershire, UK), at 630 nm, and by refer-

ence to a standard curve with concentrations between

0.05 and 6.4 ng/mL.

PGD2 was determined by initially stabilizing the pro-

stanoid in each sample, through the addition of meth-

oxylamine hydrochloride, to generate the methoxime

derivative. Samples were incubated with anti-PGD2

antibody and acetylcholinesterase-linked PGD2-meth-

oxime tracer followed by the development of product,

using Ellman’s reagent, and microplate reading at

405 nm. The PGD2 level in each sample was calcu-

lated using a standard curve with a concentration

range between 0.004 and 0.5 ng/mL.

Statistical analysis

Results were analysed using GraphPad Prism 3.0

(GraphPadPrism Software Inc., San Diego, CA, USA)

and expressed as mean ± standard deviation (S.D.).

Data were subjected to statistical examination using

the Alternate t-test and assuming Gaussian population

with different S.D. where P � 0.05 was considered

significant.

RESULTS

The emergence and development of CR EAE

Figure 1 illustrates the neurological course for the

acute phase of CR EAE in Biozzi mice preceding the

random assignment of animals to the undosed, vehicle

or (+) MK-801 treatments. Disease symptoms appeared

17 days after the initial inoculation, then peaked


(+) MK-801 influences prostaglandin levels in the CNS during experimental autoimmune encephalomyelitis 3

between 4 and 5 days later, and had completely

resolved by day 29 post-inoculation. Figure 1 also

shows the mean neurological scores, over the period of

sampling, for mice in the undosed, vehicle and drug

treatments were 3.2 ± 2.4, 2.0 ± 1.6 and 2.8 ± 2.5,

respectively, indicating tissue dissection, during the ini-

tial stages of the relapse phase of CR EAE and confirm-

ing no significant differences between the groups.

PGE2 levels in CNS tissues sampled from controland (+) MK-801-treated mice during the relapsephase of CR EAE

Figure 2 a–c shows the mean PGE2 content of the cere-

bral cortices, cerebellums and spinal cords from nor-

mals and undosed, vehicle- and (+) MK-801-treated

mice with relapse symptoms of CR EAE. The amounts

of PGE2 in the cerebral cortices from animals receiving

(+) MK-801 were reduced, but not significantly, com-

pared to normal and control tissues (Figure 2a). The

concentration of PGE2 in the cerebral cortices was

approximately tenfold higher than other areas of the

CNS and concurs with earlier findings on the distribu-

tion of the prostanoid in brain [25].

Cerebellums from undosed mice with relapsing dis-

ease contained significantly more PGE2 than levels

recorded in samples from normal animals (P < 0.001)

(Figure 2b). Tissues from mice receiving vehicle had a

lower concentration of PGE2, compared to the undosed

group, and the value was further reduced, but not sig-

nificantly, in the (+) MK-801 treatment. Similarly, the

levels of PGE2 in the spinal cords from vehicle- and (+)MK-801-treated mice were lower compared to the

undosed diseased group (Figure 2c). In contrast, compa-

rable concentrations of PGE2 were measured in spinal

tissues from undosed and normal animals.

PGD2 levels in CNS tissues sampled from controland (+) MK-801-treated mice during the relapsephase of CR EAE

The mean PGD2 content of cerebral cortices, removed

from undosed mice with relapsing disease, was raised,

but not significantly, compared to samples from nor-

mals (Figure 3a). Vehicle treatment lowered prostanoid

concentrations to normal values, and levels were fur-

ther reduced in tissues from (+) MK-801-dosed mice.

PGD2 was significantly raised in cerebellums from

undosed mice, compared to normals (P < 0.05) (3B).

Vehicle treatment significantly lowered the amount of

PGD2, compared to the undosed group (P < 0.05), but

dosing with (+) MK-801 did not significantly alter the

prostanoid compared to the control group. PGD2 levels

in spinal cords from undosed mice were significantly

less than measured in normal tissues (P < 0.02) and

a comparable value for the PG was recorded in the

vehicle treatment (3C). The concentration of PGD2 in

spinal tissues from mice receiving (+) MK-801 was sig-

nificantly reduced compared to the vehicle control

(P < 0.01). Cerebellum and spinal cord levels of PGD2

Figure 1 Neurological profile of the acute stage of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE), prior to the

assignment of mice to undosed, vehicle or drug treatments, following the remission of symptoms and during the early relapse phase of

disease. Mice were inoculated for CR EAE and assessed, from day 15 post-inoculation, for acute neurological deficits. Central nervous

system tissues were sampled from undosed mice between 33 and 49 days post-inoculation and from vehicle- and drug-treated mice

between 40 and 49 days post-inoculation. Mean neurological scores, during the sampling period, for mice in the undosed (mean day of

sampling: 42 ± 7), vehicle and drug treatments (mean day of sampling: 45 ± 4 and 44 ± 4, respectively) were 3.2 ± 2.4, 2.0 ± 1.6 and

2.8 ± 2.5, respectively.


4 C. Bolton et al.

were approximately 20-fold less than detected in cere-

bral cortex samples.

DISCUSS ION

The current study has measured PGE2 and PGD2 levels,

in specific areas of the CNS from CR EAE-sensitized

mice, during the relapse phase of the disease. In partic-

ular, the effect of treatment with the NMDA receptor

antagonist, (+) MK-801, on prostanoid concentrations

in selected CNS tissues was examined. The amounts of

PGE2 and PGD2 measured in cerebral tissues from nor-

mals and undosed, diseased mice were comparable. In

contrast, both prostanoids were increased in cerebellar

(a)

(b)

(c)

Figure 2 Prostaglandin E2 levels in the cerebral cortices (a),

cerebellums (b) and spinal cords (c) from normals and undosed,

vehicle- or (+) MK-801-treated mice during the relapse phase of

CREAE. Vehicle or (+) MK-801 was administered, at 0.3 mg/kg

body weight, to a minimum of 5 mice/treatment, for 3 days, from

the first day of relapse-associated body weight loss. Each snap-

frozen tissue was pulverized, suspended in 15% (v/v) ethanol and

centrifuged. Supernatants were added to C-18 Sep-Pak separation

columns to enable PGE2 extraction and quantitation by

immunoassay. Undosed vs. normal: ***P < 0.001.

(a)

(b)

(c)

Figure 3 PGD2 levels in the cerebral cortices (a), cerebellums

(b) and spinal cords (c) from normals and undosed, vehicle- or (+)

MK-801-treated mice during the relapse phase of CREAE. Vehicle

or (+) MK-801 was administered, at 0.3 mg/kg body weight, to a

minimum of five mice/treatment, for 3 days, from the first day of

relapse-associated body weight loss. Each snap-frozen tissue was

pulverized, suspended in 15% (v/v) ethanol and centrifuged.

Supernatants were added to C-18 Sep-Pak separation columns to

enable PGD2 extraction and quantitation by immunoassay.

Cerebellum: undosed vs. normal *P < 0.05, vehicle vs. undosed

#P < 0.05; spinal cord: undosed vs. normal **P < 0.02, (+) MK-

801 vs. vehicle ##P < 0.01.



samples from animals with relapsing symptoms and,

unexpectedly, the concentration of PGD2 was signifi-

cantly lowered by vehicle treatment. PGE2 levels in

spinal cords from undosed diseased mice were similar

to normals but the amounts of PGD2 were significantly

less. (+) MK-801 therapy did not significantly alter pro-

stanoid concentrations in cerebral or cerebellar tissues.

Similarly, PGE2 levels in spinal cords from drug-dosed

mice were unchanged. However, treatment with (+)MK-801 did significantly reduce the amounts of PGD2

in spinal tissues.

Initial studies and recent investigations by us have

described major changes in CNS prostanoid concentra-

tions in several models of EAE [20–23]. In particular,

our latest work revealed significant alterations in the

PGE2 and PGD2 content of the cerebral cortex, cerebel-

lum and spinal cord from mice, exclusively, during the

relapse rather than the acute stage of CR EAE [20]. PG

synthesis is ultimately governed by COX enzyme activ-

ity but the preceding mechanistic events that deter-

mine prostanoid generation and which occur during

the course of EAE are not defined. Studies by Nair and

Bonneau [6], using a restraint model of stress, have

suggested that brain PG production is regulated by

NMDA receptor activation that may also influence COX

enzyme expression in CNS tissues [19,26]. Moreover,

the investigations demonstrated, through the use of the

anti-glucocorticoid RU486 and (+) MK-801, that

in vivo upregulation of the NMDA receptor is achieved,

primarily, via stress-induced, elevated glucocorticoid

levels. Finally, the work established, through pharma-

cological intervention, that endogenous steroid-induced

NMDA receptor stimulation culminates in microglial

activation.

Previous studies by us and others have shown that

the neurological symptoms of acute and CR EAE are

associated with a dramatic rise in circulating glucocor-

ticoids [8–10] and, particularly in the latter model,

alterations in microglial morphology and activity [27–29]. We have also established, through the use of (+)MK-801, that NMDA receptor-dependent mechanisms

are involved in the pathogenesis of EAE and, in partic-

ular, BBB breakdown and neurological disease [2,4].

Therefore, it is feasible that PG production in the CNS,

during EAE, is controlled by glucocorticoid–NMDA

receptor-linked events that ultimately determine patho-

logical alterations, including microglial changes, in tar-

get tissues.

However, the current study has revealed that the

previously established rise in peripheral corticosteroid

levels during the relapse phase of CR EAE [10] does

not necessarily lead to a corresponding and uniform

increase in prostanoid production in CNS tissues from

diseased mice. The distribution of the NMDA receptor

in brain and spinal cord is ubiquitous with expression

on the vast majority of neurons and glial cells [30].

Interestingly, the cerebral cortex, composed predomi-

nantly of grey matter, is a particularly rich source of

the receptor [31], and the present study has recon-

firmed substantial quantities of prostanoids are gener-

ated. Despite the potential for synthesis, the amounts of

PGE2 and PGD2 in cerebral samples were not signifi-

cantly elevated during relapse and neither vehicle or

(+) MK-801 treatment appreciably altered the levels,

implying endogenous glucocorticoids do not effect an

increase in prostanoid output and production does not

appear to be governed by NMDA receptor activation.

The cerebellum is another region of the CNS richly

populated by the NMDA receptor [32]. Characteristi-

cally, the receptor consists of the NR1, NR2A to NR2D

and NR3 subunits, featuring inhibitory and stimulatory

sites for a variety of compounds including polyamines

and a range of pharmacological agents but, intrigu-

ingly, cerebellar-located NMDA receptors differ in a

variety of functional parameters, compared to those

found in other areas of the brain [1, 33]. For example,

pharmacological interactions and drug effects, via

specific subunit sites, are more apparent in cerebellar-

derived NMDA receptors. In particular, the NR2 domain

contains a steroid-binding site that facilitates selective

interaction between either locally produced neuroster-

oids or peripherally derived steroidal-type compounds

[34, 35]. Consequently, the interaction between endog-

enous glucocorticoids and cerebellar NMDA receptors

may be enhanced and thereby account for the tissue-

specific, disease-related increase in PGE2 and PGD2 con-

centrations observed in the current study.

A heightened response of cerebellar-located NMDA

receptors to the glucocorticoids may also account, in

part, for the significant effect of vehicle treatment on

PGD2 levels. Earlier work, by us, described the modula-

tory effects of ip vehicle administration on biochemical

and neurological aspects of EAE [36], and we attrib-

uted the alterations, at least in part, to an exogenous

stressor-induced elevation in glucocorticoid levels

which, as previously reported [6], would be expected to

raise prostanoid concentrations via NMDA receptor

activation. Importantly, Perez-Nievas et al. [37] have

recently demonstrated that stress, during acute EAE,

increases circulating corticosteroids and, depending on


6 C. Bolton et al.

the length of exposure, may have a positive or negative

influence on CNS prostanoid levels. Indeed, steroidal

compounds have the ability to interact directly or indi-

rectly with inhibitory or potentiating sites on the

NMDA receptor and, in particular, operate through tar-

geted interaction at the NR2-associated neurosteroid

modulatory recognition site [35]. Therefore, the unex-

pected but significant reduction in PGD2, following ip

vehicle dosing, may be related to the combined profiles

and effects of early relapse- and dosing regime-induced

stress on cerebellar-hypersensitive NMDA receptors

together with the site-specific modulatory actions of

endogenous glucocorticoids or, indeed, locally synthe-

sized neurosteroids [38].

Interestingly, (+) MK-801 treatment failed to signifi-

cantly reduce cerebellar PG concentrations, compared

to vehicle controls, suggesting a lack of NMDA receptor

involvement and implying the altered PGD2 level in the

control treatment results from either glucocorticoid-

dependent pathways that operate via non-NMDA

receptor routes or glucocorticoid-independent mecha-

nisms able to influence prostanoid production. In sup-

port of these suggestions, Garcia-Bueno et al. [39]

have shown, in CNS tissues and through the use of

pharmacological tools, that stress-induced glucocortic-

oids are able to act directly, via corticoid receptors, to

regulate PG synthesis. The studies also revealed that

catecholamines, which are raised during EAE [40],

have the ability to control prostanoid levels by reduc-

ing COX expression. Alternatively, and as previously

considered, the raised glucocorticosteroid levels, result-

ing from either disease or non-disease-related, stress-

associated effects, may preferentially and negatively

regulate prostanoid production, via NMDA receptor

interaction, and, as a result, negate the effects of (+)MK-801 administration.

Consequently, the effects of exogenous stressors, such

as ip drug administration, on levels of endogenous bio-

chemical mediators that influence PG synthesis, partic-

ularly during the relapse phase of CR EAE, may, in

addition, account for the selective, unpredicted and sig-

nificantly reduced PGD2 concentrations in spinal cords

from undosed and vehicle-treated, diseased mice. Inter-

estingly, levels of the prostanoid in spinal tissues were

further and significantly lowered after (+) MK-801

treatment indicating NMDA receptor involvement in

PGD2, but not PGE2, production.

In summary, our previous investigations established

that major changes in CNS prostanoid levels occur,

predominantly, during the relapse period of CR EAE,

which coincides with a surge in endogenous glucocor-

ticoid levels and ultimately, as shown by others, dra-

matic microglial pathology. Related in vivo studies, in a

non-immune model of stress, demonstrated that

microglial activation, via possible PG generation, was

dependent upon preceding glucocorticoid-induced

NMDA receptor-linked events. However, the current

work does not support the existence, at least during

the relapse phase of CR EAE, of a major pathway fea-

turing endogenous steroid-mediated NMDA receptor-

associated events that lead to significant alterations in

prostanoid production and which may be responsible

for the documented changes in microglial behaviour.

Finally, CR models of EAE are characterized by dra-

matic pathological changes in CNS tissues and, in par-

ticular, widespread and severe neuronal disruption

which, speculatively, and under current investigation,

may alter receptor function and potential antagonism

and thus account for the specific effects of (+) MK 801

on prostanoid levels in defined areas of the CNS [27,

41, 42].

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial sup-

port of The William Harvey Research Foundation and

The Leverhulme Trust for provisions of funds for Dr

Ayoub.

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N-methyl-D-aspartate (NMDA) receptor involvement in central nervous system prostaglandin production during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis